基于苦楝转录组测序的SSR分子标记开发  

Development of SSR Molecular Markers Based on Transcriptome Sequencing of Melia azedarach

作　　者：蔡金峰[1] 杨晓明 郁万文[1] 汪贵斌[1] 曹福亮[1] Cai Jinfeng;Yang Xiaoming;Yu Wanwen;Wang Guibin;Cao Fuliang(Co-Innovation Center for Sustainable Forestry in Southern China Nanjing Forestry University, Nanjing 210037)

机构地区：[1]南京林业大学南方现代林业协同创新中心,南京210037

出　　处：《林业科学》2021年第6期85-92,共8页Scientia Silvae Sinicae

基　　金：江苏省农业科技自主创新资金项目(CX(16)1005)。

摘　　要：【目的】基于转录组测序数据开发苦楝SSR标记,为苦楝种质资源的选育、评价和遗传改良提供理论基础和科学依据。【方法】利用Illumina HiSeqTM 2500平台对苦楝叶片进行高通量测序,分别利用Trinity、MISA软件对转录组数据进行拼接组装、序列检索及SSR分布类型和特征分析。采用Primer 3软件设计合成100对SSR引物,分别用2%琼脂糖凝胶电泳和PAGE凝胶电泳进行初步筛选和多态性筛选。【结果】共获得Unigenes 20077条,平均长度为1431.82 bp,N50为1955 bp;对Unigenes进行SSR位点的发掘和分析,发现4116条Unigenes序列中含有5469个SSR位点,SSR的发生频率为20.50%,平均分布距离为8.74 kb;单、二、三核苷酸重复单元分别占总SSR的50.23%、24.43%和22.11%;SSR重复单元以6~10次重复的最多,占总数的51.27%。筛选出16对多态性引物并进行PCR扩增,共检测到56个等位基因片段,平均每个位点含3.5个,平均有效等位基因数为2.31个,平均Shannon多样性指数I为0.861,平均多态性信息含量PIC为0.507。【结论】苦楝转录组SSR位点具有较高的出现频率和分布密度,基元类型和重复次数相对较高,具有高多态性的潜能,可以进行有目的的引物设计和开发。开发的16对SSR引物在苦楝上可检测到较高的多态信息含量,进一步丰富了楝科现有SSR标记数据库资源,可为楝科植物在基因组水平上的遗传多样性分析和分子辅助育种等提供参考。【Objective】Based on transcriptome sequencing data,SSR markers of Melia azedarach were developed to provide theoretical and scientific basis for breeding,evaluation and genetic improvement of M.azedarach.【Method】The transcriptome of M.azedarach leaves was sequenced by Illumina HiSeqTM 2500.The transcriptome data was assembled,sequenced and analyzed by the Trinity and MISA software.Using the Primer 3 software,100 pairs of SSR primers were designed and synthesized.The 2%agarose gel electrophoresis and PAGE gel electrophoresis were used for preliminary screening and polymorphic screening.【Result】A total of 20077 unigenes were assembled and clustered based on the transcriptome sequences.The average length of unigenes was 1431.82 bp and N50 was 1955 bp.Simple Sequence Repeats(SSRs)were further analyzed in the sequences of unigenes,and 5469 SSRs from 4116 unigene sequences(20.50%of total)were identified.The average distribution distance of SSRs was 8.74 kb.The analysis of repetition types indicated that mononucleotide repetition was the main one,accounting for 50.23%,followed by dinucleotide(24.43%)and trinucleotide(22.11%).In SSR repeat units,6-10 repeats were the most,accounting for 51.27%of the total.16 pairs of polymorphic primers were screened and amplified by PCR.A total of 56 alleles were detected with an average of 3.5 alleles and 2.31 effective alleles per locus.The average Shannon’s diversity index(I)and the average polymorphism information content(PIC)were 0.861 and 0.507,respectively.【Conclusion】The frequency of SSR loci,the distribution density and repetition frequency of SSR in transcriptome of M.azedarach were all at a high level.A total of 16 primer pairs developed in this study further enriched the existing SSR markers of M.azedarach,which are valuable for further studies on genetic diversity and molecular breeding of M.azedarach.

关 键 词：苦楝 转录组 EST-SSR 引物 

分 类 号：S718.46[农业科学—林学]