星天牛转录组SSR位点特征分析
Characteristics of the SSR loci in the Anoplophora chinensis transcriptome
韩小红；王伊凡；卢赐鼎；林浩宇；是雨霏；何 欢；张飞萍；梁光红
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DOI：10.7679/j.issn.2095-1353.2019.141
作者单位：福建农林大学林学院，生态公益林重大有害生物防控福建省高校重点实验室，福州 350002；福建农林大学林学院，生态公益林重大有害生物防控福建省高校重点实验室，福州 350002；福建农林大学林学院，生态公益林重大有害生物防控福建省高校重点实验室，福州 350002；福建农林大学林学院，生态公益林重大有害生物防控福建省高校重点实验室，福州 350002；福建农林大学林学院，生态公益林重大有害生物防控福建省高校重点实验室，福州 350002；福建农林大学林学院，生态公益林重大有害生物防控福建省高校重点实验室，福州 350002；福建农林大学林学院，生态公益林重大有害生物防控福建省高校重点实验室，福州 350002；福建农林大学林学院，生态公益林重大有害生物防控福建省高校重点实验室，福州 350002
中文关键词：星天牛；转录组；微卫星；重复类型；重复基元；引物
英文关键词：Anoplophora chinensis; transcriptome; SSR; repeat type; motif type; primer
中文摘要：
【目的】 为了获得星天牛Anoplophora chinensis的SSR位点信息并开发其SSR分子标记技术，进一步为其遗传多样性以及综合治理提供理论依据。【方法】 利用MISA软件，对星天牛转录组数据进行简单重复序列（SSR）位点筛选与分析；使用Primer 3软件设计引物，采用PCR扩增以及电泳检测，筛选SSR引物，开发星天牛SSR分子标记技术。【结果】 在9 325条unigene序列中共挖掘到2 360个SSR位点，出现频率为25.31%，涉及SSR位点序列1 758条，发生频率为18.85%。星天牛转录组中SSR的主要重复类型为单碱基重复，其次是三碱基重复，分别占总数的79.03%、12.54%。在核苷酸重复类型中，A/T基元种类数目最多，所占比例高达99.30%。SSR长度为10-11 bp的占比最高，为56.10%；重复次数为10次的数量最多，SSR位点数为1 188（50.34%）。重复次数和长度的分析结果对SSR位点的多态性获得了初步验证。在随机挑选序列设计的60对引物中，53对扩增产物达到预期大小，候选引物可用率高达88%，可在今后的研究中利用。【结论】 本文对星天牛SSR 位点的信息分析以及引物的设计与验证将有助于星天牛基因挖掘、种群遗传结构、遗传多样性、进化关系和综合治理的研究。
英文摘要：[Objectives] SSR (Simple Sequence Repeat) markers are a very useful method for species identification, molecular linkage mapping, and genetic diversity studies. In order to develop molecular markers for Anoplophora chinensis, SSR markers were identified based on transcriptome sequence. This study provides a basis for studies of genetic diversity and the integrated control of A. chinensis. [Methods] The software MISA was used to explore all microsatellite loci in the transcriptome database of A. chinensis. Primers were designed using the software Primer Premier 3.0, and screened using PCR amplification and electrophoresis. [Results] A total of 2 360 SSRs were identified through software analysis; a frequency of 25.31%. These had 1758 sequences, accounting for 18.85% of the total number of sequences. Most repeat types were mononucleotide motifs (79.03%) followed by trinucleotide motifs (12.54%). The dominant repeat type was A/T (99.30%). The proportion of SSR length to 10-11 bp was highest (56.10%), the maximum number of repeats was 10 and the number of SSR loci was 1188 (50.34%). These results indicate the feasibility of using polymorphism analysis to develop SSR markers for A. chinensis．A total of 60 primer pairs were randomly selected and verified by PCR amplification, 53 of which were able to amplify the expected products; a primer availability rate as high as 88% which could be used in future studies. [Conclusion]The information analysis of SSR loci and the design and validation of primers will be helpful for gene mining, and for research on population genetic structure, genetic diversity, evolutionary relationships and the integrated control of A. chinensis.