Towards understanding genetic basis of chapatti (Indian flat bread) making quality

Towards understanding genetic basis of chapatti (Indian flat bread) making quality

M. GARG, S. ARORA, R.P. Singh*, A. SINGH, J.K. ROY

National Agri-Food Biotechnology Institute

*Deptt. Of Plant Breeding and Genetics

Punjab Agricultural University, Ludhiana.

Sample ID Character Hardness Pina-D1 Pinb-D1

Chapatti

Score

CHINESE SPRING Check 48.66 a a -

PBW 343 Check 86.26 b a 7.5

WH 291 Chapatti 60.81 b a 5

SONALIKA Chapatti 66.88 a b 6

C 306 Chapatti 94.64 b a 9

LOK 1 Chapatti 67.51 b a 7

Parameter/cultivar

C306 LOK1 WH291 SONALIKA

Chapatti score Very Good Good Poor Medium Poor

Grain hardness (g) Hard Med Soft Med hard Med Soft

Seed size Med Med Small Large

Color White Brown Brown Brown

Water absorption High Med Med Med

Protein content Med Med High Med

Gluten Medium Medium High Low

Starch gelatinisation Medium High Medium High

RVA Breakdown High High Low Medium

HMW-GS-ABD Null, 20, 2+12 2*,17+18,

2+12 2*,20, 2+12 2*,7+9, 2+12

Puroindolines (5DS) PinaD1b, PinbD1a

PinaD1b, PinbD1a

PinaD1b, PinbD1a

PinaD1a, PinbD1b

11

Affymetrix GeneChip® Wheat Genome Array

7DAA 14DAA 21DAA 28DAA 35DAA Fig. 1 Caryopsis at three developmental stages- 7, 14, 21, 28, & 35 days after

anthesis (DAA)

Expression profiling of 17,344 probe

sets at three developmental stages-

7DAA, 14DAA, 28DAA

RNA EXTRACTION

3’-IVT

Hybridization

Washing, Staining and scanning

File generation and Data analysis

AGCC-MAS5,RMA, GCRMA, PILER)

{GeneSpring, Array star,

Overview of experiment

Quality control of samples RIN calculation

Correlation coefficient (r)

Positive hybridization control

Summary

Data analysis of7DAA Good vs. Poor

61,290 (Total probe sets)

Normalized & filter by percentile

37,277 (Probe sets)

Statistical data analysis

2,989 (Probe sets)

Fold change analysis

59

1027 137

Functional annotation:

Up-regulating

Catalytic subunit activity

Nutrient reservoir activity

DNA binding activity

Translational initiation factor activity

Down regulation:

Catalytic subunit activity

Magnesium ion binding activity

GO ANALYSIS:

Two fold regulation:

Molecular function: 43.6%

Biological function: 34.0%

Cellular function: 22.6%

Contd….

Data analysis of28DAA Good vs.Poor

61,290 (Total probe sets)

Normalized & filter by percentile

33,083 (Probe sets)

Statistical data analysis

527 (Probe sets)

Fold change analysis

• 194 (113↑, 81↓)

• 72 (33↑, 26↓)

• 19 (9↑, 10↓)

Functional annotation:

Up-regulating

Response towards heat (HSP-16, 26)

Lipid binding proteins and its transport (LTP3)

Down regulation:

Catalase activity (Response towards oxidative stress)

Starch biosynthesis activity (WGBSSI)

GO ANALYSIS:

Two fold regulation: Molecular process: 33.3%

Biological process: 66.6%

Contd…

30% - storage proteins

30% -no prior information on gene

function. Rest - Transcription

factors, trypsin inhibitors,

metal binding, lipid transfer, cell

wall related proteins, etc.

photosynthesis, regulatory genes

Major genes – No prior

information on gene function.

Rest - cytochrome C heme

attached protein, peroxidases.

starch synthesis, proteases

1 2

3,4 5

23

S.

No.

Probe Set ID UniGen

e ID

(DAA) Fold

change

Function

1 Ta.24736.1.S1_at NA 7 +284.7 Predicted protein (Hordeum

vulgare )

2 Ta.27778.4.S1_x_at Ta.5420

6

7 +108.1 Pre-alpha-/beta-gliadin A-II

3 Ta.7158.1.S1_at Ta.7158 7 +105.9 Transcribed locus

4 Ta.6175.1.S1_at Ta.3546

3

7 +102.3 No significant similarity in non-

redundant (nr) protein

sequences database

5 Ta.24114.14.S1_x_

at

Ta.6588

1

7 +284.7 Triticum aestivum gamma-

gliadin gene

Differentially expressed genes (up and down)

At the three development stages among two good and two poor chapatti varieties Source-Dr. Joy K Roy

24

6 Ta.6984.1.A1_at Ta.5710

0

7 -58.0 Predicted protein & also

similar with PHD zinc finger

protein-like [Oryza sativa

Japonica Group]

7 TaAffx.104444.1.S1

_at

Ta.3578

4

7 -47.5 No significant similarity

8 Ta.23013.3.S1_s_a

t

NA 7 -31.5 B22EL8 [Hordeum vulgare]

9 Ta.14507.2.S1_at Ta.5418

6

7 -29.9 Predicted protein & also similar

with nucleotide-binding protein

1 [Zea mays]

10 Ta.4957.1.S1_at Ta.4957 7 -26.6 Triticum aestivum granule bound starch synthase I (GbssI)

11 Ta.23366.2.S1_at NA

28 +59.96

Triticum aestivum TaPrx-B mRNA for peroxidase, partial cds

12 Ta.28744.1.S1_at Ta.28744

28 +35.22

Transcribed locus, Triticum aestivum cDNA

13 Ta.14050.1.S1_at Ta.1405

0

28 +37.26

Hordeum vulgare subsp. vulgare mRNA for predicted protein

Source-Dr. Joy K Roy

25

14 TaAffx.91902.1.A1_at Ta.4769

5

28

+32.62 Transcribed locus, Triticum aestivum cDNA

15 Ta.23366.2.S1_x_at NA

28 +28.88

Triticum aestivum TaPrx-B mRNA for peroxidase, partial cds

16 Ta.2025.1.S1_at

Ta.2025

28 -1727.4

Transcribed locus_Hordeum

vulgare subsp. vulgare cDNA

clone: FLbaf53o13

17 Ta.24114.7.A1_at Ta.30989

28 -92.767 Triticum aestivum granule

bound starch synthase I (GbssI)

18 TaAffx.80118.1.S1_at

Ta.48558

28 -32.63 Transcribed locus

19 Ta.14489.1.S1_at

NA

28 -28.99

S.cereale acclimation

protein/Triticum aestivum cold-

responsive protein (Wlt10)

20 Ta.27445.1.S1_at Ta.27445

28 -28.62

Medicago sativa FtsH protease

(FtsH) mRNA/Triticum aestivum

clone wlp1c.pk007.f6:fis Source-Dr. Joy K Roy

26

zEEMAN et al. Biochem. J. (2007) 401 (13–28

DAY NIGHT

Nagai Y S et al. Plant Cell Physiol 2009;50:635-643 © The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant

Physiologists. All rights reserved. For permissions, please email:

journals.permissions@oxfordjournals.org

Tian Z et al. PNAS 2009;106:21760-21765 ©2009 by National Academy of Sciences

GBSS genes

GC rich 11 EXONS 10 INTRONS

GBSS A1 gene 2781bp

GBSS D1 gene 2862bp

GBSS B1 gene

2794bp

GBSS genes

GC rich 11 EXONS 10 INTRONS

Saito et al., JARQ 44 (2), 109–115 (2010)

Good starch swelling properties-

Good for Udon noodles

Low amylose content (27-28% compared to 29%

22-23% compared to 25%)

Amylopectin matrix in

starch granule can swell

better

38

Japanese Udon Indian Chapatti Softness

39

Null GBSS A1 gene Deletion mutation -19bp

2762bp (2781bp wild type)

Null GBSS A1 gene

Insertion mutation from Turkey

(Transposable element) 2854bp (2781bp wild type)

Transp.elemt

IR IR 12bp

Target site duplication 8bp

Null GBSS B1 gene Absence (2794bp wild type)

Null GBSS D1 gene Deletion mutation 2850bp (2862bp

wild type)

Yanagisawa et al EUPHYTICA, 121-3, 209-214

GBSS D1 gene

Allele GBSS-D1e

Nakamura et al. Plant physiology

Waxy

Waxy

Normal

Nakamura et al. Plant physiology

49 49

50

51

S.No Chapatti Quality

Cultivar GBSS 4A1 5+10/ 2+12

PINa-D1

1 Good PBW550 A 5+10 b

2 Good C306 A 2+12 b

3 Good LOK1 A 2+12 b

4 Good PBW175 A 2+12 b

5 Good HI1563 A 2+12 b

6 Good K8027 A 5+10 b

7 Good HD2888 A 2+12 b

8 Good HI1500 A 2+12 b

9 Good PBW175 A 2+12 b

52

10 HI977 A 5+10 b 11 HI1563 A 2+12 b 12 HD2987 A 5+10 b 13 HD2236 A 2+12 b 14 HD2385 A 2+12 b 15 WH416 A 2+12 b 16 JWS17 A 2+12 b 17 K8434 A 2+12 b 18 HD2307 A 2+12 b 19 HD2380 A 2+12 a

20 TAWA267 A 2+12 b

53

1 Poor PBW343 P 5+10 b

2 Poor PBW621 P 2+12 b

3 Poor SONALIKA P 2+12 a

4 Poor WH291 P 2+12 b

5 Soft IITR67 P 2+12 a

6 Soft NAPHAL P A a

7 HUW629 P 5+10 b

8 UAS305 P 5+10 b

9 DHT5 P 2+12 LIGHT b

10 CPAN4202 P 2+12 b

11 UTKALIKA P 5+10 b

12 DWR39 P 2+12 b

54

• Batey et al., 1997. Cereal Chem. 74(4):497–50

3 6 9 12 15

500

1000

1500

2000

Rapid visco

analyzer’s graph

Influenced of RVA breakdown on chapatti making quality

Variety Breakdown (cP)

Final Viscosity (cP)

Set back (cP)

Peak time (min)

Pasting temp (˚C)

C306 648.7 1580.7 828.3 5.7 69.7

LOK1 752.3 1821.0 842.0 5.9 85.2

WH291 374.3 1393.0 697.7 5.4 65.8

Sonalika 504.0 1923.0 935.7 6.0 87.7

Commercial

C306

Sonalika

Lok1

WH291

High RVA breakdown in good chapatti variety (C306, Lok1)

Low RVA breakdown in poor chapatti variety (WH291)

Medium RVA breakdown in poor chapatti variety (Sonalika)

•The analysis will be repeated on the above samples and to be conducted on a larger samples to calculate correlation between RVA breakdown and good chapatti

500 bp

250 bp

668 bp

778 bp GBSSB1

GBSSB1null

F1

BC1

BC2

MAs

BC3

1st Year

2nd Year MAS

BC3F2

BC4

58

59

60