基于八角转录组序列的SSR分子标记引物开发  

Development of SSR molecular markers based on transcriptome sequencing of Illicium verum

作　　者：余兴华[1] 杨艳梅[2] 李玉祥[1] 陶永宏[1] 孟凡来[1] 滕娟[1] YU Xing-hua;YANG Yan-mei;LI Yu-xiang;TAO Yong-hong;MENG Fan-lai;TENG Juan(Wenshan State Academy of Agricultural Sciences,Wenshan,Yunnan 663099,China;Yunnan Agricultural University,Kunming 650201,China)

机构地区：[1]文山州农业科学院,云南文山663099 [2]云南农业大学,昆明650201

出　　处：《西南农业学报》2022年第6期1261-1268,共8页Southwest China Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31760417);文山州基础科学研究项目(2019WF03);文山州创新人才项目(202136);文山州政府八角高质量发展项目(202126)。

摘　　要：【目的】为解决八角(Illicium verum)缺乏的SSR分子标记,以八角转录组数据为基础,开发EST-SSR分子标记,揭示其在转录组序列中的分布类型及特征,为八角分子生物学研究奠定基础。【方法】应用SSR搜索程序从八角转录组数据中筛选SSR位点,并对SSR重复基序种类、数量、分布特征等进行统计分析。应用在线设计软件对SSR引物进行设计,采用毛细管电泳的方法检测并确定引物多态性,应用筛选到的多态性引物分析不同八角种质间的遗传关系。【结果】从44267条Unigenes中获得8210个SSR位点,含一个EST-SSR位点的占比为11.11%,含2个或2个以上EST-SSR位点的占比为3.13%,复合SSR位点的占比为2.09%。SSR发生频率为14.24%,平均每5.37 kb出现一个SSR。在检索出的SSR位点中,二核、三核苷酸是主要重复类型,二核苷酸重复中以AG/CT占比最高,为46.03%,三核苷酸重复中以GAA/CTT占比最高,为16.58%,四、五、六核苷酸的重复类型及占比相对较少。SSR重复单元的重复次数为4~40次,其中5次重复数最多,占比22.58%,再者为6次重复,占比19.83%;微卫星的长度分布为12~80 bp,其中12~15 bp的最多,占比37.39%。随机挑选的100对引物中扩增筛选出16对多态性较高的引物,其Na为3~8、Ho为0.13~0.88、He为0.54~0.88、Ne为2.03~5.82、I为0.83~1.84、引物多态性信息量指数为0.43~0.81,平均值为0.63,表明所开发的EST-SSR位点具有较高的多态性,遗传关系指出不同八角种质间具有不同的亲缘关系。【结论】基于八角转录组序列开发EST-SSR标记是可行的,其引物具有特异性好、多态性高等优点,开发的SSR标记能为八角遗传多样性分析、功能基因挖掘、遗传连锁图谱构建等提供依据。【Objective】The present paper aimed to address the lack of SSR molecular markers in Illicium verum,based on I.verum transcriptome data,EST-SSR molecular markers were developed and the distribution types and characteristics in transcriptome sequences were revealed.The purpose of this study was to lay a foundation for the study of I.verum at molecular level.【Method】SSR loci were screened from I.verum transcriptome data by SSR search program,and the types,quantity and distribution characteristics of SSR repeat motifs were subjected to statistical analyses.An online design software was used to design SSR primers,and the polymorphism result from primers was evaluated by capillary electrophoresis.The genetic relationship between different I.verum germplasm was analyzed by using selected polymorphic primers.【Result】8210 SSR loci were obtained from 44267 Unigenes,The proportion of one EST-SSR loci was 11.11%,the proportion of two or more EST-SSR loci was 3.13%,and the proportion of composite SSR loci was 2.09%.The frequency of SSR was 14.24%,with an average of one SSR every 5.37 kb.Dinucleotides and trinucleotides were the main repeat types among the SSR loci,the highest proportion of dinucleotide repeats was AG/CT,which accounted for 46.03%,the highest proportion of trinucleotide repeats was GAA/CTT,which accounted for 16.58%,and the repetition types and proportion of four,five and six nucleotides were relatively low.The number of replicates of the SSR repeat units ranged from 4 to 40 times,and 5 repetitions were the most,accounting for 22.58%,Then there were 6 repetitions,accounting for 19.83%.The length distribution of microsatellites rangeed from 12 to 80 bp,and 12-15 bp was the most,accounting for 37.39%.Sixteen pairs of primers with high polymorphism were screened by amplification of 100 pairs of primers randomly selected.Na was 3 to 8,Ho was 0.13 to 0.88,He was 0.54 to 0.88,Ne was 2.03 to 5.82,I was 0.83 to 1.84.The primer polymorphism information index was 0.43 to 0.81,with an average of 0.63,indicatin

关 键 词：八角 转录组测序 EST-SSR 引物开发 

分 类 号：S718[农业科学—林学]