Integrated Phenotypic, Nutritional, and SSR Marker Analyses Reveal Genetic Diversity and Guide Germplasm Utilization in Lotus corniculatus

Abstract Lotus corniculatus is a perennial legume valued for its roles in forage production, soil and water conservation, and landscaping. However, the limited number of improved cultivars hampers its broader utilization. In this study, we characterized the genetic variation of 23 germplasm accessions from diverse geographic origins using 12 quantitative traits and 29 simple sequence repeat (SSR) markers. Quantitative trait analysis revealed substantial variation, particularly in plant height (CV = 31.76%), leaf area (30.09%), ether extract (38.53%), and crude fiber (31.79%). Significant correlations were observed between nutritional quality and morphological traits, indicating that phenotypic selection can indirectly improve forage quality. Cluster analysis based on phenotypic and nutritional data grouped the accessions into five categories, identifying germplasms with high crude protein and ether extract (Q1), superior leaf morphology (Q2), and high total sugar content with thick stems (Q5), each offering distinct breeding advantages. Genome-wide SSR mining identified 53,364 loci, dominated by dinucleotide repeats (52.5%), with (AT/AT)ₙ as the most frequent motif. Twenty-nine highly polymorphic SSR primers generated 299 alleles (mean = 10.17 per locus; PIC = 0.740). SSR-based clustering separated the accessions into three groups broadly aligned with geographic origin. Analysis of molecular variance (AMOVA) indicated that most genetic variation resided within populations, underscoring the potential for intra-population selection. These findings provide a germplasm classification framework that integrates phenotypic performance, nutritional quality, and genetic background, enabling breeders to select complementary parental combinations for developing L. corniculatus cultivars with improved yield, quality, and adaptability.