Novel Alleles from Cicer reticulatum L. for Genetic Improvement of Cultivated Chickpeas Identified through Genome Wide Association Analysis.

The availability of wild chickpea (Cicer reticulatum L.) accessions has the potential to be used for the improvement of important traits in cultivated chickpeas. The main objectives of this study were to evaluate the phenotypic and genetic variations of chickpea progeny derived from interspecific crosses between C. arietinum and C. reticulatum, and to establish the association between single nucleotide polymorphism (SNP) markers and a series of important agronomic traits in chickpea. A total of 486 lines derived from interspecific crosses between C. arietinum (CDC Leader) and 20 accessions of C. reticulatum were evaluated at different locations in Saskatchewan, Canada in 2017 and 2018. Significant variations were observed for seed weight per plant, number of seeds per plant, thousand seed weight, and plant biomass. Path coefficient analysis showed significant positive direct effects of the number of seeds per plant, thousand seed weight, and biomass on the total seed weight. Cluster analysis based on the agronomic traits generated six groups that allowed the identification of potential heterotic groups within the interspecific lines for yield improvement and resistance to ascochyta blight disease. Genotyping of the 381 interspecific lines using a modified genotyping by sequencing (tGBS) generated a total of 14,591 SNPs. Neighbour-joining cluster analysis using the SNP data grouped the lines into 20 clusters. The genome wide association analysis identified 51 SNPs that had significant associations with different traits. Several candidate genes associated with early flowering and yield components were identified. The candidate genes and the significant SNP markers associated with different traits have a potential to aid the trait introgression in the breeding program.

Genetic Analysis of Partially Resistant and Susceptible Chickpea Cultivars in Response to Ascochyta rabiei Infection.

The molecular mechanism involved in chickpea (Cicer arietinum L.) resistance to the necrotrophic fungal pathogen Ascochyta rabiei is not well documented. A. rabiei infection can cause severe damage in chickpea, resulting in significant economic losses. Understanding the resistance mechanism against ascochyta blight can help to define strategies to develop resistant cultivars. In this study, differentially expressed genes from two partially resistant cultivars (CDC Corinne and CDC Luna) and a susceptible cultivar (ICCV 96029) to ascochyta blight were identified in the early stages (24, 48 and 72 h) of A. rabiei infection using RNA-seq. Altogether, 3073 genes were differentially expressed in response to A. rabiei infection across different time points and cultivars. A larger number of differentially expressed genes (DEGs) were found in CDC Corinne and CDC Luna than in ICCV 96029. Various transcription factors including ERF, WRKY, bHLH and MYB were differentially expressed in response to A. rabiei infection. Genes involved in pathogen detection and immune signalings such as receptor-like kinases (RLKs), Leucine-Rich Repeat (LRR)-RLKs, and genes associated with the post-infection defence response were differentially expressed among the cultivars. GO functional enrichment and pathway analysis of the DEGs suggested that the biological processes such as metabolic process, response to stimulus and catalytic activity were overrepresented in both resistant and susceptible chickpea cultivars. The expression patterns of eight randomly selected genes revealed by RNA-seq were confirmed by quantitative PCR (qPCR) analysis. The results provide insights into the complex molecular mechanism of the chickpea defence in response to the A. rabiei infection.

Iron accumulation and partitioning in hydroponically grown wild and cultivated chickpea (Cicer arietinum L).

Chickpea (Cicer arietinum L.) is a staple food in many developing countries where iron (Fe) deficiency often occurs in their population. The crop is a good source of protein, vitamins, and micronutrients. Fe biofortification in chickpea can be part of long-term strategy to enhance Fe intake in human diet to help to alleviate Fe deficiency. To develop cultivars with high Fe concentration in seeds, understanding the mechanisms of absorption and translocation of Fe into the seeds is critical. An experiment was conducted using a hydroponic system to evaluate Fe accumulation in seeds and other organs at different growth stages of selected genotypes of cultivated and wild relatives of chickpea. Plants were grown in media with Fe zero and Fe added conditions. Six chickpea genotypes were grown and harvested at six different growth stages: V3, V10, R2, R5, R6, and RH for analysis of Fe concentration in roots, stems, leaves, and seeds. The relative expression of genes related to Fe-metabolism including FRO2, IRT1, NRAMP3, V1T1, YSL1, FER3, GCN2, and WEE1 was analyzed. The results showed that the highest and lowest accumulation of Fe throughout the plant growth stages were found in the roots and stems, respectively. Results of gene expression analysis confirmed that the FRO2 and IRT1 were involved in Fe uptake in chickpeas and expressed more in roots under Fe added condition. All transporter genes: NRAMP3, V1T1, YSL1 along with storage gene FER3 showed higher expression in leaves. In contrast, candidate gene WEE1 for Fe metabolism expressed more in roots under Fe affluent condition; however, GCN2 showed over-expression in roots under Fe zero condition. Current finding will contribute to better understanding of Fe translocation and metabolism in chickpea. This knowledge can further be used to develop chickpea varieties with high Fe in seeds.

Quantitative trait loci associated with amino acid concentration and in vitro protein digestibility in pea (Pisum sativum L.).

With the expanding interest in plant-based proteins in the food industry, increasing emphasis is being placed on breeding for protein concentration and quality. Two protein quality traits i.e., amino acid profile and protein digestibility, were assessed in replicated, multi-location field trials from 2019 to 2021 in pea recombinant inbred line population PR-25. This RIL population was targeted specifically for the research of protein related traits and its parents, CDC Amarillo and CDC Limerick, had distinct variations in the concentration of several amino acids. Amino acid profile was determined using near infrared reflectance analysis, and protein digestibility was through an in vitro method. Several essential amino acids were selected for QTL analysis, including lysine, one of the most abundant essential amino acids in pea, and methionine, cysteine, and tryptophan, the limiting amino acids in pea. Based on phenotypic data of amino acid profiles and in vitro protein digestibility of PR-25 harvested in seven location-years, three QTLs were associated with methionine + cysteine concentration, among which, one was located on chromosome 2 (R2 = 17%, indicates this QTL explained 17% phenotypic variation of methionine + cysteine concentration within PR-25), and two were located on chromosome 5 (R2 = 11% and 16%). Four QTLs were associated with tryptophan concentration and are located on chromosome 1 (R2 = 9%), chromosome 3 (R2 = 9%), and chromosome 5 (R2 = 8% and 13%). Three QTLs were associated with lysine concentration, among which, one was located on chromosome 3 (R2 = 10%), the other two were located on chromosome 4 (R2 = 15% and 21%). Two QTLs were associated with in vitro protein digestibility, one each located on chromosomes 1 (R2 = 11%) and 2 (R2 = 10%). QTLs associated with in vitro protein digestibility, and methionine + cysteine concentration on chromosome 2 were identified to be co-localized with known QTL for total seed protein concentration in PR-25. QTLs associated with tryptophan and methionine + cysteine concentration co-localized on chromosome 5. The identification of QTLs associated with pea seed quality is an important step towards marker-assisted selection of breeding lines with improved nutritional quality, which will further boost the competitiveness of pea in plant-based protein markets.

Freezing stress response of wild and cultivated chickpeas.

Chickpea is an economically and nutritionally important grain legume globally, however, cold stress has adverse effects on its growth. In cold countries, like Canada where the growing season is short, having cold stress-tolerant varieties is crucial. Crop wild relatives of chickpea, especially Cicer reticulatum, can survive in suboptimal environments and are an important resource for crop improvement. In this study, we explored the performance of eleven C. reticulatum wild accessions and two chickpea cultivars, CDC Leader and CDC Consul, together with a cold sensitive check ILC533 under freezing stress. Freezing tolerance was scored based on a 1-9 scale. The wild relatives, particularly Kesen_075 and CudiA_152, had higher frost tolerance compared to the cultivars, which all died after frost treatment. We completed transcriptome analysis via mRNA sequencing to assess changes in gene expression in response to freezing stress and identified 6,184 differentially expressed genes (DEGs) in CDC Consul, and 7,842 DEGs in Kesen_075. GO (gene ontology) analysis of the DEGs revealed that those related to stress responses, endogenous and external stimuli responses, secondary metabolite processes, and photosynthesis were significantly over-represented in CDC Consul, while genes related to endogenous stimulus responses and photosynthesis were significantly over-represented in Kesen_075. These results are consistent with Kesen_075 being more tolerant to freezing stress than CDC Consul. Moreover, our data revealed that the expression of CBF pathway-related genes was impacted during freezing conditions in Kesen_075, and expression of these genes is believed to alleviate the damage caused by freezing stress. We identified genomic regions associated with tolerance to freezing stress in an F2 population derived from a cross between CDC Consul and Kesen_075 using QTL-seq analysis. Eight QTLs (P<0.05) on chromosomes Ca3, Ca4, Ca6, Ca7, Ca8, and two QTLs (P<0.01) on chromosomes Ca4 and Ca8, were associated with tolerance to freezing stress. Interestingly, 58 DEGs co-located within these QTLs. To our knowledge, this is the first study to explore the transcriptome and QTLs associated with freezing tolerance in wild relatives of chickpea under controlled conditions. Altogether, these findings provide comprehensive information that aids in understanding the molecular mechanism of chickpea adaptation to freezing stress and further provides functional candidate genes that can assist in breeding of freezing-stress tolerant varieties.

Genome-wide association analysis of stress tolerance indices in an interspecific population of chickpea.

Chickpea is a cool season crop that is highly vulnerable to abiotic stresses such as heat and drought. High temperature during early flowering and pod development stages significantly reduces the crop yield. The wild relatives of chickpeas can be potential donors for the introgression of heat and drought tolerance into cultivated chickpeas for crop improvement. Initially, 600 interspecific lines were derived from crosses between two elite cultivars, CDC Leader (kabuli chickpea) and CDC Consul (desi chickpea), and 20 accessions of Cicer reticulatum. The F5 interspecific lines were tested for agronomic and seed quality traits including reaction to ascochyta blight disease under field conditions at two locations in 2018. A subset of 195 lines were selected based on resistance to ascochyta blight and acceptable seed quality. These lines were evaluated for their performance under suboptimal conditions at Lucky Lake (2019 and 2020) and Moose Jaw (2019), Saskatchewan, Canada, and Yuma, Arizona, United States (2019-2020). The lines were grown and evaluated at two seeding dates, normal (SD1) and late (SD2) seeding dates, at each location and year. The same lines were genotyped using Cicer60K Axiom® SNP chip. The population structure was determined based on 35,431 informative SNPs using fastStructure, and the interspecific lines were clustered at a k-value of 15. Significant marker-trait associations were identified for seed yield from SD1 and SD2 seeding dates, and stress tolerance indices (ATI, K1STI, MP, SSPI, and TOL) using phenotypic values both from individual locations and combined analyses based on BLUP values. SNP marker Ca2_34600347 was significantly associated with yield from both the seeding dates. This and other SNP markers identified in this study may be useful for marker-assisted introgression of abiotic stress tolerance in chickpea.

Identification of Quantitative Trait Loci Associated with Seed Protein Concentration in a Pea Recombinant Inbred Line Population.

This research aimed to identify quantitative trait loci (QTLs) associated with seed protein concentration in a recombinant inbred line (RIL) population of pea and aimed to validate the identified QTLs using chromosome segment-introgressed lines developed by recurrent backcrossing. PR-25, an RIL population consisting of 108 F7 bulked lines derived from a cross between CDC Amarillo (yellow cotyledon) and CDC Limerick (green cotyledon), was used in this research. The RIL population was genotyped using an Axiom 90K SNP array. A total of 10,553 polymorphic markers were used for linkage map construction, after filtering for segregation distortion and missing values. The linkage map represents 901 unique loci on 11 linkage groups which covered a map distance of 855.3 Centimorgans. Protein concentration was assessed using near-infrared (NIR) spectroscopy of seeds harvested from field trials in seven station-years in Saskatchewan, Canada, during the 2019-2021 field seasons. Three QTLs located on chromosomes 2, 3 and 5 were identified to be associated with seed protein concentration. These QTLs explained 22%, 11% and 17% of the variation for protein concentration, respectively. The identified QTLs were validated by introgression lines, developed by marker-assisted selection of backcross lines for introgression of corresponding chromosome segments (~1/4 chromosome) harboring the QTL regions. Introgression line PR-28-7, not carrying any protein-related QTLs identified in this study, was 4.7% lower in protein concentration than CDC Amarillo, the lower protein parent of PR-25 which carried one identified protein-related QTL. The SNP markers located at the peak of the three identified QTLs will be converted into breeder-friendly KASP assays, which will be used for the selection of high-protein lines from segregating populations.

A chickpea genetic variation map based on the sequencing of 3,366 genomes.

Zero hunger and good health could be realized by 2030 through effective conservation, characterization and utilization of germplasm resources1. So far, few chickpea (Cicer arietinum) germplasm accessions have been characterized at the genome sequence level2. Here we present a detailed map of variation in 3,171 cultivated and 195 wild accessions to provide publicly available resources for chickpea genomics research and breeding. We constructed a chickpea pan-genome to describe genomic diversity across cultivated chickpea and its wild progenitor accessions. A divergence tree using genes present in around 80% of individuals in one species allowed us to estimate the divergence of Cicer over the last 21 million years. Our analysis found chromosomal segments and genes that show signatures of selection during domestication, migration and improvement. The chromosomal locations of deleterious mutations responsible for limited genetic diversity and decreased fitness were identified in elite germplasm. We identified superior haplotypes for improvement-related traits in landraces that can be introgressed into elite breeding lines through haplotype-based breeding, and found targets for purging deleterious alleles through genomics-assisted breeding and/or gene editing. Finally, we propose three crop breeding strategies based on genomic prediction to enhance crop productivity for 16 traits while avoiding the erosion of genetic diversity through optimal contribution selection (OCS)-based pre-breeding. The predicted performance for 100-seed weight, an important yield-related trait, increased by up to 23% and 12% with OCS- and haplotype-based genomic approaches, respectively.

Identification of heat responsive genes in pea stipules and anthers through transcriptional profiling.

Field pea (Pisum sativum L.), a cool-season legume crop, is known for poor heat tolerance. Our previous work identified PR11-2 and PR11-90 as heat tolerant and susceptible lines in a recombinant inbred population. CDC Amarillo, a Canadian elite pea variety, was considered as another heat tolerant variety based on its similar field performance as PR11-2. This study aimed to characterize the differential transcription. Plants of these three varieties were stressed for 3 h at 38°C prior to self-pollination, and RNAs from heat stressed anthers and stipules on the same flowering node were extracted and sequenced via the Illumina NovaSeq platform for the characterization of heat responsive genes. In silico results were further validated by qPCR assay. Differentially expressed genes (DEGs) were identified at log2 |fold change (FC)| ≥ 2 between high temperature and control temperature, the three varieties shared 588 DEGs which were up-regulated and 220 genes which were down-regulated in anthers when subjected to heat treatment. In stipules, 879 DEGs (463/416 upregulation/downregulation) were consistent among varieties. The above heat-induced genes of the two plant organs were related to several biological processes i.e., response to heat, protein folding and DNA templated transcription. Ten gene ontology (GO) terms were over-represented in the consistently down-regulated DEGs of the two organs, and these terms were mainly related to cell wall macromolecule metabolism, lipid transport, lipid localization, and lipid metabolic processes. GO enrichment analysis on distinct DEGs of individual pea varieties suggested that heat affected biological processes were dynamic, and variety distinct responses provide insight into molecular mechanisms of heat-tolerance response. Several biological processes, e.g., cellular response to DNA damage stimulus in stipule, electron transport chain in anther that were only observed in heat induced PR11-2 and CDC Amarillo, and their relevance to field pea heat tolerance is worth further validation.

Standardization of Aquafaba Production and Application in Vegan Mayonnaise Analogs.

Canning or boiling pulse seeds in water produces a by-product solution, called "aquafaba", that can be used as a plant-based emulsifier. One of the major problems facing the commercialization of aquafaba is inconsistency in quality and functionality. In this study, chickpea aquafaba production and drying methods were optimized to produce standardized aquafaba powder. Aquafaba samples, both freeze-dried and spray-dried, were used to make egg-free, vegan mayonnaise. Mayonnaise and analog physicochemical characteristics, microstructure, and stability were tested and compared to mayonnaise prepared using egg yolk. Chickpeas steeped in water at 4 °C for 16 h, followed by cooking at 75 kPa for 30 min at 116 °C, yielded aquafaba that produced the best emulsion qualities. Both lyophilization and spray drying to dehydrate aquafaba resulted in powders that retained their functionality following rehydration. Mayonnaise analogs made with aquafaba powder remained stable for 28 days of storage at 4 °C, although their droplet size was significantly higher than the reference sample made with egg yolk. These results show that aquafaba production can be standardized for optimal emulsion qualities, and dried aquafaba can mimic egg functions in food emulsions and has the potential to produce a wide range of eggless food products.

Thermal processing methods differentially affect the protein quality of Chickpea (Cicer arietinum).

Chickpea is a widely produced pulse crop, but requires processing prior to human consumption. Protein bioavailability and amino acid quantity of chickpea flour can be altered by multiple factors including processing method. For this reason, the protein quality of processed chickpea flour was determined using in vivo and in vitro analyses for processed chickpeas. Processing differentially affected the protein digestibility-corrected amino acid score (PDCAAS) of chickpeas with extruded chickpea (83.8) having a higher PDCAAS score than both cooked (75.2) and baked (80.03). Interestingly, the digestible indispensable amino acid score (DIAAS) value of baked chickpea (0.84) was higher compared to both extruded (0.82) and cooked (0.78). The protein efficiency ratio, another measure of protein quality, was significantly higher for extruded chickpea than baked chickpea (p < .01). In vivo and in vitro analysis of protein quality were well correlated (R 2 = .9339). These results demonstrated that under certain circumstances in vitro methods could replace the use of animals to determine protein quality.

Integrating genomics for chickpea improvement: achievements and opportunities.

KEY MESSAGE: Integration of genomic technologies with breeding efforts have been used in recent years for chickpea improvement. Modern breeding along with low cost genotyping platforms have potential to further accelerate chickpea improvement efforts. The implementation of novel breeding technologies is expected to contribute substantial improvements in crop productivity. While conventional breeding methods have led to development of more than 200 improved chickpea varieties in the past, still there is ample scope to increase productivity. It is predicted that integration of modern genomic resources with conventional breeding efforts will help in the delivery of climate-resilient chickpea varieties in comparatively less time. Recent advances in genomics tools and technologies have facilitated the generation of large-scale sequencing and genotyping data sets in chickpea. Combined analysis of high-resolution phenotypic and genetic data is paving the way for identifying genes and biological pathways associated with breeding-related traits. Genomics technologies have been used to develop diagnostic markers for use in marker-assisted backcrossing programmes, which have yielded several molecular breeding products in chickpea. We anticipate that a sequence-based holistic breeding approach, including the integration of functional omics, parental selection, forward breeding and genome-wide selection, will bring a paradigm shift in development of superior chickpea varieties. There is a need to integrate the knowledge generated by modern genomics technologies with molecular breeding efforts to bridge the genome-to-phenome gap. Here, we review recent advances that have led to new possibilities for developing and screening breeding populations, and provide strategies for enhancing the selection efficiency and accelerating the rate of genetic gain in chickpea.

Genome-Wide Association Mapping for Agronomic and Seed Quality Traits of Field Pea (Pisum sativum L.).

Genome-wide association study (GWAS) was conducted to identify loci associated with agronomic (days to flowering, days to maturity, plant height, seed yield and seed weight), seed morphology (shape and dimpling), and seed quality (protein, starch, and fiber concentrations) traits of field pea (Pisum sativum L.). A collection of 135 pea accessions from 23 different breeding programs in Africa (Ethiopia), Asia (India), Australia, Europe (Belarus, Czech Republic, Denmark, France, Lithuania, Netherlands, Russia, Sweden, Ukraine and United Kingdom), and North America (Canada and USA), was used for the GWAS. The accessions were genotyped using genotyping-by-sequencing (GBS). After filtering for a minimum read depth of five, and minor allele frequency of 0.05, 16,877 high quality SNPs were selected to determine marker-trait associations (MTA). The LD decay (LD1/2max,90) across the chromosomes varied from 20 to 80 kb. Population structure analysis grouped the accessions into nine subpopulations. The accessions were evaluated in multi-year, multi-location trials in Olomouc (Czech Republic), Fargo, North Dakota (USA), and Rosthern and Sutherland, Saskatchewan (Canada) from 2013 to 2017. Each trait was phenotyped in at least five location-years. MTAs that were consistent across multiple trials were identified. Chr5LG3_566189651 and Chr5LG3_572899434 for plant height, Chr2LG1_409403647 for lodging resistance, Chr1LG6_57305683 and Chr1LG6_366513463 for grain yield, Chr1LG6_176606388, Chr2LG1_457185, Chr3LG5_234519042 and Chr7LG7_8229439 for seed starch concentration, and Chr3LG5_194530376 for seed protein concentration were identified from different locations and years. This research identified SNP markers associated with important traits in pea that have potential for marker-assisted selection towards rapid cultivar improvement.

Iron Fortification and Bioavailability of Chickpea (Cicer arietinum L.) Seeds and Flour.

Iron (Fe) deficiency is one of the most common nutritional disorders, and is mainly due to insufficient intake of bioavailable Fe. Chickpea (Cicer arietinum L.) was examined as a potential vehicle for Fe fortification. Fortificants (FeSO4·7H2O (ferrous sulfate hepta-hydrate), FeSO4·H2O (ferrous sulfate mono-hydrate) and NaFeEDTA (ethylenediaminetetraacetic acid iron (iii) sodium salt)) were applied by a spraying and drying method. At 2000 µg g-1 iron fortificant, the fortified split desi seeds (dal), desi flour and kabuli flour supplied 18-19 mg, 16-20 mg and 11-19 mg Fe per 100 g, respectively. The overall consumer acceptability using a nine-point hedonic scale for sensory evaluation demonstrated that NaFeEDTA-fortified cooked chickpea (soup and chapatti) scored the highest among the three fortificants. Lightness (L*), redness (a*) and yellowness (b*) of Fe-fortified products changed over time. However, no organoleptic changes occurred. Fe bioavailability was increased by 5.8-10.5, 15.3-25.0 and 4.8-9.0 ng ferritin mg-1 protein for cooked split desi seeds (soup), desi chapatti and kabuli chapatti, respectively, when prepared using Fe-fortified chickpea. Desi chapatti showed significantly higher Fe bioavailability than the other two. The increase in Fe concentration and bioavailability in fortified chickpea products demonstrated that these products could provide a significant proportion of the recommended daily Fe requirement.

A reference genome for pea provides insight into legume genome evolution.

We report the first annotated chromosome-level reference genome assembly for pea, Gregor Mendel's original genetic model. Phylogenetics and paleogenomics show genomic rearrangements across legumes and suggest a major role for repetitive elements in pea genome evolution. Compared to other sequenced Leguminosae genomes, the pea genome shows intense gene dynamics, most likely associated with genome size expansion when the Fabeae diverged from its sister tribes. During Pisum evolution, translocation and transposition differentially occurred across lineages. This reference sequence will accelerate our understanding of the molecular basis of agronomically important traits and support crop improvement.

Development of a Sequence-Based Reference Physical Map of Pea (Pisum sativum L.).

Whole genome profiling (WGP) is a sequence-based physical mapping technology and uses sequence tags generated by next generation sequencing for construction of bacterial artificial chromosome (BAC) contigs of complex genomes. The physical map provides a framework for assembly of genome sequence and information for localization of genes that are difficult to find through positional cloning. To address the challenges of accurate assembly of the pea genome (∼4.2 GB of which approximately 85% is repetitive sequences), we have adopted the WGP technology for assembly of a pea BAC library. Multi-dimensional pooling of 295,680 BAC clones and sequencing the ends of restriction fragments of pooled DNA generated 1,814 million high quality reads, of which 825 million were deconvolutable to 1.11 million unique WGP sequence tags. These WGP tags were used to assemble 220,013 BACs into contigs. Assembly of the BAC clones using the modified Fingerprinted Contigs (FPC) program has resulted in 13,040 contigs, consisting of 213,719 BACs, and 6,294 singleton BACs. The average contig size is 0.33 Mbp and the N50 contig size is 0.62 Mbp. WGPTM technology has proved to provide a robust physical map of the pea genome, which would have been difficult to assemble using traditional restriction digestion based methods. This sequence-based physical map will be useful to assemble the genome sequence of pea. Additionally, the 1.1 million WGP tags will support efficient assignment of sequence scaffolds to the BAC clones, and thus an efficient sequencing of BAC pools with targeted genome regions of interest.

Correction to: Genome-wide SNP discovery for development of high-density genetic map and QTL mapping of ascochyta blight resistance in chickpea (Cicer arietinum L.).

Unfortunately there was an error in the name of the gene "Ca-AKL18" in the discussion section.

Genome-wide SNP discovery for development of high-density genetic map and QTL mapping of ascochyta blight resistance in chickpea (Cicer arietinum L.).

KEY MESSAGE: A high-density linkage map of chickpea using 3430 SNPs was constructed and used to identify QTLs and candidate genes for ascochyta blight resistance in chickpea. Chickpea cultivation in temperate conditions is highly vulnerable to ascochyta blight infection. Cultivation of resistant cultivars in combination with fungicide application within an informed disease management package is the most effective method to control ascochyta blight in chickpeas. Identifying new sources of resistance is critical for continued improvement in ascochyta blight resistance in chickpea. The objective of this study was to identify genetic loci and candidate genes controlling the resistance to ascochyta blight in recombinant inbred lines derived from crossing cultivars Amit and ICCV 96029. The RILs were genotyped using the genotyping-by-sequencing procedure and Illumina® GoldenGate array. The RILs were evaluated in the field over three site-years and in three independent greenhouse experiments. A genetic map with eight linkage groups was constructed using 3430 SNPs. Eight QTLs for resistance were identified on chromosomes 2, 3, 4, 5 and 6. The QTLs individually explained 7-40% of the phenotypic variations. The QTLs on chromosomes 2 and 6 were associated with the resistance at vegetative stage only. The QTLs on chromosomes 2 and 4 that were previously reported to be conserved across diverse genetic backgrounds and against different isolates of Ascochyta rabiei were confirmed in this study. Candidate genes were identified within the QTL regions. Their co-localization with the underlying QTLs was confirmed by genetic mapping. The candidate gene-based SNP markers would lead to more efficient marker-assisted selection for ascochyta blight resistance and would provide a framework for fine mapping and subsequent cloning of the genes associated with the resistance.

QTL sequencing strategy to map genomic regions associated with resistance to ascochyta blight in chickpea.

Whole-genome sequencing-based bulked segregant analysis (BSA) for mapping quantitative trait loci (QTL) provides an efficient alternative approach to conventional QTL analysis as it significantly reduces the scale and cost of analysis with comparable power to QTL detection using full mapping population. We tested the application of next-generation sequencing (NGS)-based BSA approach for mapping QTLs for ascochyta blight resistance in chickpea using two recombinant inbred line populations CPR-01 and CPR-02. Eleven QTLs in CPR-01 and six QTLs in CPR-02 populations were mapped on chromosomes Ca1, Ca2, Ca4, Ca6 and Ca7. The QTLs identified in CPR-01 using conventional biparental mapping approach were used to compare the efficiency of NGS-based BSA in detecting QTLs for ascochyta blight resistance. The QTLs on chromosomes Ca1, Ca4, Ca6 and Ca7 overlapped with the QTLs previously detected in CPR-01 using conventional QTL mapping method. The QTLs on chromosome Ca4 were detected in both populations and overlapped with the previously reported QTLs indicating conserved region for ascochyta blight resistance across different chickpea genotypes. Six candidate genes in the QTL regions identified using NGS-based BSA on chromosomes Ca2 and Ca4 were validated for their association with ascochyta blight resistance in the CPR-02 population. This study demonstrated the efficiency of NGS-based BSA as a rapid and cost-effective method to identify QTLs associated with ascochyta blight in chickpea.