Seed longevity is an important trait in the context of germplasm conservation and economics of seed production. The identification of populations with high level of genetic variability for seed longevity and associated traits will become a valuable resource for superior alleles for seed longevity. In this study, Genotyping-by-sequencing (GBS)-single nucleotide polymorphism (SNP) approach, simple sequence repeats (SSR) markers and agro-morphological traits have been explored to investigate the diversity and population structure of assembled 96 genotypes. The GBS technique performed on 96 genotypes of soybean (Glycine max (L.) Merrill) resulted in 37,897 SNPs on sequences aligned to the reference genome sequence. The average genome coverage was 6.81X with a mapping rate of 99.56% covering the entire genome. Totally, 29,955 high quality SNPs were identified after stringent filtering and most of them were detected in non-coding regions. The 96 genotypes were phenotyped for eight quantitative and ten qualitative traits by growing in field by following augmented design. The STRUCTURE (Bayesian-model based algorithm), UPGMA (Un-weighed Pair Group Method with Arithmetic mean) and principal component analysis (PCA) approaches using SSR, SNP as well as quantitative and qualitative traits revealed population structure and diversity in assembled population. The Bayesian-model based STRUCTURE using SNP markers could effectively identify clusters with higher seed longevity associated with seed coat colour and size which were subsequently validated by UPGMA and PCA based on SSR and agro-morphological traits. The results of STRUCTURE, PCA and UPGMA cluster analysis showed high degree of similarity and provided complementary data that helped to identify genotypes with higher longevity. Six black colour genotypes, viz., Local black soybean, Kalitur, ACC Nos. 39, 109, 101 and 37 showed higher seed longevity during accelerated ageing. Higher coefficient of variability observed for plant height, number of pods per plant, seed yield per plant, 100 seed weight and seed longevity confirms the diversity in assembled population and its suitability for quantitative trait loci (QTL) mapping.

Sixty genotypes with different seed coat colour and seed sizes were evaluated for seed longevity under both natural and accelerated ageing over seasons. The genotypes were grown during rabi, 2018, and summer, 2021, and freshly harvested seeds were used. For natural ageing, seeds were stored in a cloth bag in ambient condition and were removed at bimonthly intervals till 20 months. Accelerated ageing was carried out as per International Seed Testing Association (ISTA) guidelines. The germination percentage after natural and accelerated ageing over two seasons was determined. The correlation between two seasons of accelerated ageing and month-wise natural ageing was highly significant. The principal component analysis (PCA) using seed longevity grouped the majority of black genotypes into a separate cluster. Higher seed longevity was associated with black seed coat colour and small seed size. Microsatellite marker-based clustering also produced a separate cluster for majority of black genotypes and grouped the genotypes into a large number of clusters suggesting high diversity in the plant material. Two black seed coat colour genotypes, ACC No.369 and ACC No.39 consistently showed higher longevity under natural and both the years of accelerated ageing and serve as a source of alleles for higher seed longevity in soybean.