疣吻沙蚕转录组SSR位点鉴定及特征分析

Identification and characteristics analysis of SSR loci in the transcriptome of Tylorrhynchus heterochaetus

【目的】鉴定疣吻沙蚕转录组中简单重复序列(SSR)位点,并分析其分布规律,为该物种多态性SSR分子标记的高效开发提供理论依据。【方法】利用高通量测序技术获得疣吻沙蚕的转录组数据,经过滤、组装后使用MISA搜索鉴定SSR位点,分析其序列特征及分布规律,初步验证SSR引物的有效性,并对有效扩增引物进行多态性分析。【结果】从转录组数据组装获得79420条Unigenes,总长度为104411064 bp,N50值和N90值分别为1902和331 bp。从79420条Unigenes中鉴定到9932个SSR位点,分布在8707条Unigenes上,其中1029条至少包含1个SSR位点,SSR出现频率为8.49%,发生频率为7.43%,丰度为58.85个/Mb(未计入单核苷酸重复)。SSR重复类型共133种,重复次数为4~26次,主要集中在4~15次,长度≥20 bp的SSR约占SSR总数的20%。SSR优势重复基序为二核苷酸、单核苷酸和三核苷酸,发生频率分别为40.29%、32.12%和21.76%,其中二核苷酸重复基序以AT/TA为主,占比79.88%,单核苷酸和三核苷酸分别以A/T和AGC/GCT为主,占比分别为65.92%和29.06%。SSR位点以中等多态性的Ⅱ型为主,共有7296条,占SSR总数的80%,而高度多态性的Ⅰ型有1813条,占SSR总数的20%。筛选到11对多态性引物,每对引物平均产生3.73个多态性片段。【结论】疣吻沙蚕转录组SSR类型丰富,且具有较高的多态性,可用于SSR分子标记开发,对其种质资源评价与保护利用、种群遗传学及分子育种研究等具有实用价值。

【Objective】Simple sequence repeat(SSR)loci were detected and characterized in the transcriptome of Ty-lorrhynchus heterochaetus,the distribution pattern was analyzed to provide reference for the efficient development of SSR markers.【Method】The transcriptomic data of T.heterochaetus were obtained by high-throughput sequencing technology.After filtering and assembly,the SSR loci were searched and identified by MISA,and their characteristics and distribu-tion were analyzed.The effectiveness of SSR primers was preliminarily verified,and the polymorphism of effective amplification primers was further analyzed.【Result】Transcriptome assembly generated 79420 Unigenes(total length 104411064 bp)with a N50 length of 1902 bp and a N90 length of 331 bp.From the 79420 Unigenes,a total of 9932 SSR loci were detected and distributed on 8707 Unigenes,with SSR existence frequency of 8.49%and an occurrence frequency of 7.43%.Of which,1029 Unigenes contained more than one locus,and the SSR abundance reached 58.85 loci/Mb(single nucleotide repeats were not included).A total of 133 SSR repeat types were observed and the number of repetition ranged from 4 to 26,most of which were 4 to 15.About 20%of the SSR loci were found to be≥20 bp in length.The dominant re-peat units were dinucleotide,mononucleotide and trinucleotide,which accounted for 40.29%,32.12%and 21.76%of the total SSR loci respectively.The AT/TA was the dominant repeat unit in dinucleotide,accounting for 79.88%of this type SSR loci.The A/T and AGG/CCT were the main repeat units in mononucleotide and trinucleotides,accounting for 65.92%and 29.06%respectively.In addition,7296(80%of the total SSR)type II SSR loci with medium polymorphism were found to be predominant,and the remaining 1813(20%of the total SSR)SSR loci were type I with high polymorphism.A total of 11 pairs of SSR primers were validated to be polymorphic,and the average number of alleles per primer pair reached 3.73.【Conclusion】The SSR loci in the transcriptome of T.heterochaetus have a rich variety and high polymor-phic potential.They can be used for the development of SSR molecular markers and has practical value for the evaluation,protection and utilization of germplasm resources and the study of population genetics and molecular breeding.