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.

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.

Discovering healthcare provider behavior patterns through the lens of Medicare excess charge.

BACKGROUND: The phenomenon of excess charge, where a healthcare service provider bills Medicare beyond the limit allowed for a medical procedure, is quite common in the United States public healthcare system. For example, in 2014, healthcare providers charged an average of 3.27 times (and up to 528 times) the allowable limit for cataract surgery. Previous research contends that such excess charges may be indicative of the actual amount that providers bill to non-Medicare patients and subsequent cost-shifting behavior, where a healthcare provider tries to recoup underpayment by Medicare from privately insured, self-pay, out-of-network, and uninsured patients. OBJECTIVES: The objective of this study is to examine the drivers of a provider's excess charge patterns, especially the extent to which the degree of excess charges may be associated with physician characteristics, Medicare reimbursement policy, or socioeconomic status and demographics of a provider's patient base. METHODS: Using data from the 2014 Medicare Provider Utilization files, we identify three procedures with the highest variation in Medicare reimbursements to study the excess charge phenomenon. We then employ a two-step cluster analysis within each procedure to identify distinct provider groups. RESULTS: Each procedure code yielded distinct healthcare provider segments with specific patient demographics and related behavior patterns. Cluster silhouette coefficients indicate that these segments are unique. Three random subsamples from each procedure establish the stability of the clusters. CONCLUSIONS: For each of the three procedures investigated in this study, a sizeable number of healthcare providers serving poorer, riskier patients are often paid significantly lower than their peers, and subsequently have the highest excess charges. For some providers, excess charges reveal possible cost-shifting to private insurance. Patterns of excess charges also indicate an imbalance of market power, especially in areas with lower provider competition and access to health care, thus leading to urban-rural healthcare disparities. Our results reinforce the call for price transparency and an upper limit to overbilling.

Understanding aquaporin transport system, silicon and other metalloids uptake and deposition in bottle gourd (Lagenaria siceraria).

Aquaporins (AQPs) facilitates the transport of small solutes like water, urea, carbon dioxide, boron, and silicon (Si) and plays a critical role in important physiological processes. In this study, genome-wide characterization of AQPs was performed in bottle gourd. A total of 36 AQPs were identified in the bottle gourd, which were subsequently analyzed to understand the pore-morphology, exon-intron structure, subcellular-localization. In addition, available transcriptome data was used to study the tissue-specific expression. Several AQPs showed tissue-specific expression, more notably the LsiTIP3-1 having a high level of expression in flowers and fruits. Based on the in-silico prediction of solute specificity, LsiNIP2-1 was predicted to be a Si transporter. Silicon was quantified in different tissues, including root, young leaves, mature leaves, tendrils, and fruits of bottle gourd plants. More than 1.3% Si (d.w.) was observed in bottle gourd leaves, testified the in-silico predictions. Silicon deposition evaluated with an energy-dispersive X-ray coupled with a scanning electron microscope showed a high Si accumulation in the shaft of leaf trichomes. Similarly, co-localization of Si with arsenic and antimony was observed. Expression profiling performed with real-time quantitative PCR showed differential expression of AQPs in response to Si supplementation. The information provided in the present study will be helpful to better understand the AQP transport mechanism, particularly Si and other metalloids transport and localization in plants.

Harnessing High-throughput Phenotyping and Genotyping for Enhanced Drought Tolerance in Crop Plants.

Development of drought-tolerant cultivars is one of the challenging tasks for the plant breeders due to its complex inheritance and polygenic regulation. Evaluating genetic material for drought tolerance is a complex process due to its spatiotemporal interactions with environmental factors. The conventional breeding approaches are costly, lengthy, and inefficient to achieve the expected gain in drought tolerance. In this regard, genomics-assisted breeding (GAB) offers promise to develop cultivars with improved drought tolerance in a more efficient, quicker, and cost-effective manner. The success of GAB depends upon the precision in marker-trait association and estimation of genomic estimated breeding values (GEBVs), which mostly depends on coverage and precision of genotyping and phenotyping. A wide gap between the discovery and practical use of quantitative trait loci (QTL) for crop improvement has been observed for many important agronomical traits. Such a limitation could be due to the low accuracy in QTL detection, mainly resulting from low marker density and manually collected phenotypes of complex agronomic traits. Increasing marker density using the high-throughput genotyping (HTG), and accurate and precise phenotyping using high-throughput digital phenotyping (HTP) platforms can improve the precision and power of QTL detection. Therefore, both HTG and HTP can enhance the practical utility of GAB along with a faster characterization of germplasm and breeding material. In the present review, we discussed how the recent innovations in HTG and HTP would assist in the breeding of improved drought-tolerant varieties. We have also discussed strategies, tools, and analytical advances made on the HTG and HTP along with their pros and cons.

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.

Mapping Quantitative Trait Loci for Carotenoid Concentration in Three F2 Populations of Chickpea.

CORE IDEAS: Quantitative trait locus (QTL) analyses for carotenoids in chickpea were completed for three F2 populations. A moderate number of QTLs and candidate genes associated with carotenoid concentration in chickpea seeds were identified. Green cotyledon color is positively associated with provitamin A carotenoids. Three F2 populations derived from crosses between cultivars with green and yellow cotyledon colors were used to identify quantitative trait loci (QTLs) associated with carotenoid components in chickpea (Cicer arietinum L.) seeds developed by the Crop Development Centre (CDC). Carotenoids including violaxanthin, lutein, zeaxanthin, ß-cryptoxanthin, and ß-carotene were assessed in the F2:3 seeds via high-performance liquid chromatography (HPLC). In the 'CDC Jade' × 'CDC Frontier' population, 1068 bin markers derived from the 50K Axiom CicerSNP array were mapped onto eight linkage groups (LGs). Eight QTLs, including two each for ß-carotene and zeaxanthin and one each for total carotenoids, ß-cryptoxanthin, ß-carotene, and violaxanthin were identified in this population. In the 'CDC Cory' × 'CDC Jade' population, 694 bin markers were mapped onto eight LGs and one partial LG. Quantitative trait loci for ß-cryptoxanthin, ß-carotene, violaxanthin, lutein, and total carotenoids were identified on LG8. A map with eight LGs was developed from 581 bin markers in the third population derived from the 'ICC4475' × 'CDC Jade' cross. One QTL for ß-carotene and four QTLs, one each for ß-cryptoxanthin, ß-carotene, lutein, and total carotenoids, were identified in this population. The highest phenotypic variation explained by the QTLs was for ß-carotene, which ranged from 58 to 70% in all three populations. A major gene for cotyledon color was mapped on LG8 in each population. A significant positive correlation between cotyledon color and carotenoid concentration was observed. Potential candidate genes associated with carotenoid components were obtained and their locations on the chickpea genome are presented.

The Chickpea Early Flowering 1 (Efl1) Locus Is an Ortholog of Arabidopsis ELF3.

In climates that experience short growing seasons due to drought, heat, or end-of-season frost, early flowering is a highly desirable trait for chickpea (Cicer arietinum). In this study, we mapped, sequenced, and characterized Early flowering3 (Efl3), an ortholog of Arabidopsis (Arabidopsis thaliana) EARLY FLOWERING3 (ELF3) that confers early flowering in chickpea. In a recombinant inbred line population derived from a cross between CDC Frontier and ICCV 96029, this gene was mapped to the site of a quantitative trait locus on Ca5 that explained 59% of flowering time variation under short days. Sequencing of ELF3 in ICCV 96029 revealed an 11-bp deletion in the first exon that was predicted to result in a premature stop codon. The effect of this mutation was tested by transgenic complementation in the Arabidopsis elf3-1 mutant, with the CDC Frontier form of CaELF3a partially complementing elf3-1 while the ICCV 96029 form had no effect on flowering time. While induction of FLOWERING LOCUS T homologs was very early in ICCV 96029, an analysis of circadian clock function failed to show any clear loss of rhythm in the expression of clock genes in ICCV 96029 grown under continuous light, suggesting redundancy with other ELF3 homologs or possibly an alternative mode of action for this gene in chickpea. The 11-bp deletion was associated with early flowering in global chickpea germplasm but was not widely distributed, indicating that this mutation arose relatively recently.

Genetic Analysis of NBS-LRR Gene Family in Chickpea and Their Expression Profiles in Response to Ascochyta Blight Infection.

Ascochyta blight is one of the major diseases of chickpea worldwide. The genetic resistance to ascochyta blight in chickpea is complex and governed by multiple QTLs. However, the molecular mechanism of quantitative disease resistance to ascochyta blight and the genes underlying these QTLs are still unknown. Most often disease resistance is determined by resistance (R) genes. The most predominant R-genes contain nucleotide binding site and leucine rich repeat (NBS-LRR) domains. A total of 121 NBS-LRR genes were identified in the chickpea genome. Ninety-eight of these genes contained all essential conserved domains while 23 genes were truncated. The NBS-LRR genes were grouped into eight distinct classes based on their domain architecture. Phylogenetic analysis grouped these genes into two major clusters based on their structural variation, the first cluster with toll or interleukin-1 like receptor (TIR) domain and the second cluster either with or without a coiled-coil domain. The NBS-LRR genes are distributed unevenly across the eight chickpea chromosomes and nearly 50% of the genes are present in clusters. Thirty of the NBS-LRR genes were co-localized with nine of the previously reported ascochyta blight QTLs and were tested as potential candidate genes for ascochyta blight resistance. Expression pattern of these genes was studied in two resistant (CDC Corinne and CDC Luna) and one susceptible (ICCV 96029) genotypes at different time points after ascochyta blight infection using real-time quantitative PCR. Twenty-seven NBS-LRR genes showed differential expression in response to ascochyta blight infection in at least one genotype at one time point. Among these 27 genes, the majority of the NBS-LRR genes showed differential expression after inoculation in both resistant and susceptible genotypes which indicates the involvement of these genes in response to ascochyta blight infection. Five NBS-LRR genes showed genotype specific expression. Our study provides a new insight of NBS-LRR gene family in chickpea and the potential involvement of NBS-LRR genes in response to ascochyta blight infection.

Identification and Expression Analysis of Candidate Genes Involved in Carotenoid Biosynthesis in Chickpea Seeds.

Plant carotenoids have a key role in preventing various diseases in human because of their antioxidant and provitamin A properties. Chickpea is a good source of carotenoid among legumes and its diverse germplasm and genome accessibility makes it a good model for carotenogenesis studies. The structure, location, and copy numbers of genes involved in carotenoid biosynthesis were retrieved from the chickpea genome. The majority of the single nucleotide polymorphism (SNPs) within these genes across five diverse chickpea cultivars was synonymous mutation. We examined the expression of the carotenogenesis genes and their association with carotenoid concentration at different seed development stages of five chickpea cultivars. Total carotenoid concentration ranged from 22 µg g-1 in yellow cotyledon kabuli to 44 µg g-1 in green cotyledon desi at 32 days post anthesis (DPA). The majority of carotenoids in chickpea seeds consists of lutein and zeaxanthin. The expression of the selected 19 genes involved in carotenoid biosynthesis pathway showed common pattern across five cultivars with higher expression at 8 and/or 16 DPA then dropped considerably at 24 and 32 DPA. Almost all genes were up-regulated in CDC Jade cultivar. Correlation analysis between gene expression and carotenoid concentration showed that the genes involved in the primary step of carotenoid biosynthesis pathway including carotenoid desaturase and isomerase positively correlated with various carotenoid components in chickpea seeds. A negative correlation was found between hydroxylation activity and provitamin A concentration in the seeds. The highest provitamin A concentration including ß-carotene and ß-cryptoxanthin were found in green cotyledon chickpea cultivars.

Genome-Wide Analysis of the Aquaporin Gene Family in Chickpea (Cicer arietinum L.).

Aquaporins (AQPs) are essential membrane proteins that play critical role in the transport of water and many other solutes across cell membranes. In this study, a comprehensive genome-wide analysis identified 40 AQP genes in chickpea (Cicer arietinum L.). A complete overview of the chickpea AQP (CaAQP) gene family is presented, including their chromosomal locations, gene structure, phylogeny, gene duplication, conserved functional motifs, gene expression, and conserved promoter motifs. To understand AQP's evolution, a comparative analysis of chickpea AQPs with AQP orthologs from soybean, Medicago, common bean, and Arabidopsis was performed. The chickpea AQP genes were found on all of the chickpea chromosomes, except chromosome 7, with a maximum of six genes on chromosome 6, and a minimum of one gene on chromosome 5. Gene duplication analysis indicated that the expansion of chickpea AQP gene family might have been due to segmental and tandem duplications. CaAQPs were grouped into four subfamilies including 15 NOD26-like intrinsic proteins (NIPs), 13 tonoplast intrinsic proteins (TIPs), eight plasma membrane intrinsic proteins (PIPs), and four small basic intrinsic proteins (SIPs) based on sequence similarities and phylogenetic position. Gene structure analysis revealed a highly conserved exon-intron pattern within CaAQP subfamilies supporting the CaAQP family classification. Functional prediction based on conserved Ar/R selectivity filters, Froger's residues, and specificity-determining positions suggested wide differences in substrate specificity among the subfamilies of CaAQPs. Expression analysis of the AQP genes indicated that some of the genes are tissue-specific, whereas few other AQP genes showed differential expression in response to biotic and abiotic stresses. Promoter profiling of CaAQP genes for conserved cis-acting regulatory elements revealed enrichment of cis-elements involved in circadian control, light response, defense and stress responsiveness reflecting their varying pattern of gene expression and potential involvement in biotic and abiotic stress responses. The current study presents the first detailed genome-wide analysis of the AQP gene family in chickpea and provides valuable information for further functional analysis to infer the role of AQP in the adaptation of chickpea in diverse environmental conditions.

QTL mapping of early flowering and resistance to ascochyta blight in chickpea.

In western Canada, chickpea (Cicer arietinum L.) production is challenged by short growing seasons and infestations with ascochyta blight. Research was conducted to determine the genetic basis of the association between flowering time and reaction to ascochyta blight in chickpea. Ninety-two chickpea recombinant inbred lines (RILs) developed from a cross between ICCV 96029 and CDC Frontier were evaluated for flowering responses and ascochyta blight reactions in growth chambers and fields at multiple locations and during several years. A wide range of variation was exhibited by the RILs for days to flower, days to maturity, node of first flowering, plant height, and ascochyta blight resistance. Moderate to high broad sense heritability was estimated for ascochyta blight reaction (H(2) = 0.14-0.34) and for days to flowering (H(2) = 0.45-0.87) depending on the environments. Negative correlations were observed among the RILs for days to flowering and ascochyta blight resistance, ranging from r = -0.21 (P < 0.05) to -0.58 (P < 0.0001). A genetic linkage map consisting of eight linkage groups was developed using 349 SNP markers. Seven QTLs for days to flowering were identified that individually explained 9%-44% of the phenotypic variation. Eight QTLs were identified for ascochyta blight resistance that explained phenotypic variation ranging from 10% to 19%. Clusters of QTLs for days to flowering and ascochyta blight resistances were found on chromosome 3 at the interval of 8.6-23.11 cM and on chromosome 8 at the interval of 53.88-62.33 cM.

The CarERF genes in chickpea (Cicer arietinum L.) and the identification of CarERF116 as abiotic stress responsive transcription factor.

The AP2/ERF family is one of the largest transcription factor gene families that are involved in various plant processes, especially in response to biotic and abiotic stresses. Complete genome sequences of one of the world's most important pulse crops chickpea (Cicer arietinum L.), has provided an important opportunity to identify and characterize genome-wide ERF genes. In this study, we identified 120 putative ERF genes from chickpea. The genomic organization of the chickpea ERF genes suggested that the gene family might have been expanded through the segmental duplications. The 120 member ERF family was classified into eleven distinct groups (I-X and VI-L). Transcriptional factor CarERF116, which is differentially expressed between drought tolerant and susceptible chickpea cultivar under terminal drought stress has been identified and functionally characterized. The CarERF116 encodes a putative protein of 241 amino acids and classified into group IX of ERF family. An in vitro CarERF116 protein-DNA binding assay demonstrated that CarERF116 protein specifically interacts with GCC box. We demonstrate that CarERF116 is capable of transactivation activity of and show that the functional transcriptional domain lies at the C-terminal region of the CarERF116. In transgenic Arabidopsis plants overexpressing CarERF116, significant up-regulation of several stress related genes were observed. These plants also exhibit resistance to osmotic stress and reduced sensitivity to ABA during seed germination. Based on these findings, we conclude that CarERF116 is an abiotic stress responsive gene, which plays an important role in stress tolerance. In addition, the present study leads to genome-wide identification and evolutionary analyses of chickpea ERF gene family, which will facilitate further research on this important group of genes and provides valuable resources for comparative genomics among the grain legumes.

Genetic diversity and association mapping of iron and zinc concentrations in chickpea (Cicer arietinum L.).

Chickpea (Cicer arietinum L.) is the world's second most important pulse crop after common bean. Chickpea has historically been an important daily staple in the diet of millions of people, especially in the developing countries. Current chickpea breeding programs have mainly been directed toward high yield, biotic and abiotic stress resilience that has increased global production, but less attention has been directed toward improving micronutrient concentrations in seeds. In an effort to develop micronutrient-dense chickpea lines, a study to examine the variability and to identify SNP alleles associated with seed iron and zinc concentrations was conducted using 94 diverse accessions of chickpea. The results indicated that there is substantial variability present in chickpea germplasm for seed iron and zinc concentrations. In the current set of germplasm, zinc is negatively correlated with grain yield across all locations and years; whereas the negative correlation between iron and grain yield was only significant at the Elrose locality. Eight SNP loci associated with iron and (or) zinc concentrations in chickpea seeds were identified. One SNP located on chromosome 1 (chr1) is associated with both iron and zinc concentrations. On chr4, three SNPs associated with zinc concentration and two SNPs for iron concentration were identified. Two additional SNP loci, one on chr6 and the other on chr7, were also found to be associated with iron and zinc concentrations, respectively. The results show potential opportunity for molecular breeding for improvement of seed iron and zinc concentrations in chickpea.

Identification and characterization of Wilt and salt stress-responsive microRNAs in chickpea through high-throughput sequencing.

Chickpea (Cicer arietinum) is the second most widely grown legume worldwide and is the most important pulse crop in the Indian subcontinent. Chickpea productivity is adversely affected by a large number of biotic and abiotic stresses. MicroRNAs (miRNAs) have been implicated in the regulation of plant responses to several biotic and abiotic stresses. This study is the first attempt to identify chickpea miRNAs that are associated with biotic and abiotic stresses. The wilt infection that is caused by the fungus Fusarium oxysporum f.sp. ciceris is one of the major diseases severely affecting chickpea yields. Of late, increasing soil salinization has become a major problem in realizing these potential yields. Three chickpea libraries using fungal-infected, salt-treated and untreated seedlings were constructed and sequenced using next-generation sequencing technology. A total of 12,135,571 unique reads were obtained. In addition to 122 conserved miRNAs belonging to 25 different families, 59 novel miRNAs along with their star sequences were identified. Four legume-specific miRNAs, including miR5213, miR5232, miR2111 and miR2118, were found in all of the libraries. Poly(A)-based qRT-PCR (Quantitative real-time PCR) was used to validate eleven conserved and five novel miRNAs. miR530 was highly up regulated in response to fungal infection, which targets genes encoding zinc knuckle- and microtubule-associated proteins. Many miRNAs responded in a similar fashion under both biotic and abiotic stresses, indicating the existence of cross talk between the pathways that are involved in regulating these stresses. The potential target genes for the conserved and novel miRNAs were predicted based on sequence homologies. miR166 targets a HD-ZIPIII transcription factor and was validated by 5' RLM-RACE. This study has identified several conserved and novel miRNAs in the chickpea that are associated with gene regulation following exposure to wilt and salt stress.

Genome wide SNP identification in chickpea for use in development of a high density genetic map and improvement of chickpea reference genome assembly.

BACKGROUND: In the whole genome sequencing, genetic map provides an essential framework for accurate and efficient genome assembly and validation. The main objectives of this study were to develop a high-density genetic map using RAD-Seq (Restriction-site Associated DNA Sequencing) genotyping-by-sequencing (RAD-Seq GBS) and Illumina GoldenGate assays, and to examine the alignment of the current map with the kabuli chickpea genome assembly. RESULTS: Genic single nucleotide polymorphisms (SNPs) totaling 51,632 SNPs were identified by 454 transcriptome sequencing of Cicer arietinum and Cicer reticulatum genotypes. Subsequently, an Illumina GoldenGate assay for 1,536 SNPs was developed. A total of 1,519 SNPs were successfully assayed across 92 recombinant inbred lines (RILs), of which 761 SNPs were polymorphic between the two parents. In addition, the next generation sequencing (NGS)-based GBS was applied to the same population generating 29,464 high quality SNPs. These SNPs were clustered into 626 recombination bins based on common segregation patterns. Data from the two approaches were used for the construction of a genetic map using a population derived from an intraspecific cross. The map consisted of 1,336 SNPs including 604 RAD recombination bins and 732 SNPs from Illumina GoldenGate assay. The map covered 653 cM of the chickpea genome with an average distance between adjacent markers of 0.5 cM. To date, this is the most extensive genetic map of chickpea using an intraspecific population. The alignment of the map with the CDC Frontier genome assembly revealed an overall conserved marker order; however, a few local inconsistencies within the Cicer arietinum pseudochromosome 1 (Ca1), Ca5 and Ca8 were detected. The map enabled the alignment of 215 unplaced scaffolds from the CDC Frontier draft genome assembly. The alignment also revealed varying degrees of recombination rates and hotspots across the chickpea genome. CONCLUSIONS: A high-density genetic map using RAD-Seq GBS and Illumina GoldenGate assay was developed and aligned with the existing kabuli chickpea draft genome sequence. The analysis revealed an overall conserved marker order, although some localized inversions between draft genome assembly and the genetic map were detected. The current analysis provides an insight of the recombination rates and hotspots across the chickpea genome.