The flesh color is an important appearance and quality trait of pomelo varieties. The aim of this study was to excavate variation loci and genes significantly related to the flesh color of pomelo varieties, so as to lay a foundation for further understanding the flesh color development mechanism and marker-assisted breeding of pomelo varieties.

100 pomelo accessions were used to identify their flesh color with a color difference meter, and classified based on their phenotype results. The simplified genome sequencing of 100 pomelo accessions was carried out using GBS (genotyping-by-sequencing) technology. The eigenvalues and eigenvectors of the genotype data obtained from sequencing were calculated by GCTA software to analyze the population structure, and the genetic differentiation index (Fst) of two different flesh color populations were calculated using Plink 2.0 software. The GLM model in GEMMA software was adopted to perform genome-wide association analysis, and the variation loci significantly associated with color phenotype were identified for further allelic variation analysis. According to the citrus linkage disequilibrium (LD) distance, the candidate genes located in the 25 kb adjacent region around the variation loci that might be related to the forming of flesh color were screened out, four white and four red pomelo accessions were randomly selected to perform qPCR to obtain the candidate genes expression at different fruit development stages.

According to the phenotype of flesh color, 100 accessions were divided into two categories corresponding to white and red pomelo accessions, including 58 white flesh pomelos and 42 red flesh pomelos. Six SNP sites with Fst index greater than 0.4 and -log₁₀ (P)>9 were screened out after Fst genetic differentiation analysis and GWAS genome-wide association analysis. The genotypes of six variation SNPs sites were extracted from 100 pomelos accessions and the genotypes of these SNPs could be used to predict the flesh color of pomelos. A total of 14 genes that function annotated as isoprene synthesis, plastid development, abscisic acid signal regulation, ethylene response related genes and transcription factors were screened and selected. Genes expression analysis showed that Cg7g013760 (mitogen-activated protein kinase kinase MAPKKK17), Cg7g013840 (chloroplast porphyrinogen deaminase), Cg7g014020 (transcription factor TCP7), Cg7g014120 (chloroplast superoxide dismutase SOD3), Cg7g014190 (FAD synthetase 2) were related to the forming of flesh color in pomelos.

A total of 6 SNP sites significantly associated with flesh color in pomelos accessions were identified by GWAS and Fst analysis. A total of 14 genes within the 25 kb regions around the mutation SNPs site were screened and annotated. The gene expression analysis showed that five genes might be involved in the forming of flesh color in pomelo accessions.