茶树基因组SSR位点挖掘及荧光标记开发  

SSR Loci Mining and Development Fluorescent Markers in Camellia sinensis Genome

作　　者：李栋梁 周颖怡 王景飞 张雯婷 汤适 曾繁敬 郝心愿[2] Li Dongliang;Zhou Yingyi;Wang Jingfei;Zhang Wenting;Tang Shi;Zeng Fanjing;Hao Xinyuan(Hainan Provincial Key Laboratory for Innovation and Utilization of Tropical Special Economic Plants,Institute of Tropical Horticulture Research,Hainan Academy of Agricultural Sciences,Haikou,571100;Key Laboratory of Biology,Genetics and Breeding of Special Economic Animals and Plants(Ministry of Agriculture and Rural Affairs),National Center for Tea Plant Improvement,Tea Research Institute,Chinese Academy of Agricultural Sciences,Hangzhou,310008;National Nanfan Research Institute(Sanya),Chinese Academy of Agricultural Sciences,Sanya,572024;Baisha Provincial Forest Germplasm Bank of Tea Plant,Hainan Natural Tea Corporation Limited,Baisha,572812;Hainan Natural Tea Research Institute Corporation Limited,Baisha,572812)

机构地区：[1]海南省农业科学院热带园艺研究所,热带特种经济植物种质资源创新利用重点实验室,海口571100 [2]中国农业科学院茶叶研究所,国家茶树改良中心,农业农村部特种经济动植物生物学与遗传育种重点实验室,杭州310008 [3]三亚中国农业科学院国家南繁研究院,三亚572024 [4]海南天然茶叶有限公司,白沙茶树省级林木种质资源库,白沙572812 [5]海南天然茶叶研究院有限公司,白沙572812

出　　处：《分子植物育种》2024年第17期5670-5691,共22页Molecular Plant Breeding

基　　金：海南省自然科学基金面上项目(320MS115);三亚中国农业科学院国家南繁研究院2022年“南繁专项”(YYLH03);海南省农业科学院院本级科研项目(HAAS2022KJCX03)共同资助。

摘　　要：基于NCBI数据库公开的茶树‘舒茶早’(Camellia sinensis var.'Shuchazao')全基因组序列,通过MISA在线分析工具挖掘SSR位点,并使用Primer 3.0软件批量开发设计引物,随机挑选引物检测有效性,采用荧光标记毛细管电泳技术验证筛选多态性较高的引物。结果表明,茶树全基因组中共挖掘659053个SSR位点,相对丰度212个/Mbp。二核苷酸重复类型数量最多(469096个,占比71.18%)、长度最长(8725988 bp,占比61.34%)、出现频率最高(151.06个/Mbp)、密度最大(2809.97 bp/Mbp),三核苷酸重复类型数量(99716个,占比15.13%)、长度(2801472 bp,占比19.69%)、出现频率(32.11个/Mbp)、密度(902.14 bp/Mbp)均次之。6个串联重复的SSR基序类型最多(146844个,占比22.28%),7个串联重复的SSR基序类型数量次之(87136个,占比13.22%)。从200对随机引物中筛选出137对引物可以扩增成功,其中82对可能存在多态性,使用8个茶树栽培品种进行筛选验证,其中22对引物具有多态性,其等位基因数(Na)、有效等位基因数量(Ne)、香农遗传多样性信息指数(I)、观测杂合度(Ho)、期望杂合度(He)、固定指数(F)、多态性信息含量(PIC)平均值分别为5.091、3.387、1.347、0.591、0.675、0.122、0.634,筛选出的引物中有20对高度多态性信息引物(PIC>0.5)、2对中度多态性信息引物(0.4<PIC<0.5),引物多态性丰富。通过茶树全基因组序列批量开发多态性SSR标记引物,可为茶树种质资源分子鉴定、品种权保护及辅助育种提供技术参考。Microsatellite DNA loci were identified using the MISA online analysis tool based on the published whole genome sequence of Camellia sinensis var.'Shuchazao'in the NCBI database.SSR primers were designed in batch by Primer 3.0 software,some of them were randomly selected to test their effectiveness,high polymorphic primers were screened and verified using fluorescence-labeled capillary electrophoresis technology.The results showed that 659053 microsatellites DNA loci were detected in whole genome sequences of Camellia sinensis,with a relative abundance of 212 per Mbp.In all of the loci,dinucleotide repeats were identified with the highest number(469096,accounting for 71.18%),length(8725988 bp,accounting for 61.34%),frequency(151.06 per Mbp),and density(2809.97 bp/Mbp),followed by trinucleotide repeats,with a number,length,frequency,and density of 99716(accounting for 15.13%),2801472 bp(accounting for 19.69%),32.11 per Mbp and 902.14 bp/Mbp,respectively.The SSR motif types with 6 tandem repeats were the highest(146844,accounting for 22.28%),followed by the SSR motif types with 7 tandem repeats(87136,accounting for 13.22%).137 pairs of primers out of 200 pairs of random primers were successfully amplified.Among them,82 pairs might have polymorphism.Primer screening was conducted on 8 tea cultivars,and 22 pairs of primers showed polymorphism,with average values of allele number(Na),effective allele number(Ne),Shannon's diversity index(I),observed heterozygosity(Ho),expected heterozygosity(He),fixation index(F),and polymorphism information content(PIC)of 5.091,3.387,1.347,0.591,0.675,0.122,and 0.634,respectively.Among the 22 screened primers,20 pairs were highly polymorphic information primers(PIC>0.5)and 2 pairs were polymorphic information primers(0.4<PIC<0.5),which had rich poly morphism.Based on the whole genome sequences of Camellia sinensis,polymorphic SSR primers were developed in batch,which can be used as technical references for molecular identification,variety rights protection,and molecular-assisted breed

关 键 词：茶树(Camellia sinensis) 全基因组 SSR标记 引物多态性 

分 类 号：S571.1[农业科学—茶叶生产加工]