Genetic Variation Of Flesh Color In Pomelo Germplasm Materials

Pomelo is an evergreen fruit tree of Citrus in Rutaceae.It is one of the three basic species of Citrus,and plays an important role in the resource utilization and market consumption structure of Citrus.Pulp color is an important quality trait of pomelo and an important factor affecting the development of pomelo industry.The color formation of pomelo pulp is mainly affected by carotenoids.Carotenoids are an important pigment that determines the color of pomelo pulp at maturity.They have rich nutritional value and play an important role in animals and plants.Therefore,carotenoids,as a pigment compound that affects the appearance quality and nutritional value of pomelo,have become an important breakthrough point for breeding workers to improve pomelo breeding.In the process of carotenoid accumulation,the expression of related genes in the synthesis pathway and the role of metabolic regulators affect it.Studies have shown that carotenoid synthesis pathway-related genes often have rich sequence variations in different materials,resulting in the function of carotenoid-related genes being affected and causing color differences.At the same time,due to the long juvenile period of pomelo fruit trees,large area of hybrid population,complex genetic background and other factors,little is known about the metabolic regulation network and related genes of pulp color.However,it is time-consuming and difficult to mine and locate the related regulatory factors of pulp color by constructing genetic hybrid population.Therefore,this study targeted sequencing of 11 genes（PSY1,PDS,ZDS,LCYB2,LCYE,BCH1,ZEP,VDE,NSY,NCED3,CCD4）in the carotenoid synthesis pathway to explore the effect of sequence variation on pulp color.At the same time,combined with GBS whole genome sequencing analysis method,the correlation analysis of pomelo pulp color was carried out to explore the related sites and related genes related to pomelo pulp color,so as to provide ideas for understanding the complex phenotypic trait of pulp color and further provide reference for the breeding improvement of pomelo.The main results of this paper are as follows:（1）The flesh color identification,statistics and diversity analysis of 157 pomelo resources were carried out.The color parameters of 81 white-fleshed pomelo,46 red-fleshed pomelo and 30 yellow-fleshed pomelo were analyzed by colormeter pro colorimeter and Photoshop software.The results showed that the color indexes L,a,b,C,H and CCI varied greatly among pomelo materials with different flesh colors.The analysis of different color parameters could screen out materials consistent with the color phenotype record.At the same time,pomelo materials with different pulp colors have obvious clustering phenomenon under a,b,C,H and CCI analysis.The color difference range between different materials can be roughly clarified by color parameters such as a,b,C,H and CCI.（2）Based on the compatible sample size of the second-generation sequencing platform chip in our laboratory,96 pomelo samples were selected for targeted sequencing analysis of 11 structural genes related to carotenoid anabolism（PSY1,PDS,ZDS,LCYB2,LCYE,BCH1,ZEP,VDE,NSY,NCED3,CCD4）.Six white-fleshed pomelo,six red-fleshed pomelo and six yellow-fleshed pomelo were selected to analyze the expression patterns of the above 11 genes.The results showed that the expression patterns of 11 genes were complex and diverse in different materials,and the high expression or low expression of a single gene could not directly determine the color of pulp.In white-fleshed pomelo,the expression of PSY1 gene decreased during development,which limited the metabolic flux of upstream carotenoids and reduced pigment accumulation.In red-fleshed pomelo,the expression of upstream genes was higher,while the expression of genes related to lycopene cyclization branch was lower,which limited the process of lycopene conversion to downstream products and made the pulp red.In yellow-fleshed pomelo,the expression of downstream genes BCH1 and ZEP is low,which may lead to the accumulation ofβ-carotene and make the flesh yellow.The targeted sequencing results of11 genes showed that the sequencing data was 2.067 Gb,Q 30≥93.39%,and the average amount of data per sample was 21.53 Mb.A total of 1046 high-quality SNPs were obtained after SNP calling and filtering.Extracting the information of different gene mutation sites,it was found that the mutation frequency of LCYB2 gene was the lowest,1SNP/53.7 bp,and the mutation frequency of ZDS was the highest,1 SNP/22.8 bp.Based on the SNP of 11 genes,the population structure of 96 materials was studied.It was found that the yellow-fleshed pomelo and red-fleshed pomelo had better population clustering effect,while the division results of white-fleshed pomelo were mixed.A total of 14significant variation sites(-log₁₀P>2.5)were screened by MLM association analysis model.Among them,9 loci were located in PSY1 gene,3 loci were located in LCYE gene,and 12 loci were corresponding to the intron of the gene.Two loci were located in the exons of ZEP gene.The genotype analysis of each locus showed that there were significant differences in the flesh color difference of the materials corresponding to different genotypes,and the flesh color could be preliminarily predicted by using different genotypes.（3）GBS whole genome sequencing analysis was performed on 100 materials of red and white pomelo.After filtering and screening,121726 high-quality SNPs were obtained.Based on the filtered SNPs,there was no obvious stratification effect on white-fleshed pomelo and red-fleshed pomelo.Combined with the previous phenotypic identification results,six SNP loci(Fst>0.4 and-log₁₀P>9)on chromosome 7 were mapped by GWAS genome-wide association analysis and Fst genetic differentiation analysis,and the gene CCD4 directly involved in the carotenoid degradation pathway was found at about 55 kb upstream of the mutation site.Through the allelic variation analysis of 6 variable sites,it was found that different genotypes of these sites could be used to judge the flesh color.After annotating the adjacent regions of the significant association sites,14 genes were screened as candidate genes and found by gene expression analysis.Cg7g013760（mitogen-activated protein kinase kinase kinase kinase kinase MAPKKK17）,Cg7g013840（bile pigment pro deaminase PBGD）,Cg7g014020（transcription factor TCP7）,Cg7g014120（superoxide dismutase SOD3）,Cg7g014190（FAD synthase 2）,Cg7g014430（ethylene response transcription factor ERF119）6 genes may be involved in the pulp color formation of pomelo.