Unravelling the genetics of heat-responsive fruit traits in pepper [Capsicum annuum L.] via GWAS under subtropical climates

Pepper (Capsicum annuum L.) holds significant global importance as both a spice and a vegetable, extensively cultivated for its unique pungency, nutritional value, and diverse pharmaceutical uses. Being a warm tropical fruit vegetable, it is highly sensitive to heat stress (HS). However, temperature above 33 °C impairs its reproduction, subsequently impacting fruit yield and seed quality. Despite this vulnerability, pepper harbors substantial genetic diversity for key morphological, fruit, and seed traits. Therefore, studying this genetic diversity and identifying genome-wide candidate genes through GWAS for these traits under heat-stress conditions is crucial for advancing heat-resilient pepper improvement. We assessed the genotypic and phenotypic variance among 146 diverse accessions in two seasons (S1: 2022 and S2: 2023) under HS condition. Key morphological, fruit, and seed traits exhibited significant genotypic effects (P < 0.001) across seasons. A pooled ANOVA revealed significant genotype × season interactions for a number of traits, including leaf length (LL), number of primary branch (NPB), average fruit width (AFW), average fruit weight (AF Wt), and hundred-seed weight (HSW). The majority of traits exhibited high heritability (>60%) and high genetic advance (>20%) suitable for selection. Genome-wide SNP screening identified 292.91 million biallelic SNPs, with 1,04,354 high-quality SNPs chosen for GWAS. Population structure and PCA revealed three genetic subpopulations, with LD decay estimated at 580.21 kb, showing extended LD. The BLINK model with Bonferroni correction (−log₁₀(p) > 7.01) identified 58 significant MTAs across all chromosomes, with chr01 having highest (13) followed by chr03 (10), chr11 (6), chr05 (5), chr10 (5), chr06 (4), chr07 (4), chr12 (4), chr04 (3), chr08 (2) and chr09 (2). Fourteen MTAs were consistent across seasons, including one pleiotropic SNP that affects six traits. In-silico annotation revealed that the majority of potential SNPs were found in gene-rich locations. Our study offers significant insights into phenotypic and genetic diversity of pepper for heat tolerance. The BLINK model detected 14 consistent MTAs. Annotated putative genes were mainly associated with cell differentiation, hormonal signalling, flowering, fruit and seed biology, making them useful targets for climate-resilient breeding programs and MAS strategies.