QTL mapping of polymethoxyflavone (PMF) accumulation in citrus

Polymethoxyflavones (PMFs) are bioactive flavonoids exclusively found in the genus citrus, and they show various health-promoting activities. Researchers have studied the biosynthesis of PMFs and focused on isolation and characterization of the responsible genes. However, our knowledge about the biosynthesis of PMFs is still limited. In this study, we aimed to reveal the loci for causative factors that are responsible for PMF accumulation. We investigated the frequency distributions of PMF accumulation in F₁ hybrids derived from several patterns of parent combinations. The distribution patterns in F₁ hybrids indicated that the major gene for PMF biosynthesis was recessive. We developed single-nucleotide polymorphism (SNP) markers by taking advantage of their high abundance in genomes. SNP genotyping was performed using high-resolution melting (HRM) analysis. We successfully mapped 119 SNP markers and were able to construct a linkage map consisting of nine linkage groups (LGs) with a total genetic distance of 1504.3 cM. Quantitative trait locus (QTL) analysis for PMF accumulation was carried out with the mapped SNP markers. We found three novel QTLs: two QTLs related to nobiletin accumulation in LG1 and LG8 and one QTL related to tangeretin accumulation in LG3. We found 27 candidate genes in QTL confidence intervals, which could be associated with the biosynthesis of PMFs and suggested that flavanone 3-hydroxylase, flavonoid 3′-monooxygenase, and O-methyltransferase could be the key enzymes responsible for PMF biosynthesis. Our results provide valuable information that contributes to the elucidation of the PMF biosynthetic pathway.