Unraveling the genetics of heat tolerance in chickpea landraces (Cicer arietinum L.) using genome-wide association studies.

Chickpea, being an important grain legume crop, is often confronted with the adverse effects of high temperatures at the reproductive stage of crop growth, drastically affecting yield and overall productivity. The current study deals with an extensive evaluation of chickpea genotypes, focusing on the traits associated with yield and their response to heat stress. Notably, we observed significant variations for these traits under both normal and high-temperature conditions, forming a robust basis for genetic research and breeding initiatives. Furthermore, the study revealed that yield-related traits exhibited high heritability, suggesting their potential suitability for marker-assisted selection. We carried out single-nucleotide polymorphism (SNP) genotyping using the genotyping-by-sequencing (GBS) method for a genome-wide association study (GWAS). Overall, 27 marker-trait associations (MTAs) linked to yield-related traits, among which we identified five common MTAs displaying pleiotropic effects after applying a stringent Bonferroni-corrected p-value threshold of <0.05 [-log10(p) > 4.95] using the BLINK (Bayesian-information and linkage-disequilibrium iteratively nested keyway) model. Through an in-depth in silico analysis of these markers against the CDC Frontier v1 reference genome, we discovered that the majority of the SNPs were located at or in proximity to gene-coding regions. We further explored candidate genes situated near these MTAs, shedding light on the molecular mechanisms governing heat stress tolerance and yield enhancement in chickpeas such as indole-3-acetic acid-amido synthetase GH3.1 with GH3 auxin-responsive promoter and pentatricopeptide repeat-containing protein, etc. The harvest index (HI) trait was associated with marker Ca3:37444451 encoding aspartic proteinase ortholog sequence of Oryza sativa subsp. japonica and Medicago truncatula, which is known for contributing to heat stress tolerance. These identified MTAs and associated candidate genes may serve as valuable assets for breeding programs dedicated to tailoring chickpea varieties resilient to heat stress and climate change.

A Multi-Model Based Stability Analysis Employing Multi-Environmental Trials (METs) Data for Discerning Heat Tolerance in Chickpea (Cicer arietinum L.) Landraces.

Identifying a congenially targeted production environment and understanding the effects of genotype by environmental interactions on the adaption of chickpea genotypes is essential for achieving an optimal yield stability. Different models like additive main effect and multiplicative interactions (AMMI 1, AMM2), weighted average absolute scores of BLUPs (WAASB), and genotype plus genotype-environment (GGE) interactions were used to understand their suitability in the precise estimation of variance and their interaction. Our experiment used genotypes that represent the West Asia-North Africa (WANA) region. This trial involved two different sowing dates, two distinct seasons, and three different locations, resulting in a total of 12 environments. Genotype IG 5871(G1) showed a lower heat susceptibility index (HSI) across environments under study. The first four interactions principal component axis (IPCA) explain 93.2% of variations with significant genotype-environment interactions. Considering the AMMI stability value (ASV), the genotypes IG5862(G7), IG5861(G6), ILC239(G40), IG6002(G26), and ILC1932(G39), showing ASV scores of 1.66, 1.80, 2.20, 2.60, and 2.84, respectively, were ranked as the most stable and are comparable to the weighted average absolute scores of BLUPs (WAASB) ranking of genotypes. The which-won-where pattern of genotype plus genotype-environment (GGE) interactions suggested that the target environment consists of one mega environment. IG5866(G10), IG5865(G9), IG5884(G14), and IG5862(G7) displayed higher stability, as they were nearer to the origin. The genotypes that exhibited a superior performance in the tested environments can serve as ideal parental lines for heat-stress tolerance breeding programs. The weighted average absolute scores of BLUPs (WAASB) serve as an ideal tool to discern the variations and identify the stable genotype among all methods.

Morphological and antioxidant responses of Cicer arietinum L. genotypes exposed to combination stress of anthracene and sodium chloride.

Various mutagenic, carcinogenic pollutants such as Polycyclic Aromatic Hydrocarbons (PAHs) are released into the environment posing a negative effect on plant metabolism. All the pollutants that are emitted into the atmosphere, ultimately find their way into the plant. Soil salinity stress is one of the major determinants of crop productivity. Different plants respond differently to different abiotic stress present alone or in combination. One such combination of abiotic stress is PAHs and salinity stress. The present research aims to study the effect of the application of NaCl and Anthracene alone and in various combinations on two chickpea genotypes GPF2 and PDG4. A 21 days laboratory experiment was conducted in petriplates and growth pouches. Different concentrations of NaCl and Anthracene were given to two chickpea genotypes viz. GPF2 and PDG4, alone as well as in combinations to study morphological, physiological and antioxidant responses. Results obtained were further analyzed by using various statistical measures such as Principle Component Analysis and Two-way ANOVA. Results indicated that under the dual presence of NaCl and Anthracene, GPF2 exhibited higher activities of antioxidant enzymes and was shown to have a negative correlation with plant height and chlorophyll content. Based on the results of the present investigation, it was concluded that GPF2 was a better performing chickpea genotype towards the combined presence of Anthracene and NaCl as compared to PDG4.

Phenotypic evaluation of agronomic and root related traits for drought tolerance in recombinant inbred line population derived from a chickpea cultivar (C. arietinum L.) and its wild relative (C. reticulatum).

Drought is a major abiotic stress that drastically reduces chickpea yields. The present study was aimed to identify drought-responsive traits in chickpea by screening a recombinant inbred line population derived from an inter-specific cross between drought cultivar of GPF2 (C. arietinum L.) and drought sensitive accession of ILWC292 (C. reticulatum), at two locations in India. Twenty-one traits, including twelve morphological and physiological traits and nine root-related traits were measured under rainfed (drought-stress) and irrigated conditions (no-stress). High genotypic variation was observed among RILs for yield and root traits indicated that selection in these germplasms would be useful in achieving genetic progress. Both correlation and principal component analysis revealed that plant height, number of pods per plant, biomass, 100-seed weight, harvest index, membrane permeability index, and relative leaf water content were significantly correlated with yield under both irrigated and drought stress environments. Root length had significant positive correlations with all root-related traits except root length density in drought-stressed plants. Path analysis and multiple and stepwise regression analyses showed that number of pods per plant, biomass, and harvest index were major contributors to yield under drought stress conditions. Thus, a holistic approach across these analyses identified number of pods per plant, biomass, harvest index, and root length as key traits for improving chickpea yield through indirect selection for developing drought-tolerant cultivars. Overall, on the basis of yield components morphological and root traits, a total of 15 promising RILs were identified for their use in chickpea breeding programs for developing drought tolerant cultivars. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01218-z.

Genetic mapping of QTLs for drought tolerance in chickpea (Cicer arietinum L.).

Chickpea yield is severely affected by drought stress, which is a complex quantitative trait regulated by multiple small-effect genes. Identifying genomic regions associated with drought tolerance component traits may increase our understanding of drought tolerance mechanisms and assist in the development of drought-tolerant varieties. Here, a total of 187 F8 recombinant inbred lines (RILs) developed from an interspecific cross between drought-tolerant genotype GPF 2 (Cicer arietinum) and drought-sensitive accession ILWC 292 (C. reticulatum) were evaluated to identify quantitative trait loci (QTLs) associated with drought tolerance component traits. A total of 21 traits, including 12 morpho-physiological traits and nine root-related traits, were studied under rainfed and irrigated conditions. Composite interval mapping identified 31 QTLs at Ludhiana and 23 QTLs at Faridkot locations for morphological and physiological traits, and seven QTLs were identified for root-related traits. QTL analysis identified eight consensus QTLs for six traits and five QTL clusters containing QTLs for multiple traits on linkage groups CaLG04 and CaLG06. The identified major QTLs and genomic regions associated with drought tolerance component traits can be introgressed into elite cultivars using genomics-assisted breeding to enhance drought tolerance in chickpea.

A comprehensive analysis of Trehalose-6-phosphate synthase (TPS) gene for salinity tolerance in chickpea (Cicer arietinum L.).

Soil salinity affects various crop cultivation but legumes are the most sensitive to salinity. Osmotic stress is the first stage of salinity stress caused by excess salts in the soil on plants which adversely affects the growth instantly. The Trehalose-6-phosphate synthase (TPS) genes play a key role in the regulation of abiotic stresses resistance from the high expression of different isoform. Selected genotypes were evaluated to estimate for salt tolerance as well as genetic variability at morphological and molecular level. Allelic variations were identified in some of the selected genotypes for the TPS gene. A comprehensive analysis of the TPS gene from selected genotypes was conducted. Presence of significant genetic variability among the genotypes was found for salinity tolerance. This is the first report of allelic variation of TPS gene from chickpea and results indicates that the SNPs present in these conserved regions may contribute largely to functional distinction. The nucleotide sequence analysis suggests that the TPS gene sequences were found to be conserved among the genotypes. Some selected genotypes were evaluated to estimate for salt tolerance as well as for comparative analysis of physiological, molecular and allelic variability for salt responsive gene Trehalose-6-Phosphate Synthase through sequence similarity. Allelic variations were identified in some selected genotypes for the TPS gene. It is found that Pusa362, Pusa1103, and IG5856 are the most salt-tolerant lines and the results indicates that the identified genotypes can be used as a reliable donor for the chickpea improvement programs for salinity tolerance.

Identification of promising chickpea interspecific derivatives for agro-morphological and major biotic traits.

The wild Cicer species is well-known for having climate-resilient and productivity-enhancing traits of interest. Therefore, wide hybridization could be used as a realistic strategy for introgressing prospective traits from wild species into the cultivated gene pool. The present study was, thus, undertaken to evaluate F7 chickpea interspecific derivatives derived from Cicer reticulatum Ladiz. and C. echinospermum P. H. Davis wild annual Cicer species. As a result, a set of six interspecific crosses were advanced using the single seed descent (SSD) method of breeding. The F7 generation of these crosses was assessed in two diverse agro-ecological regions of India. The data revealed a wide range of variation with respect to seed yield and its important component traits, which resulted in the identification of the most promising derivatives carrying desirable characters as indicated by range, mean, and coefficient of variation. Further, fruitful heterosis was also estimated as promising selection criteria for identifying superior lines for earliness and high seed yield, including resistance against prevailing stresses (ascochyta blight, botrytis gray mold, dry root rot, and fusarium wilt). The superior derivatives carrying putative characters could be recommended for further breeding and selection of genetic materials for developing suitable genotypes.

Introgressing cry1Ac for Pod Borer Resistance in Chickpea Through Marker-Assisted Backcross Breeding.

The gram pod borer Helicoverpa armigera is a major constraint to chickpea (Cicer arietinum L.) production worldwide, reducing crop yield by up to 90%. The constraint is difficult to overcome as chickpea germplasm including wild species either lacks pod borer resistance or if possessing resistance is cross-incompatible. This study describes conversion of elite but pod borer-susceptible commercial chickpea cultivars into resistant cultivars through introgression of cry1Ac using marker-assisted backcross breeding. The chickpea cultivars (PBG7 and L552) were crossed with pod borer-resistant transgenic lines (BS 100B and BS 100E) carrying cry1Ac that led to the development of BC1F1, BC1F2, BC1F3, BC2F1, BC2F2, and BC2F3 populations from three cross combinations. The foreground selection revealed that 35.38% BC1F1 and 8.4% BC1F2 plants obtained from Cross A (PBG7 × BS 100B), 50% BC1F1 and 76.5% BC1F2 plants from Cross B (L552 × BS 100E), and 12.05% BC2F2 and 82.81% (average) BC2F3 plants derived from Cross C (PBG7 × BS 100E) carried the cry1Ac gene. The bioassay of backcross populations for toxicity to H. armigera displayed up to 100% larval mortality. BC1F1 and BC1F2 populations derived from Cross B and BC2F3 population from Cross C segregated in the Mendelian ratio for cry1Ac confirmed inheritance of a single copy of transgene, whereas BC1F1 and BC1F2 populations obtained from Cross A and BC2F2 population from Cross C exhibited distorted segregation ratios. BC1F1 plants of Cross A and Cross B accumulated Cry1Ac protein ranging from 11.03 to 11.71 µgg-1 in leaf tissue. Cry1Ac-positive BC2F2 plants from Cross C demonstrated high recurrent parent genome recovery (91.3%) through background selection using SSR markers and phenome recovery of 90.94%, amongst these 30% plants, were homozygous for transgene. The performance of BC2F3 progenies derived from homozygous plants was similar to that of the recurrent parent for main agronomic traits, such as number of pods and seed yield per plant. These progenies are a valuable source for H. armigera resistance in chickpea breeding programs.

Inheritance and biochemical basis of yellowing of apical leaves: a unique trait in chickpea (Cicer arietinum L.).

A unique trait, i.e. yellowing of apical/young leaves in response to low temperature and high relative humidity was identified in a chickpea genotype, ICCX110069. To determine inheritance pattern of this trait, ICCX110069 was crossed to four other genotypes, GL14050, GL14049, GL14059 and SAGL152117, that exhibited normal green apical leaves under similar environmental conditions. The F1, F2, F3, BC1F1 and BC1F2 generations were generated. A ratio of 13 normal green leaf: three yellow leaf was found to be the best fit, indicated digenic gene action with suppressor effect of normal green leaf over the expression of yellowing of apical/young leaf trait. The chlorophyll content was significantly lower, while guaiacol peroxidase activity was significantly higher in yellow leaves of ICCX110069 as compared to green leaves of the same genotype and of GL14049, indicating the competence of antioxidative defence mechanism involved with the expression of this trait.

Identification of stable heat tolerance QTLs using inter-specific recombinant inbred line population derived from GPF 2 and ILWC 292.

Heat stress during reproductive stages has been leading to significant yield losses in chickpea (Cicer arietinum L.). With an aim of identifying the genomic regions or QTLs responsible for heat tolerance, 187 F8 recombinant inbred lines (RILs) derived from the cross GPF 2 (heat tolerant) × ILWC 292 (heat sensitive) were evaluated under late-sown irrigated (January-May) and timely-sown irrigated environments (November-April) at Ludhiana and Faridkot in Punjab, India for 13 heat tolerance related traits. The pooled ANOVA for both locations for the traits namely days to germination (DG), days to flowering initiation (DFI), days to 50% flowering (DFF), days to 100% flowering (DHF), plant height (PH), pods per plant (NPP), biomass (BIO), grain yield (YLD), 100-seed weight (HSW), harvest index (HI), membrane permeability index (MPI), relative leaf water content (RLWC) and pollen viability (PV)) showed a highly significant difference in RILs. The phenotyping data coupled with the genetic map comprising of 1365 ddRAD-Seq based SNP markers were used for identifying the QTLs for heat tolerance. Composite interval mapping provided a total of 28 and 23 QTLs, respectively at Ludhiana and Faridkot locations. Of these, 13 consensus QTLs for DG, DFI, DFF, DHF, PH, YLD, and MPI have been identified at both locations. Four QTL clusters containing QTLs for multiple traits were identified on the same genomic region at both locations. Stable QTLs for days to flowering can be one of the major factors for providing heat tolerance as early flowering has an advantage of more seed setting due to a comparatively longer reproductive period. Identified QTLs can be used in genomics-assisted breeding to develop heat stress-tolerant high yielding chickpea cultivars.

Phenotypic evaluation of genetic variability and selection of yield contributing traits in chickpea recombinant inbred line population under high temperature stress.

Heat is a major abiotic stress that drastically reduces chickpea yield. This study aimed to identify heat-responsive traits to sustain crop productivity by screening a recombinant inbred line (RILs) population at two locations in India (Ludhiana and Faridkot). The RIL population was derived from an inter-specific cross between heat-tolerant genotype GPF 2 (C. arietinum L.) and heat sensitive accession ILWC 292 (C. reticulatum). The pooled analysis of variance showed highly significant differences for all the traits in RILs and most of the traits were significantly affected by heat stress at both locations. High values of genotypic coefficient of variation (19.52-38.53%), phenotypic coefficient of variation (20.29-39.85%), heritability (92.50-93.90%), and genetic advance as a percentage of mean (38.68-76.74%) have been observed for plant height, number of pods per plant, biomass, yield, and hundred seed weight across the heat stress environments. Association studies and principal component analysis showed a significant positive correlation of plant height, number of pods per plant, biomass, hundred seed weight, harvest index, relative leaf water content, and pollen viability with yield under both timely-sown and late-sown conditions. Path analysis revealed that biomass followed by harvest index was the major contributor to yield among the environments. Both step-wise and multiple regression analyses concluded that number of pods per plant, biomass and harvest index consistently showed high level of contribution to the total variation in yield under both timely-sown and late-sown conditions. Thus, the holistic approach of these analyses illustrated that the promising traits provide a framework for developing heat-tolerant cultivars in chickpea. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-00977-5.