Uncovering novel loci and developing functional Kompetitive Allele Specific PCR markers for chilling requirement in peach via genome-wide association study

Abstract Chilling requirement (CR) is a key determinant for bud dormancy release in peach [Prunus persica (L.) Batsch]. To examine the genetic basis of CR and facilitate the breeding of climate-resilient varieties, we conducted a genome-wide association study (GWAS) on a diverse panel of 213 peach accessions with their CR phenotypes. The CR phenotypic data collected over 3 years demonstrated high heritability (H2 = 0.86), indicating a strong genetic component. The GWAS analysis identified 52 SNPs associated with CR traits, with major loci clustered on chromosome 1 (17.3–21.2 and 43.7–47.3 Mb) and chromosome 2 (5.2–13.9 Mb), thereby both confirming established loci in the DAM cluster and identifying novel genetic regions. By focusing on regions exhibiting stable CR associations across years and which could be successfully validated by Kompetitive Allele Specific PCR (KASP) assays, 13 candidate CR-related genes were identified. Two highly robust KASP markers derived from loci in chromosome 1 were developed and validated. These markers effectively discriminated between low (