GWAS-driven discovery and KASP validation of flowering time and seed weight loci in lentil (Lens culinaris Medik.).

Lentil agronomic traits such as flowering time and seed weight are vital for adaptation and yield improvement. While prior quantitative trait loci (QTL) mapping and genome wide association studies (GWAS) have identified genomic regions associated with these traits, trait-linked KASP markers for routine breeding remain scarce in lentil. This study evaluated the agronomic traits of 200 lentil genotypes grown in Sanya over three years and combined with genotyping-by-sequencing (GBS) data. Analysis of variance revealed significant genotypic variation, and broad-sense heritability estimates were high for all traits. Principal component analysis (PCA) biplot separated the lentil genotypes into three groups. Correlation analysis revealed significant associations among all studied traits, except days to germination initiation (DGI) and days to germination completion (DGC). A radar plot and stability analysis identified 17 top-performing and stable genotypes for breeding programs. Single nucleotide polymorphism (SNP) filtering yielded 364,976 high-quality markers. Population structure and PCA divided the genotypes into two groups (168 in Group 1, 32 in Group 2). GWAS identified 1390 significant SNPs associated with agronomic traits across the seven lentil chromosomes using MLM, BLINK, SUPER, and FarmCPU models. Haplotype blocks refined these associations to three significant SNPs and two candidate genes, primarily putatively associated with an F-box/RNI-like superfamily protein (OsFBX330) and a CCCH-type zinc finger protein with an ARM repeat domain. This study developed and internally confirmed two KASP markers (Lcu1661 and Lcu4625) associated with flowering time and seed weight in lentil, yet external validation in independent populations is needed before use in marker-assisted breeding.