Chickpeas exhibit nutritional benefits and are recommended for sustainable diets. Proximate analysis of selected chickpea genotypes revealed that these genotypes possess high macro and micro nutrient contents and show great phytochemical potential. Findings of proximate compositions are in agreement with the studies conducted earlier on legumes by other researchers [17, 18, 19, 20, 21, 22]. As far as total polyphenols and antioxidant activity are considered, our results showed significantly high level of TPC and antiradical activity which suggests that these genotypes are having substantial phytochemical properties which can be utilized in product development to cure the inflammation and malnutrition. Our results of TPC are in consistent with earlier reports [21, 23] and also showed similarity with certain under used legumes in Korea like pigeon pea (248-300 mg), groundnut (140-358 mg), kidney bean (250-320 mg) and groundnut species (100-289 mg) as reported [24]. In general, all desi had greater antioxidant activity in comparison to kabuli chickpeas. Such discrepancies in antioxidant actions amongst genotypes were additionally found in several studies that can emerge due to genetic variations, the extraction method and external ambient like rainfall, temperature etc.[25].
Our results on frequency and Characterization of Novel EST-SSR showed that the most frequent repeat type was trinucleotide (35.29%) followed by tetra (23.5%) and di nucleotide (18%) motifs. The ample of tri nucleotide motifs in the chickpea coding sequences (35.29%) was in concurrence of inspections noted in mono and dicots [26] emulating the necessity of the coding domains to perpetuate the codons [27]. In total, 348 of the 1,778 contigs encompassed SSRs (19.6%) of which 27 contained ample fringing sequences to blue print primer pairs (Table-I). In a similar study, relatively higher level of EST-SSR (11.5%) from the assessed ESTs in Cicer arietinum [28]) as compared to SSRs (3.2%) in cereal [29] was observed. However, it should be kept in mind that the myriad of SSRs excavated out of a sequence database turns on the SSR discovery criteria, the size of the dataset and the database mining tools that are operated [30]. The 27 SSR flanking primer pairs designed in the current study amplified products in the expected size range in each of the assessed chickpea genotype and 6 of these produced polymorphisms with a median PIC (0.46) value for the 9 genotypes (Supplementary Table-I).
Regarding putative functional categorization of the novel EST-SSRs, thejoint mapping and expression studies will determine the potential usefulness of markers for traits of interest. Future approaches will integrate transcriptomics and marker development in a single step. Although the level of polymorphism within EST derived SSR markers is generally lower than within SSR derived from genomic libraries [31], the markers in our study have shown to be polymorphic across several accessions. The use of SSCP analysis may further disclose internal single nucleotide polymorphisms [32]. In future, the SSRs developed from ESTs will be mapped to determine if they co-segregate with the genetic variation explained by the trait loci as an initial step towards identifying potential candidate genes.
The high PIC value observed by us is also supported [33]. Meticulous perusal and interpretations based on primers amplification, number of alleles, repeat motifs, product size, polymorphism level and PIC values indicated that ten primers namely ICCeM012, ICCeM0049, ICCeM0070, ICCeM0078, SVP55, SVP95, SVP96, SVP146, SVP213 & SVP217 revealed their efficiency as potential markers for macro-micro nutritional trait association and polymorphism studies.
The maximum genomic similarities (0.94) among 9 genotypes were expressed by the genotypes PUSA 362 vs PUSA 1105, PUSA 1103 vs PUSA 362, PUSA1105 vs PUSA 1088, PUSA 1088 vs PUSA 1103 and JG-74 vs JG-62. However, the minimum genomic similarity (0.82) was observed between the genotypes PUSA 1053 and PUSA 1108. The similarity coefficients were employed to congregate the data following the UPGMA algorithm. The consequent depicted phenogram assembled 9 genotypes into 3 distinct clusters with different sub clusters (Figure1). Similar works have also been reported [34, 35, 36, 37, 38] utilizing different molecular markers in chickpea.
We applied an integrated approach of macro-micro nutrients and molecular diversity analysis across nine chickpea genotypes. The nutritional analysis revealed maximum variation between PUSA-362 vs PUSA-1053 for the ash, PUSA-1108 vs K-850 for moisture, PUSA-1108 vs PUSA-1103 for protein, PUSA-1105 vs PUSA-1103 for fat, PUSA-1103 vs PUSA-1088 for carbohydrate, K-850 vs PUSA-1108 for fibre, K-850 vs PUSA-1053 for TPC & antiradical activity, JG-74 vs PUSA-1053 for phytate, JG-62 vs PUSA-1053 for tannin, PUSA-1053 vs JG-74 for iron and PUSA-1053 vs PUSA-1088 for zinc contents indicating richness of PUSA-362 for the trait ash, PUSA-1108 for moisture & protein, K-850 for fibre, TPC & antiradical activities, JG-74 for phytate, PUSA-1053 for iron & zinc, PUSA-1103 for carbohydrate. On the other hand, lowest contents were reflected by the PUSA-1103 for the traits protein & fat, PUSA-1053 for ash, PUSA-1088 for carbohydrate & zinc, PUSA 1053 for TPC, phytate & antiradical activities. The findings are in conformities of the earlier nutritional availability potential for the above varieties K-850, PUSA-1108 & PUSA-1053 as reported[39, 40].
The nutritional dendrogram expressed close association between the desi genotypes (PUSA-362, PUSA-1103 & K-850) by allocating them in cluster-1 and kabuli genotypes PUSA-1053 and PUSA-1108 were allocated in clusters-3 & 4 respectively indicating significant nutritional variation in desi vs kabuli genotypes. However, the molecular dendrogram expressed distant associations amongst the desi (PUSA-362, PUSA-1103 & K-850) and kabuli (PUSA-1053 & PUSA-1108) genotypes by allocating them in clusters 1A, 1A, 3, 2B & 1B respectively and placing PUSA-362 & K-850 at the two ends. Thus, Cluster analysis based on nutritional and molecular diversities partially match to each other in principle and needs an in-depth analysis to extract the advisory conclusion.
The close perusal of nutritional observations revealed overall superiority of PUSA-1103 and K-850 over PUSA-362 in the tune of earlier studies conducted [17]. The genotype PUSA-1103 has also been reported to be a resource donor for nickel and drought resistance [41].
Thus, an intense scientific interpretation suggested that the identified novel potential resources as chickpea genotypes PUSA-1103 for higher carbohydrate and zinc, K-850 for higher antiradical activity and fibre, PUSA-1108 for protein and PUSA-1053 for higher Iron, Zinc and lower TPC and phytate contents and the 10 EST-SSR markers ICCeM012, ICCeM0049, ICCeM0070, ICCeM0078, SVP55, SVP95, SVP96, SVP146, SVP213 & SVP217 may be utilized as potential donor / marker resources for the macro-micro nutritional trait specific development of mapping populations, construction of genetic maps, marker trait associations, localization of genes /QTLs for the useful nutritional traits in chickpea. Further, the identified genotypes being agronomically adopted varieties may also be utilized by food technologist and govt sponsored product-oriented schemes for amelioration of malnutrition amongst infants, children and pregnant women.