Reaping the benefits of

genome sequence and re-

sequence information for

chickpea improvement

Mahendar Thudi

The IYP 2016 aims to

heighten public awareness of

the nutritional benefits of

pulses as part of sustainable

food production aimed

towards food security and

nutrition

Chickpea is an

important pulse

crop for food and

nutritional security

The Chickpea crop

Second most important food legume globally

Genome size= ~740 Mbp

Grown on about 13.54 m ha

across 59 countries with

13.10 m tons of total production

Marginal environments in

Sub-Saharan Africa and Asia

Kenya Ethiopia

India Myanmar

Production constraints

-1500

-1000

-500

0

500

1000

1500

2000

2500

3000

Tota

l pro

duct

ion

Dro

ught

Low te

mper

ature

Wilt

Asc

och

yta blig

ht

Pod

bore

r

Millio

n (

US

$)

To

tal p

rod

uc

tio

n

Dro

ug

ht

Sa

lin

ity

Wil

t

Asco

ch

yta

bli

gh

t

Po

d b

ore

r

Abiotic stresses

Terminal drought Salinity

Wilt Pod borer Blight

Biotic stresses

Botrytis grey mould

Breeding

Translational genomics in

agriculture (TGA) Genomics

(incl. informatics)

Genetics -logy disciplines Varshney et al. 2015 Crit Rev Plant Sci

How can we use

genomics to overcome

the problems of

international agriculture?

Varshney et al. 2014 PLoS Biology

Samples:

- 5 wild species

- 25 landraces

- 60 breeding lines

Or

- 5 wild species

- 57 Desi genotypes

- 28 Kabuli genotypes

Illumina sequencing used to generate

153.01 Gb

73.8% of the genome is captured in

scaffolds

Genome analysis predicted 28,269 genes

High levels of synteny observed between

chickpea and Medicago

> 81,845 SSRs and 4.4 million variants

(SNPs and INDELs)

Genome sequence and re-sequence

Varshney et al. 2013

Nature Biotechnology

Cross Segregating trait

ICC 4958 × ICC 1882 Drought

ICC 283 × ICC 8261 Drought

ICC 6263 × ICC 1431;

ICCV 2 × JG 11

Salinity

WR 315 × C104 Fusarium wilt (FW)

ICCV 2 × ICC 1496;

ICCV 10 × ICC 1496

Botrytis grey mould

(BGM)

ICC 506-EB × Vijay;

ICC 3137 × IG 72953;

ICC 3137 × IG 72933

Helicoverpa

ICCV 2 × JG 62 FW, BGM, Helicoverpa

and salinity

JG 62 × ICCV 05530;

Pb 7 × ICCV 04516

Ascochyta blight

ICC 995 × ICC 5912 Protein content

35 parental lines of

mapping populations

Desi 74%

Kabuli 17%

Wild [PERCENTAGE]

Segregating for abiotic, biotic and

nutritionally important traits were targeted

Drought

Salinity

Ascochyta blight

Helicoverpa

Fusarium

Botrytis grey Pod borer

35 select inbred lines sequenced

using Hi Seq 2500

3.41 trillion base pair data

916.21 million 150 bp reads

Avg. sequencing depth of 8.6X

916 million clean reads

92.19% reads with 82.18%

average coverage from each

genotype were aligned to the

reference genome CDC Frontier

Illumina

sequencing

Raw

Reads

Clean

Reads

Quality

filtering Discarded

Failed

Mapping reads to

reference genome

using SOAP

Structural

variations

Outline of data analysis

Data generation and analysis

Indels in 35 inbred lines

The maximum and minimum indel ratios were 1.03 and

0.81 in case of JAKI 9218 and ILC 3279 respectively

Identified 26 candidate genes

Jaganathan et al. 2015

Two

codon

insertions

in gene

Ca_04570

related to

7s seed

storage

gene

family

Codon insertions in genes

Structural variations

Type of

structural

variation

Pseudomolecules Total

Ca1 Ca2 Ca3 Ca4 Ca5 Ca6 Ca7 Ca8

CTX 4 2 2 4 9 10 30 6 67

DEL 92 58 42 46 75 65 53 10 441

INS 22 1 12 14 73 26 30 23 201

INV 0 1 0 2 0 8 2 0 13

ITX 0 11 2 4 4 2 4 3 30

Total 118 73 58 70 161 111 119 42 752

*CTX - inter chromosomal translocations, DEL - deletions, INS- insertions,

INV- inversions and ITX-intra chromosomal translocations,

Ca5 had large number of structural variations

large deletion

Circos diagrams were plotted based on line specific variations like SNPs, indels,

deletions and duplications using Circos software. The outer circle represents line

specific SNPs followed by line specific InDels, deletions and duplications. Inside

each circle four lines with distinct colors show genotypes (PI 489777, ICC 3137,

IG 72953 and IG 72933).

Genome wide line specific variations

PI 489777, ICC 3137, IG

72953 and IG 72933

segregate for Helicoverpa

resistance

Maximum number of line

specific variations, 78,320,

were observed in PI

489777 (68,799 SNPs and

9,521 indels), and followed

by IG 72953 (55,393 SNPs

and 7,415 indels).

Thudi et al. 2016, BMC Plant Biology

- Representing 10 countries –

India, Canada, Australia,

Ethiopia, Bangladesh,

Kenya, Myanmar, Sudan,

Burlgaria, Spain and

ICARDA;

- Based on year of release

a. before 1993

b. 1993-2002

c. 2003-2012

129 release varieties

Genome wide variation -129 lines SNPs Indels CNVs PAVs SVs

All_129 Genotypes 1,378,790 151,440 3,822 24,593 24,126

Market type (129 Genotypes) 1,378,790 151,440 3,822 24,593 24,126

Desi (88 genotypes) 1,323,116 144,046 2,954 22,819 22,398

Kabuli (41 genotypes) 1,150,365 103,036 3,273 20,818 20,406

Year wise ( 124 genotypes) 1,374,090 150,246 3,811 24,078 23,652

Before 1993 (38 genotypes) 1,172,636 123,549 2,315 19,856 19,482

1993-2002 (40 genotypes) 1,194,990 103,514 2,318 20,331 19,967

2003-2012 (46 genotypes) 1,242,979 112,370 3,511 21,137 20,776

Varieties released between 2003-2012 are more diverse

SNPs, indels, CNVs, PAVs and SVs were high on Ca4

CNV-copy number variations; PAVs- presence absence variations SVs-structural variations

Population structure and genetic

diversity

More kabuli varieties

developed after 1993

Increase in diversity in

release varieties after

2002

Chickpea reference set

(300 lines from 35 countries)

Genome wide association for drought

and heat tolerance

Reference set phenotyped

1–6 seasons

1–3 locations in India

(Patancheru, Kanpur,

Bangalore)

three locations in Africa

(Nairobi, Egerton in Kenya

and Debre Zeit in Ethiopia)

Whole genome re-sequencing

provided – 4 million SNP

Both generalized linear model (GLM) & mixed linear model

(MLM) were used for identifying markers associated with

traits of interest.

Root related traits

Root traits

Traits Seasons Root length (cm) 3

Root length density (cm cm-3

) 3

Root volume (cm3) 3

Root dry weight (g) 3

Rooting depth (cm) 3

Root surface area 3

R-T ratio(%) 3

Shoot dry weight (g) 3

Stem dry weight (g) 3

Leaf dry weight (g) 3

Projected area 2

Average diameter 2

Experiment of chickpea

root growth in ROS

Root length screening

Agronomic traits

Field phenotyping under rainfed

and irrigated environments

Heat tolerance phenotyping

Traits Seasons Traits Seasons

Morphological traits Yield related traits

Plant height (cm) 14 Pods/plant 2

Plant width (cm) 7 100 SDW (g) 10

Plant stand 7 Yield (g/m2) 3

Apical primary

branch

7 Yield (Kg/ha) 10

Apical secondary

branch

7 Yield per plant 7

Basal primay

branch

7 Production 7

Basal secondary

branch

7 Biomass 6

Teritiary branches 7 Biomass/plant 2

Phenological traits Harvest index 6

Days to flowering 13 TDM weight (g/m2) 2

Days to maturity 9 Transpiration efficiency Seeds per pod 7

13C 2

Seeds/plant 2 SPAD 2

Trait Number

of MTAs P-value PVE (%)

Root length density

(RLD, cm cm-3) 3 5.73 × 10-6 - 2.1 × 10-8 6.5 - 16.6

Root dry weight (RDW, g plant-1) 11 6.81 × 10-6 - 9.18 × 10-10 5.58 - 10.49

Root surface area (RSA, cm2

plant-1) 6 9.17 × 10-6 - 1.65 × 10-7 5.9 - 10.12

Root volume (RV, cm3 plant-1) 13 7.28 × 10-6 - 1.43 × 10-7 5.77 - 10.41

Days to 50% flowering (DF) 24 8.1 × 10-6 - 7.8 × 10-9 9.09- 20.36

Days to maturity (DM) 48 9.06 × 10-6 - 4.82 × 10-8 8.96 -21.29

100 seed weight (100SDW, g) 98 1.07 × 10-6 - 2.89 × 10-22 10.34 - 14.4

Yield (YLD, Kg/ha) 22 9.42 × 10-6 - 2.77 × 10-7 7.16 - 18.6

Biomass (BM, g) 8 1.6 × 10-6 - 6.35 × 10-8 6.29 -12.02

Harvest index (HI, %) 15 8.87 × 10-6 - 1.46 × 10-8 5.97-14.84

Delta Carbon ratio (δ13C) 1 6.02 × 10-7 20.7

249 MTAs for drought tolerance

related traits

Trait

Numb

er of

MTAs

P-value PVE (%)

Days to 50% flowering (DF) 10 3.69 × 10-6 - 1.54 ×

10-8 9.64 - 18.26

Pods per plant (PPP) 11 1.02 × 10-6 - 1.17 ×

10-8 9.19 - 17.42

100 seed weight (100SDW,

g) 16

1.07 × 10-6 - 2.89 ×

10-22 10.34 - 14.4

Yield (YLD, g) 16 2.03 × 10-6 - 7.31 ×

10-9 10.96 - 21.56

Harvest index (HI, %) 14 2.55 × 10-6 - 2.77 ×

10-8 6.94 - 14.79

Heat tolerance index (HTI) 17 8.58 × 10-6 - 7.75 ×

10-7 7.45 - 14.11

84 MTAs for heat tolerance

related traits

GWAS signals 100 seed weight

Time for coffee

GWAS signals for Days to 50%

flowering

24 significant MTAs under drought

GWAS signals root length density

Ca_09763

Ca1 Ca2 Ca3 Ca4 Ca5 Ca6 Ca7 Ca8

SNP locus Ca4_37195367 on Ca4 associated with RLD explained 16.67% PVE

Ca_09763 gene associated with correlated traits like

RSA and RV encoded for protein Aspartic protease

Root proteases are shown to play a key role in nitrogen

acquisition and drought tolerance

GWAS signals biomass

8 significant MTAs for biomass (BM), of which 6 loci were on Ca5 and one each on

Ca1 and Ca4.

two loci were in candidate genes Ca_8986 and Ca_8946 that code for key disease

resistance genes in plants (CC-NBS-LRR gene) and potassium ion transport.

Four SNP loci (Ca5_26316428, Ca5_26343334, Ca5_26421763 and

Ca5_26788879) on Ca5 were found associated with BM and HI under drought

stress.

Ca1 Ca2 Ca4 Ca5 Ca7 Ca8 Ca3 Ca6

On an average the sequencing depth on 8 parental lines was 48X and 96% of the reads generated were aligned

On an average 1135 MAGIC lines were sequenced at 7X and 96% of the reads generated were aligned

51.69 billion clean reads

(90 bp) containing 4.65

TB clean data was

generated

Whole genome re-sequencing (WGRS) of MAGIC lines

0

50,000

1,00,000

1,50,000

2,00,000

2,50,000

Ca1 Ca2 Ca3 Ca 4 Ca 5 Ca 6 Ca 7 Ca 8

Deletions Insertions

Total SNPs in genes synonymous SNPs

non-synonymous SNPs Total SNPs

Total variant count

Genotype Total

SNPs Line

specific

SNPs

Deletions Line

specific

deletions

Insertions Line

specific

insertions

Genes

deleted

Genes

duplicated

ICC 4958 340,803 3 29976 1 28429 0 23 47

ICCV 00108 125,680 49 9217 6 8907 2 30 323

ICCV 10 174,644 219 14137 20 13534 22 14 21

ICCV 97105 261,406 120 24016 6 24274 13 18 42

JAKI 9218 315,032 1 30320 0 31339 0 8 1120

JG 11 207,785 496 17690 36 17640 31 21 25

JG 130 166,662 13 12936 2 12342 3 25 17

JG 16 147,615 95 10579 10 10094 6 27 73

Genome-wide variations in parents of

MAGIC population

Deletion, insertions and duplications were high in case

of JAKI 9218

Summary Resequencing 90 elite lines provided thousand of SSRs

and SNPs

Resequencing of parental lines provided genome wide

variations for trait mapping

Resequencing of 129 lines provided insights into spacial

and temporal trends in diversity

Resequencing of reference set provided insights into

genetic diversity and population structure

Diversity existing the germplasm lines including MAGIC

population can be deployed for chickpea improvement

Acknowledgements ICRISAT

Rajeev K Varshney

Manish Roorkiwal

Anu Chitikineni

Abhishek Rathore

Dadakhalandar Doddamani

Aamir W Khan

Pooran Gaur

Hari Upadhayaya

BGI

Weiming He

Jianbo Jian

Gengyun Zhang

Jun Wang

ICAR

Narendra P Singh

SK Chaturvedi

Swapan K Datta

Funding Agencies AISRF

GCP

Thanks for your attention