白三叶转录组SSR位点特征分析及引物开发

Analysis of SSR site characteristics of Trifolium repens transcriptome and primer development

本研究旨在了解白三叶(Trifoliumrepens)SSR位点信息和序列特征,开发多态性SSR引物,区分20份白三叶材料。对白三叶不同颜色的花瓣进行转录组(RNA-seq)测序,并根据转录组测序结果中的SSR位点批量设计引物,随机选择60对引物进行多态性验证,选择多态性高的引物指纹图谱构建。结果表明,白三叶转录组中含有SSR位点的序列有24960个,出现频率为13.25%,平均约5.29 kb出现1个SSR位点;白三叶转录组SSR重复碱基类型有6种,其中三碱基重复占比最高(33.70%),其次为双碱基(28.26%)和单碱基重复(25.02%),其他碱基重复类型较低,仅占总SSR的9.01%。A/T(24.85%)、AG/CT(16.48%)和AAG/CTT(8.89%)分别为单碱基到三碱基的优势重复单元;单碱基至六碱基各基元重复次数集中在4~21次,占总数量的98.11%;序列长度变化在12~35 bp,占总数的96.43%;根据SSR位点序列设计出18594对引物,占总SSR的74.50%;60对随机引物中能扩增出条带的有52对,多态性引物17对,分别占86.7%和32.7%;以多态性较高的5对引物构建的白三叶指纹图谱,能够有效地将20个不同白三叶品种区分开。本研究中白三叶转录组SSR位点分布频率高,类型丰富,具有较高的多态性,在白三叶遗传多样性分析、分子标记辅助育种和指纹图谱构建中具有较大的应用潜力。

The main study aim was to comprehensively understand the SSR loci information and sequence characteristics of white clover(Trifolium repens),develop polymorphic SSR primers,and distinguish 20 white clover varieties.An SSR site search was conducted using the sequencing data of the petals transcriptome of different colors of T.repens,and primers were designed in batches.Sixty pairs of primers were randomly selected for polymorphism verification,and primers with high polymorphism were selected for fingerprint construction.A total of 24960 SSRs were obtained,with a frequency of 13.25%,and an average of one SSR locus appeared at approximately 5.29 kb.Six SSR repeating base types were found in the transcriptome of white clover,including the main repeation types:trinucleotides(33.70%),dinucleotides(28.26%),and mononucleotides(25.02%).The other base repeating types were lower,accounting for only 9.01%of the total SSR.The main repeating motifs were A/T(24.85%),AG/CT(16.48%),and AAG/CTT(8.89%)from mononucleotide to trinucleotide,respectively.The number of repetitions of each primitive from mononucleotide to hexanucleotide concentrated between 4 and 21 times,accounting for 98.11%of the total,and the sequence length varied between 12 and 35 bp,accounting for 96.43%of the total sequence length.A total of 18594 primers were yielded from 24960 SSRs,accounting for 74.50%.Among the 60 pairs of random primers,52 and 17 pairs of could amplify bands and polymorphic primers,accounting for 86.7%and 32.7%,respectively.The fingerprint map of white clover constructed with five pairs of primers with high polymorphism could effectively distinguish the 20 different white clover varieties.The SSR loci of the white clover transcriptome in this study were showed high frequency and rich distribution,with high polymorphism,demonstrating considerable potential for application in the analysis of the genetic diversity,molecular-marker-assisted breeding,and fingerprint profile construction of white clover.