Identification of QTLs for total flavonoid content and development of KASP markers in cultivated peanut (Arachis hypogaea L.)

Flavonoids are important nutritional components, and total flavonoid content (TFC) is a critical quality trait in peanut ( Arachis hypogaea L.) kernels. Identifying the genetic loci and candidate genes underlying TFC is essential for improving nutritional quality through molecular breeding. In this study, quantitative trait locus (QTL) mapping and genome-wide association study (GWAS) were integrated to explore the key loci of TFC in peanut. In a recombinant inbred line (RIL) population evaluated across four environments, TFC ranged from 46.96 to 131.15 mg rutin equivalents (RE)/100 g. A total of 11 QTLs associated with TFC were identified, among which qflvA03.1 and qflvA10.2 were major-effect loci, accounting for 16.95% and 11.64% of the phenotypic variation explained (PVE), respectively. In a natural population of 103 peanut accessions evaluated across three environments, TFC ranged from 63.49 to 129.27 mg RE/100 g. GWAS identified 92 significantly associated single-nucleotide polymorphisms (SNPs), explaining 9.15%-18.19% of the phenotypic variation. Four SNPs were co-localized with QTL intervals detected in the RIL population, distributed on chromosomes A06, A10, and B09, contributing 6.60%–8.9% to the PVE. Within 160 kb ( r 2 = 0.2) regions surrounding these co-localized SNPs, 33 candidate genes potentially involved in flavonoid biosynthesis were identified, including genes encoding 4-coumarate-CoA ligase, isoflavone reductase, anthocyanin 5-aromatic acyltransferase, and flavonol synthase. A Kompetitive Allele-Specific PCR marker was developed at locus A03_124522117. The KASP marker was developed from the qflvA03.1 QTL, and used to validate the association between genotype and flavonoid content. The genotypes carrying CC had significantly higher flavonoid content than those carrying TT. These findings provide insights into the genetic mechanisms underlying TFC in peanuts and establish a foundation for molecular breeding of high-quality, flavonoid-rich peanut varieties.