Cloning And Identification Of Genes Regulating Flesh Color And Fruit Shape In Pummelo

Pummelo(Citrus grandis)is one of the three basic species of citrus,with a relatively pure genetic background.China is one of the centers of origin of pummelo,forming a rich local resource and variety.Compared with other citrus which are disturbed by asexual reproduction,pummelo is mainly sexual reproduction and suitable for forward genetics research.Few studies have been conducted using pummelo for forward genetics because of the juvenile stage of about eight years and the large size of the tree.Flesh color and fruit shape are important breeding goals for variety selection and genetic improvement.Among the diversified germplasm resources of pummelo in our country,the flesh color and fruit shape show rich polymorphism,which provides valuable materials for mining flesh color and fruit shape regulatory genes.In this study,based on our pummelo genomic platform,we combined forward genetics,metabolomics and functional validation of genes to study the metabolite components of flesh color in the natural population of pummelo,identified the loci associated with red-fleshed traits in pummelo,cloned and functionally validated the genes regulating flesh color and fruit shape of pummelo.The main research results are as follows:1.Analysis of metabolite composition and associated SNP loci for flesh color in a natural population of pummelo.163 flesh samples of pummelo germplasm resources collected and preserved in the laboratory were analyzed for carotenoid composition using HPLC.The correspondence between flesh color and carotenoid composition was established by combining the flesh color and HPLC analysis results.Based on previous GWAS results,12 closely associated SNPs were validated in the natural population of pummelo.2.Genetic analysis of pummelo flesh color hybrid population.The laboratory previously constructed three hybrid populations of WF-GXMY×PS,RF-GXMY×PS and HNHY×HM-GXMY.In 2017,WF-GXMY×PS,RF-GXMY×PS and HNHY×HMGXMY populations had 134,104 and 184 fruiting individuals respectively.The fruit flesh color of the WF-GXMY×PS and RF-GXMY×PS population offspring has no color separation,while flesh color index value of the HNHY×HM-GXMY population offspring showed a skewness distribution.The fruit flesh color of the HNHY×HMGXMY population offspring was observed in 2017,2018 and 2019,and the color performance of the fruit flesh of the same individual in different years is stable,indicating that the ’HNHY’ pummelo flesh color is a heritable quantitative trait.From the results of natural population association analysis,it was identified that a SNP locus had the highest association with flesh color,and co-segregated with extreme individuals of the offspring of the HNHY×HM-GXMY population.3.Identification and functional analysis of key genes regulating pummelo flesh color.By analyzing the transcriptome data of the flesh of white flesh ’GBY’ pummelo,red flesh ’HNHY’ pummelo and the extreme individual of red flesh HP337 and white flesh HP146 in the HNHY×HM-GXMY population offspring,a candidate regulatory gene CitSDG40 was identified with an expression pattern consistent with flesh color change.The expression of CitSDG40 was analyzed by q RT-PCR in the flesh of white flesh ’WBY’ and red flesh ’QDL’ pummelo at different developmental stages and in the flesh of five pairs of white flesh and red flesh extreme individuals in the HNHY×HMGXMY population offspring.It showed that CitSDG40 expression was higher in red flesh pummelo.Moreover,CitSDG40 and phytoene synthase(PSY)gene,a critical gene of the carotenoid pathway,were physically adjacent to each other on the chromosome,and there was a partial overlap region between the two promoters.The subcellular localization results indicated that CitSDG40 was located at the nucleus.Stable overexpression of CitSDG40 in citrus Rm callus significantly increased total carotenoid content,and the expression of PSY gene and DXR gene in the MEP pathway were significantly higher in overexpression lines than in control.But there was no obvious phenotypic difference between the overexpression lines and control in Arabidopsis.Combining previous GWAS results and flesh transcriptome data,a CitPITP1 gene containing the CRAL＿TRIO domain was identified as having an expression pattern consistent with flesh color changes.The subcellular localization results indicated that CitPITP1 was located at the cytoplasm.Arabidopsis overexpressing CitPITP1 showed no significant phenotypic differences from the control.It was also found that the coding sequence of CitPITP1 can serve as a prokaryotic promoter.4.Identification and functional analysis of key genes regulating pummelo fruit shape.A total of 16 OFPs were identified in the pummelo genome by bioinformatics.A phylogenetic tree of OFPs was constructed with members of the Arabidopsis and pummelo OVATE gene families and the Slovate gene.The 16 OFPs genes in pummelo were divided into three subfamilies,ClassⅠ,ClassⅡ and ClassⅢ.The expression patterns of the 16 OFPs were analyzed using the typical pear-shaped ’GBY’ and the round ’HNHY’ pummelo.The CitOFP19 expression in ovary of round pummelo is significantly higher than that in pear-shaped pummelo.In tomato,CitOFP19 was stably overexpressed and the overexpression lines showed pear-shaped fruits,while the control fruits were round,indicating that CitOFP19 plays an important role in regulating fruit shape traits.In this study,we carefully analyzed the corresponding relationship between the color of pummelo flesh and carotenoids,and identified a SNP highly associated with flesh color.We revealed the genetic characteristics of flesh color of GXMY and HNHY pummelo,and identified a CitSDG40 gene that regulates carotenoid homeostasis in citrus.Through genome-wide analysis of the OVATE gene family,we identified a CitOFP19 gene that regulates citrus fruit shape.This study provides genetic resources and SNP locus information for molecular marker-assisted breeding as well as genomic selection for breeding.