柚果肉颜色遗传变异分析及候选基因挖掘  

Genetic Variation Analysis and Candidate Genes Mining of Regulating Flesh Color in Pomelo

作　　者：程莉 杨胜男 朱延松 王旭 赵婉彤 李仁静 李沛 苑忠杰 江东[1] CHENG Li;YANG ShengNan;ZHU YanSong;WANG Xu;ZHAO WanTong;LI RenJing;LI Pei;YUAN ZhongJie;JIANG Dong(Citrus Research Institute of Southwest University,Chongqing 400712)

机构地区：[1]西南大学柑桔研究所,重庆400712

出　　处：《中国农业科学》2023年第17期3420-3434,共15页Scientia Agricultura Sinica

基　　金：国家科技重点研发项目(2019YFD1001400);柑橘种质资源精准鉴定项目(19211142);西南大学实验技术研究项目(SYJ2023021)。

摘　　要：【目的】果肉颜色是柚类品种重要的外观性状以及品质性状,挖掘与柚果肉颜色显著相关的变异位点及相关基因,为进一步理解柚类品种果肉呈色机理以及分子标记辅助育种奠定基础。【方法】通过表型调查及色差仪测量,对100份柚类种质的果肉颜色进行评价和分类,利用GBS(genotyping-by-sequencing)测序技术对所有材料进行简化基因组测序。将测序得到的基因型数据通过GCTA软件计算特征值及特征向量分析群体结构,利用plink 2.0软件计算不同果肉颜色群体的遗传分化系数(Fst),选用GEMMA软件中GLM模型进行全基因组关联分析,选取与颜色表型显著关联的变异位点进行等位变异分析,根据柑橘LD大小对变异位点邻近位置进行基因注释,筛选出与果肉颜色形成相关的候选基因,随机选择4份白肉柚和4份红肉柚材料在不同发育时期对候选基因表达进行初步验证。【结果】根据果肉颜色将100份柚类种质分为白肉柚和红肉柚两大类,其中包含58份白肉柚和42份红肉柚。通过Fst遗传分化指数分析和GWAS全基因组关联分析共筛选到Fst系数大于0.4且-log10(P)>9的位点6个,对6个变异位点进行基因分型,根据基因型能够预测柚品种的果肉颜色。对变异位点侧翼25 kb序列进行基因注释共筛选出14个基因,其中包含类异戊二烯合成、叶绿体氧化还原反应、质体发育、脱落酸信号调节、乙烯响应等相关基因和转录因子。基因表达分析显示,Cg7g013760(促分裂原活化蛋白激酶激酶激酶MAPKKK17)、Cg7g013840(叶绿体卟啉原脱氨酶)、Cg7g014020(转录因子TCP7)、Cg7g014120(叶绿体超氧化物歧化酶SOD3)、Cg7g014190(FAD合成酶2)与果肉颜色形成存在一定关联。【结论】经过GWAS和Fst两种分析方式共鉴定到6个与果肉颜色显著关联的SNP位点,对该位点上下游25 kb的距离进行基因注释共筛选到了14个基因,通过基因表达模式分析初步判�【Objective】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.【Method】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.【Result】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

关 键 词：柚 果肉颜色 FST 关联分析 候选基因 

分 类 号：S666.3[农业科学—果树学]