The breeding for the development of Climate Resilient Chickpea is a critical initiative aimed at enhancing the productivity and adaptability of chickpea genotypes under challenging environmental conditions. Chickpea, a vital pulse crop globally, faces yield limitations due to the combined impact of heat, cold, drought, and salinity stresses. The average yields, currently far below the potential, necessitate the development of highly productive and resilient chickpea cultivars. Traditional breeding methods and modern genomic resources, including molecular markers, genetic maps, and QTL identification, have been instrumental in enhancing grain yields and stress adaptation. Marker-assisted backcrossing has successfully produced cultivars like Pusa Manav, demonstrating the effectiveness of genomic technologies. Additionally, the adoption of gene-editing technologies, such as CRISPR-Cas9, holds promise in accelerating genetic gain for stress-related traits.

1. GP 692: 1(1+0)
DOCTORAL SEMINAR II
TOPIC :
Breeding for Development of Climate Resilient
Chickpea
K. Modunshim Maring
Roll No.: 2003305002
Reg. No. : D/PBG/337/2020-21
PhD Scholar(4th Semester)
Department of Genetics & Plant Breeding
DRPCAU, Pusa
Seminar In-Charge :
Dr. Sweta Mishra
Dr. M. K. Singh
Dr. Ravikant
Department of Genetics & Plant Breeding
DRPCAU, Pusa
1

2. Introduction
2
 Scientific name – Cicer arietinum L.
 It is a self-pollinated, annual legume crop with high protein content cultivated in arid and
semi-arid areas.
 It is variously known as Gram or Bengal gram, Garbanzo or Egyptian pea.
 As per the data of 2020,
 Area of production - 14.84 Mha
 Production - 15.08 Mt
 Productivity – 1.01 t/ha.
 India is the single largest producer of chickpea in the world (65% of world production)
followed by Australia.
 In India,
 Largest Area and Production - Madhya Pradesh
 Productivity- Andhra Pradesh

3. Impact of abiotic stresses
3
 Drought at terminal stage accounts for 50% of yield losses globally.
 Yield loss was estimated to be between 10% and 15% for every 1oC above the optimum growth
temperature (10 - 30oC).
 Exposure to heat stress (>35 C) during reproductive development drastically reduced the yields,
resulting in yield losses of up to 39% due to male reproductive tissue (pollen and anther) functions
being negatively affected. (Devasirvatham et. al., 2012).
 Chickpea also suffers from cold stress when it faces chilling (3-8oC) that result in arresting the
germination process. It causes severe membrane damage due to cellular dehydration associated with
freezing during cold stress.
 Soil salinity leads to osmotic stress and ion toxicity that results in ionic imbalance.
 To overcome these issues, the new chickpea cultivars must be climate change resilient and should be
widely adaptable to a range of environments to maintain food security in near future.
Upadhyaya et.al., 2011

4. Approaches for chickpea breeding
4
BREEDING
APPROACHES
Modern(Omics)
Transgenomics
&
Genome Editing
Metabolomics
Proteomics
Transcriptomics
Genomics
Conventional

5. Conventional approaches
5
 For traits related to abiotic stress tolerance, such as root system architecture, stomatal conductance,
and canopy temperature, moderate to low heritability has been estimated. In fact, it is widely
known that the traits related to yield components and abiotic tolerance are complex and involve
numerous genes associated with their regulation.
 Chickpea breeding through conventional
approaches includes plant introduction,
hybridization (recombination), and mutation.
 These approaches allows allow the
incorporation of genes/alleles associated
with yield components and tolerance to
abiotic stress into cultivars of chickpea. Wild Cicer species as sources of alleles to abiotic stress tolerance

6. Introgression of genes for cold stress through
6
 Four crosses, “ILC 482 (C. arietinum) x ILWC 179 (C. echinospermum)” and “ILC x 48
ILWC 124 (C. reticulatum)” and their reciprocals, were made. Pedigree selection was used
to advance the material.
 ILC 482 - partially resistant to
fusarium wilt and cold,
high yielding and wide adapted
 ILWC 124 - is resistant to
fusarium wilt and cold,
 ILWC 179 - resistant to fusarium wilt,
seed beetle and cold.
 In F4, nine lines produced higher seed yield than cultigen (ILC 482). One line (No. 55),
which produced a 39% higher yield than the cultigen, was significantly different.
Singh and Ocampo, 1996

7. MAGIC Populations
7
 MAGIC – Multiparent Advanced Generation Intercross.
 The genetic diversity of multiple parents, recombined over several generations,
generates a genetic resource population with large phenotypic diversity.
 Multi-parent Advanced Generation InterCrosses (MAGIC) inter-mate multiple
inbred founders for several generations prior to creating inbred lines, resulting in a
diverse population whose genomes are fine-scale mosaics of contributions from all
founders.
 MAGIC population requires greater initial investment in capability and time.
 Careful selection of founders allows its generalizability to wider breeding population
and ensures revelance as a long-term genetic resource panel.

8. Breeding Scheme for MAGIC Population Development
8
Samineni et al., 2021
AT F8, 1136 magic
lines were
phenotyped under
ranifed, irrigated
and heat stress
conditions. 61
MAGIC lines under
HS show higher
yield than best
parent. Several
genotypes showed
higher grain yield
than best check
(JG-14) under heat
stress were
identified.

9. Mutagenesis Approaches
9
 Mutagenesis approaches is useful when the crops
 Has a narrow genetic diversity
 A small flowers
 Are very difficult to hybridize
 The developed mutants can be released directly as
cultivars, or they can be used as donors to improve a
specific trait.
 Mutagenesis has enabled diversification of the genetic
variability of chickpea with the aim of increasing yield
and tolerance to a wide range of stresses.

10. Types of Mutagens
10

11. Mutagenesis for resistance to abiotic stresses
11

12. Mean survival percentage of chickpea genotypes, in 4 different doses
of radiation
12
The effect of gamma radiation for freezing tolerance in chickpea genotypes. It was observed that
the maximum survival was in MCC741 genotype in 180 Gy dose, MCC477 genotype in 140 Gy
dose and MCC495 genotype in 180 Gy dose. Increased survival of these genotypes to more than 50
percent shows the tolerance increase in them towards freezing stress.
Akbar et. al., 2011
(Genotypes)
(Dose of
Radiation)
Albino plant after
Gamma radiation

13. Mutant cultivars of chickpea approved by the Joint FAO
13

14. Constraints in Conventional Approaches
14
 Natural genetic variability is key to responding to the effects of
climate change. But the bottlenecks events that is occurring
during domestication, narrow its genetic base. Hence, narrow
genetic base makes it difficult for breeders to produce new
elite chickpea cultivars with long-lasting tolerance to major
abiotic stresses.
 The frequency of desirable mutation is very low (0.1% of total mutation). So,
the breeder has to screen a large population to select a desirable mutation.
 Conventional approaches is time-consuming and hence it cannot ensure the for
security for the growing population.

15. Modern breeding approaches
 While the conventional approaches are appropriate for highly heritable and easy-to-
score traits, but mostly traits associated with tolerance to abiotic stresses and yield
are multigenic an have low heritability and are highly influenced by the environment.
15
 Conventional breeding does not guarantee the
requirement of food security of the growing population
under extreme environment due to climate change.
 With respect to these, modern (omics) breeding
approaches can be used as a complement to increase
selection effficiency, reduce breeding time, and identify
the specific genes that control the desired traits.

16. Genomics in Chickpea
16
 Genomics includes the development of molecular markers, which allow us
to analyse genetic diversity, develop genetic maps, and identify regions of
the genome associate with desirable traits in crops.
 The development of markers such as RAPD, SSR and SNP, etc, has allowed
the construction of several genetic maps, including high-density genetic
maps in chickpea.
 The use of genomics tools in breeding programs has allowed the
development of new chickpea cultivars through approaches such as marker-
assisted backcrossing (MABC), marker-assisted recurrent selection
(MARS), and genomic selection (GS), with the MABC approach
extensively used to develop improved cultivars of chickpea with higher
yield and tolerance to abiotic stresses in recent years.

17. List of some major QTLs associated with traits related to
abiotic tolerance in chickpea
17
PH-Plant Height, PV-Plant Vigor, GY-Grain Yield, CC-Chlorophyll Content, MSI-Membrane Stability Index, SS-Seed
Size, CT-Cold Tolerance, VER-Vernalization

18. SSR Markers
18
A SSR locus. Each repeat
unit, represented as
big dot, has a shot
sequence, e.g. AC.
The unique sequence
flanking the SSR locus
are used as primers for
PCR amplification. B.
SSR alleles differ in
the number of copies
of the repeating unit.
The PCR products
form different alleles
differ in length an are
detected by gel
electrophoresis.

19. Genetic linkage map of chickpea
19
Identification of 15
genomic regions
significantly
associated with
traits affecting
drought tolerance
in an RIL
population derived
from the cross
between ILC 588
(drought tolerant)
and ILC 3279
(susceptible)
genotypes of
chickpea.
Rehman et al., 2011

20. Genetic linkage map of chickpea
20
Rehman et al., 2011

21. Development of chickpea cultivars using MABC
21
 MABC allows one to incorporate desirable major genes/QTLs from a donor
parent into an elite cultivar or recurrent parent. In this context, the “QTL-
hotspot” region has been introgressed into several chickpea cultivars through the
MABC method.
 Ethiopia became the first country to release a climate-resilient chickpea variety,
Geletu developed using marker-assisted backcrossing efforts. (Parents : ICC
4958× JG 11 )
 Pusa chickpea 10216 is the first molecular breeding variety with enhanced
drought tolerance released in India. (Parents : ICC 4958 x Pusa 372).

22. Transcriptomics in Chickpea
22
 RNA-seq is the preferred method and has been the dominant transcriptomics
technique since 2010.
 Single-cell transcriptomics allows tracking of transcript changes over time within
individual cells.
 The transcriptome is the set of all RNA,
including coding and non-coding in a
population of cells.
 Two biological techniques are used to study
the transcriptome, namely DNA microarray,
a hybridization-based technique and RNA-
seq, a sequence-based approach.

23. DNA Microarray
23
 Microarray provides a high-throughput means to study the gene expressions of
thousands of transcripts simultaneously.
 The availability of transcriptome sequence in public domain has provided the
opportunity to develop a high-throughput resource for studying the expression of all
the transcripts in different biological contexts.
Steps in DNA Microarray

24. cDNA microarray approach to study impact of drought, cold
and salinity stresses in chickpea genotypes
24
The figure shows that the level of several transcripts
was altered by more than one of the stresses assessed,
which may indicate gene interaction pathways among
the biological responses involved in these stress
reactions. The number of differentially expressed (DE)
transcripts affected in response to high salinity was
much higher than those affected in response to cold and
drought stress in all genotypes.
Mantri et al., 2007
Figure : Relationship between the number of DE transcripts in
amongst the three abiotic stress treatments.
Issues :
• Poor quantification of lowly and highly expressed genes
• Needing to know a sequence.

25. RNA-Seq
25
 RNA sequencing is
a technique which
uses next-generation
sequencing (NGS)
to reveal the
presence and
quantify the RNA in
a sample at a given
moment, analyzing
the continously
changing cellular
transcriptome.
Workflow of RNA_Seq.

26. Transcriptomics in chickpea genotypes using RNA-Seq Approach
26
Bivanij - drought resistant
Hashem - drought sensitive
The 80 highly expressed genes of eight samples were used to
construct heat map. Scale shows high and low expressions as red
and green, respectively.
Mahdavi et al,. 2018

27. scRNA-Seq
27
RNA-Seq:
Analyze expression
of RNAs from large
populations of cells
scRNA-Seq :
Provides expression
profiles of individual
cells
WORKFLOW

28. Candidate genes identified in chickpea for improving traits
related to abiotic tolerance
28

29. Proteomics in Chickpea
29
 Proteome is the entire set of proteins produced or modified by the plant.
 Proteome is blend of the word, “protein” and “genome”.
 Proteomics generally denotes the large-scale experimental analysis of
proteins and proteomes, but often refers, specifically to protein
purification and mass spectrometry.
 It is more complicated than genomics and transcriptomics because the
plant’s genome is more or less constant, whereas proteomes differ from
cell to cell and from time to time.

30. Mass Spectrometer
30
A mass spectrometer consists of three components
•an ion source
•a mass analyzer
•a detector
The ionizer converts a portion of the sample
into ions. The ions are then targeted through the mass
analyzer and into the detector. The differences in
masses of the fragments allows the mass analyzer to
sort the ions by their mass-to-charge ratio. The
detector measures the value of an indicator quantity
and thus provides data for calculating the abundances
of each ion present.

31. Pathways involved in cell defense, signaling, and cell wall modification
under dehydration stress in chickpea ECM
31
Bhushan et al., 2007

32. Effect of salicylic acid (SA) on SDS-PAGE
32
Patel and Hemant., 2013
•The arrow indicates the
increased band intensity in
response to the drought stress
treatment.
•Drought stress was imposed
during the pre-anthesis phase.
•Genotypes Tyson and DCP 92-
3 noticed high band intensity at
treatment T2 and T1
respectively whereas, the
genotypes ICC 4958 and JG
315 showed high band intensity
on both the treatment i. e. T1
and T2.

33. Proteins or protein functions involved in conferring tolerance
to abiotic stresses to chickpea
33

34. Metabolomics in Chickpea
34
 Transcriptomics and proteomic studies reveal the complex biological processes
related to abiotic stress tolerance.
 They are still insufficient to understand the global landscape of cellular response
shown by plants under abiotic stress since most biological processes are ultimately
mediated by cell metabolites.
 In this sense, metabolomics is an emerging field of “omics” research that focuses on
the high-throughout characterization of small molecule metabolites in biological
matrices.
 Metabolites are the substrates, intermediates and products of metabolism.
Metabolomics bridges the gap between genotype and phenotype.

35. GC-MS
35
Gas chromatography–mass spectrometry (GC-MS) is an
analytical method that combines the features of gas-
chromatography and mass spectrometry to identify different
substances within a test sample.
Insides of GC-MS

36. LC-MS
36

37. Software List for Metabolamic Analysis
37

38. Logarithmic ratios of sugar and organic acids content (left), and
amino acids and amine content (right) at saline conditions
38
Black
bars:
Flowers
White
bars
:
Pods
Dias et. al., 2015

39. Chromatographic separation of 45 metabolites using GC-
QqQ-MS
39
Dias et. al., 2015

40. Advantages and Disadvantages of GC-MS and LC-MAS
40

41. Transgenomics and Genome editing in Chickpea
41
 Generally, increasing the level of various osmolytes and proteins are responsible for
conferring tolerance against abiotic stresses.
 However, to obtain a significant level of tolerance to abiotic stresses, it is necessary
to transfer several potentially useful genes to the same genotype.
 Transgenomics is targeted gene-based functional genomics tool that offers valuable
information to understand the regulatory abiotic stress tolerance in plants.
 In genome editing, specific target DNA sequence of the genome is altered by adding,
removing, or replacing DNA bases. Genome editing technology used in plants are
ZFNs, TALENs, CRISPR Cas-9, etc.

42. 42

43. Advantages and disadvantages of genome editing
43
Intended effects Unintended effects
ZFNs
Any genomic sequence is
targeted. Fewer off-target
effects.
Low efficiency
Targets only single site at one time.
Designing constructs is time consuming laborious and
expensive.
TALENs
Target any genomic sequence.
Off-target effects are limited.
Relatively low in efficiency.
Targets only single site at one time.
Designing constructs is not time consuming and are not
cost-effective.
Sensitive to target DNA methylation.
CRISPRs
Highly specific highly efficient
simultaneous of multiple sites
Target selection limited by requirement for PAM
sequence.
Off-target effects.

44. Case Study-I
44

45. Materials and Methods
45
Pusa 391, a desi chickpea variety which
is extensively cultivated in the central
zone of India has become susceptible to
Fusarium Wilt (FW).
WR 315, which harbors resistance to
fusarium wilt, was used as a donor
Parent.
The introgression lines developed in
Pusa 391 genetic background were
subjected to foreground selection using
three SSR markers (GA16, TA27 and
TA96).

46. GGE-biplot analysis
46
E1
(Indore),
E2
(Junagadh)
,
E3
(Sehore),
E4(Badnapur),
E5
(New
Delhi).
E6
(Nandyal)
AEC AEC

47. Grain Yield, AVT-1(2018-19) & AVT-2(2019-20) of IARI-AICRP
47
BGM 20211 and BGM 20212 were the top performers in yield and were highly stable across all environments and were
nominated for Advanced Varietal Trials (AVT) of AICRP. BGM 20211 was identified for release and notified as Pusa
Manav for Madhya Pradesh, Gujarat and Maharashtra states by the Central Sub- Committees on Crop Standards,

48. Case Study-II
48

49. Materials and Methods
49
 They selected the 4-coumarate ligase (4CL) and Reveille 7 (RVE7) genes, both
associated with drought tolerance for CRISPR/Cas9 editing in chickpea protoplast.
 4CL regulates the accumulation of lignin under stress conditions in several plants.
The RVE7 is a MYB transcription factor which is part of regulating circadian
rhythm in plants.
 The study was conducted to knockout the 4CL and RVE7 genes in the chickpea
protoplast to help understand the drought response mechanism in chickpea.
 This is the first study in chickpea protoplast utilizing CRISPR/Cas9 DNA free gene
editing of drought tolerance associated genes.

50. CRISPR/Cas9 DNA free gene editing in the chickpea
protoplast
50
The protoplasts of young leaves were isolated and transformed with CRISPR RNP complex
under optimum conditions. The DNA was extracted from the protoplast control and
samples are tested and sent for Sanger sequencing to detect the mutations.

51. The sequence represents ICE Analysis of RVE7 sgRNA1 and 4CL
sgRNA1 RNP Edited protoplast cell
51
“+” sign is for wild type sample. The target sites are denoted by black vertical dotted lines.
ICE – Inference of CRISPR Edits.

52. CONCLUSIONS AND FUTURE PERSPECTIVES
52
 The shifts in rainfall patterns and terminal heat stress are likely the factors most
affecting the yield and quality of cultivated chickpea.
 The introgression of desirable genes/alleles from wild germplasm into cultivated
chickpea can improve the tolerance levels to abiotic stress and their yields, which is
needed to maintain food security for the upcoming years.
 Efforts have been made to develop chickpea cultivars that are tolerant to different
abiotic stresses, which have been achieved through the combination of conventional
and molecular (omics) breeding methods. However, there are still efforts to be made.
 DNA sequencing methods should be faster, more accurate, and cheaper to obtain the
genetic information of the plants to be used in breeding programs.
 Moreover, the preservation of plant genetic resources should be a priority since it
contains the genetic variability to cope with future climatic adversities.

53. Thank You