3.1 Assessment of variance: Assessment of variance was accomplished for eleven agro-morphological characters. The results (Table-2) suggested that, for 11 agro-morphological parameters, the variance attributed by genotypes was highly significant at 1% level of significance This indicates that the genotypes associated with the agro-morphological traits in this study exhibited a considerable degree of genetic variability.
3.2 Analysis of the correlation coefficient: A statistically significant and positive association was found between the seed yield per plant and other yield-related characteristics like number of days to 50% flowering (0.509**) and number of days to maturity (0.495**), as presented in Table-3. Number of days to 50% flowering and the number of days to maturity are the traits that are positively correlated with the yield of seed. This relationship between DTF and DTM with SYP helps in the development of early-maturing varieties. Early maturing varieties can sometimes escape disease and increase seed yield. Sridhar et al., (2020) showed similar results with respect to days to maturity.
3.3 PCA assessment using phenotypic data: The mean values of the morphological data were subjected to principal component analysis (PCA). In the present study, 30 genotypes were characterized into 2 different groups by principal component analysis (Fig. 4). Group-1 having 19 genotypes namely AZAD-2, PU-13-05, PU-15-2, PU-15-23, PU-15-26, PU-15-28, PU-15-29, PU-15-30, PU-15-31, PU-15-35, PU-15-40, PU-19, PU-31,PU-35, PU-7, PU-8, PU-9, PU-IPU-2-43 and PU-KU-99-21, while group-2 had 11 genotypes namely MASH-114, NU-1, PU-07-7, PU-10, PU-15-21, PU-15-32, PU-15-34, PU-17-4, PU-40, PU-KUG216 and PU-UPU-97-1. The initial two principal components (PCs), which collectively explained 47.60% of the total variations were chosen. The eigenvalues, variability %, and cumulative contributions are presented in Table-5. The highest eigen values were shown by PC1 (2.71), followed by PC-2 (2.45), PC3 (1.23) and PC4 (1.09). In PC1, the maximum contributions were shared by three characters, namely seed yield per plant (SYP), number of pods per cluster (NPC) and number of pods per plant (NPP), which are represented by red colour. The largest contribution in the second cluster was divided among the number of branches per plant, the number of seeds per plant, and the pod length (Fig. 5).
3.4 Molecular characterization of blackgram genotypes: Molecular marker-based (DNA-based) assessment of genetic diversity is an efficient method for determining the genetic diversity present of blackgram (Vigna mungo L.) genotypes. In the present investigation, thirty different blackgram genotypes were selected for molecular characterization. The blackgram plant genomic DNA was extracted with a GENELUTE PLANT GENOMIC DNA KIT. The quality of the plant genomic DNA was examined on a 0.8% agarose gel. Thirty blackgram genotypes were molecularly characterized via polyacrylamide gel electrophoresis (PAGE), and fifteen PCR-based SSR primers were used.
3.5 Primer-based amplification
The genetic diversity of blackgram was assessed among 30 blackgram (Vigna mungo L.) genotypes by using PCR based SSR markers (Table-4). Fifteen SSR primers were used in this investigation out of fifteen, fourteen SSR primers, namely, CEDG176, CEDG156, CEDG128, CEDG199, VR303, VR102, VR216, LR738A, DMBSSR217, DMBSSR182 (Fig. 2), CEDG198, CEDG092, CEDG245 and VR-9, exhibited genetic polymorphism among the blackgram (Vigna mungo L.) genotypes.
3.6 Polymorphism information content (PIC): The ability of a marker's power to identify polymorphisms among individuals in a population is measured by its polymorphism information content (PIC), and the greater this capacity is, the greater the marker's usefulness. In genetic investigations, it is one of the flags of marker quality indicators. For codominant markers, the PIC values vary from 0 (monomorphic) to 1 (very informative, containing many alleles with similar frequencies). Polymorphic information content values provide an estimate of a locus's discriminatory power by considering both the relative frequency and the number of expressed alleles. PIC value of each SSR primer is presented in Table-4. In the present study, the polymorphic information content (PIC) values ranged from 0.18 to 0.70. The highest polymorphic information content (PIC) value was exhibited by the CEDG245 primer (0.70) followed by CEDG198 (0.65) and VR-9 (0.58).
3.7 Similarity coefficients: The similarity coefficient among thirty blackgram (Vigna mungo L.) genotypes based on SSR markers amplification was analyzed via Jaccard’s coefficient of similarity; thus, the similarity matrix is presented in Table-6. Estimates of genetic similarity derived from the binomial data generated through these markers utilizing Jaccard’s coefficient ranged from 0.13 (PU-15-30/ PU-07-7) to 0.93 (PU-KUG216 between PU-9/ PU-IPU-243) exhibiting significant diversity among the thirty blackgram genotypes and suggesting their further use as potential parents in blackgram improvement breeding programs. Based on the molecular data, a similarity score of 0.93 was found between the PU-9 and PU-IPU-2-43 genotypes indicating greater genetic similarity. The results revealed significant molecular diversity among the thirty blackgram (Vigna mungo L.) genotypes.
3.8 Cluster analysis using SSR markers: The molecular data obtained from all thirty blackgram (Vigna mungo L.) genotypes via SSR markers were analyzed with unweighted paired group method on arithmetic averages (UPGMA) method via NTSYS software, and a dendrogram was constructed from the genetic similarity coefficients to explain the genetic relationships among the blackgram genotypes (Fig. 3). Thirty blackgram (Vigna mungo L.) genotypes were grouped into two major clusters. Cluster I was further divided into two subgroups comprising twelve genotypes (Azad-2, MASH-114, PU-15-23, NU-1, PU-15-26, PU-15-28, PU-15-29, PU-07-7, PU-10, PU-13-05, PU-15-21 and PU-15-2), while the other eighteen genotypes belonged to cluster II, and were further divided into two subgroups. The eighteen genotypes in cluster II, namely, PU-15-30, PU-40, PU-15-31, PU-8, PU-9, PU-IPU-2-43, PU-KUG216, PU-UPU-97-1, PU-KU-99-21, PU-31, PU-35, PU-7, PU-12-32, PU-15-34, PU-15-35, PU-15-40, PU-17-4 and PU-19, were distantly linked to one another and there seemed to be considerable amount of genetic variation among them. The findings of this study clearly showed that the fifteen SSR primers used in the present analysis revealed a significant amount of genetic variation in the genotypes of blackgram.
3.9 PCA assessment using molecular data: The molecular data were subjected to principal component analysis (PCA) in these studies. Thirty blackgram genotypes were fall in dim-1 and dim-2. Dim-1 contributed 19.1%, while dim-2 contributed 12.7% to the total diversity (Fig. 6). Based on molecular data, two different genetic groups were identified in this study i.e. group-1 had eleven genotypes, namely, PU-19, PU-15-2, PU-13-05, PU-15-21, NU-1, PU-15-34, PU-15-35, PU-07-07, PU-10, PU-17-4 and AZAD-2, which indicated by reflected by red dots and group-2 had nineteen genotypes, namely, PU-15-32, PU-KUG216, PU-IPU-2-43, PU-8, PU-40, PU-9, PU-15-30, PU-15-23, PU-UPU-97-1, PU-KU-99-21, PU-7, PU-15-31, PU-31, PU-35, PU-15-26, MASH-114, PU-15-28, PU-15-29 and PU-15-40.
3.10 Associations of SSR markers with the traits determined using Student’s t-test: For under studied eleven quantitative traits, marker traits association was studied using Student’s t-test. In the present investigation, out of fifteen markers three markers namely., VR-102 (0.0055), CEDG-156 (0.0508) and CEDG-176 (0.0505) were associated with seed yield per plant. P values from one-tailed t tests are given in brackets. Three markers viz., VR-303 (0.0070), VR-216 (0.0258), and CEDG-156 (0.0158), displayed association with the number of branches per plant. One marker, DMBSSR-182 (0.0028), exhibited an association with the number of pods per plant. The marker namely VR-102 (0.0162) associated with the number of seeds per pod. Pod length was significantly associated with DMBSSR-182 (0.0377), CEDG-156 (0.0311) and CEDG-199 (0.0408). Three markers, namely, CEDG-092 (0.0412), CEDG-176 (0.0463) and CEDG-245 (0.0227) were associated with the number of clusters per plant. The Marker, CEDG-156 (0.0262) associated with the number of pods per cluster. Among the 15 SSRs, five, CEDG-156 (0.0078), CEDG-154 (0.0047), CEDG-092 (0.0013), VR-216 (0.0337) and CEDG-176 (0.0122), were associated with 1000 seed weight. Four markers, DMBSSR-182 (0.0008), CEDG-156 (0.0069), VR-303 (0.0487) and VR-216 (0.0211), were strongly associated with plant height. Marker, VR-303 (0.0067) was strongly associated with days to fifty percent flowering.