基于转录组测序技术的黄唇鱼SSR分子标记筛选

Development of SSR markers in Bahaba flavolabiata by transcriptome sequencing

【目的】筛选出可用于黄唇鱼(Bahaba flavolabiata)遗传资源评价及亲缘鉴定的SSR分子标记,为开展其保护遗传学研究打下基础。【方法】利用Illumina HiSeq 4000测序平台对野生黄唇鱼样本进行转录组测序,采用TGICL对转录组数据进行聚类去冗余以获得Unigene,并通过生物信息学方法进行功能注释分类、代谢通路和SSR特征分析等。【结果】黄唇鱼混合组织的转录组测序共获得13.43 Gb数据,经组装并去冗余后获得65047条Unigenes。采用BLAST对组装获得的Unigenes进行七大功能数据库(NR、NT、GO、COG、KEGG、Swissprot和Interpro)注释,结果共有55583条Unigenes被注释(占85.45%)。通过MISA对黄唇鱼的Unigene进行SSR搜索,共检测到29797个SSR位点分布于19664条Unigenes中。SSR位点分布类型及其特征分析结果显示,最主要的重复类型为二核苷酸重复基元(11855个,占39.79%),其次是单核苷酸重复基元(9695个,占32.54%)和三核苷酸重复基元(6663个,22.36%),而四核苷酸、五核苷酸和六核苷酸重复基元的数量相对较少。二核苷酸重复基元类型中重复最多的基序是AC/GT(8355条),在三核苷酸重复基元类型中重复最多的基序是AGG/CCT(1866条)。从随机选取的186对SSR引物中,最终筛选出47对扩增稳定性好且具有多态性的SSR引物,以二核苷酸和四核苷酸重复基元的扩增结果居多。【结论】通过转录组测序能有效筛选出具有多样性的黄唇鱼SSR分子标记,可为黄唇鱼的遗传多样性分析、亲缘鉴定和遗传图谱构建等后续研究提供基础资料。

【Objective】SSR markers,which could be used to evaluate genetic resources and identify genetic relatives of Bahaba flavolabiata,were selected to lay a foundation for the study of conservation genetics.【Method】Illumina HiSeq 4000 sequencing platform was used to carry out transcriptome sequencing on wild B. flavolabiata. TGICL was used to cluster and remove redundancy from transcriptome data to obtain Unigene. Functional annotation classification,metabolic pathway and SSR characteristics were analyzed by bioinformatics.【Result】Transcriptome sequencing of mixed tissues of B. flavolabiata obtained a total of 13.43 Gb data,and 65047 Unigenes were obtained after assembly and redundancy removal. Unigenes generated by BLAST were annotated in seven functional databases(NR,NT,GO,COG,KEGG,Swissprot and Interpro),and a total of 55583 Unigenes were annotated(accounting for 85.45%). Through SSR search on Unigenes of B. flavolabiata by MISA,a total of 29797 SSRs were detected distributed in 19664 Unigenes. SSR loci distribution type and its characteristic analysis results showed that,the major repeat types were dinucleotide repeat units (11855,accounting for 39.79%),followed by single nucleotide repeat units(9695,accounting for 32.54%)and trinucleotide repeat units(6663,accounting for 22.36%),while the number of tetrapotide,pentapotide and hexapotide repeat units was relatively small. The most repeated motif in dinucleotide repeats was AC/GT(8355),while the most repeated motif in dinucleotide repeats was AGG/CCT(1866). From the 186 pairs of SSR primers randomly selected,47 pairs of SSR primers with good amplification stability and polymorphism were finally selected,and the amplification results of dinucleotide and tetrauleotide repeat primitives were the majority.【Conclusion】SSR molecular markers with diversity can be effectively screened out by transcriptome sequencing,which can provide basic data for the genetic diversity analysis,genetic identification and genetic mapping construction of B. flavolabiata.