Genome-wide association study reveals SNP markers controlling drought tolerance and related agronomic traits in chickpea across multiple environments.

Chickpea, renowned for its exceptional nutritional value, stands as a crucial crop, serving as a dietary staple in various parts of the world. However, its productivity faces a significant challenge in the form of drought stress. This challenge highlights the urgent need to find genetic markers linked to drought tolerance for effective breeding programs. The primary objective of this study is to identify genetic markers associated with drought tolerance to facilitate effective breeding programs. To address this, we cultivated 185 chickpea accessions in two distinct locations in Lebanon over a two-year period, subjecting them to both irrigated and rain-fed environments. We assessed 11 drought-linked traits, including morphology, growth, yield, and tolerance score. SNP genotyping revealed 1344 variable SNP markers distributed across the chickpea genome. Genetic diversity across populations originating from diverse geographic locations was unveiled by the PCA, clustering, and structure analysis indicating that these genotypes have descend from five or four distinct ancestors. A genome-wide association study (GWAS) revealed several marker trait associations (MTAs) associated with the traits evaluated. Within the rainfed conditions, 11 significant markers were identified, each associated with distinct chickpea traits. Another set of 11 markers exhibited associations in both rainfed and irrigated environments, reflecting shared genetic determinants across these conditions for the same trait. The analysis of linkage disequilibrium (LD) highlighted two genomic regions with notably strong LD, suggesting significant interconnections among several investigated traits. This was further investigated by the correlation between major markers associated with these traits. Gene annotation of the identified markers has unveiled insights into 28 potential genes that play a role in influencing various chickpea drought-linked traits. These traits encompass crucial aspects such as blooming organ development, plant growth, seed weight, starch metabolism, drought regulation, and height index. Among the identified genes are CPN60-2, hsp70, GDSL(GELP), AHL16, NAT3, FAB1B, bZIP, and GL21. These genes collectively contribute to the multifaceted response of chickpea plants to drought stress. Our identified genetic factors exert their influence in both irrigated and rainfed environments, emphasizing their importance in shaping chickpea characteristics.

Identification, characterization, and validation of NBS-encoding genes in grass pea.

Grass pea is a promising crop with the potential to provide food and fodder, but its genomics has not been adequately explored. Identifying genes for desirable traits, such as drought tolerance and disease resistance, is critical for improving the plant. Grass pea currently lacks known R-genes, including the nucleotide-binding site-leucine-rich repeat (NBS-LRR) gene family, which plays a key role in protecting the plant from biotic and abiotic stresses. In our study, we used the recently published grass pea genome and available transcriptomic data to identify 274 NBS-LRR genes. The evolutionary relationships between the classified genes on the reported plants and LsNBS revealed that 124 genes have TNL domains, while 150 genes have CNL domains. All genes contained exons, ranging from 1 to 7. Ten conserved motifs with lengths ranging from 16 to 30 amino acids were identified. We found TIR-domain-containing genes in 132 LsNBSs, with 63 TIR-1 and 69 TIR-2, and RX-CCLike in 84 LsNBSs. We also identified several popular motifs, including P-loop, Uup, kinase-GTPase, ABC, ChvD, CDC6, Rnase_H, Smc, CDC48, and SpoVK. According to the gene enrichment analysis, the identified genes undergo several biological processes such as plant defense, innate immunity, hydrolase activity, and DNA binding. In the upstream regions, 103 transcription factors were identified that govern the transcription of nearby genes affecting the plant excretion of salicylic acid, methyl jasmonate, ethylene, and abscisic acid. According to RNA-Seq expression analysis, 85% of the encoded genes have high expression levels. Nine LsNBS genes were selected for qPCR under salt stress conditions. The majority of the genes showed upregulation at 50 and 200 µM NaCl. However, LsNBS-D18, LsNBS-D204, and LsNBS-D180 showed reduced or drastic downregulation compared to their respective expression levels, providing further insights into the potential functions of LsNBSs under salt stress conditions. They provide valuable insights into the potential functions of LsNBSs under salt stress conditions. Our findings also shed light on the evolution and classification of NBS-LRR genes in legumes, highlighting the potential of grass pea. Further research could focus on the functional analysis of these genes, and their potential use in breeding programs to improve the salinity, drought, and disease resistance of this important crop.

Genome-wide identification, characterization, and validation of the bHLH transcription factors in grass pea.

Background: The basic helix-loop-helix (bHLH) transcription factor is a vital component in plant biology, with a significant impact on various aspects of plant growth, cell development, and physiological processes. Grass pea is a vital agricultural crop that plays a crucial role in food security. However, the lack of genomic information presents a major challenge to its improvement and development. This highlights the urgency for deeper investigation into the function of bHLH genes in grass pea to improve our understanding of this important crop. Results: The identification of bHLH genes in grass pea was performed on a genome-wide scale using genomic and transcriptomic screening. A total of 122 genes were identified as having conserved bHLH domains and were functionally and fully annotated. The LsbHLH proteins could be classified into 18 subfamilies. There were variations in intron-exon distribution, with some genes lacking introns. The cis-element and gene enrichment analyses showed that the LsbHLHs were involved in various plant functions, including response to phytohormones, flower and fruit development, and anthocyanin synthesis. A total of 28 LsbHLHs were found to have cis-elements associated with light response and endosperm expression biosynthesis. Ten conserved motifs were identified across the LsbHLH proteins. The protein-protein interaction analysis showed that all LsbHLH proteins interacted with each other, and nine of them displayed high levels of interaction. RNA-seq analysis of four Sequence Read Archive (SRA) experiments showed high expression levels of LsbHLHs across a range of environmental conditions. Seven highly expressed genes were selected for qPCR validation, and their expression patterns in response to salt stress showed that LsbHLHD4, LsbHLHD5, LsbHLHR6, LsbHLHD8, LsbHLHR14, LsbHLHR68, and LsbHLHR86 were all expressed in response to salt stress. Conclusion: The study provides an overview of the bHLH family in the grass pea genome and sheds light on the molecular mechanisms underlying the growth and evolution of this crop. The report covers the diversity in gene structure, expression patterns, and potential roles in regulating plant growth and response to environmental stress factors in grass pea. The identified candidate LsbHLHs could be utilized as a tool to enhance the resilience and adaptation of grass pea to environmental stress.

Unveiling the genetic basis of Fusarium wilt resistance in chickpea using GWAS analysis and characterization of candidate genes.

Introduction: Chickpea is a legume crop that thrives in regions with semi-arid or temperate climates. Its seeds are an excellent source of proteins, carbohydrates, and minerals, especially high-quality proteins. Chickpea cultivation faces several challenges including Fusarium wilt (FW), a major fungal disease that significantly reduces productivity. Methods: In this study, a Genome-wide Association Analysis (GWAS) was conducted to identify multiple genomic loci associated with FW resistance in chickpea. We conducted a comprehensive evaluation of 180 chickpea genotypes for FW resistance across three distinct locations (Ethiopia, Tunisia, and Lebanon) during the 2-year span from 2015 to 2016. Disease infection measurements were recorded, and the wilt incidence of each genotype was calculated. We employed a set of 11,979 single nucleotide polymorphisms (SNPs) markers distributed across the entire chickpea genome for SNP genotyping. Population structure analysis was conducted to determine the genetic structure of the genotypes. Results and Discussion: The population structure unveiled that the analyzed chickpea germplasm could be categorized into four sub-populations. Notably, these sub-populations displayed diverse geographic origins. The GWAS identified 11 SNPs associated with FW resistance, dispersed across the genome. Certain SNPs were consistent across trials, while others were specific to particular environments. Chromosome CA2 harbored five SNP markers, CA5 featured two, and CA4, CA6, CA7, and CA8 each had one representative marker. Four SNPs demonstrated an association with FW resistance, consistently observed across a minimum of three distinct environments. These SNPs included SNP5826041, SNP5825086, SNP11063413, SNP5825195, which located in CaFeSOD, CaS13like, CaNTAQ1, and CaAARS genes, respectively. Further investigations were conducted to gain insights into the functions of these genes and their role in FW resistance. This progress holds promise for reducing the negative impact of the disease on chickpea production.

The interaction between drought stress and nodule formation under multiple environments in chickpea.

Environmental stresses, particularly drought, limit symbiotic nitrogen fixation in legumes, resulting in decreased yielding capacity. Drought is one of the most important constraints limiting yield potential in crops and it is the major abiotic stress that can cause more than 70% yield loss in chickpea. In this study, a total of two hundred four chickpea (Cicer arietinum L.) genotypes were selected to study the interaction between drought stress and nodule formation. This interaction was assessed by using morphological, yield and yield components. The field experiments were laid out in two locations (Terbol and Kfardan stations, Bekaa valley, Lebanon) using Alpha lattice design with two replications and two watering treatments (irrigation and rainfed) during 2016 and 2017 seasons. Parameters that were measured include days to 50% flowering (DFL), day to maturity (DM), plant height (PLH), nodule biomass (NB), nodule fresh weight (NFW), nodule dry weight (NDW), grain yield (GY), Biological yield (BY), 100 seed weight (100SW) and drought tolerance stress (DTS). The results indicated a significant variation between genotypes, environments and other morphological, yield and yield components traits. Drought stress reduced significantly the yield and the nodule's characteristics, biological and grain yield. The genotypes with the highest levels of drought tolerance, such as IG70399, IG8256, IG71832, IG70270, and IG70272, showed a minimal decrease in yield and nodule biomass. Nodule observations significantly and positively correlated with GY (0.36-0.38) under drought stress treatment. The correlation values for nodule characteristics with DFL and DM were higher under drought stress compared to irrigated conditions. This is a comparative study between drought stress and nodule formation traits associated with morphological, yield and yield components traits.

Exploring the Genetic Variability and Potential Correlations Between Nutritional Quality and Agro-Physiological Traits in Kabuli Chickpea Germplasm Collection (Cicer arietinum L.).

Chickpea is an important source of plant-based protein and mineral elements such as iron (Fe) and zinc (Zn). The development of superior high-yielding germplasm with high nutritional value becomes central for any breeding program. Chickpea biofortified and nutrient-dense seeds can contribute to mitigate many human health problems associated with protein and micronutrients deficiency. In this study, 282 advanced chickpea lines were grown under field conditions to evaluate their agronomic performances and nutritional quality value. The trial was conducted under winter planting conditions during the cropping season 2017/2018 at ICARDA-Marchouch research station, Morocco. Results revealed high genetic variation and significant differences between the tested genotypes for all studied parameters. Under field conditions, the grain yield (GY) varied from 0.57 to 1.81 (t.ha-1), and 100-seed weight (HSW) ranged from 23.1 to 50.9 g. Out of the 282 genotypes, only 4 genotypes (i.e., S130109, S130058, S130066, and S130157) combined both good agronomic performances (GY, HSW) and high nutritional quality (protein, macronutrients, and micronutrients). Protein content ranged from 18.9 to 32.4%. For the whole collection, Fe content varied from 31.2 to 81 ppm, while Zn content ranged from 32.1 to 86.1 ppm. Correlation analysis indicated that the studied traits were significantly intercorrelated, with negative correlation between protein content and Zn concentration. Positive correlations were observed between grain filling time (F2M) and the micronutrients Zn, Cu, and Mn and macroelements K and Mg. Low positive correlation was also recorded between Pr and Fe concentrations. No significant correlation was observed between Fe and Zn. Positive correlations observed between main agronomic and nutritional quality traits makes easy any simultaneous enhancement when combining these traits.

Novel Genomic Regions Linked to Ascochyta Blight Resistance in Two Differentially Resistant Cultivars of Chickpea.

Ascochyta blight (AB), caused by the fungal pathogen Ascochyta rabiei, is a devastating foliar disease of chickpea (Cicer arietinum L.). The genotyping-by-sequencing (GBS)-based approach was deployed for mapping QTLs associated with AB resistance in chickpea in two recombinant inbred line populations derived from two crosses (AB3279 derived from ILC 1929 × ILC 3279 and AB482 derived from ILC 1929 × ILC 482) and tested in six different environments. Twenty-one different genomic regions linked to AB resistance were identified in regions CalG02 and CalG04 in both populations AB3279 and AB482. These regions contain 1,118 SNPs significantly associated with AB resistance (p ≤ 0.001), which explained 11.2-39.3% of the phenotypic variation (PVE). Nine of the AB resistance-associated genomic regions were newly detected in this study, while twelve regions were known from previous AB studies. The proposed physical map narrows down AB resistance to consistent genomic regions identified across different environments. Gene ontology (GO) assigned these QTLs to 319 genes, many of which were associated with stress and disease resistance, and with most important genes belonging to resistance gene families such as leucine-rich repeat (LRR) and transcription factor families. Our results indicate that the flowering-associated gene GIGANTEA is a possible key factor in AB resistance in chickpea. The results have identified AB resistance-associated regions on the physical genetic map of chickpea and allowed for the identification of associated markers that will help in breeding of AB-resistant varieties.

Genome-wide association analysis of chickpea germplasms differing for salinity tolerance based on DArTseq markers.

Soil salinity is significant abiotic stress that severely limits global crop production. Chickpea (Cicer arietinum L.) is an important grain legume that plays a substantial role in nutritional food security, especially in the developing world. This study used a chickpea population collected from the International Center for Agricultural Research in the Dry Area (ICARDA) genebank using the focused identification of germplasm strategy. The germplasm included 186 genotypes with broad Asian and African origins and genotyped with 1856 DArTseq markers. We conducted phenotyping for salinity in the field (Arish, Sinai, Egypt) and greenhouse hydroponic experiments at 100 mM NaCl concentration. Based on the performance in both hydroponic and field experiments, we identified seven genotypes from Azerbaijan and Pakistan (IGs: 70782, 70430, 70764, 117703, 6057, 8447, and 70249) as potential sources for high salinity tolerance. Multi-trait genome-wide association analysis (mtGWAS) detected one locus on chromosome Ca4 at 10618070 bp associated with salinity tolerance under hydroponic and field conditions. In addition, we located another locus specific to the hydroponic system on chromosome Ca2 at 30537619 bp. Gene annotation analysis revealed the location of rs5825813 within the Embryogenesis-associated protein (EMB8-like), while the location of rs5825939 is within the Ribosomal Protein Large P0 (RPLP0). Utilizing such markers in practical breeding programs can effectively improve the adaptability of current chickpea cultivars in saline soil. Moreover, researchers can use our markers to facilitate the incorporation of new genes into commercial cultivars.