密叶红豆杉SSR位点分布特征及分子标记开发  

Distribution characteristics of SSR loci and development of molecular markers in Taxus fuana

作　　者：申响保[1] 朱妍洁 徐刚标[1] SHEN Xiangbao;ZHU Yanjie;XU Gangbiao(The Laboratory of Forestry Genetics,Central South University of Forestry&Technology,Changsha 410004,Hunan,China)

机构地区：[1]中南林业科技大学林木遗传育种实验室,湖南长沙410004

出　　处：《中南林业科技大学学报》2021年第4期139-147,共9页Journal of Central South University of Forestry & Technology

基　　金：“十三五”国家重点研发计划项目(2016YFC0503103)。

摘　　要：【目的】密叶红豆杉是西喜马拉雅地区特有种,为我国I级保护植物,也是全国120个极小种群野生植物拯救保护物种之一。了解种群遗传多样性是开展种群遗传资源保育的重要内容,开展密叶红豆杉SSR位点分布特征及分子标记开发可为研究其种群遗传多样性提供可靠的手段。【方法】基于高通量测序技术,采用de novo组装方法获取转录组序列数据。利用MISA 1.0软件在数据库中搜索SSR位点,再用Primer 5.0软件批量设计SSR引物。选用6个样本对设计的引物进行PCR扩增初步筛选,再用24个样本对有目的条带的引物进行多样性筛选,其扩增产物的毛细管电泳结果用GeneMarker软件判定产物的片段长度。最后分别用GenALEx 6.5软件、Genepop V4.7软件和CERVUS 3.0软件对筛选出来的引物扩增产物数据进行遗传参数分析。【结果】共获得39261条Unigene,搜索到2965个SSR位点,发生频率为6.7%,位点分布频率为1/12.43 kb。在剔除单核苷酸SSR位点不同重复类型中以三核苷酸重复数量最多,占总数65.1%;二核苷酸重复次之,占总数的34.0%。在三核苷酸重复基序中以AAG/CTT重复基序最多;二核苷酸重复基序中以AG/CT重复基序最多。选用的160对SSR引物中有92对引物扩增出目的条带,扩增率为57.5%;其中有24对引物的扩增产物具有多态性,多态位点百分比为26.1%。在这24对多态性SSR位点中,等位基因数(Na)、观测杂合度(Ho)、期望杂合度(He)、近交系数(Fis)以及多态信息含量(PIC)平均分别为4.958、0.497、0.671、0.246和0.619。主成分分析结果表明24个SSR位点散布性较好。【结论】在密叶红豆杉中占优势的SSR位点重复类型和重复基序与其它物种基本相同,但具有多态性的三核苷酸重复类型与其所占比例不一致。筛选出的24个SSR位点高度多态,可为后续密叶红豆杉种群遗传多样性研究提供丰富的标记引物。【Objective】Taxus fuana,a species of Taxus,is endemic to the western Himalayan region of China and listed in the national protected plants.It is also one of the 120 species of wild plants with very small populations in the national plan for rescue and protection.Understanding population genetic diversity is an important part of population genetic resources conservation.Developing SSR loci distribution characteristics and molecular markers of Taxus fuana can provide a reliable method to study population genetic diversity.【Method】Based on high-throughput sequencing technology of transcriptome sequences,de novo assembly method was used to obtain sequence data.The SSR loci were obtained from the database by MISA 1.0 software,and SSR primers were designed in batch by using Primer 5.0 software.Six samples were selected for PCR amplification and twenty-four samples were selected for diversity screening.The amplified products were analyzed by capillary electrophoresis,and the length of fragments was determined according to the GeneMarkers software.Subsequently,the genetic parameters of the amplified products were analyzed by GenALEx 6.5,Genepop V4.7 and CERVUS 3.0.【Result】In total,39261 Unigene were obtained.And 2965 SSR locus were found,with an occurrence frequency of 6.7%and a distribution frequency of 1/12.43 kb.Among the different types of SSR locus,the number of trinucleotide repeats was the largest,accounting for 65.1%of the total,followed by dinucleotide repeats,accounting for 34.0%.Additionally,the AAG/CTT trinucleotide repeat and AG/CT dinucleotide repeat occurred most frequently.By taking advantages of 160 pairs of SSR primers,92 pairs amplified the target bands with an amplification rate of 57.5%.Meanwhile,the products from 24 pairs exhibited polymorphism,and the percentage of polymorphic loci was 26.1%.Among the 24 pairs of polymorphic SSR locus,the average allele number(Na),observed heterozygosity(Ho),expected heterozygosity(He),inbreeding coefficient(Fis)and polymorphic information content(PIC)we

关 键 词：密叶红豆杉 转录组 SSR引物 频率 

分 类 号：S722.3[农业科学—林木遗传育种]