Shriram belge (exome sequencing) 27 2003

1

2

Exome sequencing in crop Exome sequencing in crop improvementimprovement

Shriram Ashru Belge

2012-11-105

Centre for Plant Biotechnology and Molecular Biology

College of Horticulture 22

3

Outline

Introduction Exome sequencing and its significance Strategies for crop improvement Advantages of exome sequencing Sequencing: Tools and Techniques Application of exome sequencing Limitations Conclusions

33

Human genome sequencing

•2003- Human genome sequenced ($ 2.7 billion and 13 years)

•2008- $1.5 million and 5 months

•At present- $10 000 in few days (Jonatan et al., 2013)

4

Introduction

Solanum tuberosum in 2011

Solanum lycopersicum in 2012

Elaeis guineensis 2013

5

Musa acuminata in 2012

6(http://www.123rf.com)

Central dogma of life

Intron

Exon

Exome and its significance

7

• Exon- sequence of DNA or RNA which code for protein synthesis

• Exome- entire protein coding region of haploid set of chromosome in an organism

8

• Whole human genome size 3 billion bp and 30,000 genes (www.genome.gov.in)

• The exome of the human genome consists 180,000 exons constituting about 1% of the total genome, or about 30 mb of DNA (Turner et al., 2009)

…Exome and its significance

Strategies for crop improvement

• Conventional breeding- almost exhausted

• MAS (Marker assisted selection)- lack of validated markers

9

10

• Transgenics- biosafety issues

• Cisgenics- lack of trait specific genes/markers

…strategies for crop improvement

11

Advantages of exome sequencing

• The majority of genetic disorder/disrupt due to changes in protein-coding sequences

• To identify the functional variation that is responsible for differential expression of desirable traits (Choi et al., 2009)

• To identify the coding region sequence variation in closely related species

Feature Whole genome sequencing

Exome sequencing

Sequence included Whole genome Only protein coding sequence

Sequence size Whole genome Smaller than the whole genome

Time Fairly long Faster due to smaller size

Cost Expensive Relatively cheaper

Assembly success rate Low due to highly repetitive sequences

High due to small size

Ease of analysis Low due to large data size High due to smaller data size

Information excluded No sequence information is excluded

All non coding regions excluded

12

Whole genome vs. exome sequencing

(Tang et al., 2010)

Sequencing: Tools and Techniques

13

I. cDNA synthesis

• Isolation of mRNA at specific/different stages

• cDNA synthesis by reverse transcriptase

14

Exome sequencing process

A. Sanger sequencing:-

• Sanger dideoxy chain termination method (Sanger and Coulson, 1975)

• Chemical sequencing method (Maxam and Gilbert in 1976-77)

B. Next-generation sequencing:-

1. Pyrosequencing

2. Reversible terminator-based sequencing

3. Sequencing by-ligation

4. Ion semiconductor-based nanoptical sequencing

5. DNA nanoball sequencing 15

… exome sequencing process

II. DNA sequencing

C. Next-next generation sequencing (Third generation):-

1. True single molecule sequencing(TSMS)

2. Single molecule, real time (SMRT) sequencing

3. Single-molecule RNAP motion-based real-time sequencing

4. Nanopore sequencing

16

… DNA sequencing

A. Sanger sequencing:-

• Oldest method and still in practice

• Four ddNTPs labelled with different fluorescent dye during amplification

• Laser based detection of the incorporated ddNTP in amplicon

• Poor quality at the initial 20-50 bases and poor size resolution at 600-1000 bases due to large DNA size

17

… exome sequencing process

18

Sanger sequencing

(Singh et al., 2012)

Tools:-

- ABI prism 3100

- ABI prism 3700

19

Sequencing-by-synthesis

Tools:-

- Illumina (HiSeq2500)

- Roche-454

(http://www.illuminaseq.com)

20

Exome sequencing process

Isolation of mRNA

cDNA strand synthesis

Fragmentation of cDNA strand synthesis

Ligation of adapters

Amplification

DNA sequencing and data analysing

Applications of Exome Sequencing

21

I. Exploring biodiversity:-

II. Investigating the natural evolution of crop:-

III. To study host-pathogen interaction:-

IV. Crop breeding:-

… application

22

I. Exploring biodiversity:-

• Morphological analysis and DNA fingerprinting not enough to identify closely related species (Hebert et al., 2003)

• Exome sequencing overcome the limitation

• Physcomitrella patens exome sequenced and annotated

• Compared with some green algae and flowering plants (Daniel et al., 2008)

23

… exploring biodiversity

(Lang et al., 2008)

II. Exome sequencing for investigating the natural evolution of crop:-

•Natural evolution is studied by utilizing morphological and molecular markers

•Exome sequencing technologies used for maize and rice domestication studies (Doebley et al., 2006)

•Exome sequencing is reported to be more efficient in studying crop evolution in African rice (Burk et al., 2007)

24

… application

• Evolutionary and geographical origins of cassava have remained unresolved and controversial

• Exome analysis for nuclear gene glyceraldehyde 3-phosphate dehydrogenase (G3pdh) acting on aldehydes

25

…evolution of cassava

26

…evolution of cassava

• Sequenced all the genotypes from different region and compared the exome

• 28 haplotypes identified among 212 individuals (424 alleles) examined

• Origin of Cassava:- conformed as Amazon basin and Brazil (Kenneth and Barbara,1999)

27

…evolution of cassava

BrazilAmazon basin

III. Exome sequencing to study host-pathogen interaction:-

•Virulence and susceptibility in the host-pathogen interaction can be altered by even single amino acid change (Carroll et al., 2011)

•Mapping entire genome of host for every modification of host-pathogen interaction is challenging task

•Hu et al., 2010 identified the genes involved in Plant-bacterial interaction through exome sequencing

28

…applications

• Evaluated Pseudomonas syringae (DC3000 strain) infection in Arabidopsis thaliana

• Cause bacterial speck disease and some changes occurred

• Type III secreted effectors (T3SEs) and other virulence-associated genes detected (Hu et al., 2010)

29

… study host-pathogen interaction

(http://www.mmg.mso.edu.com)

• Traditional breeding is not enough to improve disease resistance in high yielding varieties

• Exome sequencing help analysis of individual allele and QTL of genotypes

• Unique tool to test genetic markers in MAS

• TILLING and EcoTILLING, easier in germplasm collections for allelic variants in target genes

30

IV. Exome sequencing in crop breeding:-

31

• Some useful region of the genome is uncovered in exomic sequencing

Examples:-

- miRNA

- UTRs and

- pseudogenes etc

Limitations

1. MicroRNA (miRNAs) and other noncoding RNAs:-

• miR160, miR167 and miR171 could be responsible for the development of Arabidopsis thaliana root systems under N-starvation conditions

32

… limitations

2.3’- and 5’-UTR regions (Washida et al., 2009):-

3.In rice, glutelin production require cis-localization element for RNA transport process

4.Two located at the 5 ′ and 3 ′ ends of the coding sequences and the third is within the 3 ′ untranslated region (not covered in exome sequence) 33

...limitations

3. Pseudogenes (Ebert and Sharp, 2010):-

• Non coding region regulate the RNA coding region

• miRNA is produced from non coding region

34

…limitations

35

• Exome- part of genome which include only the coding region

• Knowing the exact coding region can help manage the crop more efficiently

• More efficient in new generation sequencing

• Less data is generated and it is easy to assemble and analyse

Conclusion

…conclusion

• Excellent and practical (cost and time) tool for crop improvement compared to whole genome

• Utilizing the omics and bioinformatics platform and integration of their outcome will serve for crop improvement

36

37