Next Generation Sequencing in

Crop Improvement

Dr.S.Uma,

Principal Scientist & Head,

Crop Improvement Division,

NRCB, Trichy

Fruit crops belong to several families of flowering plants.

Selection for valuable traits by classical breeding strategies is

complicated

- long duration crops

- Long Juvenile period

- clonal propagation

Genomic tools - dense genetic maps

- whole-genome sequences

.

FRUIT CROPS

Overall view of crop improvement

Several "omics" (genomics, transcriptomics, metabolomics)

aim to dissect biological events, with particular attention

- to fruit and flower development,

- plant-pathogen interaction,

- parthenocarpy,

- plant habits,

- dormancy break,

- post-harvest

- as other events correlated to plant management,

- plant productivity and

- fruit shelf-life.

State of plant genome sequencing

• 45 genome sequences publically available for 19 plant species

• 15 projects were underway

List of whole genome sequenced plants Sl.No. Category No. Crops

1 Cereals 4 Rice, Sorghum, Maize, Foxtail Millet

2 Legumes 3 Soybean, Pigeon pea, Common bean

3 Fibre crops 3 Cotton, Cannabis, Flax,

4 Tuber crops 2 Potato, Cassava

5 Vegetables 3 Tomato, Sugar beet, Cucumber

6 Fruits 10 Grapes, Papaya, Peach, Apple, Sweet Orange,

Clementine orange, Straw berry, Pear ,

Banana

7 Oil and Fuel crops 4 Mustard, Date Palm, Jatropa, Cater bean

8 Tree 4 Rose cum, Amborella, Cocoa, Poplar

9 Flowers 3 Monkey flower, Lotus, Columbine

10 Medicinal plants 2 Capsella, Selaginella

11 Fodder crops 2 Medicago, Eragrostis tef

12 Grasses 2 Switch grass, Brachypodium

13 Non flowering 1 Physcomitrella

14 Algae 1 Chlamydomonas

15 Model crop 1 Arabidopsis

Total 45

Fruit crops and their genome details

Fruit crop Scientific name Genome size Year of

completion

Annotated

Genes

Grapes Vitis vinifera 490 Mb 2007 26346

Papaya Carica Papaya 372 Mb 2008 28,629

Peach Prunus persica 227 Mb 2010 27,852

Apple Malus domestica 742.3 Mb 2010 57,386

Sweet orange Citrus sinensis 319 Mb 2010 25,376

Clementine orange Citrus clementina 296 Mb 2010 25,385

Straw berry Fragaria vesca ssp. vesca 240 Mb 2010 34,809

Banana Musa acuminata 613 Mb 2012 36,542

Banana Musa balbisiana 523 Mb 2013 36,638

Pear Pyrus spp. 577.3 Mb 2014 43,419

Why banana….?

• Kalpatharu’-’ Plant of virtues’.

• Form the basis of food and nutritional security

• Adaptation and acclimatization to wide range of agro-climatic zones

• Socio-economic importance

• Banana alone contributes 1.99% of the Indian GDP.

• India accounts for 17% of the world’sproduction

• As center of origin - wide Genetic diversity,

How diverse is the crop….?

Global Status of Banana

India - leader in global banana industry

But stand low in production of quality fruits

Nil with respect to export

Growth of Banana

Industry Country

Area (million ha) Production

(million tonnes)

World 5.14 106.53

India 0.796 28.45

Fusarium wilt

BBrMVPseudostem borerScarring Beetle

Bunchy Top

Why genomics for banana….?

Genome sequencing for model crops like Arabidopsis and rice - available

But limited synteny exists between banana and model crops .

Banana genome size (613Mb) is 4X of Arabidopsis (125Mb) and

30% larger than rice (466Mb).

Crop based uniqueness are – Non grassy monocot

- Tropical fruit crop

- Polyploidy ( 2x, 3x, 4x)

- Parthenocarpy

Diploid (2X) Triploid (3X) Tetraploid (4X) Pentaploid (5X)

Polyploidy

Non grassy monocot Parthenocarpy

Apart from these,

Good model crop for genetic studies

- as it is one of the few plant species with bi- parental

cytoplasmic inheritance

-one of the few crops where virus has integrated in the

host genome ( Banana streak virus)

Domestication

- Human interventions

Super Domestication

- Human + Biotechnology

X

M.acuminata diploid - AA M.balbisiana diploid - BB

Banana A Genome Sequencing

36,542 protein-coding genes

Combining Sanger, Roche 454 and Illumina – used DH plants

-M.Acuminata type Pahang was sequenced.

-Almost 90% of the genome (523Mb) has been sequenced.

-From this, 36,542 protein coding genes identified

- slightly higher than human genome

-Interestingly, among the sequenced plants – has highest no. of

Regulatory genes (TFs) have been identified.

Banana B Genome Sequencing(Pisang Klutuk Wulung)

Re-sequencing project - collaboration between-Lab. of Fruit Breeding and Biotechnology, Department of Biosystems, Katholieke

Universiteit Leuven, Belgium

- The Centre for Research in Biotechnology for Agriculture and the Institute of

Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur,

Malaysia.

Published by Davey et al.

BMC Genomics 2013, 14:683

Illumina Hi Seq 2000 II used

BGI, China

B-genome is 79% the size of the A-genome

36,638 predicted functional genes

Sequences which are nearly identical to the 36,542 (87%) of the

A-genome.

Valuable – B genome harbours diverse resistant genes for many

Biotic and abiotic stresses affecting banana, especially Black

streak, xanthomonas wilt and cold and drought tolerance.

It provides wide therapeutic and non-ediblle applications.

Decoding B-genome will halp in trapping trait specific genes,

regulatory genes which may not be in AA genome.

Comparison B & A Genome Sequencing

Genes for specific traits - Parthenocarphy, resistant genes etc.

Regulatory genes – Silencing the genes

Comparison of A & B Genome

Markers

Among A and B genomes,

more no. of repeats identified

in A genome.

But the SSR’s (bp’s and

kbp’s) higher in B genome.

These markers are already

being exploited For finger

printing and QTL

development.

Isolation of novel genes, promoters, miRNA etc

miRNA

Inspite of smaller genome size,

B genome contains more

repetitive elements and

more known and novel miRNA’s

but fewer conserved miRNA families

than the A genome.

High no. of mi RNA loci here could be

related to higher no. of transposable

Elements present.

These are thought to have contributed for the generation of species

Specific miRNA genes.

Several B specific miRNA predicted targets are proteins with

unknown function and appear to be present only in B genome

representing B genome distinct functional networks.

= miRNAa = small, single-stranded RNA molecules (~21-mer) that bind to complementary of one or more mRNAs (often transcription factor mRNAs)

Regulation Of Transcript Abundance By MicroRNAs

Mature miRNA within RNA-induced silencing complex (RISC)miRNA – RISC complex

binding to complementary mRNA

miRNAs either degrade or impair the translation of their target mRNAs!

Taken from http://www.ambion.com/techlib/resources/miRNA/index.html

Importance of transcriptome sequencing

WGS provides overall information about the genome

But transcriptome sequencing is essential as it provides info. On

- Gene expression under specific physiological status

(specific tissues, developmental/ disease challenge etc.)

- As it analyses only transcribed portion of the genome

- Thus helps to interpret the functional elements of the

genome.

Variety/cultivar Techniques Tissues Stage/stress No. of

expresse

d genes

Reference Laboratory

M. acuminata,

subgroup Caven- dish

‘Grand Nain’

cDNA

AFLP

Fruit Crown rot

disease

10 Lassois et al.

2011

University of Liege,

Gembloux,

Belgium

M. acuminata ssp.

burmannicoides

‘Calcutta-4,

cv. Rose, FHIA 17,

Williams’

cDNA

AFLP

Root Fusarium 76 Munro 2008 University of

Pretoria,

Pretoria, South Africa

M. acuminata ssp.

burmannicoides

‘Calcutta-4’

SSH Root Fusarium 83 Swarupa et al.

2013

IIHR, Bangalore

India

M. acuminata cv.

Manoranjitham

(AAA)

SSH Leaf Eumusae

leaf spot

805 Uma et

al.2011

NRCB, Trichy,

India

M. acuminata cv.

Karthobiumtham

(ABB)

SSH Root Nematode 256 Backiyarani et

al. 2014

NRCB, Trichy,

India

M. acuminata cv. Tuu

Gia

SAGE Leaf 5292 Coemans et

al.2005

KUL, Belgium

M. acuminata ssp.

burmannicoides

‘Calcutta-4’

EST

Sequencing

Hot and

cold stress

Fruit

ripening

220 Santos et al.

2005

EMBRAPA, Brazil

Status of EST sequencing in Banana - pre NGS Era

Variety/cultivar Techniques Tissues Stage/stress No. of

expresse

d genes

Reference Laboratory

M. acuminata ‘

Williams'

Combina- tion of

SSH and microarray

FruitFusarium

79 Van den berg

et al. 2007FABI, University of Pretoria,

SouthAfrica

M. acuminata,

cv. Grand Nain

Combination of SSH

and microarray

Fruit Early ripening of

banana

84 Man- rique-

Trujillo et al.

2007

Centro

de Investigación y de

Estudio- sAvanzados del

IPN, México

M. acuminata AAA

group

Combination of SSH

and microarray

Fruit Early ripening of

banana

265 Xu et al. 2007Institute of Tropical

Bioscience and

Biotechnology, Chinese

Academy of Tropical

Agricultural Sciences,

China

M. acuminata L.

AAA group

Combination of SSH

and microarray

Fruit Ethylene

biosynthesis

initiation in fruits.

22

Jin et al. 2009 Institute of Tropical

Bioscience and

Biotechnology, Chinese

Academy of Tropical

Agricultural Sciences,

China

M. acuminata,

Dwarf Cavandish

( AAA)

Combination of SSH

and microarray

Fruit Ethylene

biosynthesis

initiation in fruits

37 Kesari et al.

2007

NBRI, Lucknow, India

Exploitation of NGS in banana for various stresses

Variety/cultivar Techniques Tissues Stage/stres

s

No. of

expresse

d genes

Reference Laboratory

M. acuminata cv.

Manoranjitham (AAA)

Illumina

Sequencing

Leaf Eumusae

leaf spot

6195 Uma et al.

2014

NRCB, Trichy

M. acuminata, cv.

Grand Naine

Illumina

Sequencing

Leaf Eumusae

leaf spot

6212 Uma et al.

2014

NRCB, Trichy

M. acuminata cv.

Karthobiumtham

(ABB)

Illumina

Sequencing

Leaf Nematode 5736 Uma et al.

2014

NRCB, Trichy

M. acuminata cv.

Nendran

Illumina

Sequencing

Leaf Nematode 6521 Uma et al.

2014

NRCB, Trichy

M. acuminata cv. Saba Illumina

Sequencing

Leaf Drought 2546 Uma et al.

2014

NRCB, Trichy

M. balbisiana

cv. Attikol

Ion Torrent

Sequencing

Whole

Parts

Wild 3301 Uma et al.

2014

NRCB, Trichy

M. acuminata Illumina

Sequencing

- - - Ravishankar

et al. 2013

IIHR, Trichy

M. Acuminata

‘

Illumina

Sequencing

Roots Fusarium 3331 Ting-Ting

Bai et al.

2013.

SAU,Guangdong,

China

Variety/cultivar Techniqu

es

Tissue

s

Stage/stress No. of

expresse

d genes

Reference Laboratory

M. acuminata

Calcutta 4 and

Cavendish Grande

Naine

454 GS-

FLX

Titanium

technolog

y

Leaf Mycosphaerella

musicola

10,645 Passos et al.

2013

Instituto de Ciências

Biológicas, Brazil

M. acuminata

subgroup Cavendish

(AAA)

Illumina

Sequenci

ng

Root Fusarium 842 Chunqiang

Li et al. 2013

Chinese Academy of

Tropical

Agricultural

Sciences, Haikou,

China.

M. acuminata

subgroup Cavendish

(AAA)

Illumina

Sequenci

ng

Root Fusarium 5008 Chunqiang

Li et al. 2012

Guangdong

Academy of

Agricultural

Sciences,

Guangzhou 510640,

China

Musa acuminata . AAA

group,

‘Brazilian’

Illumina

Sequenci

ng

Root Fusarium 2,825 Zhuo Wang

et al. 2012

Chinese Academy of

Tropical

Agricultural

Sciences, Hainan

571101, China

Pre NGS Era - From 2005-14,

Techniques - SSH, RDA, cDNA AFLP, SAGE, DNA microarray etc.

only 12 EST- sequencing completed with an average of 600 genes.

-Information was limited and not sufficient to understand

the Host- stress interaction.

- It also limited the prediction of level of gene expression

as it needs the prior knowledge of genome sequence.

NGS Era- from 2012-14

- 12 transcriptome sequencing completed within 2 years which

led to the annotation with an average of 30,000 genes.

This is the magnitude of importance of NGS.

Musa B genome whole transcriptome

Assembled using a MIRA tool

Generated 82,413 contigs

Functionally Annotated –

BLAST2GO- SWISSPROT, TrEMBL,

COG and PlantCYC databases.

35,783 protein coding genes

1,93,826 Gene ontology terms

predicted

cv. Attikol, wild Indigenous, unique accession, donor source

Musa B genome whole transcriptome

Annotated results

Max. no transcripts were hit with

Arabidopsis gene followed by rice

Functional classification through COG indictaed

Most of the transcripts have the function of

- Post translational modification

- Protein turnover and chaperones,

-Followed by signal transduction mechanism and intre

And intracellular trafficking and vesicular

transport

-Total of 3,301 defense related genes detected

Max. no. of transcripts were involved in

-PAMP followed by

Musa B genome whole transcriptome

Total of 3,301 defense related genes

detected

Max. no. of transcripts were involved in

-PAMP followed by

-Plant hormone biosynthesis

- genes involved in cell wall modifications

SSR markers identified from this

genome has been tested in various

germplasm accessions and it clarely

distinguish the A genome B genome and

wild speccies.

Musa transcriptome for different stresses

Sigatoka

Transcriptome

Nematode

Transcriptome

Drought

Transcriptome

Fusarium wilt

Transcriptome (IIHR)

Challenged Unchallenged

Resistant C-R U-R

Susceptible C-S U-S

For each stress four different c-DNA libraries- constructed and sequenced through Illumina Platform.

Based on digital gene expression studies, unique genes expressed only In resistant cultivar under challenged conditions - identified.

Commonly expressed defense related genes expressed in all stress Conditions in respective resistant cultivars were also identified.

60.00%

62.00%

64.00%

66.00%

68.00%

70.00%

72.00%

74.00%

76.00%

78.00%

UR CR US CS

Venn diagram for genes significantly

expressed (>2 fold) in challenged and

unchallenged resistant and susceptible

cultivars

Percentage of genes expressed in challenged

and unchallenged resistant and susceptible

cultivars

Significantly expressed genes in resistant

and susceptible cultivarsTotal genes expressed in resistant and

susceptible cultivars

Heat map for significantly expressed

challenged and unchallenged resistant

and susceptible cultivars

Another tool to quick identification of resistant genes having more no. of transcripts

Allows us to speculate the possible pathways involved

in resistance mechanism/s

Schematic representation of nematode (P. coffeae)

resistant mechanism /s in banana

SSRs and SNPs developed from various Transcriptome of

Musa cultivars

Libraries

M. Eumusae Nematode ( P. coffeae) Water deficit stress

SSR SNP SSR SNP SSRSNP

Unchallenged Resistant

4825 83 11650 432 9692 451

Challenged Resistant

9153 355 3408 287 8128 351

Unchallenged Susceptible

5008 79 8250 143 8478 92

Challenged Susceptible

9239 271 7512 305 3775 334

Total 28225 788 30820 1167 30073 1228

6 transcriptome data available with us facilitated the identification of SSR and

SNP markers across R and S cvs.

In-silico polymorphic markers predicted are being validated for MAS

Bioinformatics

Databases and software developments

Genetic resources (MGIS),

Genetic data (TropGENE DB) (INIBAP, CIRAD)

EMBRAPA).

Genomic data (data_Musa)

Development of Biomoby services to improve

Interoperability and data integration of databases

Development of software tools, Genoma, for storage,

annotation and analysis of EST

Development of a web-based infrastructure for the

management of the genomics resources

Development of a web-based infrastructure based

on GBrowse to visualize sequence annotations

(BIOVERSITY)

(BIOVERSITY)

(EMBARAPA)

(CIRAD, INIBAP)

Banana Marker Database Development

(SSR)

Markers identified have been linked to their respective metabolic pathwayswhich shall narrow down the selection of primers based on our objective.

• - To develop trait specific markers for use in Marker Assisted

• breeding

• - Development of QTLs in banana

• - Can also be used for developing Musa DNA chip

Database developed is being used

- to develop High resolution map for A and B genome

With Transcriptome data obtained through NGS,

We have gained knowledge on

- gene sequence information- novel/unigenes

- role of regulatory elements- kinases, TFs

- cultivar specific gene expression

- stress specific gene expression

- highly regulated pathway

- change in metabolic activities- enzymes

- sub cellular level gene expression- e.g. mitochondria

- most influenced organ

- genes not affected by stress

- common gene expression or cascade pattern for a particular

stress

- development of stress specific marker /s etc.