Rice, wheat and barley comparative genomics to …...Rice, wheat and barley comparative genomics to identify new molecular markers for dwarfing This thesis is submitted in fulfillment

Rice, wheat and barley comparative

genomics to identify new molecular

markers for dwarfing

This thesis is submitted in fulfillment of the requirement for the degree of Bachelor of Science with Honours (Hons) in

Biotechnology by

Jingjuan Zhang

Western Australian State Agricultural Biotechnology Centre School of Biology and Biotechnology Division of Science and Engineering

Murdoch University Perth, Western Australia

November 2004

ii

Declaration

I declare that this thesis is my own account of my research and contains as its main

content work which has not previously been submitted for a degree at any tertiary

education institute.

Jingjuan Zhang

iii

ABSTRACT

By using bioinformatics, the barley semi-dwarfing gene, sdw1, was found to be

similar in its structure to that of the semi-dwarfing gene, sd1, in rice. Part of exon1,

whole of exon2 and intron 1, and part of exon3 of this gene sequence was isolated

from barley and the sequences of twelve barley varieties were compared in intron1

and exon2 regions. The similarity and differences of DNA and protein sequences

between sdw1 (barley) and sd1 (rice) were compared. The most likely GA20-oxidases

in wheat, barley and rice were analysed and discussed. The sequences of intron1 and

exon2 obtained from these experiments indicates that the respective regions are most

unlikely to be the source of the differences in sdw1 status between the varieties tested.

Further experiments will be used to develop a diagnostic marker to distinguish

different sdw1 alleles in order to modify pleiotropic effects on other traits such as long

coleoptile, strong initial vigour combined with suitable kernel shape and size.

iv

ACKNOWLEDGEMENT

This thesis was completed between September 2003 and November 2004 during my

part-time (two semesters) and full-time (one semester) study in the Division of

Biological and Engineering Science, Murdoch University.

I would like to take this opportunity to thank the people who have helped me. First of

all, I would like to express my sincerest gratitude to my supervisors, Professor Rudi

Appels and Dr. Chengdao Li, for their continuous guidance, assistance, teaching and

encouragement. Their supervision has made a significant contribution to the writing

of this essay.

Secondly, I would like to thank Sharon Westcott. She taught me many basic technical

skills and also taught me how to write the thesis. And also I would like to give my

gratitude to Dora Li, Fiona Drake-Brockman, Meredith Carter, Gabrielle Devlin,

Natasha Teakle, Danielle Cash, Julie Uhlmann, Esther Walker, Vera Limadinata etc.

In addition, I would like to give my appreciation to Natasha Teakle, Mehmet Cakir,

and my English teacher Colin Beasley and Marie Arandiga for checking my thesis.

Thirdly, I give my gratitude to Dr. Michael Francki and Mr. Dave Hodgson for their

help.

Lastly, I thank my husband Dr. Shaobai Huang and my daughter Sarah Huang for

their support.

v

List of Abbreviations 1.Units of measurement % percent °C degree of Celsius bp base pair hr hour Kb kilobase l litre mg milligram min minute ml milliliter mM millimolar ng nanogram pmol picomoles rpm revolutions per minute s second U unit μ micro μg microgram μl microlitre Vol volume 2. Abbreviations used in the text 3' hydroxyl-terminus of DNA molecule 5' phosphate-terminus of DNA molecule A adenine or adenosine C cytosine or cytidine C-terminus carboxy terminus cDNA complementary DNA chr. chromosome chs chalcone synthase dATP deoxyadenine triphosphate dCTP deoxycytosine triphosphate dGTP deoxyguanine triphosphate DNA deoxyribonucleic acid Dnase deoxyribonuclease dNTP deoxynucleoside triphosphate E.coli Escherichia coli EDTA ethylenediamine tetraacetic acid G guanine or guanosine HCl hydrochloric acid

vi

LB Luria-Bertani mRNA messenger ribonucleic acid N-terminus amino terminus NaCl sodium chloride PCR polymerase chain reaction RNA ribonucleic acid RNase ribonuclease Pro. production SDS sodium dodecyl sulfate T thymine or thymidine TBE Tris-Boric-EDTA buffer Taq Thermus aquaticus DNA plymerase TE tris-EDTA U Unit UV ultra violet X times or signal of a number + and × cross

vii

TABLE OF CONTENTS

DECLARATION ii

ABSTRACT iii

ACKNOWLEDGEMENTS

iv

LIST OF ABBREVIATIONS

v

TABLE OF CONTENTS vii

General introduction 1

Chapter1: Literature review 3

1.1 Food challenge and ‘green revolution’ 4

1.1.1 Food challenge 4

1.1.2 Dwarfing plants contribute to ‘Green revolution’ 4

1.2 Dwarfing genes in rice, barley, maize and wheat 6

1.2.1 Dwarfing genes in Rice 6

1.2.2 Dwarfing genes in Barley 7

1.2.3 Dwarfing genes in Maize 7

1.2.4 Dwarfing genes in Wheat 8

1.3 Mechanism of dwarfing genes 8

1.3.1 Mechanism of dwarfing gene 8

1.3.1.1 ent-kaurene synthesis pathway (terpene cyclases) and related genes

10

1.3.1.2 Monoxygenese pathway and related genes 12

1.3.1.3 Dioxygenase pathway and related genes 13

1.3.1.3.1 GA 20-oxidases 14

1.3.1.3.2 GA 3β-hydroxylases 14

viii

1.3.1.3.3 GA 2-oxidases 15

1.3.2 Mechanism of GA sensitive and insensitive mutants 17

1.3.3 Other GA mutants 18

1.3.4 GA-sensitive and GA-insensitive mutants in maize, wheat, rice and barley

18

1.3.4.1 GA-sensitive mutants 18

1.3.4.2 GA-insensitive mutants 19

1.4 Identify dwarfing genes by using comparative genomics 19

1.4.1 General gene comparative bioinformatics in plants 19

1.4.2 Collinearity between rice and wheat 21

1.4.3 Close relationship between wheat and barley 21

1.4.3.1 Wheat A, B and D genomes are homologous 21

1.4.3.2 Wheat shows synteny with barley 22

1.4.4 Dwarfing gene comparison in rice, wheat and barley 23

1.4.5 Synteny among several dwarfing genes among maize, rice, wheat and barley

24

1.4.5.1 Comparative analysis between Dwf2 (barley)and RhtB1, RhtD1 (wheat)

24

1.4.5.2 Colinearity between D8 (maize) and RhtD1 or RhtB1b (wheat)

25

1.5 Application of dwarf genes in conventional breeding 27

1.5.1 Utilization of dwarf genes for breeding 27

1.5.2 Challenges for breeding dwarfing variety 30

1.6 Summary of literature review 31

1.7 Aims of this study and project outline 32

1.7.1 Outline of this project 32

1.7.2 Outputs from the thesis 33

ix

Chapter 2 Material and method 34

2.1 Plant material preparation 35

2.1.1 Plant materials 35

2.1.2 DNA extraction 35

2.1.3 Fluorometric quantification of DNA 36

2.2 General methods 36

2.2.1 Primer Design 36

2.2.2 Polymerase Chain Reaction (PCR) Amplification 38

2.2.3 Agarose gel electrophoresis 38

2.2.4 DNA purification 39

2.2.4.1 DNA purification from PCR product solution 39

2.2.4.2 DNA purification from agarose gels 40

2.2.5 pGEM®-T Easy vector systems 41

2.2.5.1 Preparation of chemically competent cells 41

2.2.5.2 DNA ligation into pGEM®-T Easy 42

2.2.5.3 E.coli transformation 42

2.2.5.4 Bacteria, media and growth medium 43

2.2.5.5 Antibiotics used 43

2.2.5.6 PCR screening E. coli transformants for insert DNA

43

2.2.5.7 Restriction digest screening 44

2.2.5.8 Purification of plasmid DNA for sequencing 44

2.2.6 PCR sequencing 45

2.2.7 Analysis of sequence data 46

2.3 Genome Walker 46

x

2.3.1 Size and quality measurement of genomic DNA 46

2.3.1.1 Size of genomic DNA on a 0.5% agarose/EtBr gel 46

2.3.1.2 Purity of genomic DNA by Dra 1 digestion 47

2.3.2 Digestion of genomic DNA 47

2.3.3 Purification of DNA 47

2.3.4 Ligation of genomic DNA to Genome Walker adaptor 48

2.3.5 Primer design and PCR amplification 48

Chapter 3 Results 51

3.1 Introduction 52

3.2 Proposed gene structure of sdw1 and primers for gene isolation

52

3.2.1 Synteny between dwarfing gene sdw1 in barley and sd1in rice

52

3.2.2 Proposed structure of gene sdw1 54

3.2.3 Homology identification and primer design 55

3.3 Primers designed for sdw1 57

3.3.1 Primer Ex1F1 and Gsp4 amplified part of exon1 of sdw1 gene

57

3.3.2 Amplification of second exon by primers sdwn2 and sdwex2R

58

3.3.3 Amplification of first intron and second exon by primers sdwn1 and sdwex2R

60

3.3.4 Primers Jex3F and Jex3R2 amplified part of exon3 sequence

62

3.3.5 Size of intron2 63

3.4 Barley semi-dwarfing gene-sdw1 gene structure 65

3.5 Sequence alignment and sequence nucleotide polymorphism identification

66

3.6 Preliminary studies to analyze promoter region- Genome walker

69

xi

3.7 Sequence amplification with one primer 71

3.8 Summary of results 73

Chapter 4 General Discussion 74

4.1 Overview of research goals 75

4.2 Lack of polymorphism in intron1 and exon2 of sdw1 among twelve varieties

75

4.3 Reasons for non completion sequence on exon1, exon3 and intron2 of sdw1

77

4.4 Amplification of the 500 bp band from the genome DNA of Dhow and Gairdner

77

4.5 sdw1 sequence analysis 78

4.5.1 DNA sequence and amino acid sequence comparison between sdw1 and sd1

78

4.5.2 sdw1 amino acid comparison with the GA20-oxidases of rice, wheat and barley

82

4.5.3 sdw1 intron1 sequence analysis 83

4.6 GA20-oxidase comparative bioinformatics 84

4.7 Function of GA20-oxidases 87

4.8 Value of sdw1 in barley breeding 89

4.9 Important aspects of experimental work 90

4.9.1 Single primer positive control 90

4.9.2 Achieving effective ligation results 91

4.9.3 Using part of exon sequence to identify the correct intron band size

91

4.10 Conclusion 92

4.11 Future research 93

5. References 94

Appendices 103

xii

Appendix 1: Rice dwarfing genes 104

Appendix 2: Barley dwarfing genes 111

Appendix 3: Maize dwarfing genes 125

Appendix 4: Wheat dwarfing genes 133

Appendix 5: Figures of gene bioinformatics 134

Appendix 6: Intron1 blast 136

Appendix 7: sd1 DNA and protein sequense

137

1

Introduction

The rapidly increasing population creates a big challenge to world agriculture.

Advances in grain yield are the main ways to solve the problem due to the limit of

available and suitable land for crops. Large increases in yield have resulted from

reduced plant height (dwarfing plants), due to lodging resistance, improved harvest

index, and more efficient utilisation of the environment. Dwarfing plants have

contributed to the ‘Green Revolution’ (Milach and Federizzi, 2001).

Because of the importance of dwarfing plants, a significant amount of research has

been done to understand the biochemical basis, genetics, physiology, and pleiotropic

effects of the dwarf phenotypes in different plant species. Extensive research has been

done on cereal crops, particularly with wheat and rice, and one of the few reviews on

this topic with emphasis on all these aspects is that of Gale and Youssefian (1985) in

wheat. Besides that, because of the involvement of dwarfing genes with gibberellin

biosynthesis, fundamental research has been conducted with dwarf mutants involved

in the GA biosynthetic and signal transduction pathway. Both of these aspects have

been reviewed recently by Hedden and Kamiya (1997), Hedden and Phillips (2000),

Ross et al. (1997), Swain and Olszewski (1996), and Sakamoto et al. (2004).

The use of the dwarfing plant type has developed to the point that a thousand varieties

of different species are short in stature. Several major genes affecting plant height

have been reported for wheat, rice, and barley. From all dwarf mutants identified, the

most important ones for breeding purposes have been those from ‘Norin 10’ in wheat

2

and ‘IR8’ in rice because of their positive effects over other important agronomic

traits.

This thesis attempts to summarize the main findings about the dwarfing genes in rice,

wheat, barley and maize, by investigating the relationships between different dwarfing

genes of different crops with similar mechanisms. This project will emphasize the

importance of genome structure comparative analysis to increase the efficiency of

mapping and sequencing of dwarf genes. The results of dwarfing gene comparison

among cereals will be very useful for the isolation of new dwarfing genes. By using

gene bioinformatics and molecular biotechnology, one unknown but very valuable

gene was partly isolated in barley and the part sequences of this gene were compared

among twelve barley varieties. The differences in DNA and protein sequences of the

most likely gibberellic acid (GA) 20-oxidase genes among rice, wheat and barley, and

the value of the new isolated gene are discussed. Molecular technique approaches

were developed during this research.

3

Chapter 1

Literature Review

4

1.1 Food challenge and ‘Green Revolution’

1.1.1 Food challenge

According to United Nations estimates (1991), the world population reached 5.3

billion in 1990, increasing 70% from 1960. In 2025, the world population is predicted

to reach 9 billion. The high population will provide an enormous challenge to world

food supply.

There are many factors which contribute to the increases in crop yields, for example,

improved genetic potential, increased fertilizer usage and the expansion of the area

under irrigation. In Africa and Latin America, there are still large areas of land which

are suitable for crop production. However, in Asia and Western Europe, most of the

suitable land has been utilised (Mitchell et al., 1997). Without new land being made

available, crop yields have to increase considerably to meet the challenge of the food

requirement of the expanding world population. Between 1950 and 1990, the cereal

production of the world increased by 185% with 90% of the increase due to increased

yield and only 10% due to increased land area (Mitchell et al., 1997).

1.1.2 Dwarfing plants contribute to the ‘Green Revolution’

Dwarfing modifications have been reported since the early 1900s (Milach and

Federizzi, 2001). However, most of these modifications were described as genetically

heritable curiosities without practical applications in agriculture (Milach and

Federizzi, 2001). Nevertheless, in Japan, farmers have used wheat varieties with plant

5

height of 50-60 cm since 1873 (Milach and Federizzi, 2001). Japanese breeders

selected dwarfing wheat ‘Norin 10’ in 1935 (Hanson et al., 1982). In 1953, Voguel

from Washington State sent lines of the cross ‘Norin 10 × Brevor 14’ to Norman

Borlaug at CIMMYT in Mexico. Borlaug used them intensively to develop new types

of wheat that became responsible for the ‘Green Revolution’ (Milach and Federizzi,

2001) (Fig. 1.1).

Figure 1. 1 The processes of the ‘Green Revolution’. Before the 1960’s, rice varieties were tall and lodged early, and did not respond to the

application of nitrogen. A recessive dwarfing gene was identified from a Chinese

variety called ‘Dee-geo-woo-gen’ (DGWG). The first variety developed was ‘IR8’ in

1966 that also inherited important traits such as sturdy stems, heavy tillering, dark-

green and erect leaves from the other parent. Due to the success of this new plant type

of rice, short varieties have been well developed and are used in most of the areas

cultivated with rice (Kush, 1993). The short and sturdy stem of high yield varieties

(HYVs) with dwarfing genes allows the plants to carry the increased grain resulting

from heavy fertilizer application (Mitchell et al., 1997) and lead to the 'Green

Daruma × Fultz (US, 1917) Turkey (Japan 1925) ×

Norin 10 (1935 developed in Japan, 1946 transferred to the USA)

Norin 10

Gaines (1962)

× Brevor 14

Lines of the cross (sent by Voguel in 1953)

Norman Borlaug (CIMMYT)

Green Revolution

6

Revolution' of the 1960s and 70's. During that period, world yields almost doubled

(John Innes institute annual report, 1999).

1.2 Dwarfing genes in rice, barley, maize and wheat

1.2.1 Dwarfing genes in rice

About 75 dwarfing genes have been listed in the Oryzabase (Integrated Rice Science

Database, 2003). According to the list, d-10, d-15 and d-16; d-54 and d k5; d-55 and

d-k-6; sd-1 and d-47; d-32, d-k-4 and d-12; d-29 and d-k-1; d-30 and d-w; D-52 and d-

k-2; d56 and d-k-7; d-11 and d8; nak5 and nak1; d-6 and d34; d57 and d(x); d-27 and

d-t; d-28 and d-c; D-53 and D-L-3; d-33 and d-B; d-12 and d50; Bsv and Bs are allelic

respectively. Most of them are governed by a single recessive gene, with the

exception of three dominant genes, D-53, D-h (t)*, Bsv (Bs) and two incomplete

dominant genes, Dm1* and Ssi1 (Dm1)*. The allelic dwarfing genes at sd-1 locus

have the most practical value in rice breeding regardless of their source of origin

(Sasaki et al. 2002)

Among the dwarfing genes, both d18 (including its alleles: d18k, d18h, d18-AD and

d18dy) and d35 are known to have a deficiency in their gibberellin biosynthetic

pathway (Murakami, 1995; Futsuhara, 1995). The d1 was classified as a gibberellin –

insensitive mutant (Mitsunaga et al., 1994) and the sd1 was classified as a GA-

sensitive mutant (Sasaki et al., 2002). Forty eight dwarfing genes have been assigned

to chromosomes and the characteristics of the genes, the variety names and the

references are provided. (See Appendix 1)

7

1.2.2 Dwarfing genes in barley

The collection of barley (Hordeum vulgare L.) semi-dwarfs is a diverse group of

cultivars, breeding lines, and induced mutants in which plant height, spike density,

seed shape, or awn length are modified compared to parental lines or closely related

cultivars. Entries in the barley semi-dwarf collection were assigned a dwarf stock

number (DWS) which was preceded by the number 1001 to avoid confusion with

other sets of numbers assigned to breeding program lines (Franckowiak and Pecio,

1992). Some mutant genes have been well characterized by Davis et al (1997) and

some were assigned with GSHO numbers which were held at the USDA_ARS

National Small Grains Germplasm Research Facility, Aberdeen ID. The listing of

dwarfing genes was revised in BGS descriptions (barleygenomics website, 2002).

Three hundred and eighty five dwarfing genes are listed and some genes with the

same BGS number are alleles. One hundred and fourteen dwarfing mutants (including

alleles) have been assigned to chromosomes. Forty-three dwarfing mutants are

reported to be GA insensitive and two hundred and forty nine dwarfing mutants

reported are GA sensitive. (See Appendix 2)

1.2.3 Dwarfing genes in maize

Maize dwarfing genes were obtained from http://www.maizegdb.org/cgi-

bin/displayphenorecord.cgi?id=11041. Two hundred and nineteen dwarfing genes are

listed and thirty-four dwarfing genes have been assigned to chromosomes. Twenty-

8

three dwarfing genes are reported to be GA sensitive and eight dwarfing genes are GA

insensitive. (See Appendix 3)

1.2.4 Dwarfing genes in wheat

In wheat, twenty-four dwarfing genes (including alleles) have been reported. On

chromosome 4BS, wild type rht-b1a has five dominant dwarf alleles: Rht-B1b, Rht-

B1c, Rht-B1d, Rht-B1e and Rht-B1f. On chromosome 4DS, three dominant dwarfing

alleles (Rht-D1b, Rht-D1c and Rht-D1d) were reported to be collinear with the wild

type rht-D1a (Börner et al., 1996). Eight dwarfing mutants (including alleles) are GA

insensitive and sixteen dwarfing genes are known to be GA sensitive. Twelve

dwarfing mutants (including alleles) have been located on specific chromosomes.

(See Appendix 4)

1.3 Mechanism of dwarfing genes

1.3.1 Mechanism of dwarfing gene function

Most dwarfing genes reduce the plant height by reducing of gibberellin (GA)

production or by no response to GA. GA is a plant growth hormone and an essential

endogenous regulator of plant growth. GA sensitive dwarf mutants are defective in

different steps in the GA biosynthetic pathway and respond to the exogenous

application of gibberellinic acid (Phinney, 1984). GA insensitive dwarf mutants

accumulate GAs and are mostly unresponsive to applied GA (Ross et al., 1997).

There are some exceptions, such as barley dwarfing gene uzu, rice dwarfing gene d61,

9

and garden pea (Pisum sativum) dwarfing lka and lkb, which reduce the plant height

by brassinosterioids (BRs) (Chono et al., 2003)

Nearly 100 different gibberellins have been identified from plants and several fungi

(Sponsel, 1995) due to the structure of the GAs (19 or 20 carbons) combined with

different patterns of hydroxylation and other modifications. Some are intermediates to

‘biologically active’ GAs such as GA1, GA3, GA4 and GA7 and some are inactive

products, such as GA8, GA29 and GA34 (Hedden et al., 1978; Hedden and Phillips,

2000). The number of GAs present in any one species is limited (Bearder, 1980).

Furthermore, it is becoming apparent that only one endogenous GA may be active for

a particular organ. For example, the available data suggest that GA1, a member of the

early 13-hydroxylation pathway that originates as a branch from GA12-aldehyde

(Fig.1.2), could be the only native GA active in the control of shoot elongation in

maize (Phinney and Spray, 1982).

Figures 1.2 and 1.3 show the GA biosynthetic pathway in plants, in which three

sections are involved according to the nature of the enzymes: (1) the terpene cyclases,

ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS), catalyse the

initial cyclisation of geranylgeranyl diphosphate to ent-kaurene; (2) intermediate steps

in the pathway are catalysed by cytochrome P450 monooxygenases, including ent-

kaurene oxidase (KO) and ent-kaurenoic acid oxidase (KAO); (3) the late steps that

result in the synthesis of active GAs are catalysed by 2-oxoglutarate-dependent

dioxygenases, including 20-oxidase (20ox), 3-oxidase (3ox) and 2-oxidase (2ox). The

three sections occur in plastids, membranes outside the plastid and cytoplasm

respectively (Hedden and Phillips, 2000).

10

Figure 1.2: Biosynthetic pathway from mevalonic acid (MVA) to the GA precursor, GA12- aldehyde. GGDP is produced in plastids by the isoprenoid pathway, originating from mevalonic acid or, possibly, pyruvate/glyceraldehydes 3-phosphate (Figure is from Hedden and Kamiya, 1997; Hedden and Phillips, 2000; Hedden et al. 2002). 1.3.1.1 ent- Kaurene synthesis pathway (terpene cyclases) and related genes

The hydrophobic ent-kaurene is synthesized by the two-step cyclization of

geranylgeranyl diphosphate (GGDP) via the intermidiate, ent-copalyl diphosphate

(CDP). The enzymes that catalyze these reactions are copalyl diphosphate synthase

(CPS) and ent-kaurene synthase (KS). The biosynthesis of GGDP is from mevalonic

acid by many terpenoid pathways, which might involve pyruvate and

glyceraldehydes-3-phosphate in higher plants (Hedden and Kamiya, 1997; Hedden

CPS KS

CPS:ent-copalyl diphosphate synthase

KS: Kaurene synthase

Ent-kaurenoic acid 7β-hydroxylase (KAO) Ent-kaurenoic acid

7β-hydroxylase (KAO)

Ent-kaurene oxidase (KO)

Ent-kaurene oxidase (KO)

Ent-kaurenic oxidase (KO)

11

and Phillips, 2000; Hedden et al. 2002). The maize dwarfing gene An1 (anther ear-1)

located on 1L is a CPS gene due to its 51% sequence identity with the CPS of

Arabidopsis gene GA1 and 20% ent-kaurene content of the wild-type maize (Hedden

and Kamiya, 1997). In rice, genes OsCPS1 and OsCPS2 are located on Chromosome

2S and 2L respectively. Genes OsCPS3 and OsCPS4 are located on chromosomes 9S

and 4S respectively (Sakamoto et al., 2004). In barley, the gene HvSPSL1 was

mapped on 7H and also had orthologues bands on wheat chromosome 7L (Spielmeyer

et al., 2004).

The enzyme, ent-Kaurene Synthase (KS), was purified from the endosperm of

pumpkin (Saito et al., 1995). The isolated full-length cDNA was expressed in E. coil

as a fusion protein, with maltose-binding protein, which converted [3H] CDP to ent-

[3H] kaurene. The amino acid sequence of KS shares 51% similarity with CPS from

Arabidopsis and maize, but CPS lacks the DDXXD motif, which is proposed to be a

binding site for the divalent metal ion-diphosphate complex (Hedden and Kamiya,

1997; Hedden and Phillips, 2000; Hedden et al. 2002). In rice, OsKS1, OsKS2, OsKS3

were arranged as tandem direct repeats at 104 centimorgan (cM) on chromosome 4L.

OsKS4 was located near to the position of OsCPS4 on chromosome 4S. OsKS5,

OsKS6, and OsKS7 were arranged within 150kb at 86 cM of chromosome 2L, where

OsCPS2 is also located. OsKS8 and OsKS9 were located on chromosome 11L in the

same direction (Sakamoto et al., 2004). In barley, HvKSL1 is located on 2HL and also

the three homoloeologous gene are on the wheat group 2L. Furthermore, the HvKSL1

protein sequence has a closely related rice sequence OsKS1A (accession No.:

AY347876) (rice 4L) in the Genbank database, which encodes a KS gene (Spielmeyer

et al., 2004).

12

1.3.1.2 Monoxygenase pathway and related genes

The highly hydrophobic ent-kaurene is oxidized by membrane-bound

monooxygenases to GA12. In higher plants, the three steps from ent-kaurene to ent-

kaurenoic acid are assumed to include one enzyme, which is ent-kaurene oxidase

(KO). Kaurenoic acid 7β-hydroxylase (KAO) is supposed to be involved in another

three steps from ent-kaurenoic acid to ent-7α-hydroxy kaurenoic acid and from ent-

7α-hydroxy kaurenoic acid to GA12-aldehyde and then to GA12 (Hedden and Kamiya,

1997; Hedden and Phillips, 2000; Hedden et al. 2002). In rice, all five KO like genes

were mapped on chromosome 6L, arranged in tandem as a gene cluster (Sakamoto et

al., 2004). Barley HvKO1 gene was mapped on 7H. One KAO-like gene (OsKAO)

was identified in rice and also was mapped on chromosome 6S (Sakamoto et al.,

2004). In barley, HvKAO1 was mapped on 7HS and this probe detected three

corresponding bands in the wheat group 7S (Spielmeyer et al., 2004).

13

Figure 1.3: Gibberellin-biosynthetic pathway from GA12-aldehyde in cytoplasm. GA1, GA3, GA4 and GA7 are biologically active Gas. GA8, GA29 and GA34 are shown as dead-end branches because of their biological inactivity (Hedden and Kamiya, 1997; Hedden and Phillips, 2000). 1.3.1.3 Dioxygenase pathway and related genes

Figure 1.3 shows the stages of GA biosynthesis which results in the synthesis of

active GAs by 2-oxoglutarate-dependent dioxgenases including GA 20-oxidases, 3β-

GA34

GA5

3 β -hydroxylases

GA7

GA 20-oxidase

2,3- didehydrolases GA9

GA 2-oxidase

GA15 GA24 GA9 GA4

GA25

Non-13-hydroxylation pathway

3β-hydroxylases

GA 20-oxidase

GA 20-oxidase

GA 20-oxidase GA 20-oxidase

GA 2-oxidise

3β-hydroxylases

3β-hydroxylases

GA12-aldehyde

GA12

GA53

GA44 GA19

GA20 GA1

GA8

GA 8-catabolite

GA3

GA17

GA29 GA 29-catabolite

GA 7-oxidase

13-hydroxylases

GA 20-oxidase GA 20-oxidase GA 20-oxidase 3β-hydroxylases

GA 2-oxidise

GA 2-oxidise

3β-hydroxylases Early 13-hydroxylation pathway

GA 2-oxidase

GA34

14

hydroxylases and 2β-hydroxylases. GA 7-oxygenases catalyzes the step from GA12-

aldehyde to GA12. Except for pumpkin, this step has not been found in other species

(Hedden and Phillips, 2000). Both 13-hydroxylation and non-13-hydroxylation

pathways have been demonstrated in vegetative tissues (Hedden and Kamiya, 1997).

1.3.1.3.1 GA 20-oxidases

GA 20-oxidases are multifunctional enzymes, which can convert GA12 to GA15, GA15

to GA24, and GA53 to GA44, GA44 to GA19 (Hedden and Kamiya, 1997). GA24 was

converted to GA25 and GA9, and GA19 was converted to GA20 and GA17 by GA 20-

oxidases. In barley, two GA 20-oxidases were detected, such as Hv20ox1 in

chromosome 5H and Hv20ox3 in chromosome 3H (Spielmeyer et al., 2004). Also, the

Hv20ox1 probe identified three homoeologous bands on wheat chromosome 5BL,

5DL and 4AL, and also the Hv20ox3 probe detected the homoeologous bands on

wheat group 3L (Spielmeyer et al., 2004). In rice, four GA 20-oxidases were detected,

these are OsGA20ox1, OsGA20ox2, OsGA20ox3 and OsGA20ox4 (Sakamoto et al.,

2004). OsGA20ox1 was mapped to chromosome 3 while OsGA20ox2 (sd1) was

located on 1L. OsGA20ox3 and OsGA20ox4 were mapped to chromosome 7 and 5

respectively (Sakamoto et al., 2004).

1.3.1.3.2 GA 3β-hydroxylases

3β-hydroxylases converts the steps of GA20 to GA1, GA20 to GA5, GA5 to GA3, GA9

to GA4, GA9 to 2,3-didehydro-GA9, and 2,3-didehydro-GA9 to GA7 (Hedden and

Phillips, 2000). Rice 3β-hydroxylase genes OsGA3ox1 and OsGA3ox2 were mapped

15

on 5S and 1S respectively (Itoh et al. 2001). OsGA3ox2 was identified as the rice

dwarf gene D18 (Sakamoto et al., 2004). Barley 3β-hydroxylase- Hv3ox1 was

mapped on barley 2HL and wheat group 2, while Hv3ox2 markers were located on

barley 3HL and wheat group 3 (Spielmeyer et al., 2004).

1.3.1.3.3 GA 2-oxidases

GA 2-oxidase deactivates GAs by 2-hydroxylation (Hedden and Phillips, 2000). It

converts GA1 to GA8, GA4 to GA34, GA20 to GA29, and catalyzes GA8, GA34 and

GA29 to their catabolite type. Four GA2ox-like genes were identified in the rice

genome. These are OsGA2ox1, OsGA2ox2, OsGA2ox3 and OsGA2ox4. OsGA2ox1

and OsGA2ox4 are located on rice chromosome 5. OsGA2ox2 and OsGA2ox3 are

mapped on rice chromosome 1 (Sakamoto et al., 2004). Two GA2ox-like genes were

detected in barley and homoeologous genes in the wheat genome. Hv2ox4 is located

on 1HL in barley and group 1L in wheat. Hv2ox5 is mapped on 3HS in barley and

group 3S in wheat (Spielmeyer et al., 2004).

Table 1 summarises the candidate genes which control the GA biosynthetic pathway

and also shows the gene orthologues between different species (Spielmeyer et al.,

2004). The barley HvCPSL1 gene was suggested to be homoeologous to the OsCPS1

gene in rice. Also barley genes HvKSL1 and HvKO1 were related to OsKS1 and

OsKO1 in rice respectively. In addition, Hv20ox1 and Hv20ox3 genes in barley have

similarity with OsGA20ox1 and OsGA20ox3 in rice. The barley Hv2ox4 gene

corresponded to rice OsGA2ox4.

16

Table 1: Summary of candidate genes in GA biosynthetic pathway (Os: Oryza sativa; Hv: Hordeum vulgare; Zm: Zea mays; ( ): wheat chromosome location).

Enzymes in GA pathway

Candidate genes

Chromosome location

Comment Entry name

CPS OsCPS1 2S GA biosynthesis AP004572 OsCPS2 2L Linked with OsKS5, OsKS6, OsKS7 AP005114 OsCPS3 9S Pseudogene AP005767 OsCPS4 5S Linked with OsKS4 AL662933 ZmCPS/AN1 L37750 HvCPSL1 7H (7AL,

7BL,DL) Related to OsCPS1 AY551435

KS OsKS1 4L GA biosynthesis OSJN00255 OsKS2 4L Linked with OsKS1 and OsKS3 OSJN00255 OsKS3 4L Linked with OsKS1 and OsKS2 OSJN00255 OsKS4 4S Linked with OsCPS4 OSJN00145 OsKS5 2L Linked with OsKS6, OsKS7 and OsCPS2 AP005114 OsKS6 2L Linked with OsKS5, OsKS7 and OsCPS2 AP005114 OsKS7 2L Linked with OsKS5, OsKS6 and OsCPS2 AP005114 OsKS8 11L Linked with OsKS9 AC135398 OsKS9 11L Pseudogene AC135398 HvKSL1 2HL(2AL,

2BL, 2DL) Related to OsKS1 AY551435

HvKSL2 2HL(2AL, 2BL, 2DL)

AY551437

ZmKS AF105149 KO OsKO1 6L Arranged as a gene cluster with 5 tandem

repeats AP005471

OsKO2 6L GA biosynthesis, loss of function induces d35 AP005471 OsKO3 6L Arranged as a gene cluster with 5 tandem

repeats AP005471

OsKO4 6L Arranged as a gene cluster with 5 tandem repeats

AP005471

OsKO5 6L Arranged as a gene cluster with 5 tandem repeats

AP005471

HvKO1 7H (7AL, 7BL, 7DL)

Related to OsKO1 AY551434

KAO OsKAO 6S GA biosynthesis AP00616 HvKAO1 7H(7AS,

7DS) Barley Grd5 AF326277

ZmKAO 9S Maize d3 U32579 GA20-oxidase

OsGA20ox1 3L AC096690 (U5033)

OsGA20ox2 1L Loss of function induces sd1 AP003561 (AF465255)

OsGA20ox3 7S AP005840 OsGA20ox4 5S AC124836 Hv20ox1 5H (5BL,

5DL, 4AL) Related to OsGA20ox1 AY551428

Hv20ox3 3H (3AL, 3BL, 3DL)

Related to OsGA20ox3 AY551429

GA3-oxidase

OsGA3ox1 5S AC144738 (AB059416)

OsGA3ox2 1S Loss of function induces d18 AP002523 (AB056519)

Hv3ox1 2HL(2AL, 2BL, 2DL)

Related to Os3ox1 AY551430

Hv3ox2 3HL(3AL, 3BL, 3DL)

Related to OsGA3ox2 AY551431

GA2-oxidase

OsGA2ox1 5S AC119288 (AB059416)

OsGA2ox2 1L AP003143 (AB092484)

OsGA2ox3 1L AP003375 (AB092485)

OsGA2ox4 5L AC132485 Hv2ox4 1HL(1AL,

1BL, 1DL) Related to OsGA2ox4 AY551432

Hv2ox5 3H (3AS, 3BS, 3DS)

AY551433

17

1.3.2 Mechanism of GA sensitive and insensitive mutants

The mutants, which respond to the application of exogenous GA with stem

elongation, are called GA-sensitive mutants (Milach and Federizzi, 2001), for

example, maize dwarf-1 mutant (d1) loses the function of 3β-hydroxylase which

converts GA20 to GA1. In the d1 assay, the maize GAs show a clear break in activity

between GA1 and GA20. GA20 has less than 1% of the activity of GA1 and the

precursors GA20 are biologically inactive (Phinney and Spray, 1982). Furthermore,

after application of exogenous GA1, the dwarf phenotype can be restored, which

clearly demonstrates that the d1 gene controls the 3β-hydroxylation of GA20 to GA1

(Spray et al., 1984).

Some dwarfing mutants appear to be complete phenocopies of GA-deficient plants

but cannot be returned to a wild phenotype by GA application. These mutants are

called GA insensitive dwarfing mutants (Swain and Olszewski, 1996), such as wheat

Rht3, maize Dwarf-8 and Arabidopsis gai (Talon et al., 1990). It indicates a link

between GA action and biosynthesis. As an example, D8-1 plants accumulate GA1,

GA20 and GA8 relative to wild type control levels (Fujioka et al, 1988b). Aneuploid

analyses (Harberd and Freeling, 1989) indicate that D8-1 controls a step beyond the

synthesis of an active GA. It is presumed that there is a GA-receptor mutant or a

mutant with a block in the transduction pathway between the receptor and stem

elongation (Talon et al., 1990). In the brassinolide synthesis pathway, the receptors

have already been identified. For example, the lk and lkb mutation in pea and

dwarfing gene uzu in barley appear to block brassinolide synthesis and this, in turn,

prevents normal elongation responses (Ross et al., 1997).

18

1.3.3 Other GA mutants

Another class of GA mutants are called GA deactivation mutants. There is only one

mutation known to affect this process, which is sln in pea. It blocks two steps: GA29

to GA29-catabolite in maturing seeds (Reid et al., 1992) and GA20 to GA29 (Ross et

al., 1995). The two blocked steps have different enzymes. It appears that sln is a

regulatory gene controlling two steps in GA catabolism.

There is another GA mutant group called full responses in high doses of exogenous

GA. These mutants represent short stature and reduced response in low doses of

exogenous GA and appear similar to wild type in saturated GA level, for example, lgr

in pea (Ross et al., 1995).

1.3.4 GA-sensitive and GA-insensitive mutants in maize, wheat, rice and barley

1.3.4.1 GA-sensitive mutants

In maize, the d1, d2, d3, and d5 dwarfing genes have been characterized as recessive

mutations that correspond to defective enzymes for the GA biosynthetic pathway

(Fujioka et al., 1988a; Hedden and Kamiya, 1997). Other classes of sensitive mutants

in maize are shown in Appendix 3. In wheat, there are 16 sensitive dwarfing mutants

accounting for 80% of the total dwarfing mutants (Gale and Youssefian, 1985). Rht4,

Rht6, Rht7, Rht8, Rht11 and Rht17 are recessive genes; Rht12 is a strong dominant

gene and remaining are partial or semi-dominant genes. In rice, sd1 from ‘DGWG’

controls the step of GA53 to GA20 and has been extensively used in developing rice

19

semi-dwarfing varieties throughout the world (Sasaki et al., 2002). Rice dwarf gene

d18 controls the step from GA20 to GA1 (Sakamoto et al., 2004). In barley, GA

sensitive dwarfing gene sdw1 (denso) and Gpert (ari-e) genes are well used in

America and Europe (Ivandic et al., 1999).

1.3.4.2 GA-insensitive mutants

Only a few mutants have been identified that belong to those with no response to the

exogenous application of GA, compared with the much higher number of GA-

sensitive mutants. However, some of the GA insensitive mutants have been widely

used in plant breeding, such as the Rht1 and Rht2 genes from ‘Norin’ in wheat

(Milach and Federizzi, 2001). Rice insensitive mutant d1 encodes GTP-binding

protein which is associated with gibberellin signal transduction (Ashikari et al., 1999;

Ueguchi et al., 2000). Barley Dwf2 is a dominant (GA3) insensitive dwarfing mutant

and has a strong effect on the phenotype and this mutant may be too short for

breeding purposes (Ivandic et al., 1999). In maize, the GA insensitive mutants such as

D8 and D9 have been extensively studied but not used for breeding (Harberd and

Freeling, 1989; Winkler and Freeling, 1994). Moreover, D8 is a GA insensitive

mutant and is also collinear to Rht1 and Rht2 genes (Peng et al., 1999). The GA

insensitive dwarfing mutant br2 in maize may have a yield advantage, especially in

high-density plantings (Multani et al., 2003).

1.4 Identification of dwarfing genes by using comparative genomics

1.4.1 General gene comparative bioinformatics in plants

20

In the past 15 years, scientists have discovered high levels of conservation of gene

content and gene orders over millions of years of evolution within grasses, crucifers,

legumes, some trees and Solanaceae crops (Gale and Devos, 1998). Since the mid

1980s, when restriction fragment length polymorphism (RFLP) analysis was first

applied to plants (tomato and maize in the United States and wheat in the United

Kingdom), it has become clear that complementary DNA RFLP probes could be

cross-mapped to provide anchors that allowed genomes to be compared (Gale and

Devos, 1998). One study showed that the tomato and potato maps were very similar

(Bonierbale, 1988) and another showed the high homology between rice and wheat

(Sorrells et al., 2003) (Fig. 1.4). Conservation of gene orders, but not intergenic

sequences, over millions of years appears to be the rule within plant families.

Figure 1.4: The colinearity between rice and wheat (Sorrells et al., 2003).

21

By comparative genetics, it has become possible to map and isolate genes in

corresponding regions. Nevertheless, breaks in complete correspondence occured in

or near the target regions (Gale and Doves, 1998; Francki et al., 2004). These

indicate that all genes may not be colinear at the micro level where synteny is often

interrupted by insertions, deletions and inversions (Carver and Stubbs, 1997; Francki

et al., 2004; Li et al., 2004). One of the few studies to date in which contiguous DNA

sequences have been compared showed complete co-linearity through three genes in

the ~20 kb sh2-A1 regions in rice and sorghum (Appels et al., 2003; Chen et al.,

1997). (See Appendix 5: Figure 1)

1.4.2 Collinearity between rice and wheat

From the homology between rice and wheat, rice chromosome 1 (R1) has large

similarity with wheat chromosome 3 group (W3). Similarly, both R2 and R3 are

related to W6 and W4, R4 and R7 are homoeologous with W2; R5 and R10 are

related to W1, and R6 and R8 are related to W7. Some rice regions are related to

more than one wheat chromosome, and also some wheat chromosome regions are

related to all rice chromosomes (Fig. 1.4 above) (Sorrells et al., 2003).

1.4.3 Close relationship between wheat and barley

1.4.3.1 Wheat A, B and D genomes are homologous

22

Triticum aestivum has three genomes A, B, and D and is treated as seven

homeologous groups according to detailed RFLP linkage maps (Chao et al., 1989;

Devos and Gale ,1993; Xie et al., 1993; Nelson et al., 1995a, b; Van et al., 1995;

Marino et al., 1996) and physical maps ( Gill et al., 1996). Gene synteny and

colinearity is relatively well conserved among Triticeae genomes (Sandhu and Gill,

2002). The first direct evidence came from the isozyme marker analysis in the early

1980s (Hart, 1987). Recently, comparisons of the high-density genetic linkage map

have shown that the gene order and relative recombination are so conserved among

Triticeae species that it is possible to construct an accurate consensus map (Van et al.,

1995). According to the results of Faris et al. (1999), of the 93 probes that detected

group 5 loci, 57 (61%) detected loci on all three wheat homeologous chromosome 5

chromosomes (See Appendix 5: Table 1). These observations strongly suggest that the

structural and functional organization of Triticeae genomes is very similar.

1.4.3.2 Wheat shows synteny with barley

The homoeologous relationship of the barley and wheat chromosomes was presented

by Islam and Shepherd (1981). Barley chromosomes 1, 2, 3, 4, 5, 6, and 7 of the

Burnham and Hagberg system (Singh and Tsuchiya 1982) were found to correspond

to chromosomes of wheat homoeologous groups 7, 2, 3, 4, 1, 6, and 5, respectively.

The homology of the wheat and barley chromosomes led to the adoption of an

alternative nomenclature of the barley chromosomes, which is based on genomic

relationship (Linde et al., 1997). According to the homologous chromosomes with

wheat, the nomenclature of barley chromosome number is followed by the genomic

symbol H (Shepherd and Islam, 1992) and in terms of the coherent system of genome

23

symbolisation in the Triticeae, the symbol I is applied in connection with the barley

genome (Löve, 1984). On this basis, the barley chromosomes 1 to 7 can be assigned

Triticeae synonyms as follows:

Barley 1 = 7H, barley 2 = 2H, barley 3=3H, barley 4 = 4H, barley 5=1H, barley 6 =

6H, barley 7 = 5H.

The most comprehensive evidence of the genetic relationship between barley and

wheat A, B, and D chromosomes has come from recent comparative mapping studies

(Devos and Gale, 1993; Devos et al., 1993b; Namuth et al., 1994; Van et al., 1995;

Dubcovsky et al., 1996; Kunzel et al., 2000; Francki et al., 2004; Li et al., 2004),

where the map location of molecular markers has been compared in the barley and

wheat genomes. (See Fig. 4.4)

1.4.4 Dwarfing gene comparison in rice, wheat and barley

Table 2 Rice, wheat and barley dwarfing gene bioinformatics.

Barley

Chromosome

1HL 2HS 3HL 4HS 5HL 7HS

Mapped dwarfing genes

fs2, ertb Eam1, yst4, gai sdw1, uzu Dwf2, brh2 cud1, lax-a, ari-e

brh1

Wheat Syntenic regions (chromosome)

4BS 4DS 5AL 7AS

Mapped dwarfing genes

Rht-B1 Rht-D1 Rht12 Rht9

Phynotype Semidwarf Altered seed shape

Semidwarf Semidwarf Semidwarf Semidwarf

Rice

Syntenic regions (chromosome)

5 4 1 3 11 12 6

Mapped dwarfing genes

d1 d3; d11 sd1; d2; d61 d14 d27 d33 d21

Syntenic region (chromosome)

7

Mapped dwarfing genes

d6; d7

24

Table 2 shows the genome synteny among rice, wheat and barley crops according to

the genome comparative maps. The dwarfing genes that were mapped on different

crops are likely to be related. As shown in table 2, barley 1HL is related to rice

chromosome 5. The dwarfing genes fs2 and ertb mapped on 1HL (barley) might have

homology with d1 (rice 5). Barley 2HS has colinearity with a large syntenic region of

rice chromosome 4 and a small syntenic region of rice chromosome 7. The dwarfing

genes-Eam1, yst4 and gai mapped on 2HS (barley) might be related to rice dwarfing

gene d3, d11 (rice chr.4), and d6, d7 (rice chr. 7). 3HL (barley) shows synteny with

rice chromosome 1. The sdw1 and uzu dwarfing genes (barley chr. 3HL) might have

synteny with sd1, d61 and d2 (rice chr. 1). Barley 4HS has orthologues with wheat

4BS and 4DS, and rice chromosome 3 and 11 regions. As a result, Dwf2, brh2

mapped on 4HS (barley) might relate to Rht-B1 and Rht-D1 (wheat 4BS and 4DS);

d14 (rice chr.3), d27 (rice chr. 11). Barley 5HL is collinear with wheat 5AL and rice

large syntenic region chromosome 12. The dwarfing genes cud1, lax-a and ari-e

mapped on 5HL (barley 5HL) might be related to d33 (rice chr. 12). Barley 7HS is

syntenic to wheat 7AS and rice chromosome 6. The dwarfing gene brh1 (barley 7HS)

might have a relationship with Rht9 (wheat 7AS) and d21 (rice chr. 6).

1.4.5 Synteny among several dwarfing genes among maize, rice, wheat and barley

1.4.5.1 Comparative analysis between Dwf2 (barley) and RhtB1, Rht D1 (wheat)

Dwf2 is a dominant gibberellic acid (GA3) insensitive dwarfing gene from mutant

‘93/B694’ and causes a very short growth phenotype in barley (Ivandic et al., 1999).

Dwf2 was mapped on the short arm of barley 4H to marker XhvOle and distally to

25

RFLP marker Xmwg2299. Due to barley 4H having similarity to wheat chromosome

4, Dwf2 might have co-linearity with Rht3 (Rht-B1c) or Rht10 (Rht-D1c). Both Rht3

and Rht10 are GA-insensitive dwarfing genes (Börner, 1996) within the Triticeae.

Figure 1.5 Comparative map of Dwf2, Rht-B1c and Rht-D1c between barley and wheat (Börner et al., 1997).

The distance (33.1 cM) between Xmwg634 and Dwf2 in barley 4HS is similar to the

distance (30.6 cM) between Xmwg634 and Rht3 (Rht-B1C), whereas Xmwg634 is

closely linked with Rht10 (Rht-D1C) (Fig. 1.5).

Xgwm165 was mapped on barley chromosome 4HL at a distance of 39.6 cM from the

dwarfing gene Dwf2 (Fig. 1.5). Comparatively, Xgwm165 was mapped on wheat 4DL

at a distance of 28 cM from Rht10 (Rht-D1c) (Börner et al., 1997). This indicates that

Dwf2 might have homology with RhtB1 and RhtD1.

1.4.5.2 Colinearity between maize dwarfing gene D8 and wheat dwarfing gene

RhtD1b or RhtB1b

26

Maize dwarfing gene D8 is a GA insensitive dwarf mutant, which is located on maize

chromosome 1L Bin10. RFLP marker umc107 and phyA are closely linked to D8

(Fig. 1.6). According to the comparative map between rice and maize (Wilson et al.,

1999), maize bin10 is related to rice chromosome 3L. In addition, umc107 and phyA

loci were found on rice chromosome 3L (Fig. 1.6). RFLP marker-psr1871 (Pki)

closely linked with umc107 in rice was found on wheat 4DS and 4BS and tightly

linked with Rht-D1b and C15-1 (C15-1 is one of the Rht-B1b (Rht1) homoeoalleles)

respectively. Xpsr821 (phyA) was found on 4DS closely linking with Rht-D1b as

well. According to the colinearity between the regions, C15-1, Rht-D1b and D8-1

might be homoeologous (Peng et al., 1999).

RhtD1b and RhtB1b are GA insensitive dwarfing genes. The wild type RhtD1a and

d8 appear to be more closely related to GAI than to RGA (RGA is an Arabidopsis

gibberellin signalling protein that is closely related to GAI (Silverstone et al., 1998),

Fig.1.7). Compared with the wild-type sequence, similar to GAI, the dwarfing mutants

D8, RhtB1b and RhtD1b represent the sequence deletions and substitutions (Fig. 1.8),

which affect the mechanism of gibberellin acid.

Figure 1.6 Homology between d8 and Rht-D1b or Rht-B1b (Peng et al., 1999).

27

Figure 1.7 Amino-acid structural features of Rht-D1a and d8 (From Peng et al., 1999).

Figure 1.8 Dominant mutant alleles (D8, RhtB1b and RhtD1b) encode proteins compared with their wild type mutants (From Peng et al., 1999). 1.5 Application of dwarf genes in conventional breeding

1.5.1 Utilization of dwarf genes for breeding

28

Although several dwarfing genes have been identified and studied in different species,

only a few have had wide application for plant improvement (Table 3). Twenty-four

dwarfing genes were identified in wheat (Bǒrner, 1996). The most universally

accepted dwarfing genes are Rht1 (RhtB1b) and Rht2 (RhtD1b) from wheat

germplasm developed by CIMMYT and are present in around 90% of the world areas

of semi-dwarf wheats (Dalrymple, 1986). In addition, Rht8 and Rht9 genes from

‘Akakomugi’ have been extensively used in Europe (Gale and Youssefian, 1985).

There are about 75 dwarfing genes listed by the Oryzabase (Integrate Rice Science

Database, 2003). Even though a large number of semi-dwarf high-yielding rice

varieties have been developed, semi-dwarfing genes of practical importance have

been limited only to the sd1 locus (Futshura and Kikuchi, 1997). Different alleles of

this locus have been used in America and Asian countries (Rutger, 1983).

The reasons for the success of the Rht1 (RhtB1b), Rht2 (RhtD1b) and sd1 genes are

that they have allowed the varieties with desirable semi-dwarf plant height to combine

with significant increases in spikelet fertility and yield (Gale and Youssefian, 1985).

The particular advantage of the semi-dwarf gene was that the harvest index increased

due to biomass shifting from vegetative production to grain production (Gent, 1995;

Hong et al., 1998; Jiang et al., 1995). Their positive pleiotropic effects on different

traits made these genes very attractive for breeding purposes. Great efforts have been

made to transfer them to suitable genetic backgrounds early when they were first

identified.

29

A short-statured barley mutant stock containing the sdw gene was obtained from

Norway in 1957 and transferred to American germplasm (Rasmusson et al., 1973).

This gene has been wildly used in breeding programs in the United States and Canada

(Rasmusson, 1991). The denso (sdw’s allele) and Gpert (air-e) (from ‘Gold Promisse

cultivar) dwarfing genes are present in the European barley germplasm and have been

used for variety development (Ivandic et al., 1999). Nearly 80% of 147 Chinese

barley dwarf varieties and entries were derived from three dwarf landraces: ‘Chiba

Damai’, ‘Xiaoshan lixiahuang’, and Cangzhou Luodamai’ (Zhang, 1994). The gene in

the all three sources is the dwarfing gene uzu (Zhang, 2000). Zhang (2000) analysed

two crosses (“Xiaoshan lixiahuang” × Bowman; “Cangzhou Luodamai” × Bowman)

and showed that the uzu gene in chromosome 3HL is linked in coupling to genes for

dense spike and long spike.

Table 3: Inheritance, chromosome location, and marker association of important dwarfing genes in cereal improvement (S: sensitive; I: insensitive; The genetic distance (cM) between the marker and dwarfing gene are indicated in bracket in marker linkage column).

Species Gene Source Response to GA

Inheritance Chromosome location

Marker linkage Key references

Hordeum vulgare L.

denso (sdw1.a)

Triumph (Jotun)

S Recessive 3HL WG110(12.8cM) OPH7-800(31.7cM)

Barus et al. (1993) Laurie et al. (1993)

uzu Xiaoshan LiXiahuang

I Recessive 3HL WG889B (Bin 6) Chono et al., (2003)

GPert (air-e) Golden Promise

S Recessive 5H -- Thomas et al.(1984)

Oryza sativa L.

Sd-1 Dee-geo-woo-gen

I Recessive 1 Xrg220(0.3cM) Spielmeyer et al. 2002

Triticum aestivum L.

Rht1(Rht4BS-b1)

Norin 10 I Part. dominant

4BS Xprs144(11.0cM) Xmwg634 (30cM)

Konzak (1987) Bŏrner et al. (1997)

Rht2(Rht 4DS-b1)

Norin10 I Part. dominant

4DS Xprs921(0.8 cM) Smwg734 (1.5 cM)

Konzak (1987) Bŏrner et al(1997)

Rht8 Mara, Sava S Recessive 2DS -- Konzak(1987) Rht9 Mara S Recessive 7BS -- Konzak

(1987) Rht12 Karcag522 S Dominant 5AL β-amy-

A1(2.5cM) X[rs1201(15cM)

Konzak(1987) Konzun et al. (1997)

Rht14 Castelporxiano S Semidominant Unknown -- Konzak (1987)

30

1.5.2 Challenges for breeding dwarfing varieties

Although major dwarfing genes are easily transfered by crosses, breeding semi-dwarf

varieties using these genes has not always been so straightforward (Milach and

Federizzi, 2001). One of the reasons is that many of the dwarfing genes do not have

positive pleiotropic effects on grain yield. In sorghum (Windscheffel et al., 1973;

Campbell et al., 1975), pearl millet (Bidinger and Raju, 1990; Rai and Rao, 1991),

and oat (Meyers et al., 1985), dwarfing genes have generally negative effects on grain

yield. One way of avoiding this problem was first suggested by Law et al. (1978) with

the “tall-dwarf” model for breeding wheats carrying dwarfing genes with negative

effects on yield. In this model, the authors suggested fixing the dwarfing genes in

populations at an early stage, followed by positive selection for height in subsequent

generations. Testing this hypothesis in pearl millet, Bidinger and Raju (1993) were

able to produce short-stature hybrids with increased grain yield.

Negative effects on grain yield may be due to poor seedling establishment and low

early vigour that has been associated with the presence of the GA-insensitive genes

(Niklas and Paolillo, 1990). In the presence of these genes, cell elongation in juvenile

leaf and stem tissue is decreased (Hoogendoorn et al., 1990), as a result of shorter

coleoptile and small initial vigour (Richards, 1992). To minimize this problem,

Rebetzke et al. (1999) studied the relationship of plant height and coleoptile length,

and tried to find ways to breed shorter Australian wheat varieties with longer

coleoptile. They found that height and coleoptile length appeared to be largely under

independent genetic control among GA-sensitive wheat, which indicates that GA-

sensitive Rht genes could be used to select for short height and longer coleoptile

31

wheats with improved establishment and seedling vigour. A similar problem

identified in rice has also been overcome with the development of semi-dwarf

germplasm that can produce long coleoptile (Dilday et al., 1990).

Some studies show that different alleles of a same gene have different traits, for

example, the barley sdw (denso) gene. The gene sdw and denso are different alleles

(Hellewell et al., 2000; Mickelson and Rasmuson, 1994). The sdw gene is wildly

accepted for feed barley production and has not been utilized as a malting barley in

America while the denso gene has been involved in malting barley breeding programs

and contributed to many malting barley cultivars with short stature traits (Mickelson

and Rasmuson, 1994). Both of them have been reported to have lower yield, seed

weight and percentage of plump kernels than tall isolines (Hellewell et al., 2000;

Mickelson and Rasmuson, 1994). However, some varieties with the sdw gene show

increased grain yield, such as UC 828 (Gallagher et al., 1996) and Royal (Rasmusson

et al., 1994). The grain yield of varieties (lines) with sdw gene seems unsteady. It

seems to be very important to select their alleles with positive rather than negative

effects on traits by using molecular markers in order to achieve barley breeding.

1.6 Summary of literature review

This chapter illustrates the ‘Green Revolution’ history during the 1960s and 70’s was

due to that the most important dwarfing genes have been incorporated into new crop

varieties. The review has shown the huge number of dwarfing genes that have been

identified. It has also explained the different biosynthetic contributions of some

dwarfing genes in plant physiology and emphasized the homoeologous relationship of

32

different dwarfing genes belonging to different crops. It states the contribution of the

dwarfing genes to conventional breeding and the difficulty of utilization of dwarfing

genes due to its negative pleiotropic effects.

1.7 Aims of this study and project outline

This project is based on an extensive database search relating to the bioinformatics of

rice-wheat-barley comparative genomics and the data available on chromosome

regions controlling agronomic traits. It targets the barley semi-dwarfing gene sdw and

attempts to identify the corresponding gene in rice by bioinformatics, to obtain the

barley gene sequence information from the conserved region. This project aims to

find polymorphisms among sdw alleles in barley varieties to develop a diagnostic

marker in order to improve barley traits and study relationships between the sdw gene

and dormancy.

1.7.1 Outline of this project

* Isolate selected genes identified from wheat and barley using the information from

rice as well as known syntenic relationships between rice-wheat-barley

* Determine the sequence of at least one gene from barley

* Identify polymorphisms in barley parental lines for crosses in which height

information is available on the progeny from the cross. While polymorphisms are

identified, mapping work will be done to locate the gene studied to the height

33

characteristic segregating among the double haploid progeny of the cross. Breeding

lines will also be screened to assess the value of sequence information for breeding.

1.7.2 Outputs from the thesis

* Exposure to high-level comparative genomics and bioinformatics

* Gene discovery and characterisation in wheat and barley

* Relate knowledge from rice genome to problems in wheat and barley breeding.

34

Chapter 2

Materials and Method

35

2.1. Plant material preparation

2.1.1 Plant materials

Barley materials provided by Dr. Chengdao Li were planted in small pots in the

glasshouse. The names of donors were as follows:

Table 2.1 Barley varieties used

Line Origin Malt/feed Pedigree Height Alexis European Malt Br. 1622/Triumph Short Sloop SA Malt Sloop Med Galleon SA Feed (Clipper/Hiproly *3)/(Proctor/CI 3576) Short Haruna Nijo Japan Malt Satsuko Nijo/(K-3/G-65) Tall Kaputar QLD Feed 5604/1025/3/Emir/ Shabet//CM67/4 F3 Bulk Hip Short Tallon QLD Malt Triumph/Grimmett Short Baudin WA Malt Stirling/Franklin Short AC Metcalf Canada Malt TR226/Manley Med Dhow SA Malt Skiff/Haruna nijo Short Yagan CIMMYT Feed Yagan Short Gairdner WA Malt Onslow/Franklin sib Semi-short Stirling WA Malt Dampier //(A14) Prior/Ymer/3/ Piroline Med

2.1.2 DNA extraction

Fresh and healthy leaves were harvested from three week old seedlings from each

plant. The samples were frozen in liquid nitrogen and crushed with a micro-pestle to

a fine powder. 600 µl DNA extraction buffer was added to each sample and the

material was homogenized efficiently using the micro-pestle.

600 µl phenol/chloroform/iso-amyl alcohol (25:24:1) was added to each sample and

mixed thoroughly by inversion for 5 seconds and placed on ice, then centrifuged for

10 minutes at 14000 rpm. The upper aqueous phase was transferred to a fresh tube.

Another 600 µl of phenol/chloroform/iso-amyl alcohol (25:24:1) was added and

36

mixed thoroughly by inversion for 5 seconds and place on ice, then centrifuged for 5

minutes at 14000 rpm. The upper aqueous phase was transferred to a fresh tube.

60 µl 3 M sodium acetate (pH 4.8) and 600 µl isopropanol were added to each sample

and mixed by inversion. DNA was precipitated for 2 minutes at room temperature and

centrifuged for 5 minutes at 14000 rpm. The DNA pellet was washed by adding 1 ml

70% ethanol and centrifuged for 2 minutes at 14000 rpm after the supernatant was

discarded. This ethanol wash was repeated. The DNA pellets were dried thoroughly

and resuspended in 30 µl R40 (2 µl of RNase A, 5ml of 1x TE buffer) solution.

2.1.3 Fluorometric quantification of DNA

An accurate measurement of DNA concentration in solution was determined by

fluorescence in a Hoefer TKO 100 DNA fluorometer by comparison to a known DNA

standard. The standard was made up to 100 μg/ml using a 1 mg/ml calf thymus

genomic DNA, 10x TNE buffer (Tris 100 mM, EDTA Na2.H2O 10 mM, NaCl 2M)

and distilled water. The assay solution was prepared using 0.1 μg/ml H33258 in 1x

TNE buffer (0.2 M MaCl, 10 mM Tris-Cl, 1 mM EDTA pH 7.0) together with

distilled water. 2 µl of the unknown DNA sample was added to 2 ml assay solution

and the fluorescence released at 460 nm was measured relative to the DNA standard.

2.2 General methods

2.2.1 Primer design

37

The bioinformatics analysis of the rice-barley genome was part of a large project

carried out by Chengdao Li et al. (2004). Some primer sequences were designed in this

thesis based on the sequence of rice dwarf gene sd1 and its conserved regions with

wheat and maize EST available from the Gene Bank Nucleotide sequence database. The

sequence number of rice dwarfing gene-sd1 was AF465255 named as Oryza sativa

cultivar Nipponbare gibberellin-20 oxidase gene. The conserved regions are with

Sorghum bicolour (AF249881.1), Triticum aestivum mRNA sequence (Y14007.1), Zea

mays (PCO130576) (Section 3.2.3). The primers sdw1-sdw8 and sdwn1, sdwn2 and

sdwex2R were designed by Chengdao Li (Table 2.2). Primers Ap1 and Ap2 were from

the Universal GenomeWalkerTM Kit (2000, Cat: K1807-1).

Table 2.2 Primers used for PCR reactions

Primer names Sequence (5’-3’) Sdw1 CCATCATGTCTGTCCAGTGGCAA Sdw2 CTTCTCATCTCCAATCTCATGG Sdw3 GAAGGAGAGGGTCTCCTTCCA Sdw4 TGGAAGGAGACCCTCTCCTTC Sdw5 TCAACATCGGCGACACCTTCATG Sdw6 CATGAAGGTGTCGCCGATGTTGA Sdw7 CCTGTGCAGGCAGCTCTT Sdw8 CGTTCCGTTCGGCGATCAGCT Sdwn1 CGGACTACGAGCCAATGG Sdwn2 GAGTACTGCGGGAAGATGAA Sdwex2R CATGAAGGTGTCGCCGATGT Ex1F1 ACGGGTTCTTCCAGGTGTC Ex1F2 AAGCTGCCGTGGAAGGAGAC Ex1F3 GTCGCCGACTACTTCTCCAG Jex3R1 CACAGGAAGAAGGCCAGCGAC Jex3R2 CAGGTGAAGTCCGGGTAGTG Jex3R3 TCAGCTGGCCGCCTCGACCTGC Jex3R4 GTTCGTTCCGTTTCGTTCCGTTC Jex3F CTAACGGACGGTACAAGAGC Sdwn2RF ACATCGGCGACACCTTCATG Jex3FR GCTCTTGTACCGTCCGTTAG Gsp1 GTAGTAGTTGCACCGCATGATGGAG Gsp2 GTACTCCTGGTACACCCTCCTGCAATC Gsp3 GCACGTGTTCTCTCCTCAGTACTCTGC Gsp4 GTGGATATATTACCCCATTGGCTCGTAG Ap1 GTAATACGACTCACTATAGGGC Ap2 ACTATAGGGCACGCGTGGT Sdw2RF ACATCGGCGACACCTTCATG Jex3FR GCTGTTGTACCGTCCGTTAG

38

2.2.2 Polymerase Chain Reaction (PCR) condition

PCR was used extensively in this project, either to check for screening bacterial

colonies or for sequencing. A typical PCR reaction was comprised of 1.5 mM MgCl2,

1x PCR buffer II, 200 μM dNTP, 1.2 U AmpliTaq DNA Polymerase and 0.4 μM of

each primer, in a final volume of 25 µl with sterile distilled water. PCR cycling

conditions consisted of an initial denaturation step of 94°C for 4 min, followed by 30-

35 cycles of 94°C for 1 min, 55°C for 45 sec, 72°C for 1 min, and a final extension

cycle at 72°C for 5 min. Reagents used for PCR were supplied by Perkin-Elmer

(AmpliTaq DNA Polymerase, 10x PCR buffer II (500 mM KCl, 100 mM Tris-HCl pH

8.3) and 25 mM MgCl2), Promega (dNTPs) and GeneWorks (PCR primers). PCR

amplification was carried out in a Perkin Elmer PCR system 200 thermal cycler. The

PCR products were run on agarose or low melt agarose gels. According to the size,

the gel slice was cut for cloning sequencing. If it was a single band, direct sequencing

could be performed by using QIA quick PCR purification kit (Qiagen Tech. Cat. No.

28104)

2.2.3 Agarose gel electrophoresis

Agarose gel electrophoresis was carried out in either a Bio-Rad Mini Sub DNA cell or

Bio-Rad wide Mini Sub DNA cell apparatus (Sambrook and Russell 2001). 10 µl

aliquot of the PCR product was mixed with 2 µl 6x gel loading buffer (0.25%

bromophenol blue in 40% sucrose) and loaded onto 1.0-2.0% agarose gel prepared

with 1x TBE buffer (90 mM Tris-acetate, 90 mM Boric acid and 2 mM EDTA, pH

8.0). The gels ranged from 1.0-2.0% depending on the desired separation and

39

resolution of DNA fragments. Electrophoresis was done under constant voltage (70-

100 V) for 30-110 min or until the dye had migrated ¾ of the way down the gel. DNA

fragments were size fractioned using 100 bp low (GSI Genemed Synthesis, Inc., Cat

no. 81-0100) or mid standard DNA markers (GSI Genemed Synthesis, Inc., Cat no.

81-0100) and visualised under UV light on a UVP Video Imager or a Gel Doc 1000

(BIORAD company) after that the gel was stained with 1 μg/ml ethidium bromide

(Sigma-Aldrich) for 15-20 min.

2.2.4 DNA purification

2.2.4.1 DNA purification from PCR product solution

DNA was purified from 50-80 µl PCR product solution if there was a single fragment

of interest present on the gel using QIA quick PCR purification kit (Qiagen Tech. Cat.

No. 28104). Five volumes of buffer PB were added to one volume of the PCR sample

and mixed, and then transferred to a QIAquick spin column in a 2 ml collection tube.

The column was centrifuged for 30-60 sec at 14000 rpm and the flow-through was

discarded. After being washed by 0.75 ml PE buffer (96-100% ethanol was added

before using), the column was placed in a clean 1.5 ml centrifuge tube. 20 µl

sterilized H2O was carefully added to the centre of the column to elute DNA and then

the column with the tube was centrifuged for 1 min at 14000 rpm. The DNA solution

in the centrifuge tube contained the purified single fragment DNA. After the DNA

was quantified with a fluorometer or run on an agarose gel, the DNA was ready for

sequencing.

40

2.2.4.2 DNA purification from agarose gels

If there were more than one DNA fragment in PCR products, Gel DNA purification

method was used. DNA was purified from low melting point (LMP) agarose gel using

the Ultraclean DNA Purification Kit (Gene Works, Cat No. 12100-300). DNA

fragments were run on 1.0% LMP agarose gel containing 1x TBE buffer. Following

eletrophoresis and staining with ethidium bromide, the gel was viewed under UV light

and the band of interest was cut out with a clean razor blade and placed into a 1.5 ml

microcentrifuge tube. The volume of gel was estimated by weight (1 g = 1 ml). Three

gel volumes of UltraSALT were added and the sample incubated in a water bath for 5

min at 55 °C to melt the agarose. 10 µl of UltraBIND silica matrix was added and the

samples mixed regularly during 5 min incubation at room temperature to allow the

DNA to bind. The UltraBIND / DNA complex was pelleted by centrifugation at

14000 rpm for 20 sec, the supernatant discarded and the pellet washed by re-

suspension in an equal volume of UltraWash solution. The tube was then centrifuged

at 14000 rpm for 20 sec, the supernatant discarded and the pellet dried by evaporation

in a Speedvac (Savant Instruments). The DNA was eluted from the UltraBind matrix

by resuspending the pellet in 2 volumes of pre-heated sterile distilled water (80°C)

and incubating the solution in a 55°C water bath for 5 min. Samples were centrifuged

for 1 min at 14000 rpm and the supernatant containing the DNA was transferred to a

clean 0.2 ml microcentrifuge tube. The DNA concentration was measured on an

agarose gel due to the difficulty of measuring with a fluorometer. If the amount of

DNA template was enough, the template could be sequenced. If not, the segment was

cloned into a vector as described in the following section.

41

2.2.5 pGEM®-T Easy vector systems

2.2.5.1 Preparation of chemically competent cells

Rubidium chloride Escherichia coli competent cells were prepared as described in the

Promega Protocols and Applications Guide (1995). JM 109 E. coli cells were streaked

on a LB medium plate and incubated at 37°C for 24 hours. After the incubation a

single colony was then inoculated into 5 ml LB broth (bactotryptome, NaCl and yeast

extract) and incubated at 37°C on a shaker at 225 rpm. The next morning the entire

culture was used to inoculate 250 ml of LB broth. Cells were grown in a 1 L flask

until the OD600 was between 0.4-0.6. The cells were chilled on ice for 15 min,

followed by centrifugation in a pre-cooled centrifuge (4°C) at 4,500 x g for 5 min

(Beckman JA14 rotor). Cells were then resuspended in 0.4 volumes of cold TfbI

buffer (30 mM potassium acetate, 10 mM CaCl2, 50 mM MnCl, 100 mM RbCl and

15% glycerol) and cooled on ice for 15 min. Cells were pelleted by centrifugation at

4,500 x g for 5 min at 4°C and resuspended in cold TfbII buffer (10 mM MOPS, 75

mM CaCl2, 10 mM RbCl and 15% glycerol adjusted to pH 6.5). The samples were

kept on ice for 1 hour and then transferred to 1.5 ml Eppendorf tubes in 100 µl

aliquots. Tubes were subsequently snap frozen in liquid nitrogen and stored at -80°C

until use.

42

2.2.5.2 DNA ligation into pGEM®-T Easy

Ligation of DNA into pGEM®-T Easy was done using the kit reagents according to

the manufacturer’s instructions. A typical ligation reaction consisted of 1 µl T4 DNA

ligase (3 Weis units/ μl), 5 µl of 2 x T4 DNA Ligase buffer (300 mM Tris-HCl pH

7.8, 100 mM MgCl2 100 mM dithiothreitol (DTT), 10 mM ATP), 1 µl pGEM®-T

Easy plasmid DNA, X ng insert DNA (depending on the desired ratio of vector:insert)

was made up to a final volume of 10 µl with sterile distilled water. The ligation

reactions were incubated at 14°C overnight and stored at 4°C until needed.

2.2.5.3 E.coli transformation

E.coli JM109 high efficiency competent cells (>1 x 108 cfu/μg DNA) were

transformed according to the protocols and applications guide provided by the

manufacturer (Promega). Competent cells stored at -80°C were thawed on ice for 5

min. 20 µl aliquots were transferred to chilled 1.5 ml eppendorf tubes containing 2 µl

of the DNA ligation mix. The samples were mixed gently by flicking and left on ice

for 30 min. Cells were then heat shocked at 42°C for 45 sec and placed on ice for 5

min. 1 ml chilled LB was added and the mixture warmed to room temperature and

then incubated at 37°C for 1 hour on a shaker at 225 rpm. After incubation, 100 µl of

each transformation mix was plated on LB agar plates containing 100 μg/ml

ampicillin, 100 µl IPTG, 20 µl X-gal and incubated overnight at 37°C.

43

2.2.5.4 Bacteria, media and growth medium

E.coli - JM 109 (recA1, endA1, gyr96, thi, hsdR17 (rk-mk

+), relA1, supE44, ∆(lac-

rpAB) bacteria were grown in either Luria Broth (LB) (10 Bacto-tryptone, 5 g Bacto-

yeast extract, 5 g NaCl2) or on LB agar plates (15 g of agar added to 1L LB broth,

autoclaved at 121°C and 15 psi for 20 min and poured into plates one the solution

cooled to 50°C). Ampicillin (100 μg/ml) was added to the broth/plates for selection.

Plates were stored at 4°C until needed.

2.2.5.5 Antibiotics used

Ampicillin was made up to a final concentration of 100 mg/ml by adding a weighed

amount of powder to distilled water as outlined in (Sambrook et. al., 2001). The

mixture was poured through a 0.22 μm filter and stored in ready-made aliquots at -

20°C. The antibiotic was then added to autoclaved media (final concentration of 100

μg/ml) after it had cooled to 50°C. Antibiotics were used fresh to prevent false

positives that can occur because of degradation when stored at 4°C for extended

periods.

2.2.5.6 PCR screening of E. coli transformants for insert DNA

Screening of E. coli colonies for the presence of pGEM®-T plasmids containing DNA

inserts was done by PCR. Individual E. coli colonies were placed into separate

microcentrifuge tubes containing 20 µl LB, and 3 µl aliquot of this was used as a

44

template for PCR. The reaction was comprised of 1.5 mM MgCl2, 1x PCR buffer II,

200 μM dNTP, 1.2 U AmpliTaq DNA Polymerase and 0.4 μM each of modified T7 &

and SP6 primers in a final volume of 25 μl. Thermal cycling conditions were an initial

denaturation period of 4 min at 94°C, followed by 30 cycles of 94°C for 30 sec, 55°C

for 45 sec, 72°C for 1 min, and a final extension cycle of 72°C for 5 min. For colonies

found to contain inserted DNA fragments, 5 µl aliquot of the bacteria/water mix was

inoculated into 5 ml LB broth containing 100 μg/ml ampicillin and grown overnight

at 37°C.

2.2.5.7 Restriction digest screening

Restriction digests were used regularly to screen for plasmids, to release inserts for

further analysis. Typically 20 μl reactions were set up to screen plasmid for presence

of an insert. The reactions consisted of 2 μl of 10 X enzyme buffer, 2 μl of the

particular plasmid DNA, 0.5 μl of 20,000 U/ml of enzyme and the final volume made

up to 20 μl with sterile water. Reagents were mixed and then the DNA was left to

incubate at 37ºC for 1-2 hours. Samples were analyzed by comparing undigested

DNA and digested DNA on a 1% agarose gel.

2.2.5.8 Purification of plasmid DNA for sequencing

Plasmid DNA was purified from fresh E. coli cells which were cultured overnight at

37°C by using QIArep DNA spin kit (QIAGEN, Cat. No. 27104). The broth was

transferred to 1.5 ml microcentrifuge tubes and centrifuged for 1 min at 14000 rpm to

45

pellet the cells. The cells were resuspended in 250 µl of resuspension buffer P1 (50

mM Tris-HCl, pH 8.0; 10 mM EDTA containing 100 μg/ml Rnase A). 150 µl of lysis

buffer P2 (0.2 N NaOH, 1% SDS) was added, the mixture gently inverted 4-6 times

and incubated at room temperature for 5 min. 350 µl of neutralisation buffer N3 (3 M

potassium acetate, pH 5.3) was added, the mixture inverted gently 4-6 times and

centrifuged at 14000 rpm for 10 min. The supernatant was transferred to a QIAprep

DNA spin column, centrifuged at 14000 rpm for 1 min and the flow-through

discarded. Bound DNA was washed with 0.5 ml of PB buffer, centrifuged for 1 min at

14000 rpm and the flow-through discarded. The DNA was washed again with 0.75 ml

of PE, centrifuged for 1 min at 14000 rpm, the flow-through discarded and the column

was centrifuged for a further 1 min to remove residual wash buffer. The column was

placed into a new 1.5 ml microcentrifuge tube, 50 µl of sterile distilled water was

applied directly to the membrane, the tube incubated at room temperature for 1 min

and DNA eluted from the membrane by centrifugation at 14000 rpm for 1 min. The

flow-through contained the plasmid DNA.

2.2.6 PCR sequencing

The method of PCR sequencing was the same for either PCR product DNA or

Plasmid DNA with only different primers used for each. Cycle sequencing was

carried out using either Rhodamine or Big dye Terminator Dye with AmpliTaq DNA

Polymerase, FS (Perkin Elmer), as described in the ABI PrismTM Dye Terminator

Cycle Sequencing Ready Reaction Kit technical manual. For the PCR reaction the

following reagents were used: 4 µl Terminator Ready Reaction Mix, 300-400 ng/µl

plasmid DNA or 10-20 ng PCR product DNA (the amount depended on the size of the

46

DNA fragment on gels), 1 µl of 3.2 pmoles primer (T4 or SP6 or other primer else),

made up to 10 µl with sterile distilled water. The sequencing reactions were carried

out in a Perkin Elmer GeneAmp PCR System 2400 thermal cycle under the following

conditions: 1 cycle of 96°C 2 min; 25 cycles of 96°C for 10 sec, 50-60°C for 5 sec,

60°C for 4 min. The completed reactions were transferred to a 0.5 ml centrifuge tube

containing 1 µl 3 M sodium acetate (pH 5.4), 1 µl 125 mM EDTA (disodium salt) and

25 µl 95% ethanol. Samples were incubated on ice for 20 min and then centrifuged at

14,000 rpm for 30 min. The supernatant was removed, the pellet washed with 125 µl

of 80% ethanol and dried in a Speedvac. The sequencing gels were prepared,

electrophoresed and the data collected by Ms. Frances Briggs (SABC).

2.2.7 Analysis of sequence data

DNA sequence data was analysed and edited using the SeqEd TM version 1.0.3

software (Applied Biosystems). Edited sequences were converted into FASTA format

for Bioedit and NCBI databases and programs.

2.3 Genome Walker (Clontech)

2.3.1 Size and quality measurement of genomic DNA

2.3.1.1 Size of genomic DNA on a 0.5% agarose/EtBr gel

1 µl of experimental genomic DNA (0.1 μg/μl) and 1 µl of original genomic DNA

(concentrated) were loaded on a 0.5% agarose/EtBr gel in 0.5X TBE, along with

47

DNA size markers, such as 1-kb ladder or l/Hind lll digest. Genomic DNA was bigger

than 50 kb with minimum smearing.

2.3.1.2 Purity of genomic DNA by Dra 1 digestion

A 0.5-ml reaction tube was set up containing 5 µl experimental genomic DNA, 1.6 µl

Dra l (10 units/μl), 2 µl 10X Dra l restriction buffer and 11.4 µl deionized water. A

control digestion without enzyme was also prepared. After gently mixing, these

reactions were incubated at 37ºC overnight. 5 µl of each reaction were run on a 0.5%

agarose/EtBr gel along with 0.5 µl of experimental genomic DNA as a control.

2.3.2 Digestion of genomic DNA

Five reactions: DL1, DL2, DL3, DL4 and a positive control were set up. Each

reaction combined 25 µl genomic DNA (0.1 μg/μl), 8 µl restriction enzyme, 10 µl

restriction enzyme buffer and 57 µl deionized H2O in a separate 1.5-ml tube. After

gently mixing, these reactions were incubated at 37ºC overnight (16-18 hr). 5 µl of

each reaction was removed and run on a 0.5% agarose/EtBr gel to determine whether

digestion was complete.

2.3.3 Purification of DNA

An equal volume (95 μl) of phenol was added to each reaction tube and then mixed

gently. After spinning briefly, the upper aqueous layer was transferred into a fresh 1.5

ml tube and the lower organic layer was discarded. An equal volume (95 μl) of

48

chloroform was added to each tube and mixed gently. The upper aqueous layer was

transferred into a fresh 1.5 ml tube and the lower organic layer was discarded. Two

volumes (190 µl) of ice cold 95% ethanol, 1/10 volume (9.5 μl) of 3 M NaOAc (pH

4.5), and 2 µl of glycogen (10 μg/µl) were added to each tube and mixed gently. Each

reaction was centrifuged at 14000 rpm for 10 min and the supernatant was discarded.

The pellets were washed in 100 µl of ice-cold 80% ethanol and air-dried. Finally, the

pellets were dissolved in 20 µl of water.

2.3.4 Ligation of genomic DNA to Genome Walker adaptor (Clontech)

Five ligation reactions including a positive control were set up. Each reaction

combined 10 µl of digested, purified DNA, 1.9 µl Genome Walker adaptor (25 μM),

3.0 µl 10X ligation buffer and 0.5 µl T4 DNA ligase (6 units/μl). These reactions were

incubated at 16ºC overnight in a water bath. To stop the reactions, these reactions

were incubated at 70ºC for 5 min. The reactions were diluted by adding 72 µl of 10

mM TE (10/1, pH 7.4), then vortexed at slow speed for 10-15 sec and stored at 4ºC.

The Genome Walker adaptor was prepared by adding equi-molar amounts of

AdaptorF (5' GTA ATA CGA CTC ACT ATA GGG CAC GCG TGG TCG ACG

GCC CGG GCA GGT) and AdaptorR (5'PO4-ACC TGC CC-NH2), then incubated at

37ºC for 10 min.

2.3.5 Primer design and PCR amplification

Nested PCR primers (Table 2.2) were designed by using Primer 3 software (Rozen

and Skaltsky, 1998). These primers were based on the obtained sequence of sdw1

49

gene in barley. The nested adaptor primers were AP1 (5' GTA ATA CGA CTC ACT

ATA GGG C) and AP2 (5'ACT ATA GGG CAC GCG TGG T).

Amplifications of genomic DNA fragments were performed by using designed

specific primers (GSP1-4) and adaptor primers. PCR amplifications were carried out

in a total volume of 25 µl using 1 µl of digested and ligation template DNA, 0.4 μM

of each primer, 1.2 U Taq polymerase, 1.5 mM MgCl2 and 5 µl of 5X PCR buffer

with dATP, dCTP, dGTP and dTTP, and water. Samples were amplified in Perkin

Elmer PCR system 200 thermal cycler with the following cycle profile: 1 min at 94ºC,

45 sec at annealing step, 1 min at 72ºC. The annealing temperature in the first cycle

was 60ºC; this was reduced by 1ºC in each of next 4 cycles, and was continued at

55ºC for the remaining 40 cycles. PCR amplifications were followed by 5 min

incubation at 72ºC.

After the first amplification, PCR products were diluted 50 fold with distilled water,

and 1µl aliquot of this was used as a template for the second amplification reaction.

PCRs were performed in a total volume of 25 µl by using a nested specific primer

(Table 2.2) and a nested adaptor primer AP2 as described above. Amplifications were

performed for 35 cycles with the following cycle profile: 1 min at 94ºC, a 45 sec

annealing step, 1 min at 72ºC. The annealing temperature in the first cycle was 65ºC;

this was reduced by 1.0ºC for the next four cycles, and was continued at 60ºC for 30

cycles. PCR amplifications were followed by 5 min incubation at 72ºC.

Aliquots of 5 μl of PCR product from both the first and nested PCR amplifications

were electrophoresed on 1.5% agarose gels and stained with ethidium bromide

50

(Signa-Aldrich) to determine the approximate size of the products relative to a size

standard. The single band from the second nested PCR was purified and sequenced.

51

Chapter 3

Results

Isolation of a gibberellic acid (GA) 20-oxidase gene from barley and searching sequence nucleotide polymorphism between different varieties

52

3.1 Introduction

In this chapter, one gibberellin metabolic pathway gene (GA 20-oxidase) from barley

was identified by using a comparative map between barley and rice. The gene

sequence was partly isolated by PCR primers which were designed based on

conserved gene regions. Parts of the gene sequence were sequenced from twelve

barley varieties with different height traits. The results presented include the

bioinformatics between barley and rice, the part sequences of this gene, the gene

structure, the sequence nucleotide polymorphism of different varieties, preliminary

studies of Genome Walker and improved PCR technique approach.

3.2 Proposed gene structure of sdw1 and primers for gene isolation

3.2.1 Syntany between dwarfing gene sdw1 in barley and sd1 in rice

According to the synteny among several dwarfing genes that were mapped in maize,

rice, wheat and barley respectively, sdw1 in barley is the most likely orthologue of

sd1 in rice. The gene, sdw1, is a GA-sensitive semi-dwarfing gene that was mapped

on barley chromosome 3H. (http://barleygenomics.wsu.edu/arnis/linkage_maps/maps-

svg1.html). The gene, sd1, is also a GA-sensitive semi-dwarfing gene, which was

mapped on rice chromosome 1 (Rice JRGP RFLP 2000, Gramene). According to the

rice and barley genome chromosome comparative map (Fig. 3.1), in total, twenty-six

common markers have been placed on the two maps of barley 3H and rice

chromosome 1. According to RFLP markers, sdw1 was mapped on barley 3H (bin 13)

and close to the marker, R1545. Similarly, sd1 was mapped on rice chromosome 1

53

and close to the same marker, R1545 (Fig. 3.2). In addition, several RFLP markers

near sdw1 gene were mapped in rice chromosome 1 and close to sd1 gene. This

indicates the synteny between sdw1 and sd1 (Figure 3.2).

Figure 3.1 Comparative map of barley chromsome 3H (left) and rice chromosome 1 (right). Collinear loci are connected with drawn lines, syntenous but non-collinear loci by broken lines. Map distances are given in recombination units (From Smilde et. al, 2001).

sdw1

54

Figure 3.2 The relationship between barley semi-dwarfing gene-sdw1 and rice semi-dwarfing gene-sd1 (The map of barley chromosome 3H was from http://barleygenomics.wsu.edu/arnis/linkage_maps/maps-svg1.html and the map of rice 1 was from Smilde et. al, 2001. ABG499, C191, R1545, ABC161, C742, R1014 are conserved markers mapped in both map. Spielmeyer et al., 2002).

3.2.2 Proposed structure of gene sdw1

Due to the barley-rice synteny, the semi-dwarfing gene sdw1 from barley might have

similar gene structure to the rice semi-dwarfing gene sd1. The sd1 gene (NCBI:

AF465255, Appendix 7) was isolated from Oryza sativa cultivar Nipponbare and

encodes a gibberellin-20 oxidase gene. The whole sequence is 6590 bp (Appendix 7)

which includes three exons (2430-2986 bp; 3089-3410 bp; 4882-5172 bp) and two

introns (2987-3088 bp; 3411-4881 bp) (Fig.3.3). The purpose of this research was to

find the sdw1 gene structure and sequence, and find polymorphisms among different

Rice chro.1

C191 (141.6 cM) AP003252 ABG499 (144.0 cM) Sd1(149.1-151.0 cM; Spielmeyer et al., 2002) R1545(158.5cM) ABC161 (160.7cM) C742 (164.1cM) AP003246 R1014 (170.0 cM)

AP003561 AP003379 AP003346 AP003431 AP003407

Barley 3H

sdw1

ABC161

R 1014

R1545

C 742

Bin 16

ABG499 C191

55

sdw1 alleles, furthermore, utilization of new alleles of sdw1 to modify pleiotropic

effects on other traits such as preharvest sprouting.

Figure 3.3 The sd1 gene structure and proposed barley sdw1 gene structure based on rice genome sequence (sd1 gene structure was from NCBI: AF465255).

3.2.3 Homology identification and primer design

Primer sequences were based on the sequence of rice dwarf gene sd1 and its

conserved regions with wheat, maize, barley and sorghum EST which were available

at the Gene Bank Nucleotide Sequence Database. The sd1 gene structure provided in

Figure 3.4 was Oryza sativa cultivar Nipponbare gibberellin-20 oxidase (Sd-1) gene

(NCBI No.: gi|19422258|gb|AF465255.1|). After a blast search, there were several

ESTs from different species in the three exon regions, such as Triticum aestivum

mRNA for gibberellin 20-oxidase, Zea mays PCO130567 mRNA sequence, Sorghum

bicolor BAC 170F8, Arabidopsis thaliana and Mycobacteriophage sequence

conserved region. According to the conserved region of the sd1 sequence and these

ESTs, several primers were designed. The proposed primer locations are shown in

Fig. 3.5

Exon1 1472bp

3089---3410 321bp

Exon2 Exon3

Intron1 102bp

Intron2 2430---2986 557bp

4882---5172 291bp

56

Figure 3.4: Rice sd1 gene (NCBI No. AF465255) blast [Triticum aestivum mRNA for gibberellin 20-oxidase, clone S44B (accession No. TAY14007) (conserved region: 2868-2906; 3224-3328; 3388-3411; 4881-4932); Zea mays PCO130567 mRNA sequence, (accession No. AY105651.1) (conserved region : 3258-3334; 3375-3411); Mycobacteriophage PG1, complete genome(accession No. AF547430.1) (conserved region: 2507-2528); Arabidopsis thaliana complete sequence from clone GSLTFB57ZG08 of Flowers and buds of strain col-0 of Arabidopsis (accession No. BX830073) (conserved region: 2877-2902); Triticum aestivum mRNA for wga20, complete cds (accession No. AB005555) (conserved region: 4882-4932; 4941-4974); Sorghum bicolor BAC 170F8, complete sequence (accession No. AF503433) (conserved region:4995-5020)]. Figure 3.5 Predicted structure of Barley sdw1 and primer designing.

0 1 2 3 4 5 6kb

40-50 50-80 80-200

Triticum aestivum mRNA TAY14007 Zea mays PCO130567 mRNA sequence AY551429 Mycobacteriophage PG1, complete genomeAF547430.1 Arabidopsis thaliana Full-length cDNA BX830073.1 Triticum aestivum mRNA for wga20, complete cds AB005555.1 Sorghum bicolor BAC 170F8, complete sequence AF503433.1

2868-2906; 3224-3328; 3388-3411

4881-4932

3258-3334 3375-3411

2507-2528

2877-2902

4882-4932 4941-4974

4995-5020

Exon1

2432-2986

Exon2

3089-3410 Exon3 EEEExon3

4882-5172

exon3 exon1 exon2

Sdw1 2232--2254

Intron1 102bp

Intron2 1472bp

Sdw8 5187-5172

Sdw7 4920-4903

Sdw6 3410-3388

Sdw5 3388-3410

Sdw2 2412-24333 Sdw3

2896-2885

Sdw4 2885-2906

2430---2986 557bp

3089---3410 321bp

4882---5172 291bp

sdwn2 3102-3121

sdwex2R 3410-3392

Exon1F1 2707-2726

Exon1F2 2877-2896

Exon1F3 2937-2956

Sdwn1 (sdwn1R) 2968-2985

Jex3R1 4970-4950

Jex3R2 5042-5023

Jex3R3 5272-5150

Jex3R4 5200-5178

Jex3F 4888-4911

Gsp1

Gsp2

Gsp3 Gsp4

57

3.3 Primers designed for sdw1

3.3.1 Primers Ex1F1 and Gsp4 amplified part of exon1 of sdw1 gene

After the sequence of the first intron was obtained (Section 3.3.3), reverse primers

were designed within this intron to perform Genome Walker to amplify the exon1 and

the promoter region. The primers were Gsp1, Gsp2, Gsp3, and Gsp4. Due to no

successful results from the Genome Walker (see Section 3.6), normal PCRs were

performed in exon1. The primer pairs Ex1F1 and Gsp4 amplified a correct size band

which was approximately 300 bp (Fig. 3.6). After optimizing the PCR, the same 300

bp single band for both varieties (Alexis and Sloop) was amplified. The sequence of

this band also matched exon1 of sd1 in rice. The sequence results of both varieties in

this region and the sequence alignment with sd1 were shown in Fig. 3.7. The

alignment sequence comfirmed that the part of exon1 in barley was the correct

sequence of sdw1 gene. The sequence of sdw1 in this region was six bp longer than

sd1. There were also thirty-two bp differences between them.

A B

M Al Sl ck

300bp

Al Al Sl Sl ck1 ck2 sloop M

Ex1F1+Gsp4;

MgCl2: 1.2mM 1.5mM 1.2mM 1.0mM

Ex1F1+Gsp4

Mg:1.5μl; Ex1F1+Gsp4;

56.9; 55.5; 52.7; 50.7ºC

300bp

58

Figure 3.6 Ex1F1 + Gsp4 PCR results (A) and optimizing PCR (B) (Al: Alexis; Sl: Sloop; ck Negative control; ck1: MgCl2 1.2 mM negative control; ck2: MgCl2 1.5 mM negative control; M: ladder).

The sequence of Ex1F1+Gsp4 in Alexis and Sloop TACGGGTTCTTCCAGGTGTCCGGGCACGGCGTGGACAACGCCCTGGCGCGCGCGGCGCTGGACGGCGCGAGCGGGTTCTTCCGTCTGCCGCTGGCCGAGAAGCAGCGCGCGCGGCGCATCCCGGGGACCGTGTCCGGGTACACGAGCGCGCACGCCGACCGGTTCGCCTCCAAGCTCCCCTGGAAGGAGACCCTCTCCTTCGGCTTCCACGACCGCGCCGGCGCCGCCGCGCCCGTGGTGGCGGACTACTTCACCAGCACCCTCGGGCCGGACTACGAGCCAATGGGGTAATATATCCACA

The alignment of part of exon1 of sdw1 in Alexis with the exon1 of sd1 (data base) in rice Query: 2 acgggttcttccaggtgtccgggcacggcgtggacaacgccctggcgcgcgcggcgctgg 61 ||||||||||||||||||||| ||||||||| ||| ||| ||||||||||| ||||| | Sbjct: 2707 acgggttcttccaggtgtccgagcacggcgtcgacgccgctctggcgcgcgccgcgctcg 2766 Query: 62 acggcgcgagcgggttcttccgtctgccgctggccgagaagcagcgcgcgcggcgcatcc 121 ||||||| |||| |||||||| || ||||| |||||||||| |||||||| ||| ||| Sbjct: 2767 acggcgccagcgacttcttccgcctcccgctcgccgagaagcgccgcgcgcgccgcgtcc 2826 Query: 122 cggggaccgtgtccgggtacacgagcgcgcacgccgaccggttcgcctccaagctcccct 181 |||| ||||||||||| ||||| ||||| ||||||||||| ||||||||||||||||| | Sbjct: 2827 cgggcaccgtgtccggctacaccagcgcccacgccgaccgcttcgcctccaagctcccat 2886 Query: 182 ggaaggagaccctctccttcggcttccacgaccgcgccggcgccgccgcgcccgtggtgg 241 ||||||||||||||||||||||||||||||||| |||||||||| ||||| || | Sbjct: 2887 ggaaggagaccctctccttcggcttccacgacc------gcgccgccgcccccgtcgtcg 2940 Query: 242 cggactacttcaccagcaccctcgggccggactacgagccaatggggtaat 292 | ||||||||| ||||||||||||| || |||| || |||||||||||||| Sbjct: 2941 ccgactacttctccagcaccctcggccccgacttcgcgccaatggggtaat 2991 Figure 3.7 The 291 bp exon1 sequence of Ex1F1 + Gsp4 and the alignment with exon1 of sd1( sd1: NCBI No.: AF465255.1; I: sequence in the inton1; blue: forward primer Ex1F1; red: reverse primer Gsp4; Query: sdw1 sequence; Sbjct: sd1 sequence).

3.3.2 Amplification of second exon by primers sdwn2 and sdwex2R

This pair of primers (sdwn2 and sdwex2R) were expected to amplify exon2 which

was about 300 bp. The PCR result (Fig. 3.8 A) showed that there was a bright 300 bp

band in all three varieties (Alexis, Sloop and Stirling). After PCR optimizing, a single

band was obtained from Alexis (Fig. 3.8 B) and also from eight other varieties (data

not shown). This band was sequenced and was matched with rice semi-dwarfing gene

sd1 exon2 sequence. The exon2 sequence of sdw1 was three base pairs longer than

59

that of sd1. In addition, there were twenty-one base pair differences in this region

between sdw1 and sd1. This sequence was shown in Fig. 3.9.

A B Figure 3.8 PCR results of primers sdwn2+sdwex2R (AL: Alexis; SL: Sloop; ST: Stirling; ck: Negative control; M: Ladder).

Query: 4 agtactgcgggaagatgaaggagctgtcgctgaggatcatggagctgctggagctgagcc 63 ||||||||| | ||||||||||||||||||||| ||||||||| || ||||||||||||| Sbjct: 3103 agtactgcgaggagatgaaggagctgtcgctgacgatcatggaactcctggagctgagcc 3162 Query: 64 tgggcgtggagaagcgcgggtactaccgggacttcttcgcggacagcagctccatcatgc 123 ||||||||||| || || |||||| |||| |||||||||||||||||||| ||||||| Sbjct: 3163 tgggcgtggag---cgaggctactacagggagttcttcgcggacagcagctcaatcatgc 3219 Query: 124 ggtgcaactactacccgccgtgcccggagccggagcgcacgctgggcacgggcccgcact 183 ||||||||||||||||||| ||||||||||||||||| ||||| |||||||||||||||| Sbjct: 3220 ggtgcaactactacccgccatgcccggagccggagcggacgctcggcacgggcccgcact 3279 Query: 184 gcgaccccacggcgctcaccatcctcctccaggacgacgtgggcgggctggaggtcctcg 243 |||||||||| || |||||||||||||||||||||||||| ||||| || |||||||||| Sbjct: 3280 gcgaccccaccgccctcaccatcctcctccaggacgacgtcggcggcctcgaggtcctcg 3339 Query: 244 tcgacggcgactggcggcccgtccgccccgtccccggcgccatggtcatcaacatcggcg 303 |||||||||| ||||| |||||| |||||||||||||||||||||||||||||||||||| Sbjct: 3340 tcgacggcgaatggcgccccgtcagccccgtccccggcgccatggtcatcaacatcggcg 3399 Query: 304 acaccttcatg 315 ||||||||||| Sbjct: 3400 acaccttcatg 3410 Figure 3.9 Data search of the sequences amplified by sdwn2+sdwex2R (Query: The sequence of sdwn2+sdwex2R of Alexis; Sbjct: The sequence of sd1 exon2).

M AL SL ST ck

sdwn2+sdwex2R 300bp

300bp

Alexis

sdwn2+sdwex2R

M ck

60

3.3.3 Amplification of first intron and second exon by primers sdwn1 and sdwex2R

The pair of primers sdwn1 and sdwex2R was proposed to amplify the first intron and

the second exon. However, the size of the band was uncertain because it included an

intron of unknown size. After amplification, there was a bright 500 bp band (Fig.3.10

A), which might include the intron1. After optimizing the PCR, a 500 bp single band

was obtained (Fig. 3.10 B). Nested PCR by sdwn2+sdwex2R indicated this band was

the correct band which include the exon2 (Fig.3.10 C).

A B

M AL SL ST ck AL SL ST ck

sdwn2+sdwex2R sdwn1+sdwex2R

500bp

M ck

sloop

sdwn1+sdwex2R

500bp

61

C Figure 3.10 (A, B, C): sdwn1+sdwex2R PCR results (AL: Alexis; SL: Sloop; ST: Stirling; ck: Negative control; M: Ladder).

The sequencing results confirmed the band was the correct one and the intron1 of

sdw1 was 173 bp compared to the 103 bp of sd1 in rice. As shown in Figure 3.11, the

sequence of sdwn1+sdwex2R included the exon2 sequence of sdwn2+sdwex2R.

Therefore, the first intron of sdw1 should be the correct sequence.

NTTCGGACTACGAGCCAATGGGGTAATATATCCACTCGCCCACAGCCCTATCCGGCCAG CACGAACCAATCCCCGCCACTGCATTTTTTAATTTTTTTTGCTTCCGCGCGATCGTACG TTCGATCGGCGCCCACGTAGTACTGTACGTACGCAGGCAGAGTACTGAGGAGAGAACAC GTGCGATGATGATTGCAGGAGGGTGTACCAGGAGTACTGCGGCAAGATGAAGGAGCTGT Sdwn2+sdwex2R: GAGTACTGCGGCAAGATGAAGGAGCTGT CGCTGAGGATCATGGAGCTGCTGGAGCTGAGCCTGGGCGTGGAGAAGCGCGGGTACTAC CGCTGAGGATCATGGAGCTGCTGGAGCTGAGCCTGGGCGTGGAGAAGCGCGGGTACTAC CGGGACTTCTTCGCGGACAGCAGCTCCATCATGCGGTGCAACTACTACCCGCCGTGCCC CGGGACTTCTTCGCGGACAGCAGCTCCATCATGCGGTGCAACTACTACCCGCCGTGCCC GGAGCCGGAGCGCACGCTGGGCACGGGCCCGCACTGCGACCCCACGGCGCTCACCATCC GGAGCCGGAGCGCACGCTGGGCACGGGCCCGCACTGCGACCCCACGGCGCTCACCATCC TCCTCCAGGACGACGTGGGCGGGCTGGAGGTCCTCGTCGACGGCGACTGGCGGCCCGTC TCCTCCAGGACGACGTGGGCGGGCTGGAGGTCCTCGTCGACGGCGACTGGCGGCCCGTC CGCCCCGTCCCCGGCGCCATGGTCATCAACATCGGCGACACCTTCATGANNNNN CGCCCCGTCCCCGGCGCCATGGTCATCAACATCGGCGACACCTTCATGANNNNN Figure3.11 The sequence of 500 bp fragment of Sloop amplified by sdwn1+sdwex2R (500 bp fragment of Sloop including intron1 and exon2) (Blue colour: primer sdwn1; Purple colour: Primer sdwn2; red colour: sdwex2R; I: intron1).

M ck

sloop sdwn1+sdwex2R purified PCR DNA band (500bp) 2ul

Nested PCR to test if the 500bp band of sloop sdwn1+sdwex2R PCR is right

sdwn2+sdwex2R

Nested PCR band 300bp

62

3.3.4 Primers Jex3F and Jex3R2 amplified part of exon3 sequence

The primer pairs Jex3F and Jex3R2 amplified the expected band for exon3 (Fig. 3.12

A). After optimizing, a single band was obtained (Fig. 3.12 B). After purification of

the PCR products, this single band was sequenced. The result was blasted and

matched with sd1 exon3 (Fig. 3.13). There were only seven base pairs different from

the same region of sd1.

A B Figure 3.12: The PCR results of the primer pair Jex3F and Jex3R2 (Al: Alexis; Sl: Sloop; A: primary PCR; B: optimizing PCR, ck: negative control; M: Ladder) Figure 3.13: The sequence alignment of Jex3F+Jex3R2 in barley with the exon3 sequence of sd1 in rice (Blue: primer Jex3F; red: primer Jex3R2).

150bp

Jex3F+Jex3R2 sequence 159 bp of Alexis TCTAACGGACGGTACAAGAGCTGCCTGCACCGGGCGGTGGTGAACCGGCGGCAGGAGCGGCGGTCGCTGGCCTTCTTCCTGTGCCCGCGCGAGGACCGGGTGGTGCGGCCGCCGCCGAGCCTGAGGAGCCCGCGGCACTACCCGGACTTCACCTGAGNN Jex3F+Jex3R2 sequence alignment Query: 11 ggtacaagagctgcctgcaccgggcggtggtgaaccggcggcaggagcggcggtcgctgg 70 |||| ||||||||||||||| ||||||||||||||| ||||| ||||||||||||||||| Sbjct: 4898 ggtataagagctgcctgcacagggcggtggtgaaccagcggcgggagcggcggtcgctgg 4957 Query: 71 ccttcttcctgtgcccgcgcgaggaccgggtggtgcggccgccgccgagc 120 | ||||||||||||||||| |||||| ||||||||||||||||||||||| Sbjct: 4958 cgttcttcctgtgcccgcgggaggacagggtggtgcggccgccgccgagc 5007

M Al Sl Al Sl Al Sl M Alxis ck

3F+3R1 3F+3R2 3F+3R3

Jex3F+Jex3R2

200bp

200bp

63

3.3.5 Size of intron2

The primer pair sdwn2 and Jex3R2, which were proposed to include the exon2,

intron2 and part of exon3, amplified two large fragments which were 1.9 kb and 1.2

kb by using Alexis genome DNA as a template (Fig. 3.14 A). The two fragments of

Alexis were cut out and served as templates in nested PCRs. After nested PCR by

sdwn2+ sdwex2R (Fig. 3.14 B) and Jex3F+Jex3R2 (Fig. 3.14 C), both of the two

large bands had a 300 bp band amplified by primers sdwn2 and sdwex2R (the same

size as the exon2) and a 150 bp band amplified by Jex3F and Jex3R2 (the same size

as the part of exon3). Furthermore, the sequences of the nested PCR fragment were

identical to the sequence obtained from the genomic DNA (data not shown). This

indicates that both the 1.9 kb and 1.2 kb fragments might include intron 2 and the

proposed size of intron2 should be 1450 bp and 750 bp.

A B

1.9kb

1.2kb

M Al Sl Dh Gai ck

Sdwn2+Jex3R2

1kb

sdwn2+ex3R2 PCR bands of Alexis M 1.9kb 1.2kb ck

nested PCR of sdwn2+sdwex2R

300bp

64

1kb

C D M E F

Figure 3.14 sdwn2+Jex3R2 PCR products (A), nested PCR by sdwn2+sdwex2R (B) and Jex3F+Jex3R2 (C), 1.9 kb and 1.2 kb fragments bulking up (D, E) and the purified DNA from the gel (F) ( Al: Alexis; Sl: Sloop; Dh: Dhow; Gai: Gairdner; ck: negative control; M: Ladder; Pro+Gsp: Purified DNA from the 2nd PCR of Genome Walker).

To sequence the two fragments, bulking up of the PCR (Fig 3.14 D, E), DNA gel

extraction (Fig 3.14 F) and cloned into a plasmid vector. However, no insert clone

was obtained. A new pair of primers (sdwex2RF and Jex3FR) was designed closest to

the two terminals of intron2 and the PCRs were performed (Fig. 3.15). Two bright

750bpof Sloop M 1.9K 1.2K loading 1μl purified Alexis DNA

1.2kb

sdwn2+Jex3R2; 1.9kb and 1.2kb of Alexis for cloning

1.9kb

sdwn2+Jex3R2

Pro+Gsp3

1kb

M Alexis ck

sdwn2+Jex3R2

1.9kb

1.2kb 1kb

sdwn2+Jex3R2 PCR bands of Alexis M 1.9kb 1.2kb ck

Nested PCR of Jex3F+Jex3R2

65

bands (800 bp and 1.4 kb) were obtained from genomic DNA of Alexis, Sloop and

Stirling. Sequencing of a 800 bp fragment was attempted but at present the data was

not available and requires further work. Consequently, intron2 size of sdw1 could be

either about 800 bp or 1400 bp. It indicated that there might be two copies of sdw1

gene.

Figure 3.15 The PCR products of primers sdwex2RF and Jex3FR (Al: Alexis; Sl: Sloop; St: Stirling; D7: Dhow5+7 Pro.; S7: Sloop 5+7 Pro.; 5+7: primers sdw5 and sdw7; ck: Negative control ; ck1: sdwex2RF negative control; ck2: Jex3FR negative control; sdwex2RF X: primer sdwex2RF only; Jex3FR X: primer Jex3FR only; M: Ladder).

3.4 Barley semi-dwarfing gene-sdw1 gene structure

So far, the proposed sdw1 structure in barley is similar to sd1, and should include

three exons and two introns. The partial sequence of the first exon of sdw1 was 6 bp

longer than the same region of exon1 of sd1. The second exon of sdw1 was 3 bp

longer than the exon2 of sd1. The part of the third exon size obtained from this

experiment was the same size as the part exon3 of sd1. The first intron of sdw1 was

173 bp compared with 102 bp of the intron1 of sd1 and the second intron might be

M Al Sl St D7 S7 ck Al Sl St D7 S7 ck Al Sl St D7 S7 ck Sl ck1 Sl ck2

sdwex2RF+Jex3FR

Jex3FR X MgCl2: 1.5 mM

MgCl2: 1.2 mM MgCl2: 1.0 mM

66

800 bp or 1400 bp. Therefore, according to the results, the sdw1 gene structure could

be as follows (Fig. 3.16 or Fig. 3.17).

Figure 3.16 The proposed sdw1 gene structure 1. Figure 3.17 The proposed sdw1 gene structure 2.

3.5 Sequence alignment and sequence nucleotide polymorphism identification

Partial sequence comparison was performed in twelve different barley varieties.

Among the twelve extracted genomic DNA of donors, ten varieties had the 500 bp

single bright band by using the primers sdwn1 and sdwex2R (see Fig. 3.18).

However, Dhow and Gairdner did not have the same band by using the DNA

extraction as other varieties (Fig. 3.19 A and B). After the PCR amplifications were

repeated by using different DNA extractions from the same varieties, the same single

bands were obtained from the two varieties (Fig. 3.20). To avoid confusion with other

varieties, the genomic DNA of all other varieties extracted the first time were tested

(intron1 and exon2). No difference was found from the different DNA extractions

Exon1 Exon2 Exon3 ~800bp

Intron1 173bp

Intron2 ~563bp

325bp

~291bp

Exon2 Exon1 Exon3

Intron2 ~1400bp

Intron1 173bp

~563bp

325bp

~291bp

67

among other varieties in this region because the same size bands were obtained (Fig.

3.19 B). The discussion of this is presented in section 4.4.

Figure 3.18 An example of primers sdwn1 and sdwex2R PCR results in different varieties (AC: AC Metcalf; Ba: Baudin; Ga: Galleon; Ha: Haruma Nijo; Ka: Kaputor; Ta: Tallon; ck: Negative control; M: Ladder). A B Figure 3.19 The exception of the two varieties: Dhow and Gairdner missing 500 bp fragment. (’: DNA from different DNA exactions; Sl: Sloop; St: Stirling; Gai; Gairdner; Fr: Franklin; Ya: Yagan; Dh: Dhow; Al: Alexis; AC: AC Metcalf; Gal: Galleon; Ka: Kaputor; Ta: Tallon; ck: Negative control; sdwn1X: one primer sdwn1 only; sdwex2R: one primer sdwex2R only; ckn1: Negative control of sdwn1X; ck2r: Negative control of sdwex2R; M: Ladder).

M Ac Ba Ga Ha Ka Ta ck

sdwn1+sdwex2R

500bp

500bp

M Sl’ St’ Gai Fr Ya Dh ck M Al’ AC’ Ba’ Dh Fr’ Gal’ Gai Gai’ Ka’ Ta’ Ya’ ck AL’ ckn1 AL’ ck2r

sdwn1+sdwex2R

500bp

sdwn1+sdwex2R

sdwn1X

sdwex2R

68

Figure 3.20 The same single bands was obtained from Dhow and Gairdner by using different extractions of DNA. (’: DNA from different extractions of DNA; Sl: Sloop, as a positive control; Gair: Gairdner; D: Dhow; ck: Negative control; M: Ladder).

The sequences in intron1 and exon2 of sdw1 in twelve varieties were obtained (Dhow

and Gairdner’s DNA from different extractions). After sequence alignment, no

polymorphism was found (Table 3.1). In the region of part of exon1, the sequences of

Alexis and Sloop were obtained and also there was no difference between these two

varieties. In the part region of exon3, only Alexis was sequenced due to the limited

time. The obtained sequence of sdw1 from barley was presented in table 3.1, which

included part of exon1, the whole of intron1 and exon2, and part of exon3.

M Sl Gair’ D’ Sl Gair’ D’ Sl Gair’ D’ ck Sl Gair’ D’ Sl Gair’ D’ Sl Gair’ D’ ck

56.7°C

71.9°C 69.6°C 67.6°C 59.9°C

58.2°C

500bp

sdwn1+sdwex2R; Gradient PCR

69

Table 3.1 The alignment of twelve varieties in part of exon1, all of intron1 and exon2 and part of exon3 (Green: primer Ex1F1; Purple: primer sdwn1; N: primer Gsp4; Blue: primer sdwn2; Brown: primer sdwex2R; Yellow: primer Jex3F; red: primer Jex3R2).

Varieties Sequence region

Sequences

Alexis, Sloop

Part of exon1 TACGGGTTCTTCCAGGTGTCCGGGCACGGCGTGGACAACGCCCTGGCGCGCGCGGCGCTGGACGGCGC GAGCGGGTTCTTCCGTCTGCCGCTGGCCGAGAAGCAGCGCGCGCGGCGCATCCCGGGGACCGTGTCCG GGTACACGAGCGCGCACGCCGACCGGTTCGCCTCCAAGCTCCCCTGGAAGGAGACCCTCTCCTTCGGC TTCCACGACCGCGCCGGCGCCGCCGCGCCCGTGGTGGCGGACTACTTCACCAGCACCCTCGGGCCGG ACTACGAGCCAATGGG

Alexis, Ac Metcalf, Baudin, Dhow, Franklin, Gairdner, Galleon, Haruna Nijo, Kaputar, Sloop, Stirling, Yagon

Intron1 GTAATATATCCACTCGCCCACAGCCCTATCCGGCCAGCACGAACCAATCCCCGCCACTGCATTTTTTA ATTTTTTTTGCTTCCGCGCGATCGTACGTTCGATCGGCGCCCACGTAGTACTGTACGTACGCAGGCAG AGTACTGAGGAGAGAACACGTGCGATGATGATTGCAG

Exon2 GAGGGTGTACCAGGAGTACTGCGGCAAGATGAAGGAGCTGTCGCTGAGGATCATGGAGCTGCTGGAGC TGAGCCTGGGCGTGGAGAAGCGCGGGTACTACCGGGACTTCTTCGCGGACAGCAGCTCCATCATGCGG TGCAACTACTACCCGCCGTGCCCGGAGCCGGAGCGCACGCTGGGCACGGGCCCGCACTGCGACCCCAC GGCGCTCACCATCCTCCTCCAGGACGACGTGGGCGGGCTGGAGGTCCTCGTCGACGGCGACTGGCGGC CCGTCCGCCCCGTCCCCGGCGCCATGGTCATCAACATCGGCGACACCTTCATGA

Intron2 No result yet Alexis Part of exon3 TCTAACGGACGGTACAAGAGCTGCCTGCACCGGGCGGTGGTGAACCGGCGGCAGGAGCGGCGGTCGCT

GGCCTTCTTCCTGTGCCCGCGCGAGGACCGGGTGGTGCGGCCGCCGCCGAGCCTGAGGAGCCCGCGGC ACTACCCGGACTTCACCTGAGNN

3.6 Preliminary studies to analyze promoter region- Genome Walker

To obtain the full sequence of exon1, Genome Walker technique was used. Based on

the sequence of intron1 and exon2, Gsp1, Gsp2, Gsp3 and Gsp4 primers were

designed by using Primer 3 (Rozen and Skaltsky, 1998) according to the requirement

of Genome Walker. Initially, the DNA concentration of twelve varieties was

measured and 100 ng/μl concentration DNA was made up. 2 μl of 50 ng/μl of

Genome DNA was run on the gel with the comparison of the stock genome DNA

(Fig. 3.21) to ensure there was no RNA. After digestion by restriction enzyme PVU II

and purification by phenol and chloroform, 5 μl of digest DNA and 1 μl of purified

DNA were run on the gel (Fig. 3.22). After ligation with Adaptor1, PCR reactions

were done. Fig. 3.23 shows the PCR results of the first PCR and Fig. 3.24 shows the

second PCR results. An 800 bp single bright band was obtained from the nested PCR

70

by using Ap1+Gsp2 PCR products. Unfortunately, the sequence result of this band

seemed to be a mixed template and sub-cloning of segments will be required to

resolve the sequence.

M Orig 50ng/μl Orig 50ng/μl GDL1 GDL2 GDL3 GDL4

Dhow-1 Gairdner-1

run 2 μl genomic DNA

Gairdner

Digested (5 μl, left) and purified (1μl, right) Genomic DNA

Figure 3.22 An example of 1μl purified DNA by comparison with 5μl digested DNA.

Figure 3.21 50 ng/μl of Genome DNA (right) by comparison with stock concentrated DNA (left) (M: Ladder).

M SDL1 ck SDL1 ck M S1 S2 ck

Figure 3.23: The first PCR of Genome Walker (SDL1: Sloop digested purified and ligated genome DNA; Ap1: forward promer1; Gsp1-2: reverse primers; ck: Negative control; M: Ladder)

Figure 3.24: The nested PCR of Genome Walker (S1: Ap1+Gsp1 PCR pro.; S2: Ap1+Gsp2 PCR pro.; Ap2: forward promer1; Gsp4: reverse primers; ck: Negative control; M: Ladder).

Ap1+Gsp1 Ap1+Gsp2

Ap2 + Gsp4

71

3.7 Sequence amplification with one primer

During the experiments, several sequences obtained were amplified by one primer

only. For example, the primer pair (sdw2+sdw3) amplified a proposed size fragment

(about 500 bp) of exon1 by using sdw1+sdw3 PCR products (Fig. 3.25 A). After the

band was excised from the gel and cloned (Fig. 3.25 B), the sequence was amplified

by primer sdw3 only (Fig. 3.25 C).

A B C Figure 3.25: A PCR example of single primer amplification (Blue: sdw3 primer; M: Ladder) Another example is a fragment amplified by primers sdw5 and sdw7. In this case, one

primer positive control was set up when trying to amplify intron2 (Fig. 3.26 A). As a

result, sdw7 could not amplify by itself and two bands were found that might not be

amplified by sdw5, which were about 300 bp and 550 bp fragments (Fig. 3.26 B).

After the band was excised, cloned and sequenced (Fig. 3.26 C, D), 300 bp fragment

s115

500bp

M Sdw2+sdw3 ~500bp insert clone screening M Sdw1+sdw3 PCR Products of Stirling

~488bp

Nested PCR by sdw2+sdw3

s111 s122

s121 s120

s119 s118

s117 s116 s114

s113 s112

Insert clone

The ~500 bp sequence of stirling Amplified by sdw3 only TGAAGGAGAGGGTCTCCTTCCAGGCAGCCTCCCGGCGCAGCCAGAGGGCACGCCGTGCCCACATGCCGCCTGGAGCCCCTGAGGCTCCCCTGTGGCCCATCTTCTGGCCCATGGTTCCTTCCGGTGCGTGGATTTTCTCTATTTTTTATCAGAATTTTCCACGACACTTAAATATTCATTTTCCTGCACACAAGAAAACCAGACAGCCAGCTCTTCTGAAAACAACATCAGTTCGGGTTAGTTTCATTCAAATCATGCAAGAATGGAGCCAAAGCAATAGCAAAAGTGTTTGTAAAAGTCGATACGTTTGAGACGTATCAAGCGGCTCCTTGCGTGAAGCTGACAAAGACGCGACGTCGTCCAAGCTAACCCGTGGCGGGTTGGGCGAACCGAGCCGGCTCGACGAGGTCAGTGCAGGTGTCCAGCTCGGGAACCGTCATTCCAATGTTGCCGATGAAGACATGGATCTTGTGGAAGGAGACCCTCTCCTTCA

72

was amplified by sdw7+sdw5 and a 550 bp fragment was amplified by sdw5 only

(Fig. 3.27).

A B C

D E Figure 3.26 nested PCR amplified by primers sdw5 and sdw7 by using Sdw5+8 PCR Products of Alexis (5+7: primers sdw5 and sdw7; 5X: primer sdw5 only; 7X: primer sdw7 only; ck: Negative control; M: Ladder). Figure3.27 The sequences of ~300 bp and ~550 bp fragments (Blue: sdw5; red: sdw7).

Alexis

The sequence of ~550bp insert clone amplified by sdw5 only TTCAACATCGGCGACACCTTCATGACCGCTCGAGTTCGCCAGCTGCCGCACGCGCACATCGTCTTCGTCCTCCAACACAACGATCGCGATATCCCCTCTGAACAATCCTCTTGCACGCCACCGGTCCCTCGCCCCGCCGCACTATGGACCTCCTATTGAGGCAACGATCTTGACAATGAGTACTAGAGGTACGAACGAGGGGCGAAGCCTAGCTACTACGCAAGTGTGTCACTTGGATTTAGCGAGTTTGGGCCCCTCTCGAAGAGGTAAAAATCATACGTCTCGTGCTTGGAGGCTCTGTGTTTTCTATGGGAGGAGGTGATTGCAATAGGGTGTTGAACCCTTGTCCCGGAGCTCAAGGCAGGCTTATATAGAGTGTGTCGCGCCTCATTAGTGATCCCTTTCACGGGCTTTAAATGAATGTAACTATGCCAGATACTAGTAACGAGCACCATTACAATTGTTGTTATAGGTTTCAAATAAATAGAGCTATCATAATTAGACCATTTGAAACTTTGAGAGAGCATGAAGGTGTCGCCGATGTTGAA

The sequence of ~300bp insert cone amplified by sdw7+sdw5 TCCTGTGCAGGCAGCTCTTGCTCAAACTCATCATAAGCAAGCACCCGAGGTGATCCGTATATGAGTTCCGTGGGGAGAACTTCCTCCACCCCATAGACTTGGGAGAAGGGCGTCTGGCCGGTGGCACGATTTGGTGTCGTTCTGAGTGACCAAAGAACCGTCGGCAGCTTATCAATCCAACGCCTCCCGCACTTGCGCAGCTTGTCGAAAGTTCTTGTCTTGAGGCCTCGCAGTCATTCAACATTCGCCCTATCCTATTGTCCGTTGCTCCTTGGGTGAGCAACGGACGCGAAATAGATCTGCTGCCAAGGTCTTGGACATACTGCATGAAGGTGTCGCCGATGTTGA (sdw5)

Alexis

300bp

M 550bp insert clone screening M 300bp insert clone screening

M 5+7 5X 7X ck M sdw5+7 ck sdw5 ck M Sdw5+sdw7

s01 s02 s03 s04 s05 s06 s07 s08 s12 s11 s10 s09

M13F+ M13R; sdw5+sdw7

S301 S302 s303 s304 s31 S32 S33 s34

M13F+M13R; sdw5+sdw7

500bp Cut bands

DNA:3μl of 1/50 sdw5+8 PCR prducts of Alexis

500bp

73

3.8 Summary of results

The barley semi-dwarf gene - sdw1 was probably collinear with the rice semi-

dwarfing gene - sd1, as shown by comparative mapping between barley and rice. The

part sequence of exon1, whole sequence of exon2 and part of exon3 sequences of

sdw1 in barley isolated from the experiments were matched with sd1 except some

minor base pair differences. Intron1 of sdw1 was completely different to of sd1. The

sdw1 gene structure was similar to sd1 but the size of introns and exons were

different. No sequence nucleotide polymorphism was found among barley varieties

within intron1 and exon2 in the experiments. Due to limited time, the sequences of the

promoter region and 277 bp of exon1 could not be obtained by Genome Walker. Also

the bands which were proposed to include the intron 2 could not be cloned and

sequenced. According to the experiments, setting a single primer reaction as a control.

74

Chapter 4

General Discussion

75

4.1 Overview of research goals

Most of the research goals were achieved in this project. Firstly, dwarfing genes were

searched on database and listed (Appendix 1-4) for the four main cereal crops which

were rice, wheat, barley and maize. Some dwarfing genes were listed with

chromosome location. Different dwarfing genes encode different enzymes which

control different steps of the GA biosynthetic pathway (Fig. 1.2 and Fig. 1.3). A few

dwarfing genes could control more than one step such as sln in pea, which blocks two

steps: GA29 to GA29-catabolite in maturing seeds (Reid et al., 1992) and GA20 to GA29

(Ross et al., 1995). Some dwarfing mutants are GA sensitive and some are GA

insensitive. By comparative mapping among rice, wheat, barley and maize, three pairs

of dwarfing genes were identified to be homologues to each other: D8 (maize) and

Rht1 (RhtB1b, wheat), Rht2 (RhtD1b, wheat); Dwf2 (barley) and Rht1 (RhtB1b,

wheat), Rht2 (RhtD1b, wheat). In particular, the orthologue between sd1 (rice) and

sdw1 (barley) was discovered using bioinformatics (Chengdao Li contributed to this

discovery). By using the sd1 gene as an anchor, part of exon1, the whole of exon2 and

intron1, and part of exon3 of the sdw1 gene were isolated from barley. This result has

not yet been reported in the literature. The sequence results obtained from this project

showed that no polymorphism was found in the intron1 and exon2 region among

twelve barley varieties. Continued research needs to be done on this work as it will

significantly contribute to improving barley breeding.

4.2 Lack of polymorphism in intron1 and exon2 of sdw1 among twelve varieties

The sequences were obtained from twelve barley varieties (Dhow and Gairdner’s

DNA from different extractions) by using sdwn1 and sdwex2R primers (amplifying

76

intron1 and exon2). The sequencing results were identical across the 12 barley

varieties (shown in Table 3.1) without a single base pair difference. The sequence

nucleotide polymorphism could be in other regions, such as exon1, exon3 and intron2,

whose sequences have not been completely obtained. Identifying sequence nucleotide

polymorphism requires further research.

Another possibility is that there is no polymorphism in the three exons and two

introns of the sdw1 gene in barley. The polymorphism might be in the promoter

region (5’ untranslated region (UTR) or leader sequence) and 3’ UTR (trailer

sequence) region, which control the expression of the gene (Twyman, 1998). 5’UTR

controls the connection between mRNA and ribosomes, and also affects attenuator

control. 3’UTR influences the stability of mRNA.

Four mutants of the sd1 gene were found in rice: sd1-1, sd1-2, sd1-3 and sd1-4

(Sasaki et al., 2002; Sakamoto et al., 2004). These sd1 alleles were from Dee-geo-

woo-gen, Jikkoku, Calrose76 and Reimei respectively. The wild type parent of Dee-

geo-woo-gen is Woo-gen. Calrose is the wild type parent of Calrose76. The wild type

of Reimei is Fujiminori. The source of Jikkoku is unknown. As already reported

(Sasaki et al., 2002), sd1-1 was caused by a 383-bp deletion in exon1, intron1 and

exon2, which induced a frame shift and created a premature stop codon, whereas the

other three sd1 alleles had single nucleotide substitutions, which induced amino acid

changes (sd1-2, sd1-3, and sd1-4). N’ 349 amino acid, aspartic acid (D), in sd1

protein sequence was replaced by histidine (H) in sd1-2 line in exon3. N’ 266 leucine

(L) in exon2 and 94 glycine (G) in exon1 were changed by phenylalanine (F) and

77

valine (V) in sd1-3 and sd1-4 lines respectively. All these four alleles code for an

inactive OsGA20ox2 (Sasaki et al., 2002).

4.3 Reasons for non completion on sequence of exon1, exon3 and intron2 of sdw1

To find a gene sequence, it is very common to use the conserved region to design

primers. Most of the primers used in this research were designed based on conserved

nucleotide regions. Some primers positioned in exons worked successfully in the

experiments. However, some of primers were designed just based on the sd1 intron

from rice and UTR sequence such as sdw1, sdw2, sdw8 and Jex3R4 because neither

suitable exon sequence nor conserved region could be used at both the beginning and

ending regions of sd1. According to the experiments, the primers located in introns

did not work very well and no correct sequence was obtained. That is one of the

reasons for non-completion of the sequence of exon1 and exon3 of sdw1. Another

reason for the missing part of exon1 is that the Genome Walker was not successful.

The single band obtained from the nested PCR (Fig. 3.24) was sequenced as a mixed

template. The part of exon1 and promoter region of sdw1 might be involved in the

mixed template. If time had been allowed, the band could have been cloned and

sequenced. With respect to the missing part of exon3, the 5’ and 3’ primer RACE

approach could be used to obtain the missing part of exon3 (Schramm et al., 2000).

This method is also suitable to be used in the isolation of the missing exon1. Due to

limited time, the fragments of 800 bp and 1400 bp, which might be the intron2 of

sdw1, could not be cloned and sequenced. This requires further research and

experiment.

78

4.4 Amplification of the 500 bp from the genomic DNA of Dhow and Gairdner

PCRs carried out using sdwn1 and sdwex2R primers did not initially amplify the 500

bp band from the two varieties-Dhow and Gairdner (Fig. 3.19 A) while a single bright

band was amplified in the other ten varieties. When using different extractions of

DNA from the same varieties, the single and same size bands were obtained. The

DNA of the other ten varieties extracted at the same time was tested by using different

extractions of DNA to avoid confusion between varieties. But no confusion was

found. It was unlikely that the genome DNA of these two varieties was degraded. As

can be seen in Fig. 3.19 B, besides the 500 bp fragment, the other bands of Dhow are

the same as other varieties. The possibility is that sequence variation may be present

in these two barley varieties in the primer sequence regions.

4.5 sdw1 sequence analysis

4.5.1 DNA sequence and amino acid sequence comparison between sdw1 and sd1

Partial homology sequence of the rice semi-dwarf gene sd1 in rice was isolated from

barley as a candidate gene for sdw1. In total, 943 bp sequences of sdw1 were

obtained. Except for 173 bp of intron1, 770 bp sequences from exon1, 2 and 3 show

high similarity with the sd1 cDNA. In total, the sequence similarity between sdw1 and

sd1 is 88.3%. As can be seen from Fig. 4.1 and 4.2, in exon1, according to the

obtained sequence from barley, thirty-two base pairs were changed and six base pairs

were added in sdw1. Sequence similarity was 86.7%. Consequently, eight amino acids

were replaced and two additional amino acids were inserted (Fig. 4.2). Amino acid

79

similarity was 88.2%. In exon 2, twenty-one base pairs were changed in sdw1

compared with sd1 and three base pairs were inserted to the sdw1 sequence (Fig. 4.1).

Sequence similarity was 92.6%. As a result, seven amino acids were replaced and one

amino acid was added to the sequence. Amino acid similarity was 92.6% (Fig. 4.2).

In exon3, nineteen base pairs were altered (Fig. 4.1) and six amino acids were

replaced (Fig. 4.2). The sequence similarity was 87.7% and the amino acid similarity

was 88.3%. This indicates that exon2 of sdw1 might be more conserved compared

with the other exons because of the greatest similarity (92.6%) of its amino acid

sequences between sdw1 and sd1.

80

Frame Up: sd1 cDNA sequence; Down: sdw1 cDNA sequence; -: Missing part

1 ATGGTGGCCGAGCACCCCACGCCACCACAGCCGCACCAACCACCGCCCATGGACTCCACC

------------------------------------------------------------

61 GCCGGCTCTGGCATTGCCGCCCCGGCGGCGGCGGCGGTGTGCGACCTGAGGATGGAGCCC

------------------------------------------------------------

121 AAGATCCCGGAGCCATTCGTGTGGCCGAACGGCGACGCGAGGCCGGCGTCGGCGGCGGAG

------------------------------------------------------------

181 CTGGACATGCCCGTGGTCGACGTGGGCGTGCTCCGCGACGGCGACGCCGAGGGGCTGCGC

------------------------------------------------------------

241 CGCGCCGCGGCGCAGGTGGCCGCCGCGTGCGCCACGCACGGGTTCTTCCAGGTGTCCGAG

-------------------------------------ACGGGTTCTTCCAGGTGTCCGGG

301 CACGGCGTCGACGCCGCTCTGGCGCGCGCCGCGCTCGACGGCGCCAGCGACTTCTTCCGC

CACGGCGTGGACAACGCCCTGGCGCGCGCGGCGCTGGACGGCGCGAGCGGGTTCTTCCGT

361 CTCCCGCTCGCCGAGAAGCGCCGCGCGCGCCGCGTCCCGGGCACCGTGTCCGGCTACACC

CTGCCGCTGGCCGAGAAGCAGCGCGCGCGGCGCATCCCGGGGACCGTGTCCGGGTACACG

421 AGCGCCCACGCCGACCGCTTCGCCTCCAAGCTCCCATGGAAGGAGACCCTCTCCTTCGGC

AGCGCGCACGCCGACCGGTTCGCCTCCAAGCTCCCCTGGAAGGAGACCCTCTCCTTCGGC

481 TTCCACGACC------GCGCCGCCGCCCCCGTCGTCGCCGACTACTTCTCCAGCACCCTC

TTCCACGACCGCGCCGGCGCCGCCGCGCCCGTGGTGGCGGACTACTTCACCAGCACCCTC

541 GGCCCCGACTTCGCGCCAATGGGGAGGGTGTACCAGAAGTACTGCGAGGAGATGAAGGAG

GGGCCGGACTACGAGCCAATGGGGAGGGTGTACCAGGAGTACTGCGGCAAGATGAAGGAG

601 CTGTCGCTGACGATCATGGAACTCCTGGAGCTGAGCCTGGGCGTGGAG---CGAGGCTAC

CTGTCGCTGAGGATCATGGAGCTGCTGGAGCTGAGCCTGGGCGTGGAGAAGCGCGGGTAC

661 TACAGGGAGTTCTTCGCGGACAGCAGCTCAATCATGCGGTGCAACTACTACCCGCCATGC

TACCGGGACTTCTTCGCGGACAGCAGCTCCATCATGCGGTGCAACTACTACCCGCCGTGC

721 CCGGAGCCGGAGCGGACGCTCGGCACGGGCCCGCACTGCGACCCCACCGCCCTCACCATC

CCGGAGCCGGAGCGCACGCTGGGCACGGGCCCGCACTGCGACCCCACGGCGCTCACCATC

781 CTCCTCCAGGACGACGTCGGCGGCCTCGAGGTCCTCGTCGACGGCGAATGGCGCCCCGTC

CTCCTCCAGGACGACGTGGGCGGGCTGGAGGTCCTCGTCGACGGCGACTGGCGGCCCGTC

841 AGCCCCGTCCCCGGCGCCATGGTCATCAACATCGGCGACACCTTCATGGCGCTGTCGAAC

CGCCCCGTCCCCGGCGCCATGGTCATCAACATCGGCGACACCTTCATGGCTCTGTCTAAC

901 GGGAGGTATAAGAGCTGCCTGCACAGGGCGGTGGTGAACCAGCGGCGGGAGCGGCGGTCG

GGACGGTACAAGAGCTGCCTGCACCGGGCGGTGGTGAACCGGCGGCAGGAGCGGCGGTCG

961 CTGGCGTTCTTCCTGTGCCCGCGGGAGGACAGGGTGGTGCGGCCGCCGCCGAGCGCCGCC

CTGGCCTTCTTCCTGTGCCCGCGCGAGGACCGGGTGGTGCGGCCGCCGCCGAGCCTGAGG

1021 ACGCCGCAGCACTACCCGGACTTCACCTGGGCCGACCTCATGCGCTTCACGCAGCGCCAC

AGCCCGCGGCACTACCCGGACTTCACC---------------------------------

1081 TACCGCGCCGACACCCGCACGCTCGACGCCTTCACGCGCTGGCTCGCGCCGCCGGCCGCC

------------------------------------------------------------

1141 GACGCCGCCGCGACGGCGCAGGTCGAGGCGGCCAGC

------------------------------------ Figure 4.1 sdw1 cDNA sequence alignment with the cDNA of sd1 (accession No.: AF465255)(sd1 cDNA length:1158, starts at: 1, ends at 1158; sdw1 obtained cDNA length: 769, starts at: 278, ends at:1047, missing 6 bp from 889-894; blue: exon1, black: exon2 and purple: exon3; red: conserved region in exon1; dark blue: conserved region in exon2; green: conserved region in exon3).

81

Frame 1 Up: sd1 Protein Sequence; down: sdw1 protein sequence

1 M V A E H P T P P Q P H Q P P P M D S T

- - - - - - - - - - - - - - - - - - - -

21 A G S G I A A P A A A A V C D L R M E P

- - - - - - - - - - - - - - - - - - - -

41 K I P E P F V W P N G D A R P A S A A E

- - - - - - - - - - - - - - - - - - - -

61 L D M P V V D V G V L R D G D A E G L R

- - - - - - - - - - - - - - - - - - - -

81 R A A A Q V A A A C A T H G F F Q V S E

- - - - - - - - - - - - - G F F Q V S G

101 H G V D A A L A R A A L D G A S D F F R

H G V D N A L A R A A L D G A S G F F R

121 L P L A E K R R A R R V P G T V S G Y T

L P L A E K Q R A R R I P G T V S G Y T

141 S A H A D R F A S K L P W K E T L S F G

S A H A D R F A S K L P W K E T L S F G

161 F H D R - - A A A P V V A D Y F S S T L

F H D R A G A A A P V V A D Y F T S T L

181 G P D F A P M G R V Y Q K Y C E E M K E

G P D Y E P M G R V Y Q E Y C G K M K E

201 L S L T I M E L L E L S L G V E - R G Y

L S L R I M E L L E L S L G V E K R G Y

221 Y R E F F A D S S S I M R C N Y Y P P C

Y R D F F A D S S S I M R C N Y Y P P C

241 P E P E R T L G T G P H C D P T A L T I

P E P E R T L G T G P H C D P T A L T I

261 L L Q D D V G G L E V L V D G E W R P V

L L Q D D V G G L E V L V D G D W R P V

281 S P V P G A M V I N I G D T F M A L S N

R P V P G A M V I N I G D T F M A L S N

301 G R Y K S C L H R A V V N Q R R E R R S

G R Y K S C L H R A V V N R R Q E R R S

321 L A F F L C P R E D R V V R P P P S A A

L A F F L C P R E D R V V R P P P S L R

341 T P Q H Y P D F T W A D L M R F T Q R H

S P R H Y P D F T - - - - - - - - - - -

361 Y R A D T R T L D A F T R W L A P P A A

- - - - - - - - - - - - - - - - - - - -

381 D A A A T A Q V E A A S

- - - - - - - - - - - - Figure 4.2 Alignment amino acid sequence of sd1 (accession No. AF465255) and sdw1 (sd1 protein sequence length: 392, stats at: 1 ends at 392; sdw1 obtained protein sequence length: 256, starts at: 94 ends at: 349, missing A and L in exon3; Blue: missing region in exon1, exon2 and exon3; red: exon1 conserved region; green: exon2 conserved region; dark blue: exon3 conserved region; black: different amino acids; purple: sdw1 insert region).

4.5.2 sdw1 amino acid comparison with the GA20 oxidases of rice, wheat and barley

The GA20-oxidases shown in Figure 4.3 were the most highly conserved amino acid

sequences with sdw1 among rice, wheat and barley through data BLAST. As shown

82

in Figure 4.3, sdw1 shared 88.3% amino acid similarity with rice sd1 (OsGA20ox2).

In contrast, it shared 57% with GA20-oxidase-1 (Hv20ox1, accession No:

AY551428.1) mapped on chromosome 7 (5H), 54% with GA20-oxidase-3 (Hv20ox3,

accession No: AY551429.1) mapped on chromosome 3H in barley and 56% with

wheat EST (accession No: AY1007). This shows the sdw1 gene is a GA20-oxidase

and is also different from other GA20-oxidases genes published in barley and wheat. It

further confirms the homology between sdw1 and sd1. As can be seen in Figure 4.3,

GA20-oxidases-1 (Hv20ox1) in barley is the same gene as the wheat EST AY14007

because it shared 97% similarity with the wheat EST. By comparison, GA20-oxidases-

3 (Hv20ox3) only shared 59% amino acid similarity with the wheat EST. All these

three amino sequences (Hv20ox1, Hv20ox3 and wheat EST AY14007) are different

from sd1 due to the significant differences from sd1 in the amino acid sequences. The

similarity of these three genes compared with sd1 was 50%, 47% and 50%

respectively. The comparative bioinformatics of those genes are discussed in Part 4.6.

83

1 11 21 31 41 51 wheat --------------------------MVQPVFDAAVLSGRADIPSQFIWPEGESPTPDAA Hv20ox1 --------------------------MVQPVFDAAVLSGRTDIPSQFIWPEGESPTPDAT Hv20ox3 --------------------------MASLVFDAAVLSRKEDIPPQFIWPADEAPSVDGV sd1_seq -MVAEHPTPPQPHQPPPMDSTAGSGIAAPAAAAVCDLRMEPKIPEPFVWPNGDARP-ASA Hv sdw1 ------------------------------------------------------------ 61 71 81 91 101 111 wheat EELHVPLIDIGGMLSGDPAAAAEVTRLVGEACERHGFFQVVNHGIDAELLADAHRCVDNF Hv20ox1 EEMHVPLIDIGGMLSGDPRAAAEVTRLVGEACERHGFFQVVNHGIDAQLLADAHRCVDAF Hv20ox3 EEIVVPVVDLAGFLAGDDAGLNELV----AACERHGFFQVVNHGVDPALLAKAYRCCDAF sd1_seq AELDMPVVDVGVLRDGDAEGLRRAAAQVAAACATHGFFQVSEHGVDAALARAALDGASDF Hv sdw1 -----------------------------------GFFQVSGHGVDNALARAALDGASGF 121 131 141 151 161 171 wheat FTMPLPEKQRALRHPGESCGYASSFTGRFASKLPWKETLSFRSCPS---DPALVVDYIVA Hv20ox1 FTMPLPEKQRALRRPGESCGYASSFTGRFASKLPWKETLSFRSCPS---DPALVVDYIVA Hv20ox3 YALPLAEKQRAQRRLGENHGYAGSFVGRFGSKLPWKETMSFNCSAAPE-SARKVVDYFVG sd1_seq FRLPLAEKRRARRVPGTVSGYTSAHADRFASKLPWKETLSFGFHDRA--AAPVVADYFSS Hv sdw1 FRLPLAEKQRARRIPGTVSGYTSAHADRFASKLPWKETLSFGFHDRAGAAAPVVADYFTS 181 191 201 211 221 231 wheat TLGEDHRRLGEVYARYCSEMSRLSLEIMEVLGESLGVGR-AHYRRFFEGNDSIMRLNYYP Hv20ox1 TLGEDHRRLGEVYARYCSEMSRLSLEIMEVLGESLGVGR-AHYRRFFEGNESIMRLNYYP Hv20ox3 VLGEEYRHMGDVWQEYCNEMTRLALDVTEVLAACLGLDR-GALRGFFAGDDSLMRLNHYP sd1_seq TLGPDFAPMGRVYQKYCEEMKELSLTIMELLELSLGVER-GYYREFFADSSSIMRCNYYP Hv sdw1 TLGPDYEPMGRVYQEYCGKMKELSLRIMELLELSLGVEKRGYYRDFFADSSSIMRCNYYP 241 251 261 271 281 291 wheat PCQRPLETLGTGPHCDPTSLTILHQDNVGGLQVHTEGRWRSIRPRADAFVVNIGDTFMAL Hv20ox1 PCQRPLETLGTGPHCDPTSLTILHQDDVGGLQVHTDGRWRSIRPRADAFVVNIGDTFMAL Hv20ox3 PCKKPHLTLGTGPHHDPTALTLLHQDDVGGLEVFTGGAWRAVRPRSDAFVVNIGDTFSAL sd1_seq PCPEPERTLGTGPHCDPTALTILLQDDVGGLEVLVDGEWRPVSPVPGAMVINIGDTFMAL Hv sdw1 PCPEPERTLGTGPHCDPTALTILLQDDVGGLEVLVDGDWRPVRPVPGAMVINIGDTFMAL 301 311 321 331 341 351 wheat SNGRYKSCLHRAVVNSRVPRKSLAFFLCPEMDKVVAPPGTLVDAAN-PRAYPDFTWRSLL Hv20ox1 SNGRYKSCLHRAVVNSRVPRKSLAFFLCPEMDKVVAPPGTLVDAAN-PRAYPDFTWRSLL Hv20ox3 TNGRHVSCLHRAVVNGSLARRSLTFFLNPQLDRPVTPPAELLAIDGRPRVYPDFTWREFL sd1_seq SNGRYKSCLHRAVVNQRRERRSLAFFLCPREDRVVRPP----PSAATPQHYPDFTWADLM Hv sdw1 SNGRYKSCLHRAVVNRRQERRSLAFFLCPREDRVVRPP----PSLRSPRHYPDFT----- 361 371 381 391 401 wheat DFTQKHYRADMKTLEVFSSWIVQQQQPQPART--361---- Hv20ox1 DFTQKHYRADMKTLEVFSSWVVQQQ-PASART-360----- Hv20ox3 EFTQKHYRSDSRTLDAFVAWINQGHTG-355---------- sd1_seq RFTQRHYRADTRTLDAFTRWLAPPAADAAATAQVEAAS 389 Hv sdw1 -231------------------------------------- Figure 4.3 Alignment of the amino acid sequence of gibberellic acid 20- (GA20-)oxidase (wheat EST: AY1007; Hv20ox1: accession No. AY551428.1; Hv20ox3: accession No. AY551429.1; sd1: accession No.: AF465255; red: Similarity with wheat EST AY1007; dark blue: Similarity with rice sd1; Hv: Hordeum vulgare).

4.5.3 sdw1 intron1 sequence analysis

The sdw1 intron1 sequence was blasted through NCBI web site but no matching

sequence was found. This indicates that the sdw1 gene sequence has not been reported

so far. (See Appendix 6)

84

4.6 GA20-oxidases comparative bioinformatics

The results in Chapter 3 indicate sdw1 might have synteny with sd1. Hv20ox1

(located on 5HL in barley) has almost the same amino acid sequence as wheat EST

AY14007 (97% similarity) and so is likely to be the same gene. Furthermore, the

Hv20ox1 probe identified three homoeologous bands on the long arm of chromosome

5BL, 5DL and 4AL in wheat (Spielmeyer et. al., 2004). Even though the wheat EST

(AY14007) does not have a gene name and has not been mapped, it could be said that

wheat EST (AY14007) might be on chromosome 5BL, 5DL and 4AL. Synteny

between barley chromosome 5H and rice chromosome 3 (Fig. 4.4) suggests that

Hv20ox1 and rice OsGA20ox1 are orthologous genes. Hv20ox1 and OsGA20ox1

protein sequences are also more closely related to each other than to other GA20ox

sequences (Fig. 4.5). Hv20ox1 (accession No. AY551428.1) shares 68.6% amino acid

sequence similarity with OsGA20ox1 (accession No. U50333) (Fig. 4.5).

Figure 4.4 Comparative mapping results among wheat 5DL, barley 5H and rice 3 (the comparative map was modified from Li et al., 2004)

OsGA20ox1

Bin 12 Bin13 Bin14 Bin15 CDO506, Hv20ox1, ABC309

AY1007

85

1 11 21 31 41 51 Hv20ox1 ---MVQP----VFDAAVLSGRTDIPSQFIWPEGESPTPDATEEMHVPLIDIGGMLSGDPR OsGA20ox1 MSMVVQQEQEVVFDAAVLSGQTEIPSQFIWPAEESPGSVAVEELEVALIDVG---AGAER 61 71 81 91 101 111 Hv20ox1 AAAEVTRLVGEACERHGFFQVVNHGIDAQLLADAHRCVDAFFTMPLPEKQRALRRPGESC OsGA20ox1 SS--VVRQVGEACERHGFFLVVNHGIEAALLEEAHRCMDAFFTLPLGEKQRRSGARGRTA 121 131 141 151 161 171 Hv20ox1 GYASSFTGRFASKLPWKETLSFR-SCPSDP---ALVVDYIVATLGEDH-RRLGEVYARYC OsGA20ox1 -YASSFTGRFASKLPWKETLSFRYSSAGDEEGEEGVGEYLVRKLGAEHGRRLGEVYSRYC 181 191 201 211 221 231 Hv20ox1 SEMSRLSLEIMEVLGESLG-VG-RAHY-RRFFEGNESIMRLNYYPPCQRPLETLGTGPHC OsGA20ox1 HEMSRLSLELMEVLGESLGIVGDRRHYFRRFFQRNDSIMRLNYYPACQRPLDTLGTGPHC 241 251 261 271 281 291 Hv20ox1 DPTSLTILHQDDVGGLQVHTDGRWRSIRPRADAFVVNIGDTFMALSNGRYKSCLHRAVVN OsGA20ox1 DPTSLTILHQDHVGGLEVWAEGRWRAIRPRPGALVVNVGDTFMALSNARYRSCLHRAVVN 301 311 321 331 341 351 Hv20ox1 SRVPRKSLAFFLCPEMDKVVAPPGTLVDAANPRAYPDFTWRSLLDFTQKHYRADMKTLEV OsGA20ox1 STAPRRSLAFFLCPEMDTVVRPPEELVDDHHPRVYPDFTWRALLDFTQRHYRADMRTFQA 361 371 Hv20ox1 FSSWVVQQ---QPASART OsGA20ox1 FSDWLNHHRHLQP-TIYS

Figure 4.5 Protein sequence comparison between Hv20ox1 (accession No. AY551428.1) and OsGA20ox1 (accession No. U50333) (red: conserved region; Hv: Hordeum vulgare; Os: Oryza sativa). Although Hv20ox3 was mapped on barley 3HL, it is not orthologous to OsGA20ox2

(sd1), as shown (Fig.4.3) by only 47% protein similarity. However, Hv20ox3 has

considerably higher sequence similarity to rice OsGA20ox3 (accession No.

AP005840.4) located on chromosome 7S. Hv20ox3 shared 72% similarity of rice

OsGA20ox3 (Fig. 4.6). The syntenic relationship between barley chromosome 3HL

and rice chromosome 7S remains unclear. It is possible that either OsGA20ox3 or

Hv20ox3 has moved to a non-syntenic region in a small-scale translocation event.

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1 11 21 31 41 51 Hv20ox3 MASLVFDAAVLSRKEDIPPQFIWPADEAPSVDG--VEEIVVPVVDLAGFLAGDDAGLNEL OsGA20ox3 MAAVVFDAAILSKQEAIPAQFVWPADEAPAADDGVVEEIAIPVVDLAAFLASGGIGR-DV 61 71 81 91 101 111 Hv20ox3 VAACERHGFFQVVNHGVDPALLAKAYRCCDAFYALPLAEKQRAQRRLGENHGYAGSFVGR OsGA20ox3 AEACERHGFFQVVNHGVDPALLAEAYRCCDAFYARPLAEKQRARRRPGENHGYASSFTGR 121 131 141 151 161 171 Hv20ox3 FGSKLPWKETMSFNCSAAPESARKVVDYFVGVLGEEYRHMGDVWQEYCNEMTRLALDVTE OsGA20ox3 FDCKLPWKETMSFNCSAAPGNARMVADYFVDALGEEYRHMGEVYQEYCDVMTRLALDVTE 181 191 201 211 221 231 Hv20ox3 VLAACLGLDRGALRGFFAGDDSLMRLNHYPPCKKPHLTLGTGPHHDPTALTLLHQDDVGG OsGA20ox3 VLAVALGLGRGELRGFFADGDPVMRLNHYPPCRQPHLTLGTGPHRDPTSLTLLHQDDVGG 241 251 261 271 281 291 Hv20ox3 LEVFTG------GAWRAVRPRSDAFVVNIGDTFSALTNGRHVSCLHRAVVNGSLARRSLT OsGA20ox3 LQVLPDDAAAAAGGWRAVRPRADAFVVNIGDTFAALTNGRHASCLHRAVVNGRVARRSLT 301 311 321 331 341 351 Hv20ox3 FFLNPQLDRPVTPPAELLAIDGRPRVYPDFTWREFLEFTQKHYRSDSRTLDAFVAWINQ- OsGA20ox3 FFLNPRLDRVVSPPPALVDA-AHPRAFPDFTWREFLEFTQRHYRSDTNTMDAFVAWIKQR 361 Hv20ox3 -GHTG---- OsGA20ox3 NGYESLDKY

Figure 4.6 The protein sequence alignment of Hv20ox3 (accession No. AY551429.1) and OsGA20ox3 (accession No. AP005840.4) (red: conserved region; Hv: Hordeum vulgare; Os: Oryza sativa).

Figure 4.7 summarizes the GA20-oxidase relationship in barley, wheat and rice.

Barley Hv20ox1 is collinear to rice OsGA20ox1 (Li et al., 2004; Spielmeyer et al.,

2004) and wheat EST AY14007. Hv20ox1 was mapped on barley 5H and very close

to CDO506 which was next to the marker Abg391 (Li et al., 2004). Rice OsGA20ox1

was mapped close to marker Abg391 as well (Spielmeyer et al., 2004). Therefore, the

orthologue gene in wheat (AY14007) might have a similar location which is next to

the marker Abg391. The sdw1 gene identified in this research is related to rice

OsGA20ox2 gene. According to the comparative map between barley 3H and rice 1,

sdw1 might be close to the marker R1545 as this marker is the closest conserved

marker to OsGA20ox2 (sd1) in rice (Fig. 3.1 and 3.2). The Hv20ox3 gene has

orthologues with rice OsGA20ox3 (Spielmeyer et al., 2004; Sakamoto et al., 2004)

and Hv20ox3 has been mapped on barley 3HL and wheat group 3L ( Spielmeyer et

al., 2004). However, its specific chromosome location is hard to predict even through

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its homology to the OsGA20ox3 gene because no orthologues have been found

between barley 3HL and rice 7S (Spielmeyer et al., 2004).

Figure 4.7 The orthologues of GA20-oxidase genes in barley, wheat and rice (References : a: Spielmeyer et al., 2004; b: Li et al., 2004; c: Sakamoto et al., 2004; d: NCBI blast; e:from this research; f: Spielmeyer et al., 2002; Marker (I): predicted region and proposed linking marker; Hv: Hordeum vulgare; Os: Oryza sativa).

4.7 Function of GA20-oxidases

The gene function of GA20-oxidase in barley is not very clear as the genes were only

recently published (May of 2004). However, their orthologue genes in rice have been

studied extensively. As expected, GA20-oxidase genes in barley might have a similar

gene function to their homologous genes in rice.

As shown in Figure 1.3, several steps of the GA biosynthetic pathway (GA53 to GA20)

are controlled by GA20-oxidase. As shown by the research (Spielmeyer et al., 2002;

Sakamoto et al., 2004), OsGA20ox2 (sd1) gene controls the step from GA53 to GA44.

This is because the levels of GA44, GA19, GA20, GA1, GA29, and GA8 in sd1-1 were

lower than in the original strain, whereas the amount of GA53 in sd1-1 was slightly

higher (Spielmeyer et al., 2002; Sakamoto et al., 2004). Therefore, the amount of

AY551429 OSJNBa0050F 10 No AF465255 A551428 AY14007 U50333 Accession No.

unknown R2829 R1545 R1545 Abg391 Abg391 Abg391 Closed markers

3HL 7S 3HL 1L 5HL 4AL 5BL 5DL 3L Chromosome location

orthologues

Barley wheat rice

GA20-oxidase gene: Hv20ox1ab AY14007d OsGA20ox1ab sdw1e OsGA20ox2(sd1) f Hv20ox3a OsGA20ox3 (OSJNBa0050F 10) ac

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GA1 is reduced and dwarfing is a result of consequence. This indicates that the sd1

orthologues gene-sdw1 (barley) might control the step from GA53 to GA44.

In rice, OsGA20ox (1-3) genes were expressed in immature and mature panicles at

different levels (Sakamoto et al., 2004). Both articles (Sasaki et al., 2002; Sakamoto

et al., 2004) mentioned that OsGA20ox2/SD1 transcript was accumulated in stems. In

contrast, the result of OsGA20ox1 content in organs was different. Sasaki et al. (2002)

concluded that OsGA20ox1 was preferentially expressed in the panicles, but Sakamoto

et al. (2004) found that OsGA20ox1 was expressed in all vegetative organs according

to their results. In addition, OsGA20ox3 was expressed in the panicles (Sakamoto et

al., 2004) but its expression was not observed in any vegetative organs. It was

suggested by Sakamoto et al. (2004) that OsGA20ox2/sd1 is the dominant GA20ox in

stems and that OsGA20ox1 could be also involved in GA biosynthesis in vegetative

organs.

In barley, GA20ox might correspond to low seed dormancy (Li et al., 2004). It has

been observed that GA20 in seeds with low dormancy was five times higher than that

from seeds with high dormancy (Fernandez et al., 2002). Furthermore, Hv20ox1,

which is collinear with OsGA20ox1, was mapped within the seed dormancy/PHS QTL

region (Li et al., 2004). This confirmed the role of Hv20ox1. In addition, OsGA20ox1

expressed in the panicles (Sasaki et al., 2002) might contribute to the seed domancy

traits. This indicates that its orthologue gene Hv20ox1 might be expressed in the

panicles and control some seed traits.

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4.8 Value of sd1 in rice breeding and sdw1 in barley breeding

Short-stature cultivars have been developed by several breeders worldwide to reduce

lodging and increase grain yield. As described in Chapter 1, among the seventy-five

dwarfing genes in rice, semi-dwarfing gene sd1 is the most commonly utilized gene in

the rice breeding system (Futshura and Kikuchi, 1997). Its recessive character results

in a shortened culm with improved lodging resistance and a greater harvest index,

allowing for increased use of nitrogen fertilizers (Spielmeyer et al., 2002). The sd1

gene was first identified in the Chinese variety Dee-geo-woo-gen (DGWG), and was

crossed in the early 1960s with Peta (tall) to develop the semi-dwarf cultivar IR8

(Kush, 1993), which produced record yields throughout Asia and formed the basis for

the development of new high-yielding semi-dwarf plant types. Since the 1960s, sd1

has remained the predominant semi-dwarfing gene present in current rice cultivars

(Spielmeyer et al., 2002).

Three hundred and five semi-dwarfing genes are listed (Appendix 2; some of them are

alleles) in barley, but only sdw (denso), uzu and Gpert (air-e) have been utilized as

important dwarfing genes in barley improvement (Rasmusson, 1991; Ivandic et al.,

1999; Zhang, 2000) and also sdw (denso) gene has been wildly used in breeding

programs in the United States, Canada (Rasmusson, 1991) and Europe (Mickelson

and Rasmusson, 1994). In barley breeding, it seems that sdw (denso) has many alleles

which have significant different effects on agronomic traits. For example, sdw and

denso are different alleles with important different characteristics on the same

chromosome location (Hellewell et al., 2000; Mickelson and Rasmuson, 1994). The

sdw gene is widely accepted for feed barley while the denso gene is used for malting

90

barley production (Mickelson and Rasmuson, 1994). The alleles sdw and denso derive

from different sources. The gene sdw is from Jotun and denso is from Triumph. Both

have similar traits, such as late heading, low seed weight and high β–glucan content

(Hellewell et al., 2000). Both of them have also been reported to have lower yield,

seed weight and percentage of plump kernels than tall isolines (Hellewell et al., 2000;

Mickelson and Rasmuson, 1994). On the other hand, some varieties with the sdw gene

display increased grain yield, such as UC 828 (Gallagher et al., 1996) and Royal

(Rasmusson et al., 1994). The grain yield of varieties (lines) with sdw gene depends

on culture regions (Rasmusson et al., 1994) and weather because most of them

present later heading (Hellewell et al., 2000; Gallagher et al., 1996; Rasmusson et al.,

1994). Therefore, it is critical to obtain the sdw sequence to distinguish the sdw alleles

in order to modify the agronomic traits of feeding and malting barley cultivars. The

sdw1 sequences gained from this and further research will be very useful in

diagnosing the sequence nucleotide polymorphism of different sdw alleles.

4.9 Important aspects of experimental work

4.9.1 Single primer positive control

An important finding from this research is that one primer can be used to amplify

bands in PCRs, especially, as the primers were designed in conserved region. This

might be caused by duplicated region in the barley genome. Several sequences were

amplified by single primers in the experiments, such as sdw3 (Fig. 3.25), sdw5 (Fig.

3.27), sdw8 (data not shown) and sdwex2R (Fig 3.19 B). This suggests that, when

PCRs are being prepared, it is necessary to set up a single primer reaction to

91

distinguish the bands which are amplified by a pair of primers rather than a single

primer pair.

4.9.2 Achieving effective ligation results

When the insert fragment becomes larger, it is more difficult to clone. Fig. 3.14 F

shows two large fragments which are 1.2 kb and 1.9 kb. No insert clone has been

found for either fragment. The DNA concentration was measured using a Fluoro-

meter before cloning and it was less than 10 ng/μl. However, for those large

fragments, the concentration should be more than 100 ng/μl. This might be the main

reason that no insert was cloned in this experiment. Too much DNA might be lost

from gel extraction as the pH value of UltraSALT might not be less than 7.5 because

the solution color was orange. The pH of UltraSALT should therefore be adjusted

before using the solution.

4.9.3 Using part of exon sequence to identify correct intron band size

The sequence of sdw1 is expected to include two intron regions in barley and these

intron sizes could be much different between rice and barley. Therefore, the band size

which includes introns could not be estimated. In this case, the primer pairs, which

were in this region, were used to perform nested PCR to test if the fragment was

correct. For example, the size of intron1 between exon1 and exon 2 (Fig. 3.10 A) was

not known. The primers sdwn2 and sdwex2R were used to amplify the bands which

were amplified by sdwn1 and sdwex2R to identify and then sequence the correct band

(Fig. 3.10 C). Ultimately, the correct fragment and sequence of intron1 was identified.

92

The same method was used to identify the size of intron2. In this experiment, two

nested PCRs were carried out and the nested PCRs amplified the expected single

bands. The sequences of these bands were identical to the sequences from genomic

DNA PCRs. This evidence confirmed that both 1.9 kb and 1.2 kb might be the correct

bands which include intron2. This indicates that there might be two copies of the

sdw1 gene in barley.

4.10 Conclusion

In conclusion, the barley semi-dwarfing gene, sdw1, has been hypothesized to have

orthologues with rice semi-dwarfing gene, sd1, by comparative mapping of barley 3H

and rice chromosome 1. By using sd1 gene as an anchor, part of exon1, all of exon2

and intron1, and part of exon3 of sdw1 gene were isolated from barley. Due to the

homology with sd1, sdw1 is predicted to have a similar biosynthetic pathway to

OsGA20ox2 (sd1), which controls the step from GA53 to GA44. Twelve varieties were

screened and sequenced in part of sdw1 sequences, but no polymorphism was found

so far. sdw (denso) is one of the most important genes in barley breeding as it has

already contributed to the improvement of many feeding and malting barley varieties.

The development of diagnostic markers would be significant to barley breeding if the

mutations are identified in future research. The research from this thesis allowed

technique development in bioformatics, primer design, DNA extraction and

quantification, optimizing PCRs, sequence analysis, cloning and some software

operation.

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4.11 Future research

At the completion of this project, 277 bp of exon1 and 128 bp of exon3 of sdw1 are

yet to be detected. Future work could involve isolating these partial sequences and

publishing the complete gene sequence. Also, other barley varieties need to be

screened for sequence nucleotide polymorphism identification. In addition, the

promoter region would be important to assay for polymorphism among dwarf and

non-dwarf varieties of barley. This would be extremely valuable to the Australian

breeding programs and to improve barley varieties.

94

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http://barleygenomics.wsu.edu/arnis/linkage_maps/maps-svg1.html

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Appendices

104

Appendix 1: Rice dwarfing and semi-dwarfing genes

Chro. location

Gene symbol Name of gene characteristic Names of variety GA response

locus reference Gene reference Spontanous or induced by chemical or ray

1 d18 (d25) http://www.grs.nig.ac.jp/rice/oryzabase/genes/quickSearchResultAction.do?category=symbo

1 d-18k Kotaketamanishiki dwarf HO 563 Kotake- tamanishiki(KY) s Yoshimura et al. 1992 Shinbashi et al. 1976;7:170, Suge 1990; 8:121

S

1 d18-AD Akibare-waisei dwarf s Itoh,et,al 2002; Itoh ,et,al 2001 I

1 d18-dy Waito-c s http://www.grs.nig.ac.jp/rice/oryzabase/genes/quickSearchResultAction.do?category=symbol

1 d-2 Ebisu dwarf A-26 Ebisu(HK) ldeta et al. 1994b Nagao & Takahashi 1963; 7:104 S

1 d-10 (d15, d16) Kikeibanshinriki or toyohikaribunwai tillering dwarf

N-70 Toyohikari bunwai(HK) HO 548 Kikeibanshinriki(KY)

Iwata et al. 1989b Iwata & Omura 1971a. 7:31 S

1 d-54 (d k5) Dwarf Kkyushu-5 CM 719 Kinmaze Kinmaze mutant(KY) Iwata et al. 1989b Iwata et al. 1979a; 7:41

1 d-26(t) 7237 dwarf 7237(Jodon's marker)50,53 Hsieh 1960; 7:15 S

1 d-55 (d-k-6) Dwarf Kyushu-6 CM 296 do. (KY) Iwata et al 1989b Iwata et al. 1979a; 7:41 C

1 sd -1 (d-47) Dee-geo-woo-gen semidwarf /sd-1 a Taichung native- 1(HK),SC 2, 3,4,5(NA)

s Yu et al. 1992, 1995; Ogi et al. 1993 ; Cho et al. 1994a,b; Maeda et al. 1995; Kikuchi & Ikehashi 1986

Tsai 1991b,c Tanisaka et al 1994 Murai et al. 1995 Nakamura et al. 1995

Aquino & Jennings 1966; 7:2. Suh & Heu 1978; 7:183. Tsai 1991; 8:143

Ror C

1 d-61 dm-type dwarf Wu,et al 1999

105

1 Dm-1* Short culm showing dm-type Incomplete dominant dwarf

http://www.grs.nig.ac.jp/rice/oryzabase/genes/quickSearchResultAction.do?category=symbol

1 Ssi1 (Dm1)* short second internode 1 (dmtype) Incomplete dominant dwarf

Saeda & Kitano 1992; Wu, et al., 1996; 1997;1999.

1 ssi2* short second internode 2 (dmtype) Wu, et al, 1996; 1997.

1 d-18h Hosetsu-waisei or akibare dwarf N-71 Hosetsu waisei(HK) s Suge 1990 Kinoshita et al. 1974; 7:69 S

1 OsGA3ox2 GA 3betahydroxylase2 http://www.grs.nig.ac.jp/rice/oryzabase/genes/quickSearchResultAction.do?category=symbo

2 d-32(d-k-4, d-12) Dwarf Kyushu-4 M 9, M 49 Norin 8 mutant (KY) Morishima 1990 Iwata & Omura 1970; 7:30 R

2 d-5 Bunketsu-waito tillering dwarf do. Ideta et al. 1992,1995 Nagao & Takahashi 1963; 7:104 S

2 d-29 (d-k-1) Short uppermost internode dwarf M 92 Norin 8 mutant(KY) Iwata & Omuta 1977; 7:34 R

2 d-30(d-w) Waisei-shirasasa dwarf HO 539 Waisei-shirasasa (KY) Iwata et al. 1989a Iwata & Omura 1971b; 7:32 S

3 d-20 Hayayuki dwarf M-48 Hayayuki waisei(HK) Takamure & Kinoshita 1991 Takamure el al. 1991 Kinoshita et al. 1974; 7:69 R

3 d-14 (d10) Kamikawabunwai tillering dwarf N-57 Kamikawa-bunwai Takahashi & Kinoshita 1974; 7L187 S

3 d-52 (d-k-2) Dwarf Kyushu-2 CM 45 do. (KY) Iwata et al. 1977; 7:40 C

106

3 d-56 (d-k-7) Dwarf Kyushu-7 CM 298 do. (KY) Iwata et al. 1979b; 7:42 C

3 Mi Minute grain http://www.grs.nig.ac.jp/rice/oryzabase/genes/quickSearchResultAction.do?category=symbo

4 d-3 Bunketsu-waito tillering dwarf A-12 Bunketsu- waito(HK) Nagao & Takahashi 1963; 7:104 S

4 d-11 (d8) Shinkane-aikoku or nohrin 28 dwarf N-58 Norin-28 wai(HK), HO 556 Shikane x Aikoku (KY)

Yoshimura et al. 1992; Yu et al. 1992,1995 Iwata & Omura 1971b; 7:32 S

4 d-31 Taichung-155-irradiated dwarf D-155-8 Yen et al. 1968; 7:216 unknown

4 d-42(t) Liguleless dwarf M-341 Norin 8 mutant(HK), H106 Hsieh & Yen 1966; 7:19, Kinoshita &Shinbashi 1982; 7:63

R

4 nak5 (nak1) narrow leaf5 http://www.grs.nig.ac.jp/rice/oryzabase/genes/quickSearchResultAction.do?category=symbo

5 sd-7 (t) Semidwarf-7 (D56-31) D56-31 Tsai 1989,1991a Tsai 1989; 8:141 C

5 d-1 Daikoku dwarf A-23 Daikoku ; (HK), HO 532 ; Daikoku(KY)

I Mitsunaga et al. 1994 Nagao & Takahashi 1963; 7:104 S

5 sdg(t)* Semidwarf (BRGPC) BRGPC Liang et al. 1994; Zhu & Xie 1989 Zhu & Zie 1989; 8:160

5 OsGA3ox1 GA 3betahydroxylase1 http://www.grs.nig.ac.jp/rice/oryzabase/genes/quickSearchResultAction.do?category=symbo

6 d-58(t) Small grained dwarf Takamure & Kinoshita 1986, 1991,1994, Takamure 1994

Takamure & Kinoshita 1986; 8:128 R

107

6 d-4 Bunketsu-waito tillering dwarf do. Nagao & Takahashi 1963; 7:104 S

6 d-9 Chinese dwarf N-60 Cugokuto waisei(HK) Kinoshita & Takamure 1990 Nagao & Takahashi 1963; 7:104 S

6 d-21 Aomorimochi-14 dwarf J-14 Aomorimochi-14 waisei(HK) Kinoshita et al. 1974; 7:69 R

7 d-6 (d34) Ebisumochi dwarf or tankanshirasasa dwarf

A-25 Ebisumochi(HK), HO 540 Tankan-shira- sasa(KY)

Ideta et al. 1994a Nagao & Takahashi 1963; 7:104 S

7 d-7 Heiei-daikoku or cleistogamous dwarf N-7 Heiei-daikoku(HK) Nagao & Takahashi 1963; 7:104 S

7 d-60 [sd (t)] Dwarf (Hokuriku 100) Koshihikari Tomita et al. 1989, Tanisaka et al. 1990 Tomita et al. 1989; 8:136, Tanisaka et al. 1990; 8:134

R

7 Ssv* Eiguchi & Sano 1995

8 d-51 Dwarf Kyushu-8 CM 1305 Kinmaze mutant(KY) Iwata et al. 1983; 7:44 C

9 d-57 (d(x)) dwarf Yen & Hsieh 1968; 7: 215

10 OsDIM* Tice DIM protein Tao et al 1997

11 d-27(d-t) Bunketsuto tillering dwarf HO 568 Bunketsuto(KY) Abenes et al. 1994; Yu et al. 1992,1995 Iwata & Omuta 1977; 7:34 S

11 d-28(d-c) Chokeidaikoku or long stemmed dwarf HO 534 Chokeidaikoku(KY) Iwata et al. 1978b; 7:39 S

108

11 D-53 (D-L-3) Dwarf Kyushu-3; dominant dwarf LT 15 Norin 8 mutant(KY) Iwata et al. 1977; 7:40 R

12 d-33(d-B) Bonsaito dwarf HO 565 Bonsaito(KY) Ishikawa et al. 1991a,b

Iwata & Omura 1975; 7:33, Ishikawa et al. 1991; 8:37

S

0 D-h(t) Dominant dwarf Koh & Heu 1993

0 d-12 (d50) Yukara dwarf or filkei 71 dwarf N-26 Yukara waisei(HK) Murai et al. 1990 Futsuhara 1968; Takahashi et al. 1968; 7:188, Murai et al. 1990; 7:101

C or R (Fujiminori)

0 d-13 Short grained dwarf M-15 Norin 8 mutant(HK) Takahashi et al. 1968; 7:188 R

0 d-17(t) Slender dwarf I-17 Slender dwarf(HK) Takahashi & Kinoshita 1974; 7L187 S

0 d-19(t) Kamikawa dwarf N-56 Kamikawa waisei(HK) Kinoshita et al. 1974; 7:69 S

0 d-22(t) Jokei 6549 dwarf N-61 jokei 6549 waisei(HK) Takahashi & Kinoshita 1974; 7:187 S

0 d-23(t) Ah-7 dwarf AH-7(HK) Takahashi & Kinoshita 1974; 7:187 S

0 d-24(t) m-7 dwarf M-7 Norin 8 mutant(HK) Kinoshita & Shinbashi 1982; 7:63 R

0 d-35(t) Tanginbozu dwarf N-77 tanginbozu(HK) s Suge 1990 Shinbashi et al. 1975, 1976; 8:111, 7:170, Suge 1975, 1978, 1990; 8:118, 119, 121

S

0 d-49(t) Reimei dwarf Reimei (Fukei) Futsuhara 1968; 8:23 R

109

0 d-59 (t) Dwarf (DM 107-4) Awan & Cheema 1988; 8:4 unknown

0 sd1-a semidwarf-1:a Tsai 1991c; Kinoshita, 1991.

0 sd-2 Semidwarf-2 (CI111033) D 66 Foster & Rutger 1978a, b; 8:21, 22

0 sd-3 Semidwarf – 3 (CI9858) CI 9858 Foster & Rutger 1978b; 8:22 C

0 sd-4 Semidwarf-4 (CI11034) D 23, D 24, D 25 Mackill & Rutger 1979; 8:57 C

0 sd-5 Semidwarf-5 (Short labella) Short Labelle Mckenzie & Rutger 1986 Mckenzie & Rutger 1986; 8:63 C

0 sd-6 (t) Semidwarf-6 (R -34) R-34 Hu 1987 Hu 1987; 8:34 C

0 sd-8 (t) semidwarf-8 AB-60e'-3 Tsai 1994 Tsai 1994a

0 sd-9(t)* semidwarf Ginbozu Tanisaka et al 1994 Tanisaka et al. 1994

0 sd10(t)* semidwarf-10 (Kinmaze) Kuroki & Tanisaka 1997

0 sd(t)* semidwarf independent from sd-I Tanaka et al. 1992 Tanaka et al. 1992

0 sd(t)* semidwarf Fujisaka 5go Yabu et al. 1994 Yabu et al. 1994

110

0 Bsv (Bs) Black streaked dwarf resistance; dominant dwarf

http://www.grs.nig.ac.jp/rice/oryzabase/genes/quickSearchResultAction.do?category=symbol

0 adl1 adaxialized leaf-1 http://www.grs.nig.ac.jp/rice/oryzabase/genes/quickSearchResultAction.do?category=symbol

0 rgp1 http://www.grs.nig.ac.jp/rice/oryzabase/genes/quickSearchResultAction.do?category=symbol

0 rt root growth inhibition http://www.grs.nig.ac.jp/rice/oryzabase/genes/quickSearchResultAction.do?category=symbol

0 Ydv Yellow dwarf disease resistance http://www.grs.nig.ac.jp/rice/oryzabase/genes/quickSearchResultAction.do?category=symbol

The factors of mutation: GA response: C: Chemical mutagen induced S: sensitive to GA R: Radiation induced I: insensitive to GA S: Spontaneous mutation

111

Appendix 2: Barley dwarfing genes

Chro. location

Gene symbol Phenotype (gene characteristic) Name of variety Original stock GA response GBS reference DWS No. Spontanous or induced

1H dsp.an triple or broad awn, semicompact spike OUM125 Akashinriki S GSHO1723 1034

1H dsp.ab semicompact spike Mut. 2663 Donaria S GSHO1715 1178

1H pyr.aw fusiform or pyramid-shaped spike Hja80089 Hja80001 cross S GSHO2436 1249

1H dsp.ab Dense spike ab Mut.2663 Donaria S GSHO1715 1339

1HL ert-b semicompact spike ert-b2 Gull S 224 1094

1HL glo-e altered seed shape glo15 Bonus S 1151

1HL fst2 (fs2) semidwarf or reduced plant height BGS 208 Osichi-hen unknown 208 1171

1HL cud2 curly leaves and short culms OUM 112 unknown 229 1366

1HS cur5 (cu5) curly leaves long basal rachis internode SA6102-2-1-3-1 Glenn S 231 1071

2H ert-j semicompact spike ert-j31 Bonus I 90 1103

2H ert-q semicompact spike, malformed spike ert-q101 Bonus S 91 1110

2H ert-u long basal rachis internode, reduced awn length ert-u56 Bonus I 92 1114

2H ert-zd brachytic growth habit, long basal rachis internode ert-zd159 Bonus unknown 93 1122

2H com2.g(bir2) brachytic growth habitanched spike,Ea BIR2 CIMMYT freak unknown 71 1358

2H? Zeo.j semicompact spike SA121- 4-5 Glenn S GSHO1612 1053

2HL yst4 semidwarf or reduced plant height SA2105-2-2-2-1 Glenn S 85 1059

2HL eog(lin) early maturity BGS057 Club Mariout S 57 1085

2HL fol-a narrow leaf 2235/64 Foma S 73 1144

2HL glo-c altered seed shape 1080/72 Villa unknown 72 1148

2HL zeo compact spike with short rachis internodes Mult. Dom. M.D. Stock S 82 1163

2HL cur4(cu4) curly leaves BGS 460 KM11B S 460 1181

2HL abr long basal rachis internode lax-3 Bonus S 472 1280

2HL Zeo compact spike with short rachis internodes Mut. 2657 Donaria S 82 1340

2HL LKS1 (LK) reduced awn length Ridawn Eagle.awn unknown 75 1348

2HL LKS1 (LK) reduced awn length CI 13311 S 75 1350

2HS sld2.b Slender dwarf 1 OUM142-uz Akashinriki S 83 1039

112

2HS brh.y brachytic growth habit,cu 10001 Bido I GSHO1688 1230

2HS abr. long rachis internodes, long basal rachis internode lax-63 Bonus S 1296

2HS sld.d semidwarf or reduced plant height 80T-5899-2-13 Two-rowed Glacier unknown 1368

2HS sdw.aw altered seed shape dwf mx Morex unknown GSHO2446 1369

3H? mnd3.d low number of rachis internodes,nld 2.1msg2MM Chr.2 Mult.Rec S 1347

3HL sld1 (dw-1) semidwarf or reduced plant height OUM148 Akashinriki I 126 1042

3HL cur2 (cu2) curly leaves and short culms BGS114 Choshiro-hen S 114 1080

3HL lnt1 (lnt) reduced tillering OUJ 40B Mitake unknown 118 1083

3HL uzu (uz) uz 3.1msg5MM Chr.3 Mult. Rec. I 102 1087

3HL ert-c fusiform or pyramid-shaped spike, delayed heading ert-c1 Gull S 134 1095

3HL ert-ii semidwarf or reduced plant height, semicompact spike

ert-ii79 Bonus I 135 1102

3HL gra-a semidwarf with premature spike emergence 1024/65 S 131 1145

3HL sld (dw-1) semidwarf or reduced plant height 862PK Plena S 126 1153

3HL sdw2 (sdw-b) semidwarf or reduced plant height, leafy basal rachis node

437MG Mg4170 S 133 1154

3HL sdw semidwarf or reduced plant height Triumph Diamant S 518 1168

3HL cur2 (cu2) cuc 2010 Carina unknown 114 1182

3HL sdw semidwarf or reduced plant height Jotun derv. Jotun S 518 1234

3HL sdw semidwarf or reduced plant height M21 Jotun unknown 518 1235

3HL sdw semidwarf or reduced plant height M66-102 Jotun unknown 518 1236

3HL gra-a semidwarf with premature spike emergence, early maturity

OR-SS-2 Tokak unknown 131 1237

3HS brh.ad altered seed shape, compact spike with short rachis internodes

17:16:01 Birgitta S GSGO1671 1008

3HS lzd curly leaves, mat OUM 005 Akashinriki S 125 1027

3HS lc early maturity, fusiform or pyramid-shaped spike BGS111 Club Mariout S 111 1082

3HS ari-a aro ari-a6 Bonus unknown 132 1124

3HS brh.w brachytic growth habit, long basal rachis internode 7101 Volla S GSHO1687 1211

3HS dsp.ar compact spike with short rachis internodes 7114 Volla S GSHO1726 1217

3HS sld.h semidwarf or reduced plant height, much reduced vigor

XV2334-6R Indian Dwarf S 1238

3HS Pyr.i fusiform or pyramid-shaped spike, compact spike with short rachis internodes

Hja79010 Pokko S GSHO 1582 1243

3HS als1.a als1.a Club Mariout Mariout unknown 111 BGS 111

113

4H sid single elongated internode 17:09:05 Birgitta I 180 1001

4HL brh2 (br2) brachytic growth habit BGS157 Svanhals I 157 1079

4HL sid single elongated internode, uz OUX 052 R101 Akashinriki I 180 1090

4HL ari-l brachytic growth habit ari-l3 Bonus I 157 1135

4HL lks5 (lk5) reduced awn length BGS 172 CI 5641 S 172 1143

4HL min-En semidwarf or reduced plant height,reduced awn length,brachytic growth habit

BGS161 Taisho-mugi unknown 161 1361

4HL brh2 brachytic growth habith2 br2 Svanhals unknown 5 BGS 157

4HS brh.m brachytic growth habit,altered seed shape 17:18:02 Birgitta S GSHO1678 1010

4HS sld.e semidwarf or reduced plant height, narrow leaf Ant567 Dickson I GSHO2480 1050

4HS sdw.u semidwarf or reduced plant height SA6102-2-4-8-2 Glenn S GSHO2462 1072

4HS glo-a altered seed shape 1343/63 Proctor S 168 1146

4HS sld.f reduced tillering, much reduced vigor OB165 Glenn S gsho 2481 1240

4HS Zeo.h compact spike with short rachis internodes. Ea Wa11094-81 (Mo1) Morex I GSHO1611 1259

4HS mnd1 mnd1 CIho 2328 Mesa unknown 519 BGS519

5H D Controlling the rough and smooth awn GBC695 GBC695, after treated crossed with Mars

S

5HL brh.j altered seed shape,brachytic growth habit 17:13:06 Birgitta S GSHO1675 1005

5HL Zeo.f compact spike with short rachis internodes 18:15:41 Birgitta S GSHO1610 1026

5HL brh.r brachytic growth habit OUM133 Akashinriki S GSHO1083 1036

5HL brh.s brachytic growth habit OUM135 Akashinriki S GSHO1684 1037

5HL mnd4 many elongated internodes, long basal rachis internode

OUM168 Akashinriki S GSHO1798 1048

5HL nld narrow leaf OUJ 054 Nagoaka dwarf S 323 1084

5HL cud1 (cud) curly leaves and short culms OUM 120 Akashinriki S 324 1088

5HL ert-g semicompact spike ert-g24 Bonus S 330 1099

5HL ert-n reduced tillering ert-n51 Bonus unknown 331 1107

5HL ari-e brachytic growth habit, reduced awn length ari-e1 Bonus S 328 1128

5HL ari-h reduced awn length ari-h127 Foma S 329 1131

5HL ari-e (GPert) semicompact spike, reduced awn length Golden Promise Maythorpe S 328 1159

5HL ari-e (GPert) semicompact spike, reduced awn length Midas Golden Promise S 328 1161

114

5HL ari-e (GPert) semicompact spike, reduced awn length Clansmar Golden Promise S 328 1166

5HL sdw.ah semidwarf or reduced plant height 7063 Volla S GDHO2442 1208

5HL lax-a long rachis internodes,triple or broad awn lax-a8 Bonus S 474 1278

5HL com1.a brachytic growth habitanched spike lax-270 Foma S 473 1322

5HL pyr.af Pyramidatum. af Mut. 4158 Haisa S GSHO1718 1345

5HL ari-e (GPert) reduced awn length Fleet Golden Promise S 328 1349

5HL dsk semidwarf or reduced plant height OUM 299 unknown 322 1363

5HS glo-b curly leaves and short culms 1345/72 Villa S 336 1147

6HL lg compact spike with short rachis internodes OUM113 Akashinriki S 258 1033

6HL cur3 (cu3) curly leaves OUM301 Akashinriki S 263 1091

6HL ert-e compact spike with short rachis internodes, semidwarf or reduced plant height

ert-e17 Bonus S 266 1097

6HL lax-b low number of rachis internodes lax-b1 Bonus S 268 1279

6HL lax-c long rachis internodes,triple or broad awn lax-c21 Bonus S 475 1285

7H mbd Multimode and brachytic growth habitanched dwarf

93-597 unknown

7HL lks2 (lk2) reduced awn length Mult. Recess. M.R. stock S 10 1142

7HS dsp.ak semicompact spike,brachytic growth habit 18:09:03 Birgitta unknown GSHO1721 1020

7HS sdw.j semidwarf or reduced plant height, malformed spike

OUM073 Akashinriki S GSHO2453 1030

7HS brh1.t brachytic growth habit OUM136 Akashinriki I 1 1038

7HS brh1 (br) brachytic growth habit BGS001 Himalayd I 1 1078

7HS l semicompact spike MN1111 Chinese barley S 1081

7HS wnd, l spiral or curved upper peduncle,semicompact spike OUM 309 R754 Kogen-mugi I 23 1092

7HS ert-a compact spike with short rachis internodes,semidwarf or reduced plant height

ert-a6 Gull I 28 1093

7HS ert-d compact spike with short rachis internodes ert-d7 Gull S 29 1096

7HS ert-m semicompact spike, malformed spike ert-m34 Bonus unknown 30 1106

7HS ari-i brachytic growth habit ari-i38 Bonus; Himalaya (CIho 1312)

I 1132

7HS brh1 (br) brachytic growth habit 035AR Aramir I 1 1152

7HS brh1.x brachytic growth habit 7125 Volla S 1 1224

7HS Pyr.g fusiform or pyramid-shaped spike, delayed heading Hj64202 Hja80001 cross S GSHO1581 1242

115

7HS brh.z brachytic growth habit Hja80001 Aapo I GSHO1689 1246

7HS fs3 semidwarf or reduced plant height OUM 382 Kobin Katagi unknown 24 1362

7HS brh1.ae brachytic growth habitachytic 1 FN52 Steptoe FN53

0 brh.g brachytic growth habit 17:10:01 Birgitta I GSHO1672 1002

0 brh,h brachytic growth habit,cu 17:11:03 Birgitta unknown GSHO1673 1003

0 brh.i brachytic growth habit 17:12:01 Birgitta I GSHO1674 1004

0 brh.k brachytic growth habit, altered seed shape 17:14:04 Birgitta S GSHO1676 1006

0 brh.l semicompact spike,brachytic growth habit 17:15:02 Birgitta S GSHO1677 1007

0 sdw.ax semidwarf or reduced plant height 17:17:02 Birgitta S GSHO2447 1009

0 brh.n altered seed shape,brachytic growth habit 17:19:02 Birgitta S GSHO1679 1011

0 brh.o brachytic growth habit,altered seed shape 17:20:02 Birgitta S GSHO1680 1012

0 sdw.ay semidwarf or reduced plant height 18:02:04 Birgitta S GSHO2448 1013

0 semidwarf or reduced plant height 18:03:02 Birgitta unknown 1014

0 sdw.g semidwarf or reduced plant height 18:04:01 Birgitta unknown GSHO2450 1015

0 semidwarf or reduced plant height 18:05:6I Birgitta unknown 1016

0 sdw.h low number of rachis internodes,semidwarf or reduced plant height

18:06:03 Birgitta S GSHO2451 1017

0 pyr.ai fusiform or pyramid-shaped spike 18:07:04 6-rowed S GSHO2433 1018

0 dsp.aj semicompact spike,brachytic growth habit 18:08:04 6-rowed S GSHO1720 1019

0 18:10:2III 6-rowed unknown 1021

0 sdw.i semidwarf or reduced plant height 18:11:04 Birgitta S GSHO2452 1022

0 brachytic growth habit 18:12:04 Birgitta I 1023

0 pyr.al semicompact spike 18:13:02 6-rowed I GSHO2434 1024

0 18:14:11 6-rowed unknown 1025

0 altered seed shape, semicovered caryopsis OUM058 Akashinriki S 1028

0 dsp.am semidwarf or reduced plant height OUM070 Akashinriki S GSHO1722 1029

0 sdw.k semidwarf or reduced plant height OUM097 Akashinriki S GSHO2454 1031

0 dsp.an fusiform or pyramid-shaped spike OUM105 Akashinriki unknown 1032

0 brh.q brachytic growth habit OUM131 Akashinriki I GSHO1682 1035

0 semidwarf or reduced plant height OUM143 Akashinriki S 1040

116

0 sdw.l semidwarf or reduced plant height OUM145 Akashinriki S GSHO2455 1041

0 semicompact spike OUM149 Akashinriki unknown 1043

0 sdw.m semidwarf or reduced plant height OUM155 Akashinriki S GSHO2456 1044

0 sdw.n semidwarf or reduced plant height OUM158 Akashinriki S GSHO2457 1045

0 curly leaves, reduced tillering OUM163 Akashinriki S 1046

0 semidwarf or reduced plant height OUM165 Akashinriki unknown 1047

0 sdw.o semidwarf or reduced plant height Mo1 Morex S GSHO2458 1049

0 semidwarf or reduced plant height A201- 1-2-1 Glenn unknown 1051

0 sdw.p semidwarf or reduced plant height, reduced tillering

A210-2-2-1 Glenn S GSHO2459 1052

0 semidwarf or reduced plant height SA1104- 2-1-2-2 Glenn unknown 1054

0 sdw.q semidwarf or reduced plant height SA9109-2-1-3-1 Glenn S GSHO2460 1055

0 semidwarf or reduced plant height SA1113-2-3-3-3 Glenn unknown 1056

0 semidwarf or reduced plant height SA1101-12-2-3-6 Glenn unknown 1057

0 SA2105-2-1-2-2 Glenn unknown 1058

0 sdw.r semidwarf or reduced plant height SA2108-2-4-1-3 Glenn S GSHO2461 1060

0 reduced tillering SA4104-2-2-5-3 Glenn S 1061

0 sld.j semidwarf or reduced plant height, much reduced vigor

SA4114-2-4-2-1 Glenn S GSHO2485 1062

0 sld.k mul, much reduced vigor SA4117-2-1-1-1 Glenn S GSHO2486 1063

0 SA5101-2-3-4-1 Glenn unknown 1064

0 reduced tillering SA5104-2-2-3-1 Glenn S 1065

0 sdw.s semidwarf or reduced plant height SA5113-2-3-6-3 Glenn S 1066

0 SA5114-2-2-1-1 Glenn unknown 1067

0 sdw.t semidwarf or reduced plant height SA5115-2-3-1-2 Glenn S 1068

0 premature ripening SA5115-2-4-2-1 Glenn S 1069

0 SA5115-2-1-9-2 Glenn unknown 1070

0 sdw.v semidwarf or reduced plant height SA6103-2-1-10-1 Glenn S 1073

0 reduced tillering SA6112-2-3-1-2 Glenn S 1074

0 semidwarf or reduced plant height SA8104-12-1-2-1 Glenn S 1075

0 reduced tillering SA8104-12-2-3-1 Glenn S 1076

117

0 early maturity CI3410 Estate S 1077

0 mnd3 (mnd) many elongated internodes,narrow leaf 2.1msg2MM Chr.2 Mult. Rec. unknown 1086

0 sca reduced awn length OUN206 Akashinriki S 128 1089

0 ert-f compact spike with short rachis internodes ert-f18 Bonus S 560 1098

0 ert-h semicompact spike ert-h25 Bonus unknown 561 1100

0 ert-i semicompact spike, semidwarf or reduced plant height,long basal rachis internode

ert-i27 Bonus I 1101

0 ert-k semicompact spike ert-k32 Bonus S 562 1104

0 ert-l semicompact spike ert-l12 Maja S 563 1105

0 ert-o early maturity ert-o16 Maja S 1108

0 ert-p semicompact spike ert-p44 Bonus unknown 564 1109

0 Ert-r compact spike with short rachis internodes ert-r52 Bonus I 332 1111

0 ert-s semidwarf or reduced plant height ert-s50 Bonus S 565 1112

0 ert-t hr, reduced awn length ert-t55 Bonus I 566 1113

0 ert-v semicompact spike, malformed spike ert-v57 Bonus S 567 1115

0 ert-x semicompact spike, reduced awn length ert-x58 Bonus S 568 1116

0 ert-y semicompact spike ert-y59 Bonus unknown 569 1117

0 ert-z narrow leaf, reduced awn length ert-x71 Bonus S 570 1118

0 ert-za semicompact spike, altered seed shape ert-za102 Bonus I 571 1119

0 ert-zb semidwarf or reduced plant height ert-zb137 Bonus S 572 1120

0 ert-zc semicompact spike ert-zc149 Bonus unknown 573 1121

0 ert-ze semicompact spike ert-zce105 Bonus S 574 1123

0 ari-b semidwarf or reduced plant height. delayed heading

ari-b11 Bonus S 550 1125

0 ari-c reduced awn length ari-c2 Bonus S 1126

0 ari-d reduced awn length, semidwarf or reduced plant height

ari-d15 Bonus S 1127

0 ari-f reduced awn length ari-f21 Bonus S 551 1129

0 ari-g reduced awn length, narrow leaf ari-g24 Bonus unknown 89 1130

0 ari-j reduced awn length, semidwarf or reduced plant height

ari-j32 Bonus S 552 1133

0 ari-k reduced awn length ari-k504 Bonus S 553 1134

0 ari-m brachytic growth habit, altered seed shape ari-m28 Bonus S 554 1136

118

0 ari-n brachytic growth habit ari-n12 Bonus S 555 1137

0 ari-o reduced awn length, long basal rachis internode ari-o40 Bonus I 556 1138

0 ari-p reduced awn length, delayed heading ari-p27 Bonus unknown 557 1139

0 ari-q reduced awn length, narrow leaf ari-q280 Foma S 558 1140

0 ari-r curly leaves, delayed heading ari-r14 Foma S 559 1141

0 glo-d altered seed shape, curly leaves 1114/66 Donoria s 1149

0 glo-d altered seed shape glo13 Bonus unknown 1150

0 sdw.w semidwarf or reduced plant height 392JK S 1155

0 sdw.x semidwarf or reduced plant height 392JK/Bow GSHO2463 1155

0 brh.u brachytic growth habit, narrow leaf 409 JK S GSHO1685 1156

0 sdw.y semidwarf or reduced plant height, narrow leaf 421JK S GSHO2464 1157

0 sdw.av semidwarf or reduced plant height 555DK unknown GSHO2445 1158

0 semidwarf or reduced plant height MM23/45 Maris Mink unknown 1160

0 semidwarf or reduced plant height Maris Mink Deba Abed S 1162

0 fusiform or pyramid-shaped spike, delayed heading HA21 Pirkka S 1164

0 sdw.f semidwarf or reduced plant height Betina Vada S GSHO2449 1165

0 semidwarf or reduced plant height Sherpa Abed Denso unknown 1167

0 Zeo.e compact spike with short rachis internodes HI-902-1 Rubin S GSHO1609 1169

0 sdw.ac semidwarf or reduced plant height 7004 Volla unknown 1170

0 sdw.aa semidwarf or reduced plant height, narrow leaf M-63-HE-607 Spartan S 1170

0 semidwarf or reduced plant height KM1053-2001 Diamant unknown 1172

0 sdw.ab semidwarf or reduced plant height KM-1053-2004 Diamant S GSHO2440 1173

0 semidwarf with premature spike emergence HE-MN-C292 Valticky/Kneifl S 1174

0 semidwarf or reduced plant height HE-M3 Koral S 1175

0 brh.v semidwarf with premature spike emergence, brachytic growth habit

HE2816 I GSHO1686 1176

0 early maturity, shrunken endosperm Mut. 2612 Donaria S 1177

0 dsp.ae Dense spike ae Mut. 4014 Haise I GSHO1717 1179

0 dsp.ah Dense spike ah Mut. 4841 Saale I 1180

0 2020 Carina unknown 1183

119

0 long rachis internodes 2021 Carina S 1184

0 semidwarf or reduced plant height 2025 Carina unknown 1185

0 semicompact spike 2026 Carina unknown 1186

0 leaf necrosis or necrotic spot 6002 Villa unknown 1187

0 6006 Villa S 1188

0 6012 Villa unknown 1189

0 6015 Villa unknown 1190

0 low number of rachis internodes, semidwarf or reduced plant height

7004 Volla S 1191

0 long rachis internodes 7014 Volla S 1192

0 sdw.ad reduced awn length, semidwarf or reduced plant height

7015 Volla S 1193

0 semicompact spike 7039 Volla S 1194

0 semicompact spike 7040 Volla unknown 1195

0 7042 Volla unknown 1196

0 7043 Volla unknown 1197

0 sdw.ae semidwarf or reduced plant height, reduced awn length

7044 Volla I 1198

0 semicompact spike 7046 Volla unknown 1199

0 semidwarf or reduced plant height 7048 Volla S 1200

0 7050 Volla unknown 1201

0 sdw.af reduced awn length, semidwarf or reduced plant height

7052 Volla S 1202

0 sdw.ag semidwarf or reduced plant height 7055 Volla S GSHO2441 1203

0 7056 Volla S 1204

0 semidwarf or reduced plant height 7057 Volla unknown 1205

0 low number of rachis internodes, semidwarf or reduced plant height

7059 Volla S 1206

0 semidwarf or reduced plant height 7060 Volla S 1207

0 7082 Volla unknown 1209

0 long rachis internodes 7100 Volla S 1210

0 compact spike with short rachis internodes 7107 Volla I 1212

0 compact spike with short rachis internodes 7109 Volla S 1213

0 7110 Volla unknown 1214

120

0 dsp.ap compact spike with short rachis internodes 7112 Volla S GSHO1724 1215

0 dsp.aq compact spike with short rachis internodes 7113 Volla S GSHO1725 1216

0 compact spike with short rachis internodes 7115 Volla I 1218

0 dsp.as semicompact spike 7116 Volla S 1219

0 dsp.at compact spike with short rachis internodes 7117 Volla S GSHO1727 1220

0 long rachis internodes 7119 Volla S 1221

0 semidwarf or reduced plant height 7120 Volla S 1222

0 long rachis internodes 7121 Volla S 1223

0 semidwarf or reduced plant height 7130 Volla S 1225

0 semicompact spike 7170 Volla unknown 1226

0 long rachis internodes, shrunken endosperm 7175 Volla S 1227

0 long rachis internodes 7176 Volla S 1228

0 sdw.ai semidwarf or reduced plant height, low number of rachis internodes

7177 Volla S 1229

0 dsp.au compact spike with short rachis internodes 10002 Bido S GSHO1728 1231

0 narrow leaf 10009 Bido S 1232

0 reduced awn length, low number of rachis internodes

10015 Bido S 1233

0 semicompact spike, low number of rachis internodes

OB129 Glenn S 1239

0 semidwarf or reduced plant height Aapo Tab7990 S 1241

0 fusiform or pyramid-shaped spike Hja79564 Pokko S 1244

0 pyr.av fusiform or pyramid-shaped spike Hja79588 Pokko S GSHO2435 1245

0 brh.aa brachytic growth habit Hja80051 Hja80001 cross S GSHO1668 1247

0 sdw.aj semidwarf or reduced plant height Hja80075 Hja80001 cross S 1248

0 early maturity NC20 CI 15225 1250

0 dsp.ax compact spike with short rachis internodesn,brachytic growth habit,triple or broad awn

FDN shaped CI 6880 S 1251

0 pry.aa semicompact spike Betzes erect. CI 10871 S GSHO2431 1252

0 early maturity Tokak CI11216 unknown 1253

0 semidwarf or reduced plant height Abed Denso PI 361039 S 1254

0 semidwarf or reduced plant height Diamant CI 15226 unknown 1255

121

0 dsp.ay compact spike with short rachis internodes Wa11005-81 WA9037-75 S GSHO1729 1256

0 semidwarf or reduced plant height Wa11048-81 WA9044-75 unknown 1257

0 sdw.al semidwarf or reduced plant height Wa11048-81(A1) Advance I 1258

0 brh.ab brachytic growth habit,altered seed shape Wa14355-83(Mo4) Morex S GSHO1669 1260

0 sdw,am semidwarf or reduced plant height Wa14367-83(Ma1) Manker S 1261

0 sdw.an semidwarf or reduced plant height Wa14369-83(L1) Larker S 1262

0 early maturity Wa14371-83(L2) Larker S 1263

0 sdw.aq semidwarf or reduced plant height,curly leaves Wa14387-83(N1) Norbert S 1264

0 sdw.ap semidwarf or reduced plant height Wa14389-83(H1) Harrington S 1265

0 early maturity,semidwarf or reduced plant height Wa14168-84 Morex S 1266

0 early maturity Wa14198-84 Robust unknown 1267

0 semicompact spike Wa14210 Lamont S 1268

0 semidwarf or reduced plant height Wa14212 Lamont unknown 1269

0 semidwarf or reduced plant height Wa14287 Andre S 1270

0 low number of rachis internodes Wa14239-84 Andre S 1271

0 sdw.ar semidwarf or reduced plant height Wa14351-83(Mo2) Morex S 1272

0 semidwarf or reduced plant height Wa14353-83(Mo3) Morex S 1273

0 dsp.az compact spike with short rachis internodes Wa16228-85 Hazen I 1274

0 sdw.as semidwarf or reduced plant height Wa16235-85 Hazen S 1275

0 sdw.at altered seed shape,semidwarf or reduced plant height

Wa16239-85 Hazen S 1276

0 brh.ac brachytic growth habit.altered seed shape 4028 Mo6/4*Triumph I GSHO1670 1277

0 lax-f long rachis internodes lax-7 Bonus S 1281

0 semidwarf or reduced plant height,reduced awn length

lax-11 Bonus S 1282

0 long rachis internodes,delayed heading lax-13 Bonus S 1283

0 lax-e long rachis internodes lax-16 Bonus unknown 1284

0 long rachis internodes lax-22 Bonus S 1286

0 lax-h long rachis internodes, partial sterility lax-26 Bonus S 1287

0 lax-j long rachis internodes lax-29 Bonus S 1288

0 lax-j long rachis internodes lax-49 Bonus S 1289

122

0 lax-i long rachis internodes,partial sterility lax-50 Bonus unknown 1290

0 long rachis internodes lax-52 Bonus S 1291

0 long rachis internodes lax-53 Bonus S 1292

0 long rachis internodes lax-58 Bonus S 1293

0 lax-1 long rachis internodes,partial sterility lax-60 Bonus S 1294

0 lax-62 Bonus S 1295

0 long rachis internodes,long basal rachis internode lax-66 Bonus S 1297

0 lax-n long rachis internodes lax-67 Bonus S 1298

0 lax-n long rachis internodes lax-68 Bonus S 1299

0 long rachis internodes lax-69 Bonus S 1300

0 long rachis internodes lax-72 Bonus S 1301

0 lax-o lax-79 Bonus S 1302

0 lax-m long rachis internodes lax-80 Bonus S 1303

0 long rachis internodes lax-82 Bonus I 1304

0 lax-k lax-84 Bonus S 1305

0 long rachis internodes,partial sterility lax-85 Bonus S 1306

0 long rachis internodes,delayed heading lax-86 Bonus S 1307

0 lax-209 Foma unknown 1308

0 lax-ff long rachis internodes lax-216 Foma S 1309

0 long rachis internodes lax-221 Foma S 1310

0 lax-ef long rachis internodes lax-225 Foma S 1311

0 long rachis internodes,much reduced vigor lax-228 Foma S 1312

0 sld.l triple or broad awn,son lax-231 Foma S GSHO2487 1313

0 low number of rachis internodes,long rachis internodes

lax-234 Foma S 1314

0 nld,long rachis internodes lax-235 Foma S 1315

0 lax-ef long rachis internodes,semidwarf or reduced plant height

lax-237 Foma I 1316

0 lax-ef long rachis internodes lax-238 Foma I 1317

0 lax-hf long rachis internodes,curly leaves lax-244 Foma S 1318

0 lax-jf long rachis internodes lax-253 Foma S 1319

123

0 lax-jf long rachis internodes lax-255 Foma S 1320

0 bir3 brachytic growth habitanched spike lax-257 Foma S 1321

0 long rachis internodes lax-281 Foma S 1323

0 long rachis internodes,delayed heading lax-294 Foma S 1324

0 comi brachytic growth habitanched spike lax-296 Foma S 1325

0 long rachis internodes,narrow leaf lax-298 Foma S 1326

0 lax-mf long rachis internodes,curly leaves lax-302 Foma S 1327

0 long rachis internodes,partial sterility lax-304 Foma S 1328

0 long rachis internodes,partial sterility lax-305 Foma S 1329

0 lax-207 Foma S 1330

0 long rachis internodes lax-312 Foma S 1331

0 long rachis internodes,leaf necrosis or necrotic spot,narrow leaf

lax-315 Foma S 1332

0 long rachis internodes lax-316 Foma S 1333

0 long rachis internodes lax-321 Foma S 1334

0 lax-nf long rachis internodes lax322 Foma unknown 1335

0 lax-327 Foma unknown 1336

0 low number of rachis internodes Mut. 2249 Donaria unknown 1337

0 reduced awn length Mut. 2254 Donaria S 1338

0 dsp.ac Dense spike ac Mut. 2654 Donaria I GSHO1716 1341

0 com.j brachytic growth habitanched spike.nal Mut.3165 Donaria S GSHO1701 1342

0 uc3 little or no tille Mut. 3170 Donaria S 1343

0 pyr.ad Pyramidatum. ad Mut. 4009 Haisa S GSHO2432 1344

0 compact spike with short rachis internodes,mat Mut. 4551 Haisa S 1346

0 early maturity,low number of rachis internodes CI 16192 S 1351

0 tall CI 14995 S 1352

0 tall,long rachis internodes CI 16337 S 1353

0 Zeo.d compact spike with short rachis internodes msg39dm,MSS361 PI 1 S GSHO1608 1354

0 early maturity KI1031 S 1355

0 long rachis internodes,delayed heading CI16126 S 1356

124

0 dsp.ba many elongated internodes,semicompact spike UT1713-1 UT1713 S GSHO1730 1357

0 low number of rachis internodes Lamont Lamont unknown 1359

0 min-en semidwarf or reduced plant height,reduced awn length,uz

BGS160 Kairyo-bozu-mugi unknown 160 1360

0 long rachis internodes FN280 unknown 1364

0 cu curly leaves and short culms 6395 S 1365

0 semicompact spike nar2a Steptoe unknown 1367

0 compact spike with short rachis internodes 88Ab539 Morex-SD winter unknown 1370

0 compact spike with short rachis internodes Schuyler unknown 1371

0 delayed heading ND12463-1 unknown 1372

0 little or no tille Uniculm-3 unknown 1373

0 little or no tille Uniculm-5 unknown 1374

0 dwf1 Vegetative dwarf CI15624 S

0 Dwf2 single dwarf tiller H930-36 Klages/Mata. I 542

0 1974E Digenic dwarf Aiganqi Aiganqi unknown

1. GA response: S: sensitive to GA I: insensitive to GA 2. Chromosome location: 0 = unknown

125

Appendix 3: Maize dwarfing genes

locus Genes: full name

variation stock GA response reference spontanous or

induced 1L br2 brachytic2 (mi1) small plant

br2 brachytic2 unknown

1L an1 anther ear1 anther ear1 116G; 116GA

S

1L D8 dwarf plant8 dwarf plant8 121C

I

1L mpl1 miniplant1 miniplant1

120F; 120G; 125B

I

1L d*-N1352B dwarfN1352B dwarfN1352B d*-1352B (per Neuffer, MG) d*-N135B

unknown

1L d*-N1883 dwarfN1883 dwarfN1883 d*-1883 (per Neuffer, MG) d*-N1883

Unknown

1L d*-N454A dwarfN454A dwarfN454A d*-454A (per Neuffer, MG) d*-N454A

Unknown

1S et*-N617 etchedN617 etchedN617 et*-617 (per Neuffer, MG)

Unknown

1S and 4L d*-3 dwarf candidate3 dwarf candidate3

I

2S d5 dwarf plant5 dwarf plant5 214C

S

2 wrp1 wrinkled plant1 wrinkled plant1

unknown

2S d*-3685 dwarf candidate3685 dwarf candidate3685 d*-3685 dwarf candidate3685 (locus)

S

2S cp*-N1319A collapsedN1319A collapsedN1319A cp*-1319A (per Neuffer, MG)

Unknown

2S d*-N155B dwarf N155B dwarf N155B d*-155B (per Neuffer, MG)

S

2S d*-N208B dwarfN208B dwarfN208B d*-208B (per Neuffer, MG)

Unknown

2L d10 dwarf plant10 dwarf plant10 D*-2428 (per Neuffer, MG) 206C

Unknown

2S lil1 lilliputian1 lilliputian1 defective seedling GG11 (per Gavazzi, G); des*-11 (per Gavazzi, G); des*-24 (per Gavazzi, G); des*-GG11 (per Gavazzi, G); des*-GG24 (per Gavazzi, G); des11 (per Gavazzi, G) 202J

S

2S ptd*-N901A pittedN901A pittedN901A ptd*-901A (per Neuffer, MG)

Unknown

3S d1 dwarf plant1 dwarf plant1

302A;302AA;302AB

S

3L na1 nana plant1 nana plant1

I

3L yd2 yellow dwarf2 yellow dwarf2

Unknown

3L d*-N210 dwarfN210 dwarfN210 d*-210 (per Neuffer, MG) d*-N210

Unknown

3L d*-N282 dwarfN282 dwarfN282 d*-282 (per Neuffer, MG) d*-N282

I

4S na3 nana3 nana3 equal to d*-156A (per Neuffer, MG); smp*-156A (per Neuffer, MG); smp*-N156A

Unknown

5S d*-6 dwarf candidate6 dwarf candidate6 *-6 dwarf candidate6 (locus)

S

5S ad*-N664 adherentN664 adherentN664 ad*-664 (per Neuffer, MG)

Unknown

5 td1 thick tassel dwarf1 thick tassel dwarf1

Unknown

126

6L smp*-N272A small plantN272A small plantN272A equal to smp*-272A (per Neuffer, MG)

Unknown

6L d*-9 dwarf candidate9 dwarf candidate9 d*-9 dwarf candidate9 (locus)

S

8L clt1 clumped tassel1 clumped tassel1 equal to Clt*-985 (per Neuffer, MG)

Unknown

8L d12 dwarf12 dwarf12 d*-203D (per Neuffer, MG); d*-N203D d*-N203D

Unknown

8L d*-N394 dwarfN394 dwarfN394 d*-394 (per Neuffer, MG)

Unknown

9S d3 dwarf plant3 dwarf plant3

917F; 917FA;916I; 917FB;917FC;917FD 917FF;917FG; 917FH

S

9S d*-3010 dwarf candidate3010 dwarf candidate3010

Unknown

unknown d2 dwarf plant2 dwarf plant2 (locus) allelic to d3 917FA

Unknown

d9 dwarf plant9 dwarf plant9 equal to D*-2319 (per Neuffer, MG); D*-N2319

I

nl*-N371B narrowleafN371B narrowleafN371B equal to nl*-371B (per Neuffer, MG)

Unknown nl*-N625 narrowleafN625 narrowleafN625 equal to nl*-625 (per Neuffer, MG)

Unknown

Unknown nl*-N657C narrowleafN657C narrowleafN657C equanl to nl*-657C (per Neuffer, MG)

Unknown

Unknown nld*-N2275 narrowleaf dwarfN2275

narrowleaf dwarfN2275 equal to nld*-2275 (per Neuffer, MG)

Unknown

Unknown o*-N1479 opaqueN1479 opaqueN1479 equal to o*-1479 (per Neuffer, MG)

Unknown

unknown o*-N948B opaqueN948B opaqueN948B equal to o*-948B (per Neuffer, MG)

Unknown

Unknown pg*-N1980 palegreenN1980 palegreenN1980 equal to pg*-1980 (per Neuffer, MG)

Unknown

Unknown sdw*-N1594 semidwarfN1594 semidwarfN1594 equal to D*-N1594 (per Neuffer, MG); Sdw*-1594 (per Neuffer, MG)

Unknown

Unknown smp*-N1005B small plantN1005B small plantN1005B equal to smp*-1005B (per Neuffer, MG)

Unknown

Unknown smp*-N1143B small plantN1143B small plantN1143B equal to smp*-1143B (per Neuffer, MG)

Unknown

Unknown smp*-N152 small plantN152 small plantN152 equal to smp*-152 (per Neuffer, MG)

Unknown

Unknown smp*-N153B small plantN153B small plantN153B equal to d*-N153B (per Neuffer, MG); smp*-153B (per Neuffer, MG)

Unknown

Unknown smp*-N154B small plantN154B small plantN154B equal to d*-N154B (per Neuffer, MG); smp*-154B (per Neuffer, MG)

Unknown

Unknown smp*-N183B small plantN183B small plantN183B equal to smp*-183B (per Neuffer, MG)

Unknown

Unknown smp*-N216 small plantN216 small plantN216 equal to smp*-216 (per Neuffer, MG)

Unknown

Unknown smp*-N240 small plantN240 small plantN240 equal to smp*-240 (per Neuffer, MG)

Unknown

Unknown smp*-N276B small plantN276B small plantN276B equal to d*-N276B (per Neuffer, MG); smp*-276B (per Neuffer, MG)

Unknown

Unknown smp*-N279A small plantN279A small plantN279A equal to smp*-279A (per Neuffer, MG)

I

Unknown smp*-N306 small plantN306 small plantN306 equal to smp*-306 (per Neuffer, MG)

Unknown

Unknown smp*-N328 small plantN328 small plantN328 equal to d*-N328 (per Neuffer, MG); smp*-328 (per Neuffer, MG)

S

127

Unknown smp*-N377A small plantN377A small plantN377A equal to smp*-377A (per Neuffer, MG)

Unknown

Unknown smp*-N390 small plantN390 small plantN390 equal to smp*-390 (per Neuffer, MG)

Unknown

Unknown smp*-N452B small plantN452B small plantN452B equal to smp*-452B (per Neuffer, MG)

Unknown

Unknown smp*-N586B small plantN586B small plantN586B smp*-586B (per Neuffer, MG)

Unknown

Unknown smp*-N600B small plantN600B small plantN600B smp*-600B (per Neuffer, MG)

Unknown

Unknown smp*-N630C small plantN630C small plantN630C smp*-630C (per Neuffer, MG)

S

Unknown smp*-N655B small plantN655B small plantN655B smp*-655B (per Neuffer, MG)

Unknown

Unknown smp*-N706A small plantN706A small plantN706A smp*-706A (per Neuffer, MG)

Unknown

Unknown smp*-N732B small plantN732B small plantN732B smp*-732B (per Neuffer, MG)

Unknown

Unknown smp*-N784 small plantN784 small plantN784 smp*-784 (per Neuffer, MG)

Unknown

Unknown smp*-N811C small plantN811C small plantN811C smp*-811C (per Neuffer, MG)

Unknown

Unknown smp*-N831B small plantN831B small plantN831B smp*-831B (per Neuffer, MG)

Unknown

Unknown smp*-N976B small plantN976B small plantN976B smp*-976B (per Neuffer, MG)

Unknown

Unknown sms*-N252A small seedlingN252A small seedlingN252A sms*-252A (per Neuffer, MG)

Unknown

Unknown sms*-N259B small seedlingN259B small seedlingN259B smp*-N259B (per Neuffer, MG); sms*-259B (per Neuffer, MG)

Unknown

Unknown sms*-N369B small seedlingN369B small seedlingN369B sms*-369B (per Neuffer, MG)

Unknown

Unknown sms*-N385B small seedlingN385B small seedlingN385B sms*-385B (per Neuffer, MG)

Unknown

Unknown sms*-N566 small seedlingN566 small seedlingN566 sms*-566 (per Neuffer, MG)

Unknown

Unknown sms*-N587B small seedlingN587B small seedlingN587B sms*-587B (per Neuffer, MG)

Unknown

Unknown sms*-N650B small seedlingN650B small seedlingN650B d*-N650B (per Neuffer, MG); sms*-650B (per Neuffer, MG)

Unknown

Unknown sms*-N774C small seedlingN774C small seedlingN774C sms*-774C (per Neuffer, MG)

Unknown

Unknown sms*-N890B small seedlingN890B small seedlingN890B d*-N890B (per Neuffer, MG); sms*-890B (per Neuffer, MG)

Unknown

unknown sms*-N937B small seedlingN937B small seedlingN937B sms*-937B (per Neuffer, MG)

Unknown

Unknown stk*-N368A streakedN368A streakedN368A stk*-368A (per Neuffer, MG)

Unknown

Unknown tlr*-N2444 tilleredN2444 tilleredN2444 Tlr*-2444 (per Neuffer, MG)

Unknown

Unknown ty*-N1315C tinyN1315C tinyN1315C d*-N1315C (per Neuffer, MG); ty*-1315C (per Neuffer, MG)

Unknown

Unknown ty*-N236B tinyN236B tinyN236B sms*-N236B (per Neuffer, MG); ty*-236B (per Neuffer, MG)

Unknown

ty*-N327 tinyN327 tinyN327 ty*-327 (per Neuffer, MG)

Unknown

Unknown ty*-N396 tinyN396 tinyN396 d*-N396 (per Neuffer, MG); ty*-396 (per Neuffer, MG)

Unknown

Unknown ty*-N438B tinyN438B tinyN438B ty*-438B (per Neuffer, MG)

Unknown

Unknown d*-N528B dwarfN528B dwarfN528B d*-528B (per Neuffer, MG); ty*-528B (per Neuffer, MG)

Unknown

128

Unknown ty*-N626A tinyN626A tinyN626A ty*-626A (per Neuffer, MG)

Unknown

Unknown ty*-N766A tinyN766A tinyN766A sms*-N766A (per Neuffer, MG); ty*-766A (per Neuffer, MG)

Unknown

Unknown ty*-N780A tinyN780A tinyN780A ty*-780A (per Neuffer, MG)

Unknown

Unknown lld1 lethal dwarf1 lethal dwarf1 lld1 lethal dwarf1 (locus)

Unknown

Unknown nld1 narrow leaf dwarf1 narrow leaf dwarf1 nld*-2346 (per Neuffer, MG)

Unknown

Unknown d*-4 dwarf candidate4 dwarf candidate4 d*-4 dwarf candidate4 (locus)

S

Unknown blh*-N2403B bleachedN2403B bleachedN2403B blh*-2403B (per Neuffer, MG)

Unknown

Unknown d*-N1013B dwarfN1013B dwarfN1013B d*-1013B (per Neuffer, MG)

S

Unknown d*-N1074C dwarfN1074C dwarfN1074C d*-1074C (per Neuffer, MG)

Unknown

Unknown d*-N1095B dwarfN1095B dwarfN1095B d*-1095B (per Neuffer, MG) d*-N1095B

Unknown

Unknown d*-N1161C dwarfN1161C dwarfN1161C d*-1161C (per Neuffer, MG)

Unknown

Unknown d*-N1168B dwarfN1168B dwarfN1168B d*-1168B (per Neuffer, MG)

Unknown

Unknown d*-N1219B dwarfN1219B dwarfN1219B d*-1219B (per Neuffer, MG)

Unknown

Unknown d*-N1226B dwarfN1226B dwarfN1226B d*-1226B (per Neuffer, MG)

Unknown

Unknown d*-N1296B dwarfN1296B dwarfN1296B d*-1296B (per Neuffer, MG)

S

Unknown d*-N1319C dwarfN1319C dwarfN1319C d*-1319C (per Neuffer, MG)

Unknown

Unknown d*-N1336C dwarfN1336C dwarfN1336C d*-1336C (per Neuffer, MG)

S

Unknown d*-N137C dwarfN137C dwarfN137C d*-137C (per Neuffer, MG)

Unknown

Unknown d*-N1380C dwarfN1380C dwarfN1380C d*-1380C (per Neuffer, MG)

Unknown

Unknown d*-N1384C dwarfN1384C dwarfN1384C d*-1384C (per Neuffer, MG)

Unknown

Unknown d*-N1452 dwarfN1452 dwarfN1452 D*-1452 (per Neuffer, MG)

I

Unknown d*-N149 dwarfN149 dwarfN149 d*-149 (per Neuffer, MG)

Unknown

Unknown d*-N150C dwarfN150C dwarfN150C d*-150C (per Neuffer, MG)

Unknown

Unknown d*-N157B dwarfN157B dwarfN157B d*-157B (per Neuffer, MG) d*-N157B

S

Unknown d*-N1591 dwarfN1591 dwarfN1591 D*-1591 (per Neuffer, MG)

Unknown

Unknown d*-N164A dwarfN164A dwarfN164A d*-164A (per Neuffer, MG)

Unknown

Unknown d*-N166B dwarfN166B dwarfN166B d*-166B (per Neuffer, MG)

Unknown

Unknown d*-N1882 dwarfN1882 dwarfN1882 d*-1882 (per Neuffer, MG)

Unknown

Unknown d*-N188B dwarfN188B dwarfN188B d*-188B (per Neuffer, MG)

Unknown

Unknown d*-N1895 dwarfN1895 dwarfN1895 d*-1895 (per Neuffer, MG) d*-N1895

Unknown

Unknown d*-N1896 dwarfN1896 dwarfN1896 d*-1896 (per Neuffer, MG)

Unknown

129

Unknown d*-N190B dwarfN190B dwarfN190B d*-190B (per Neuffer, MG)

Unknown

Unknown d*-N1925 dwarfN1925 dwarfN1925 d*-1925 (per Neuffer, MG)

Unknown

Unknown d*-N1957 dwarfN1957 dwarfN1957 d*-1957 (per Neuffer, MG)

Unknown

Unknown d*-N197A dwarfN197A dwarfN197A d*-197A (per Neuffer, MG)

Unknown

Unknown d*-N1996 dwarfN1996 dwarfN1996 d*-1996 (per Neuffer, MG)

Unknown

Unknown d*-N2023 dwarfN2023 dwarfN2023 D*-2023 (per Neuffer, MG)

Unknown

Unknown d*-N2234 dwarfN2234 dwarfN2234 d*-2234 (per Neuffer, MG)

Unknown

Unknown d*-N2248A dwarfN2248A dwarfN2248A d*-2248A (per Neuffer, MG)

Unknown

Unknown d*-N2249A dwarfN2249A dwarfN2249A d*-2249A (per Neuffer, MG)

Unknown

Unknown d*-N2250A dwarfN2250A dwarfN2250A d*-2250A (per Neuffer, MG)

Unknown

Unknown d*-N2251 dwarfN2251 dwarfN2251 d*-2251 (per Neuffer, MG)

Unknown

Unknown d*-N2252 dwarfN2252 dwarfN2252 d*-2252 (per Neuffer, MG)

Unknown

Unknown d*-N2253 dwarfN2253 dwarfN2253 d*-2253 (per Neuffer, MG)

Unknown

Unknown d*-N2254 dwarfN2254 dwarfN2254 d*-2254 (per Neuffer, MG)

Unknown

Unknown d*-N2255 dwarfN2255 dwarfN2255 d*-2255 (per Neuffer, MG)

Unknown

Unknown d*-N2256 dwarfN2256 dwarfN2256 d*-2256 (per Neuffer, MG)

Unknown

Unknown d*-N2277 dwarfN2277 dwarfN2277 d*-2277 (per Neuffer, MG)

Unknown

Unknown d*-N2278 dwarfN2278 dwarfN2278 d*-2278 (per Neuffer, MG)

Unknown

Unknown d*-N2295 dwarfN2295 dwarfN2295 d*-2295 (per Neuffer, MG) d*-N2295

Unknown

Unknown d*-N2296 dwarfN2296 dwarfN2296 d*-2296 (per Neuffer, MG)

Unknown

Unknown d*-N230A dwarfN230A dwarfN230A d*-230A (per Neuffer, MG)

Unknown

Unknown d*-N235A dwarfN235A dwarfN235A d*-235A (per Neuffer, MG)

Unknown

Unknown d*-N2362A dwarfN2362A dwarfN2362A d*-2362A (per Neuffer, MG)

Unknown

Unknown d*-N2363B dwarfN2363B dwarfN2363B d*-2363B (per Neuffer, MG)

Unknown

Unknown d*-N2367A dwarfN2367A dwarfN2367A d*-2367A (per Neuffer, MG)

Unknown

Unknown d*-N2371 dwarfN2371 dwarfN2371 d*-2371 (per Neuffer, MG)

Unknown

Unknown d*-N2373B dwarfN2373B dwarfN2373B d*-2373B (per Neuffer, MG)

Unknown

Unknown d*-N2374 dwarfN2374 dwarfN2374 d*-2374 (per Neuffer, MG)

Unknown

Unknown d*-N2377 dwarfN2377 dwarfN2377 d*-2377 (per Neuffer, MG)

Unknown

Unknown d*-N237A dwarfN237A dwarfN237A d*-237A (per Neuffer, MG)

Unknown

Unknown d*-N2383B dwarfN2383B dwarfN2383B d*-2383B (per Neuffer, MG)

Unknown

130

Unknown d*-N254 dwarfN254 dwarfN254 d*-254 (per Neuffer, MG)

Unknown

Unknown d*-N257C dwarfN257C dwarfN257C d*-257C (per Neuffer, MG)

Unknown

Unknown d*-N262C dwarfN262C dwarfN262C d*-262C (per Neuffer, MG)

Unknown

Unknown d*-N266B dwarfN266B dwarfN266B d*-266B (per Neuffer, MG)

Unknown

Unknown d*-N274 dwarfN274 dwarfN274 d*-274 (per Neuffer, MG)

Unknown

Unknown d*-N275 dwarfN275 dwarfN275 d*-275 (per Neuffer, MG)

Unknown

Unknown d*-N287B dwarfN287B dwarfN287B d*-287B (per Neuffer, MG)

Unknown

Unknown d*-N293B dwarfN293B dwarfN293B d*-293B (per Neuffer, MG)

S

Unknown d*-N299B dwarfN299B dwarfN299B d*-299B (per Neuffer, MG) d*-N299B

S

Unknown d*-N301 dwarfN301 dwarfN301 d*-301 (per Neuffer, MG)

unknown

Unknown d*-N302C dwarfN302C dwarfN302C d*-302C (per Neuffer, MG)

Unknown

Unknown d*-N305 dwarfN305 dwarfN305 d*-305 (per Neuffer, MG)

S

Unknown d*-N307C dwarfN307C dwarfN307C d*-307C (per Neuffer, MG)

Unknown

Unknown d*-N309 dwarfN309 dwarfN309 d*-309 (per Neuffer, MG)

Unknown

Unknown d*-N353B dwarfN353B dwarfN353B d*-353B (per Neuffer, MG)

Unknown

Unknown d*-N360A dwarfN360A dwarfN360A d*-360A (per Neuffer, MG)

S

Unknown d*-N373B dwarfN373B dwarfN373B d*-373B (per Neuffer, MG)

Unknown

Unknown d*-N374 dwarfN374 dwarfN374 d*-374 (per Neuffer, MG)

Unknown

Unknown d*-N389A dwarfN389A dwarfN389A d*-389A (per Neuffer, MG)

Unknown

Unknown d*-N403B dwarfN403B dwarfN403B d*-403B (per Neuffer, MG) d*-N403B

Unknown

Unknown d*-N408A dwarfN408A dwarfN408A d*-408A (per Neuffer, MG)

S

Unknown d*-N429A dwarfN429A dwarfN429A d*-429A (per Neuffer, MG)

Unknown

Unknown d*-N434 dwarfN434 dwarfN434 d*-434 (per Neuffer, MG)

Unknown

Unknown d*-N436B dwarfN436B dwarfN436B d*-436B (per Neuffer, MG)

Unknown

Unknown d*-N518A dwarfN518A dwarfN518A d*-518A (per Neuffer, MG) d*-518A

Unknown

Unknown d*-N522B dwarfN522B dwarfN522B d*-522B (per Neuffer, MG)

Unknown

Unknown d*-N524D dwarfN524D dwarfN524D d*-524D (per Neuffer, MG)

Unknown

Unknown d*-N526B dwarfN526B dwarfN526B d*-526B (per Neuffer, MG)

Unknown

Unknown d*-N553D dwarfN553D dwarfN553D d*-553D (per Neuffer, MG)

Unknown

Unknown d*-N597A dwarfN597A dwarfN597A d*-597A (per Neuffer, MG)

S

Unknown d*-N604 dwarfN604 dwarfN604 d*-604 (per Neuffer, MG) d*-N604

Unknown

131

Unknown d*-N621C dwarfN621C dwarfN621C d*-621C (per Neuffer, MG)

Unknown

Unknown d*-N629B dwarfN629B dwarfN629B d*-629B (per Neuffer, MG) d*-N629B

Unknown

Unknown d*-N649B dwarfN649B dwarfN649B d*-649B (per Neuffer, MG)

Unknown

Unknown d*-N661B dwarfN661B dwarfN661B d*-661B (per Neuffer, MG)

Unknown

Unknown d*-N679 dwarfN679 dwarfN679 d*-679 (per Neuffer, MG)

Unknown

Unknown d*-N681B dwarfN681B dwarfN681B d*-681B (per Neuffer, MG)

Unknown

Unknown d*-N699B dwarfN699B dwarfN699B d*-699B (per Neuffer, MG) d*-N699B

Unknown

Unknown d*-N707B dwarfN707B dwarfN707B d*-707B (per Neuffer, MG)

Unknown

Unknown d*-N719B dwarfN719B dwarfN719B d*-719B (per Neuffer, MG)

Unknown

Unknown d*-N720F dwarfN720F dwarfN720F d*-720F (per Neuffer, MG)

Unknown

Unknown d*-N758C dwarfN758C dwarfN758C d*-758C (per Neuffer, MG)

Unknown

Unknown d*-N776B dwarfN776B dwarfN776B d*-776B (per Neuffer, MG)

Unknown

Unknown d*-N777A dwarfN777A dwarfN777A *-777A (per Neuffer, MG)

Unknown

Unknown d*-N808 dwarfN808 dwarfN808 d*-808 (per Neuffer, MG)

Unknown

Unknown d*-N809 dwarfN809 dwarfN809 d*-809 (per Neuffer, MG)

Unknown

Unknown d*-N838A dwarfN838A dwarfN838A d*-838A (per Neuffer, MG)

Unknown

Unknown d*-N841 dwarfN841 dwarfN841 D*-841 (per Neuffer, MG)

Unknown

Unknown d*-N871D dwarfN871D dwarfN871D d*-871D (per Neuffer, MG)

Unknown

Unknown d*-N885B dwarfN885B dwarfN885B d*-885B (per Neuffer, MG)

Unknown

Unknown d*-N905B dwarfN905B dwarfN905B d*-905B (per Neuffer, MG)

Unknown

Unknown d*-N979B dwarfN979B dwarfN979B d*-979B (per Neuffer, MG)

Unknown

Unknown d*-N984 dwarfN984 dwarfN984 D*-984 (per Neuffer, MG)

Unknown

Unknown d*-N987B dwarfN987B dwarfN987B D*-987B (per Neuffer, MG)

Unknown

Unknown d*-N994B dwarfN994B dwarfN994B d*-994B (per Neuffer, MG) d*-N994B

Unknown

Unknown d*-N998B dwarfN998B dwarfN998B d*-998B (per Neuffer, MG) d*-N998B

Unknown

Unknown de*-N1431 defectiveN1431 defectiveN1431 de*-1431 (per Neuffer, MG); fl*-N1431 (per Neuffer, MG)

Unknown

Unknown de*-N950 defectiveN950 defectiveN950 cp*-N950 (per Neuffer, MG); de*-950 (per Neuffer, MG)

Unknown

Unknown dst*-N1377 distortedN1377 distortedN1377 Dst*-1377 (per Neuffer, MG); Sdp*-N1377 (per Neuffer, MG)

Unknown

Unknown flk*-N1398B fleckedN1398B fleckedN1398B flk*-1398B (per Neuffer, MG)

Unknown

Unknown des*-GG22 defective seedling GG22

defective seedling GG22 des22 (per Gavazzi, G)

Unknown

Unknown d*-N2498 dwarfN2498 dwarfN2498 dwarf*-2498 (per Neuffer, MG)

Unknown

132

Unknown d*-N2506 dwarfN2506 dwarfN2506 d*-2506 (per Neuffer, MG)

Unknown

Unknown yg*-N2449 yellowgreenN2449 yellowgreenN2449 Yg*-2449 (per Neuffer, MG); Ys*-N2449 (per Neuffer, MG)

Unknown

Unknown d*-N2480 dwarfN2480 dwarfN2480 d*-2480 (per Neuffer, MG)

Unknown

Unknown d*-N2468 dwarf N2468 dwarf N2468

Unknown

Unknown d*-8 dwarf candidate8 dwarf candidate8

Unknown

Unknown flk*-N1399B fleckedN1399B fleckedN1399B flk*-1399B (per Neuffer, MG)

Unknown

Unknown gtl*-N2297 gritty leafN2297 gritty leafN2297 gtl*-2297 (per Neuffer, MG)

Unknown

sh*-N2219 shrunkenN2219 shrunkenN2219 sh*-2219 (per Neuffer, MG)

Reference: http://www.maizegdb.org/cgi-bin/displayphenorecord.cgi?id=11041

133

Appendix 4: Wheat dwarfing genes

Chro. location gene remark dwarfing gene phenotype source GA response reference Sponstanous(s) or induced(I)

2A Rht7 Rht7 recessive Bersee Mutant S Worland et al. (1980)

2DS Rht8 Rht8 semi dominant Mara, Sava S Worland & Law (1986)

4BS Rht-B1b Rht1 semi dominant Norin 10 I Gale & Youssefian(1985)

4BS Rht-B1c Rht3 semi dominant Tom Thumb I Gale & Youssefian(1985)

4BS Rht-B1d Rht1S (semi) dominant Saitama 27 I Worland & Petrovic(1988)

4BS Rht-B1e Rht Krasnodari 1 semi dominant Krasnodari 1 I Worland (1986)

4BS Rht-B1f Rht T. aeth. semi dominant W6824D; W 6807C; T. aethiopicum I Bŏrner et al. (1995)

4DS Rht-D1b Rht2 semi dominant Norin 10 I Gale & Youssefian(1985)

4DS Rht-D1c Rht10 (semi) dominant Ai-bian 1 I Bŏrner & Mettin (1988)ŏ

4DS Rht-D1d Rht Aibian 1a semi dominant Ai-bian 1a I Bŏrner et al. (1991)

5A Rht12 Rht12 dominant Karcag 522 S Sutka & Kovacs (1987)

7BS Rht9 Rht9 semi dominant Mara S Worland & petrovic,(1986)

unknown Rht4 Rht4 recessive Burt M937 S Konzak(1982)

unknown Rht5 Rht5 semi dominant Marfed M1 S Konzak(1982)

Unknown Rht6 Rht6 recessive Burt S Konzak(1982)

Unknown Rht11 Rht11 recessive Karilik 1 S Konzak (1987)

Unknown Rht13 Rht13 part. Dominant Magnif 41M1 S Konzak (1987)

unknown Rht14 Rht14 semi dominant Castelporziano S Konzak (1987)

Unknown Rht15 Rht15 part. recessive Durox S Konzak (1987)

Unknown Rht16 Rht16 semi dominant Edmore M1 S Konzak (1987)

Unknown Rht17 Rht17 recessive Chris M1 S Konzak (1987)

Unknown Rht18 Rht18 semi dominant Icaro S Konzak (1987)

Unknown Rht19 Rht19 semi dominant Vic M1 S Konzak (1987)

unknown Rht20 Rht20 part. Dominant Burt M860 S Konzak (1987)

GA response: S: Sensitive to GA; I: Insensitive to GA.

134

Appendix 5: Figures of gene bioinformatics

Figure 1. The relationships in the shrunken 2 (Sh2) - A1 region across species. The region remains intact across maize, sorghum, and rice but rearranges in wheat. The X1 and X2 loci code for transcription factors and maintain their relationship to A1 and Sh2 loci, respectively (Appels, 2003)

135

Table 1 RFLP clones, the restriction enzymes used to map them, the number of loci each clone detected on group 5 chromosomes and their specificity to group 5 chromosomes. (From Faris, 1999)

136

Appendix 6: Intron1 blast

RID: 1095757482-30466-108534670260.BLASTQ4

Query=

(173 letters)

Database: All GenBank+EMBL+DDBJ+PDB sequences (but no EST, STS,

GSS,environmental samples or phase 0, 1 or 2 HTGS sequences)

2,600,233 sequences; 11,806,403,425 total letters

If you have any problems or questions with the results of this search please refer to the BLAST FAQs

No significant similarity found. For reasons why, click here.

Lambda K H 1.37 0.711 1.31 Gapped Lambda K H 1.37 0.711 1.31 Gap Penalties: Existence: 5, Extension: 2 Number of Sequences: 2600233 Number of Hits to DB: 2,161,950 Number of extensions: 85398 Number of successful extensions: 4487 Number of sequences better than 10.0: 0 Number of HSP's better than 10.0 without gapping: 0 Number of HSP's gapped: 4487 Number of HSP's successfully gapped: 0 Number of extra gapped extensions for HSPs above 10.0: 4487 Length of query: 173 Length of database: 11,806,403,425 Length adjustment: 21 Effective length of query: 152 Effective length of database: 11,751,798,532 Effective search space: 1786273376864 Effective search space used: 1786273376864 A: 0 X1: 11 (21.8 bits) X2: 15 (30.0 bits) X3: 25 (50.0 bits) S1: 12 (25.0 bits) S2: 19 (38.2 bits)

137

Appendix 7: sd1 DNA and protein séquense AF465255. Oryza sativa cult...[gi:19422258]

LOCUS AF465255 6590 bp DNA linear PLN 14-MAR-2002 DEFINITION Oryza sativa cultivar Nipponbare gibberellin-20 oxidase (Sd-1) gene, complete cds. ACCESSION AF465255 VERSION AF465255.1 GI:19422258 KEYWORDS . SOURCE Oryza sativa (japonica cultivar-group) ORGANISM Oryza sativa (japonica cultivar-group) Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; Ehrhartoideae; Oryzeae; Oryza. REFERENCE 1 (bases 1 to 6590) AUTHORS Yoon,U.H., Eun,M.Y., Lee,J.S., Yun,D.W., Kim,H.I. and Hahn,J.H. TITLE Map-based cloning of semidwarf gene, Sd-1, in rice JOURNAL Unpublished REFERENCE 2 (bases 1 to 6590) AUTHORS Yoon,U.H., Eun,M.Y., Lee,J.S., Yun,D.W., Kim,H.I. and Hahn,J.H. TITLE Direct Submission JOURNAL Submitted (02-JAN-2002) Cytogenetics, National Institute of Agricultural Science and Technology, 249 Seodun-dong, Suweon 441-707, Korea FEATURES Location/Qualifiers source 1..6590 /organism="Oryza sativa (japonica cultivar-group)" /mol_type="genomic DNA" /cultivar="Nipponbare" /db_xref="taxon:39947" /chromosome="1" gene <2430..>5172 /gene="Sd-1" mRNA join(<2430..2986,3089..3410,4882..>5172) /gene="Sd-1" /product="gibberellin-20 oxidase" CDS join(2430..2986,3089..3410,4882..5172) /gene="Sd-1" /note="semidwarf" /codon_start=1 /product="gibberellin-20 oxidase" /protein_id="AAL87949.1" /db_xref="GI:19422259" /translation="MVAEHPTPPQPHQPPPMDSTAGSGIAAPAAAAVCDLRMEPKIPE PFVWPNGDARPASAAELDMPVVDVGVLRDGDAEGLRRAAAQVAAACATHGFFQVSEHG VDAALARAALDGASDFFRLPLAEKRRARRVPGTVSGYTSAHADRFASKLPWKETLSFG FHDRAAAPVVADYFSSTLGPDFAPMGRVYQKYCEEMKELSLTIMELLELSLGVERGYY REFFADSSSIMRCNYYPPCPEPERTLGTGPHCDPTALTILLQDDVGGLEVLVDGEWRP VSPVPGAMVINIGDTFMALSNGRYKSCLHRAVVNQRRERRSLAFFLCPREDRVVRPPP SAATPQHYPDFTWADLMRFTQRHYRADTRTLDAFTRWLAPPAADAAATAQVEAAS" ORIGIN 1 ctagatcaga gcacacagag aaaaaaatat agacaccttg gaaatttgtc acaaagagac 61 aaggtgactc aacaggccct ccaaactgaa aatttaatta cttgctcaag atttaaatat 121 aactacccat ccagttttta atatataaag ttgttaactt ttaaacatat atatgtttca 181 ctgttcttat aatgtatttt atcattaaac atactttaaa acatatggct tatgtttttg 241 aatatttata ttaaaaattt taaataagat taatgatcaa acgtatattt actagttaac 301 gacatcatgt attaaaaatc ggaggaggta tagaagtatg ttctcctttc ttgtaaacat 361 aggttgatct gtatatttgt ttttgtctta ttttgttttt tcattgatct caccattaaa 421 caggtggcct ccaaaaatgc atgcagccat gtatcttccc agtccatgaa attaatcttg 481 aattattata aattaaaaac atattaggat ttgatatatg aaaggtataa tggtagcaga 541 tctatcatag aaaacctata acacgtagat gaccgagtag agaaaaaaga tatacccaac 601 ataatcaagt accttgttaa ttagtaagaa gtaagaaaac catataaata caagcatttg 661 ggtgaagcta gaatgggaac tatattacca tgtatcccat acatatctat tacgcactta 721 atacctgaat tattcgtgta acgaagaaca tgctttggta aaaaataaaa tatttggacc 781 gtataccatg cggttatcgc agttatcacg tgcggtaata ttctcagttt ttcaaaccgc 841 ggtataactt gcaataaccg cgtggttttc gcggtaacca ccaaaccgtg gggctgtggt 901 aaccccacct aaaacgattt ggtaaaccct agtcagagct agcttgatcg tggctccgcc 961 ctctgcatct cctcatggtc acaagatcaa tttagaacct cttataagct gttgaaccat 1021 cagtactaca gtcccaagat taacatattt tttaaaatat caaaattgct catgatgaat 1081 tgattaccgt tcatgtgcct gtatggtgca tgggtggtat agtgcaccgt gacctgtact 1141 gtgctagatg ttccttccaa ccttagtact tcacgaaaag gagaggaacc atttccctgt 1201 caccttcctg caacatggtc aggcataaac caaacacgat cgaagcgaat cggcgatacc 1261 acacaatagc cgcgcgtcgc aaacaacaat tccgcggcta gctacttccg acctccgaaa

138

1321 ctactgcgag cccaagtggg tacggtttta gtgcaaatag ggtaagtctt gtttacatca 1381 tatcggtttc attttggtac acgaatggag aagaaatgaa agagatcgaa aaaaggaaga 1441 gctcgctgtg tatctgtctc gtaacagccc cggtgttaca cgtgctctaa gagagattaa 1501 ttaaatcgat aagctaccag aggtttagtt ttccacgtgt taattagatt ggaaagcgag 1561 agaaattaaa aatagcgagt aaaaatagag ataaccttat tgctattttg ttttttttcc 1621 agcaacaaac ttatctttca ggctagttta ggcgatcgct tagattccgc atcgtccttt 1681 tcactatttt ttttctgtca gtgacaatgt gaaaatttat tggacagacg actagcttgt 1741 ggtactagct aggaaattcc ctatcctcga tatgaacaac ttactcaact cagtagagta 1801 gcaaatgccc aagaaagccc gagtcaatct atttggaaat ccaatctatt ttctcgtatt 1861 cgtgtgggaa atcaagctat actagttgaa attcaccgga agaaatgcac gcacttcaat 1921 ataccaaaat tgcaaaggag aatcattcga ttaacagtgg aattcaacca agaaatgaaa 1981 aggtatatat aggaaatgca ctccaaccac caaccaataa gtgattccgg gcaatcaatt 2041 ctatccgcga gttgtgggtc tgttcagatt cattatatta gaacgcgtca cgtaatggat 2101 ggagtattat acaacaccat tggttttgcc actagtgtta actctaatac atgggggtta 2161 gttttacctt taaacttggt ctaaaaggat ggacatatgg caatgcaatt gcatgggggt 2221 cattgattcg accatcatgt ctgtccagtg gcaaccccct ccctcatccc ctgtggtggg 2281 ccccccacgg cgctcgtctt ctcccctgtt acaaataccc caccctcctg cccagacagc 2341 tcgccctgca cacacacaca cactcacact cacacacgct ctcaactcac tcccgctcaa 2401 cacagcgctc acttctcatc tccaatctca tggtggccga gcaccccacg ccaccacagc 2461 cgcaccaacc accgcccatg gactccaccg ccggctctgg cattgccgcc ccggcggcgg 2521 cggcggtgtg cgacctgagg atggagccca agatcccgga gccattcgtg tggccgaacg 2581 gcgacgcgag gccggcgtcg gcggcggagc tggacatgcc cgtggtcgac gtgggcgtgc 2641 tccgcgacgg cgacgccgag gggctgcgcc gcgccgcggc gcaggtggcc gccgcgtgcg 2701 ccacgcacgg gttcttccag gtgtccgagc acggcgtcga cgccgctctg gcgcgcgccg 2761 cgctcgacgg cgccagcgac ttcttccgcc tcccgctcgc cgagaagcgc cgcgcgcgcc 2821 gcgtcccggg caccgtgtcc ggctacacca gcgcccacgc cgaccgcttc gcctccaagc 2881 tcccatggaa ggagaccctc tccttcggct tccacgaccg cgccgccgcc cccgtcgtcg 2941 ccgactactt ctccagcacc ctcggccccg acttcgcgcc aatggggtaa ttaaaacgat 3001 ggtggacgac attgcatttc aaattcaaaa caaattcaaa acacaccgac cgagattatg 3061 ctgaattcaa acgcgtttgt gcgcgcagga gggtgtacca gaagtactgc gaggagatga 3121 aggagctgtc gctgacgatc atggaactcc tggagctgag cctgggcgtg gagcgaggct 3181 actacaggga gttcttcgcg gacagcagct caatcatgcg gtgcaactac tacccgccat 3241 gcccggagcc ggagcggacg ctcggcacgg gcccgcactg cgaccccacc gccctcacca 3301 tcctcctcca ggacgacgtc ggcggcctcg aggtcctcgt cgacggcgaa tggcgccccg 3361 tcagccccgt ccccggcgcc atggtcatca acatcggcga caccttcatg gtaaaccatc 3421 tcctattctc ctctcctctg ttctcctctg cttcgaagca acagaacaag taattcaagc 3481 ttttttttct ctctcgcgcg aaattgacga gaaaaataag atcgtggtag gggcggggct 3541 ttcagctgaa agcgggaaga aaccgacctg acgtgatttc tctgttccaa tcacaaacaa 3601 tggaatgccc cactcctcca tgtgttatga tttatctcac atcttatagt taataggagt 3661 aagtaacaag ctactttttt catattatag ttcgtttgat tttttttttt taaagttttt 3721 ttagttttat ccaaatttat tgaaaaactt agcaacgttt ataataccaa attagtctca 3781 tttagtttaa tattgtatat attttgataa tatatttatg ttatattaaa aatattacta 3841 tatttttcta taaacattat taaaagccat ttataatata aaatggaagg agtaattaat 3901 atggatctcc cccgacatga gaatattttc cgatggtgtg acgacgccat gtaagcttcg 3961 gtgggcctgg acggccagag gtgccaacag ccacgtccaa caacccctgg gtccccccct 4021 aacactccaa acagtagtga gtagtgtctc gtcgcgtttt agtatttgat gacaaacaaa 4081 gtgtgagttg agttagccac caccaacttg cacacgagca catacatttg tgtccattct 4141 cgccagtcac ttccatctct agtcctaact cctatctagc gatgtaagcg gataatttca 4201 tcatccgtat ataaacctgt ttgttatagt taatttccta tataatacta taacagtata 4261 cattttaaaa gaaaacaaaa ttaggataaa caggccctgc tcctatccat ccatggcact 4321 tggaaggacc agactcggtc atgccatgcc aagccaagat atgggttatg gaagagtaga 4381 gaagaggaga gatgagagat aagcatgcgt tctcctcctc gttggatgtg tattttggag 4441 ggatttgtgt agtagtagca gcggcgccgc ggggacggat gcggatggtg gcgctttcgg 4501 tggcgttttc ccgggggggt tttggtttgg cgcttggggg ggatggcatg gcgcggcgtg 4561 cggctgcacg ccacacacac gcgcgcgcac gcacgtacgt cgtcgtcgcc gcgggcggac 4621 ggtagcttag ggtggtgtgt tccgcgcgcg ggcgcggatt gttccatgcc gatcgatttg 4681 gcgccaccct cgccgcggct cttgtcgcgt cgtgcgcctc tctcgcgcgg tttgtccttg 4741 tcgcgttgct cagccggcga cgggggcacg gacattggcg atgtagccct gcacgtgtcg 4801 gcctctccgt tgatgaatga tgatgtatgt atgtattttt ttttgtctga aggaatttgt 4861 ggggaattgt tgtgtgtgca ggcgctgtcg aacgggaggt ataagagctg cctgcacagg 4921 gcggtggtga accagcggcg ggagcggcgg tcgctggcgt tcttcctgtg cccgcgggag 4981 gacagggtgg tgcggccgcc gccgagcgcc gccacgccgc agcactaccc ggacttcacc 5041 tgggccgacc tcatgcgctt cacgcagcgc cactaccgcg ccgacacccg cacgctcgac 5101 gccttcacgc gctggctcgc gccgccggcc gccgacgccg ccgcgacggc gcaggtcgag 5161 gcggccagct gatcgccgaa cggaacgaaa cggaacgaac agaagccgat ttttggcggg 5221 gcccacgccc acgtgaggcc ccacgtggac agtgggcccg ggcggaggtg gcacccacgt 5281 ggaccgcggg ccccgcgccg ccttccaatt ttggacccta ccgctgtaca tattcatata 5341 ttgcaagaag aagcaaaacg tacgtgtggg ttgggttggg cttctctcta ttactaaaaa 5401 aaatataatg gaacgacgga tgaatggatg cttatttatt tatctaaatt gaattcgaat 5461 tcggctcatg gatttcgcga atgtggatgg tggatgcccg cctcgatgaa tccgctttgt 5521 ccgatagaga aatttgaatt taaatccggg acctggattt tgcaatgtgg acgggtgtgc 5581 tttgcgaaat ctgctttgtt cgatagcgct gcacaaaaca tgcggtgggc cctgcatgag 5641 aatccgcttc ttctttgttg ccttggtagg cgaaatcgta tatggtccca acgattttct 5701 ttgtttggtt tcaacataaa tgggagtttt tatgaattta ggcttatcta catcagagct 5761 actcctaact tgtgatatga tgaaccaatc gtgttcttct catacttgtt taagttggcc 5821 aatataggat taatgcagag tatccaaggg ttttaagatg gatctagtta agatttggag 5881 aacataatct acaatcatca gcaacactaa ttataactaa atcaacttgc cttttgagtt

139

5941 ctccgcaaat atcagaacgc ttttttcttt ttttttcttt tttttttttg aggaggtggg 6001 gagcacaaaa tcggagtgaa attcgggatt cccattcaac cacttccaac catgccaaat 6061 cccggatggt ttttgtttct tggcactaag tgatgggtca cgttttcaca gtagttgata 6121 acttgcaact tgcaatcact ctatcttcag ctgctccact ggattcaacg tccgtaggag 6181 cagtaacttg tcacaatgct gagcagaaaa taaccgctag gaatatcaaa ttgcacaaaa 6241 ttataatgtc actgttgagt gatgagcagt aactcatgaa agaatccaaa gttcccatga 6301 gttccagaat gtttgactga tatgacagac aaacttttgt aaggttctcc taaataacac 6361 aaagaaattt ctcgtacact acatggctgt atggattgaa cataatttgc ctgtgacact 6421 tgtcacactt gtgacacttc caggttccaa tacaacctta cagcaatgga gcaagagcac 6481 agcaagaaga gaatatagag tgcatttgtt tggcaagaaa gaaaaaaaat gattctacaa 6541 ttacatcaag gcttatgcct tttcagaatc aacaagaata gtatgcatat