Deciphering the Genetic Basis of Polymethoxylated Flavones Biosynthesis in Citrus reticulata via the Integration of Genome-Wide Association Studies and RNA-seq

Polymethoxylated flavones (PMFs) accumulate abundantly in mandarin fruits and confer significant health benefits. However, the genetic basis underlying this trait is largely unknown. Here, we employed 198 mandarin germplasms to reveal the genetic basis of PMF accumulation. On the basis of their genetic diversity, these mandarins were categorized into five distinct subgroups. Genomic analysis indicated that pummelo introgression significantly reduced PMF contents. By GWAS, 21 PMF-associated SNPs were identified. Genotype analysis revealed that the homozygous alternative genotype (A) is superior for high PMF content and occurs predominantly in ancient mandarins. Integrating GWAS with RNA-seq identified six PMF biosynthesis-related candidate genes, among which PfkB and FBD reside within pummelo introgression sites. Transient overexpression and silencing of those two genes significantly increased and decreased the PMF content, respectively. This study revealed that pummelo introgression is a major factor influencing PMF biosynthesis and provides new insights for genetic improvement of PMFs in citrus.