Integrated high-density genetic maps in multi-parent F2 mapping populations provide a framework to unravel the genomic basis of agro-qualitative and metabolic traits in pepper (Capsicum annuum)

Pepper (Capsicum spp.) is a major vegetable crops renowned for its nutritional value and for the content of bioactive compounds with beneficial health effects. Due to its extensive agro-qualitative variability, this crop has a wide range of food and non-food uses, which drive its commercial value. In this study, quantitative trait loci (QTL) mapping and candidate gene analyses were used dissect the genetic architecture of key traits impacting overall pepper fruit quality. Two intra-specific F2 populations (n = 226) were developed by crossing highly phenotypically divergent and genetically distant parents. Using double digest restriction-site associated DNA (ddRAD-seq), over 5,000 non-distorted SNP markers were used to construct two high density genetic maps spanning approximately 2.500 cM and defining over 4,500 bins. Metabolomic profiling focused on three classes of primary and secondary metabolites including sugars (glucose, fructose, sucrose), organic acids (quinic, malic, succinic, citric, and ascorbic acid) and carotenoids (β-carotene). Simultaneously, 45 fruit agro-morphological and colour traits were assessed through automated phenotyping tools and digital analysis of over 2500 fruit section. In total, 200 QTLs were detected via inclusive composite interval mapping, with 113 QTLs co-localizing into 23 pleiotropic clusters. Eighteen candidate genes involved in the regulation of fruit organization, differentiation, and associated metabolic pathways were identified within QTL peak regions. Notably, a major hotspot at the base of chromosome 3 was found to coordinate increased sugar content and fruit size with decreased citric acid. Additional pleiotropic QTLs on chromosomes 5, 9, and 12 simultaneously increased malic acid, sucrose, and citric acid alongside fruit size parameters. Overall, agronomic and morphological traits followed a predominantly additive model, whereas many metabolite-related traits exhibited dominance or overdominance. The work aims to bridge the gap in the limited research undertaken in Capsicum to incorporate QTL analysis for agronomic, morphological, and metabolite aspects fundamental for determining the organoleptic and market-related characteristics of the fruit. Indeed, the genetic basis of many of these traits remains still largely unknown. Results from this study provide novel insight to define roadmaps toward genetic improvement for quality in pepper.