Genome-wide conserved non-coding microsatellite (CNMS) marker-based integrative genetical genomics for quantitative dissection of seed weight in chickpea.

Phylogenetic footprinting identified 666 genome-wide paralogous and orthologous CNMS (conserved non-coding microsatellite) markers from 5'-untranslated and regulatory regions (URRs) of 603 protein-coding chickpea genes. The (CT)n and (GA)n CNMS carrying CTRMCAMV35S and GAGA8BKN3 regulatory elements, respectively, are abundant in the chickpea genome. The mapped genic CNMS markers with robust amplification efficiencies (94.7%) detected higher intraspecific polymorphic potential (37.6%) among genotypes, implying their immense utility in chickpea breeding and genetic analyses. Seventeen differentially expressed CNMS marker-associated genes showing strong preferential and seed tissue/developmental stage-specific expression in contrasting genotypes were selected to narrow down the gene targets underlying seed weight quantitative trait loci (QTLs)/eQTLs (expression QTLs) through integrative genetical genomics. The integration of transcript profiling with seed weight QTL/eQTL mapping, molecular haplotyping, and association analyses identified potential molecular tags (GAGA8BKN3 and RAV1AAT regulatory elements and alleles/haplotypes) in the LOB-domain-containing protein- and KANADI protein-encoding transcription factor genes controlling the cis-regulated expression for seed weight in the chickpea. This emphasizes the potential of CNMS marker-based integrative genetical genomics for the quantitative genetic dissection of complex seed weight in chickpea.

Effects of gamma irradiation on chickpea seeds vis-a-vis total seed storage proteins, antioxidant activity and protein profiling.

The present work describes radiation—induced effects on seed composition vis—à—vis total seed proteins, antioxidant levels and protein profiling employing two dimensional gel electrophoresis (2D—GE) in kabuli and desi chickpea varities. Seeds were exposed to the radiation doses of 1,2,3,4 and 5 kGy. The total protein concentrations decreased and antioxidant levels were increased with increasing dose compared to control seed samples. Radiation induced effects were dose dependent to these seed parameters while it showed tolerance to 1 kGy dose. Increase in the dose was complimented with increase in antioxidant levels, like 5 kGy enhanced % scavenging activities in all the seed extracts. Precisely, the investigations reflected that the dose range from 2 to 5 kGy was effective for total seed storage proteins, as depicted quantitatively and qualitative 2D—GE means enhance antioxidant activities in vitro.

Genetic characterization of the acetohydroxyacid synthase (AHAS) gene responsible for resistance to imidazolinone in chickpea (Cicer arietinum L.).

KEY MESSAGE: A point mutation in the AHAS1 gene leading to resistance to imidazolinone in chickpea was identified. The resistance is inherited as a single gene. A KASP marker targeting the mutation was developed. Weed control in chickpea (Cicer arietinum L.) is challenging due to poor crop competition ability and limited herbicide options. A chickpea genotype with resistance to imidazolinone (IMI) herbicides has been identified, but the genetic inheritance and the mechanism were unknown. In many plant species, resistance to IMI is caused by point mutation(s) in the acetohydroxyacid synthase (AHAS) gene resulting in an amino acid substitution preventing herbicide attachment to the molecule. The main objective of this research was to characterize the resistance to IMI herbicides in chickpea. Two homologous AHAS genes namely AHAS1 and AHAS2 sharing 80 % amino acid sequence similarity were identified in the chickpea genome. Cluster analysis indicated independent grouping of AHAS1 and AHAS2 across legume species. A point mutation in the AHAS1 gene at C675 to T675 resulting in an amino acid substitution from Ala205 to Val205 confers the resistance to IMI in chickpea. A KASP marker targeting the point mutation was developed and effectively predicted the response to IMI herbicides in a recombinant inbred (RI) population of chickpea. The RI population was used in molecular mapping where the major locus for the reaction to IMI herbicide was mapped to chromosome 5. Segregation analysis across an F2 population and RI population demonstrated that the resistance is inherited as a single gene in a semi-dominant fashion. The simple genetic inheritance and the availability of KASP marker generated in this study would speed up development of chickpea varieties with resistance to IMI herbicides.

Technological properties, antioxidant activity and total phenolic and flavonoid content of pigmented chickpea (Cicer arietinum L.) cultivars.

Chickpeas are rich sources of highly nutritious protein and dietary fibre; the health benefits of consuming legumes such as antioxidant activity (AoxA) could be effective for the expansion of their food uses. The technological properties and antioxidant potential of five pigmented chickpea cultivars were evaluated. Protein content of the grains varied from 24.9 to 27.4 g/100 g sample (dw). The cooking time (CT) of the whole grains ranged from 90.5 to 218.5 min; the lowest CT corresponded to Black ICC3761 cultivar. The total phenolic content (TPC) and AoxA [oxygen radical absorbance capacity (ORAC) value] varied from 1.23 to 1.51 mg GAE/g sample (dw) and from 5011 to 5756 µmol TE/100 g sample (dw), respectively; Red ICC13124 showed the highest ORAC value. The differences in technological properties and AoxA among cultivars could be used in chickpea breeding programmes. Chickpea cultivars could contribute significantly to the management and/or prevention of degenerative diseases associated with free radical damage.

Effect of sprouting on antioxidant and inhibitory potential of two varieties of Bengal gram (Cicer arietinum L.) against key enzymes linked to type-2 diabetes.

Type 2 diabetes is a chronic metabolic disease, and the current treatment for type 2 diabetes targets oxidative stress and postprandial hyperglycemia via the inhibition of α-glucosidase and α-amylase, key enzymes linked to type 2 diabetes. In the present study, two varieties of sprouted and non-sprouted Bengal gram (white coated and brown coated) extracts were assayed for total phenolic content, DPPH radical scavenging activity, total antioxidative capability and the inhibition of α-glucosidase and α-amylase activity. Sprouting increased the total phenolic content in both the varieties of Bengal gram and exhibited significant DPPH radical scavenging activity and antioxidant capability compared with that of non-sprouted Bengal gram. Sprouting also increased the inhibitory potential of Bengal gram against α-glucosidase and α-amylase compared with the non-sprouted variety. The overall results suggest that increased antioxidant and inhibitory potential of sprouted Bengal gram against α-glucosidase and α-amylase makes them desirable for dietary management/prevention of diabetes. This finding also provides essential information for the development of sprouted Bengal gram-derived antidiabetic products.

Identification and morphological characterization of promising kabuli chickpea genotypes for short-season environment in central India.

Chickpea (Cicer arietinum L.) is an important food legume crop grown in arid and semi-arid regions of the world. In India, kabuli chickpea is grown in central India in ~0.5 million ha, predominantly under short winter (<110 days). Efforts are underway to select promising genotypes at the Food Legume Research Platform (FLRP), Amlaha, located in intensive kabuli chickpea growing area of India. Sixty-four kabuli chickpea lines were evaluated for agronomic traits during 2017-2018 and 2018-2019 crop seasons at FLRP following simple 8 X 8 lattice design with two replications. The analysis of variance over two years revealed significant variation exists for days to flowering, plant height, maturity period, biomass, seed size and seed yield. It was observed that with similar maturity time (106 days), FLIP09-432C produced 2273 kg/ha, which out-yielded the popular variety in central India, JGK-3 by 15%. The breeding lines, FLIP09- 436C, FLIP09-171C, FLIP09-373C and FLIP09-247C were also found promising for earliness (104-110 days), and high yielding with the good yield ability (1003-2273 kg/ha). These promising genotypes for a short duration with good yield have been selected and can be used for various chickpea breeding programmes to develop high yielding varieties in central India.

Unraveling genetics of semi-determinacy and identification of markers for indeterminate stem growth habit in chickpea (Cicer arietinum L.).

Chickpea (Cicer arietinum L.) is predominantly an indeterminate plant and tends to generate vegetative growth when the ambient is conducive for soil moisture, temperature and certain other environmental conditions. The semi-determinate (SDT) types are comparatively early, resistant to lodging and found to be similar in their yield potential to indeterminate (IDT) lines. Indeterminate and semi-determinate genotypes are found to be similar during early stage, which makes it difficult to distinguish between them. Thus, there is a need to identify molecular markers linked either to indeterminate or semi-determinate plant types. The present study was carried out to study the genetics of semi-determinacy and identify molecular markers linked to stem growth habit. The study was undertaken in the cross involving BG 362(IDT) × BG 3078-1(SDT). All F1 plants were indeterminate, which indicates that indeterminate stem type is dominant over semi-determinate. In further advancement to F2 generation, F2 plants are segregated in the ratio of 3(Indeterminate): 1(Semi-determinate) that indicates that the IDT and SDT parents which are involved in the cross differed for a single gene. The segregation pattern observed in F2 is confirmed in F3 generation. The parental polymorphic survey was undertaken for molecular analysis using total of 245 SSR markers, out of which 41 polymorphic markers were found to distinguish the parents and were utilized for bulked segregant analysis (BSA). The segregation pattern in F2 indicates that the IDT (Indeterminate) and SDT (Semi-determinate) parents which are involved in the cross differed for single gene. The segregation pattern of F2 and F3 derived from the cross BG 362 (IDT) × BG 3078-1 (SDT) confirmed the genotypic structure of the newly found SDT genotype BG 3078-1 as dt1dt1Dt2Dt2. Three SSR markers TA42, Ca_GPSSR00560 and H3DO5 were found to be putatively linked to Dt1 locus regulating IDT stem growth habit. Our results indicate that the SSR markers identified for Dt1 locus helps to differentiate stem growth habit of chickpea in its early growth stage itself and can be efficiently utilized in Marker Assisted Selection (MAS) for changed plant type in chickpea.

Assembling a puzzle of dispersed retrotransposable sequences in the genome of chickpea (Cicer arietinum L.).

Several repetitive elements are known to be present in the genome of chickpea (Cicer arietinum L.) including satellite DNA and En/Spm transposons as well as two dispersed, highly repetitive elements, CaRep1 and CaRep2. PCR was used to prove that CaRep1, CaRep2, and previously isolated CaRep3 of C. arietinum represent different segments of a highly repetitive Ty3-gypsy-like retrotransposon (Metaviridae) designated CaRep that makes up large parts of the intercalary heterochromatin. The full sequence of this element including the LTRs and untranslated internal regions was isolated by selective amplification. The restriction pattern of CaRep was different within the annual species of the genus Cicer, suggesting its rearrangement during the evolution of the genus during the last 100 000 years. In addition to CaRep, another LTR and a non-LTR retrotransposon family were isolated, and their restriction patterns and physical localization in the chickpea genome were characterized. The LINE-like element CaLin is only of comparatively low abundance and reveals a considerable heterogeneity. The Ty1-copia-like element (Pseudoviridae) CaTy is located in the distal parts of the intercalary heterochromatin and adjacent euchromatic regions, but it is absent from the centromeric regions. These results together with earlier findings allow to depict the distribution of retroelements on chickpea chromosomes, which extensively resembles the retroelement landscape of the genome of the model legume Medicago truncatula Gaertn.

Resequencing of 429 chickpea accessions from 45 countries provides insights into genome diversity, domestication and agronomic traits.

We report a map of 4.97 million single-nucleotide polymorphisms of the chickpea from whole-genome resequencing of 429 lines sampled from 45 countries. We identified 122 candidate regions with 204 genes under selection during chickpea breeding. Our data suggest the Eastern Mediterranean as the primary center of origin and migration route of chickpea from the Mediterranean/Fertile Crescent to Central Asia, and probably in parallel from Central Asia to East Africa (Ethiopia) and South Asia (India). Genome-wide association studies identified 262 markers and several candidate genes for 13 traits. Our study establishes a foundation for large-scale characterization of germplasm and population genomics, and a resource for trait dissection, accelerating genetic gains in future chickpea breeding.

Ecology and genomics of an important crop wild relative as a prelude to agricultural innovation.

Domesticated species are impacted in unintended ways during domestication and breeding. Changes in the nature and intensity of selection impart genetic drift, reduce diversity, and increase the frequency of deleterious alleles. Such outcomes constrain our ability to expand the cultivation of crops into environments that differ from those under which domestication occurred. We address this need in chickpea, an important pulse legume, by harnessing the diversity of wild crop relatives. We document an extreme domestication-related genetic bottleneck and decipher the genetic history of wild populations. We provide evidence of ancestral adaptations for seed coat color crypsis, estimate the impact of environment on genetic structure and trait values, and demonstrate variation between wild and cultivated accessions for agronomic properties. A resource of genotyped, association mapping progeny functionally links the wild and cultivated gene pools and is an essential resource chickpea for improvement, while our methods inform collection of other wild crop progenitor species.

Evaluation of a miniaturized NIR spectrometer for cultivar identification: The case of barley, chickpea and sorghum in Ethiopia.

Crop cultivar identification is fundamental for agricultural research, industry and policies. This paper investigates the feasibility of using visible/near infrared hyperspectral data collected with a miniaturized NIR spectrometer to identify cultivars of barley, chickpea and sorghum in the context of Ethiopia. A total of 2650 grains of barley, chickpea and sorghum cultivars were scanned using the SCIO, a recently released miniaturized NIR spectrometer. The effects of data preprocessing techniques and choosing a machine learning algorithm on distinguishing cultivars are further evaluated. Predictive multiclass models of 24 barley cultivars, 19 chickpea cultivars and 10 sorghum cultivars delivered an accuracy of 89%, 96% and 87% on hold-out sample. The Support Vector Machine (SVM) and Partial least squares discriminant analysis (PLS-DA) algorithms consistently outperformed other algorithms. Several cultivars, believed to be widely adopted in Ethiopia, were identified with perfect accuracy. These results advance the discussion on cultivar identification survey methods by demonstrating that miniaturized NIR spectrometers represent a low-cost, rapid and viable tool. We further discuss the potential utility of the method for adoption surveys, field-scale agronomic studies, socio-economic impact assessments and value chain quality control. Finally, we provide a free tool for R to easily carry out crop cultivar identification and measure uncertainty based on spectral data.

Genomic and phenotypic analysis of Vavilov's historic landraces reveals the impact of environment and genomic islands of agronomic traits.

The Vavilov Institute of Plant Genetic Resources (VIR), in St. Petersburg, Russia, houses a unique genebank, with historical collections of landraces. When they were collected, the geographical distribution and genetic diversity of most crops closely reflected their historical patterns of cultivation established over the preceding millennia. We employed a combination of genomics, computational biology and phenotyping to characterize VIR's 147 chickpea accessions from Turkey and Ethiopia, representing chickpea's center of origin and a major location of secondary diversity. Genotyping by sequencing identified 14,059 segregating polymorphisms and genome-wide association studies revealed 28 GWAS hits in potential candidate genes likely to affect traits of agricultural importance. The proportion of polymorphisms shared among accessions is a strong predictor of phenotypic resemblance, and of environmental similarity between historical sampling sites. We found that 20 out of 28 polymorphisms, associated with multiple traits, including days to maturity, plant phenology, and yield-related traits such as pod number, localized to chromosome 4. We hypothesize that selection and introgression via inadvertent hybridization between more and less advanced morphotypes might have resulted in agricultural improvement genes being aggregated to genomic 'agro islands', and in genotype-to-phenotype relationships resembling widespread pleiotropy.

Genome-wide high-throughput SNP discovery and genotyping for understanding natural (functional) allelic diversity and domestication patterns in wild chickpea.

We identified 82489 high-quality genome-wide SNPs from 93 wild and cultivated Cicer accessions through integrated reference genome- and de novo-based GBS assays. High intra- and inter-specific polymorphic potential (66-85%) and broader natural allelic diversity (6-64%) detected by genome-wide SNPs among accessions signify their efficacy for monitoring introgression and transferring target trait-regulating genomic (gene) regions/allelic variants from wild to cultivated Cicer gene pools for genetic improvement. The population-specific assignment of wild Cicer accessions pertaining to the primary gene pool are more influenced by geographical origin/phenotypic characteristics than species/gene-pools of origination. The functional significance of allelic variants (non-synonymous and regulatory SNPs) scanned from transcription factors and stress-responsive genes in differentiating wild accessions (with potential known sources of yield-contributing and stress tolerance traits) from cultivated desi and kabuli accessions, fine-mapping/map-based cloning of QTLs and determination of LD patterns across wild and cultivated gene-pools are suitably elucidated. The correlation between phenotypic (agromorphological traits) and molecular diversity-based admixed domestication patterns within six structured populations of wild and cultivated accessions via genome-wide SNPs was apparent. This suggests utility of whole genome SNPs as a potential resource for identifying naturally selected trait-regulating genomic targets/functional allelic variants adaptive to diverse agroclimatic regions for genetic enhancement of cultivated gene-pools.

Genome-wide identification and tissue-specific expression analysis of UDP-glycosyltransferases genes confirm their abundance in Cicer arietinum (Chickpea) genome.

UDP-glycosyltransferases (EC 2.4.1.x; UGTs) are enzymes coded by an important gene family of higher plants. They are involved in the modification of secondary metabolites, phytohormones, and xenobiotics by transfer of sugar moieties from an activated nucleotide molecule to a wide range of acceptors. This modification regulates various functions like detoxification of xenobiotics, hormone homeostasis, and biosynthesis of secondary metabolites. Here, we describe the identification of 96 UGT genes in Cicer arietinum (CaUGT) and report their tissue-specific differential expression based on publically available RNA-seq and expressed sequence tag data. This analysis has established medium to high expression of 84 CaUGTs and low expression of 12 CaUGTs. We identified several closely related orthologs of CaUGTs in other genomes and compared their exon-intron arrangement. An attempt was made to assign functional specificity to chickpea UGTs by comparing substrate binding sites with experimentally determined specificity. These findings will assist in precise selection of candidate genes for various applications and understanding functional genomics of chickpea.

Extraction and characterization of chickpea (Cicer arietinum) albumin and globulin.

Albumin and globulin fractions of 1 Desi and 2 Kabuli varieties of chickpeas (Cicer arietinum) were extracted with water and salt solutions (K(2)SO(4) and NaCl). The extractable yields and particularly the albumin-globulin ratio varied greatly with the extraction medium and chickpea variety. Depending on the procedure employed, albumin could be extracted as a major fraction of chickpea proteins. Higher levels of essential amino acids and sulfur containing amino acids were found in albumins than in globulins of all chickpeas investigated. The common structural characteristics of both Kabuli and Desi chickpea albumins and globulins were clearly identified by densitometric profiles of their sodium dodecyl sulfate polyacrylamide gel patterns. Albumins contained subunits with higher molecular weights than those of globulins. The in vitro digestibility of the chickpea proteins by papain, pepsin, chymotrypsin, and trypsin indicated that globulins were more susceptible to proteolytic hydrolysis.

Interspecific relationships of the genus Cicer L. (Fabaceae) based on trnT-F sequences.

The trnT-F region in chloroplasts was sequenced to elucidate interspecific phylogenetic relationships in the genus Cicer. Twenty-five species representing four sections and two outgroups were analyzed. A phylogenetic analysis revealed three major clades in the genus Cicer. Inferred phylogenetic relationships support multiple origins of annual species in the genus Cicer. Low variation within the most perennial species in the sequence regions suggests they may have originated during a period of rapid diversification after the genus arose. High levels of sequence divergence, biogeographical patterns and morphological traits between African and Eurasian groups of species suggest that Cicer may have independently diverged on each continent. Phylogenetic analysis of sequence data did not support the monophyly of the currently recognized sections and indicated the need for a revision of the infrageneric classification.