翘嘴鳜转录组测序及SSR新标记的开发与应用

Transcriptome Sequencing and Development and Application of Novel SSR Markers for Siniperca chuatsi

为探讨翘嘴鳜转录组测序及SSR位点的分布和序列特征,为翘嘴鳜的SSR新标记开发及遗传研究提供便利,本研究利用高通量测序技术平台Illumina HiSeq 2500对翘嘴鳜(Sinipreca chuatsi)肌肉组织进行转录组测序及数据组装和分析,并对获得的Unigenes进行SSR检索和分析。研究共获得41533736条高质量Reads,组装为138728条平均长度为808.94 bp的非冗余Unigenes,其中57009条(41.09%)Unigenes至少于4个蛋白数据库(Nr,Swissprot,KEGG和COG)中的一个库内获得注释,7125条(5.14%)Unigenes在4个数据库中均获得注释。采用Misa、mreps和trf软件对上述138728条Unigenes进行检索,3个软件均能检索到的SSR位点共4692个,分布于4662条Unigenes,出现频率为3.36%。其优势重复基元为单核苷酸、二核苷酸和三核苷酸,分别占转录组总SSR的62.66%、24.87%、10.83%。二核苷酸重复基元中又以AC(CA)/GT(TG)为优势重复基元,三核苷酸重复基元则以AGG(GGA,GAG)/CCT(CTC,TCC)为主。使用Primer 5.0对这4962条SSR序列进行引物设计,随机选取80对进行扩增有效性和产物多态性检验。结果显示41对引物可以扩增得到清晰稳定的目标条带,有效扩增率为51.25%;其中23对可扩增得到稳定可重复的多态性产物。利用此23个SSR位点对24份翘嘴鳜材料进行遗传参数分析,期望杂合度(He)和多态信息含量(I)分别为0.198~0.888和0.175~0.857。本研究结果表明,翘嘴鳜转录组测序产生的Unigenes信息既可实现批量功能基因的挖掘,也可作为SSR标记开发的有效来源;新开发的翘嘴鳜SSR位点可应用于翘嘴鳜的遗传多样性分析、种质资源保护以及遗传育种等研究。

The purpose of this study was to explore the transcriptome sequencing,the distribution and sequence characteristics of SSR loci,and to provide convenience for the development of new SSR markers and genetic research of Siniperca chuatsi.The transcriptome of Sinipreca chuatsi muscle tissue was sequenced by the high-throughput sequencing technology platform Illumina Hiseq 2500.Then transcriptome data was assembled,and SSR analysis was carried out in obtained unigenes.A total of 41533736 high-quality reads were obtained,and 138728 non-redundant unigenes were assembled with an average length of 808.94 bp.Among them,57009 unigenes(41.09%)were at least annotated in one of Nr,Swissprot,KEGG or COG database,and 7125 unigenes(5.14%)were annotated in all four databases.For SSRs discovery,all unigenes was retrieved by using Misa,mreps and trf software to search for their common SSRs.Totally,4692 SSR loci discovered were distributed in 4662 unigenes,and at a occurrence frequency of 3.36%.Among these SSR loci,the mononucleotide,dinucleotide and trinucleotide repeats were the main types,accounting for 62.66%,24.87%and 10.83%,respectively.And among these repeat motifs,AC(CA)/GT(TG)was the dominant repeat motifs in the dinucleotide repeat motifs,and AGG(GGA,GAG)/CCT(CTC,TCC)was the dominant repeat motifs in trinucleotide repeat motifs.Using Primer 5.0 to design primers for these 4692 SSR sequences,and 80 pairs of them were randomly selected for test of PCR validity and SSR polymorphism.The results showed that 41 pairs of primers could amplify clear and stable target bands with an effective amplification rate of 51.25%,and 23 pairs of primers could amplify stable and repeatable polymorphic products.The expected heterozygosity(He)and polymorphic information content(PIC)were 0.198~0.888 and 0.175~0.857,respectively.The results showed that the unigenes information from transcriptome sequencing of Siniperca chuatsi can not only be used to excavate functional genes in batches,but also be used as an effective source of SSR marker development.The newly developed SSR loci can be used for genetic diversity analysis,germplasm resource protection and genetic breeding of Siniperca chuatsi.