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林业科学 ›› 2015, Vol. 51 ›› Issue (11): 40-49.doi: 10.11707/j.1001-7488.20151106

• 论文与研究报告 • 上一篇    下一篇

杉木转录组SSR挖掘及EST-SSR标记规模化开发

文亚峰1, 韩文军2, 周宏3, 徐刚标2   

  1. 1. 中南林业科技大学风景园林学院 长沙 410004;
    2. 中南林业科技大学林学院 长沙 410004;
    3. 广东省韶关市林业局 韶关 512000
  • 收稿日期:2015-06-01 修回日期:2015-07-23 出版日期:2015-11-25 发布日期:2015-12-08
  • 基金资助:
    国家自然科学基金项目(30972357); 湖南省自然科学基金项目(10JJ2018)。

SSR Mining and Development of EST-SSR Markers for Cunninghamia lanceolata Based on Transcriptome Sequences

Wen Yafeng1, Han Wenjun2, Zhou Hong3, Xu Gangbiao2   

  1. 1. College of Landscape Architecture, Central South University of Forestry and Technology Changsha 410004;
    2. College of Forestry, Central South University of Forestry and Technology Changsha 410004;
    3. Shaoguan Forestry Administration, Guangdong Province Shaoguan 512000
  • Received:2015-06-01 Revised:2015-07-23 Online:2015-11-25 Published:2015-12-08

摘要: [目的]为解决杉木SSR标记数量不足、已开发的位点多态性较差等问题,以杉木转录组测序数据为基础,结合多重PCR技术批量挖掘SSR,规模化开发EST-SSR位点,为杉木分子遗传学研究奠定良好基础。[方法]杉木转录组序列数据(Accession:SRX151872)从NCBI的SRA数据库下载。利用CLC和CMiB软件批量挖掘SSR位点;利用四色荧光标记通用引物多重PCR(multiplex-PCR)技术实现SSR标记的规模化开发。[结果]杉木转录组de novo assembly序列拼接共得到35633个contigs,总长度31.5 Mb,其中最小拼接长度155 bp,最大23794 bp,平均长度884 bp。得到2156个SSR位点,分布于1822个contigs中,其中256个contigs中包含1个以上SSR位点,复合型SSR数量为118个, SSR平均分布密度为68.4个/Mb。不同SSR重复单元(motif)中,三核苷酸SSR重复单元数量最多,占总数的41.7%。批量引物设计得到1582个有效位点的引物对,占SSR位点总数的73.4%。利用四色荧光标记通用引物多重PCR检测技术,对35个候选标记位点进行多态性检测,其中28个位点具有多态性,多态性位点比例达到80%,检测位点多态信息含量(PIC)平均值为0.573,表明所开发的EST-SSR位点具有很高的多态性。PCA分析结果表明, 28个EST-SSR多态性位点具有很强的鉴别杉木不同地理种源,甚至同一种源不同单株的能力。[结论]将转录组SSRs挖掘和四色荧光标记通用引物多重PCR技术相结合,成功建立杉木EST-SSR高效开发流程和方法,得到较多高质量的EST-SSR标记位点,这些位点已用于后续杉木遗传多样性保护研究。与传统SSR标记位点开发技术相比较,转录组海量序列为高质量多态性位点的选择可提供充足的数据保证。四色荧光标记通用引物基因分型结果清晰、稳定可靠,不但试验成本仅为原来的10%~15%,而且结合多重PCR扩增技术,可使试验效率提高5~6倍。新方法的建立和应用不仅能促进杉木分子遗传学相关研究,而且对其他非模式生物或新物种SSR标记开发也具有重要的参考作用。

关键词: 杉木, 微卫星标记, EST-SSR, 转录组, 序列从头拼接

Abstract: [Objective] Chinese fir (Cunninghamia lanceolata) is an important timber species distributed mainly in southern China. Current genetic analyses of this species lag behind other conifer species due to the limitation of available molecular markers. Accordingly, transcriptome sequence data were used to improve the efficiency of SSR development for the species. [Method]Utilizing Chinese fir transcriptome sequences from the Sequence Read Archive (SRA) database of NCBI. CLC and CMiB software were used to assemble sequence reads, to mine SSRs and design PCR amplicon primers for contigs that contained SSRs. Four universal fluorescent labeling primers and multiplex PCR were used to accomplish genotyping for polymorphic loci. [Result]De novo assembly produced 35633 contigs, the total length was 31.5 Mb, of which mini-and max-contigs were 155 bp and 23794 bp, respectively, with an average length of 884 bp. In total, 2156 SSRs were identified distributed in 1822(5.11%) contigs, with threshold repeat numbers of 6, 5, 4, 3 and 2 for di-, tri-, tetra-, penta-and hexa-SSRs, respectively. 256 contigs contained one or more SSRs, and the numbers of compound SSR contigs was 118. The average SSR density was 68.4 SSRs·Mb-1. The most common SSR types were tri-SSRs (41.7%), followed by hexa-(29.8%), penta-(12.7%), di-(11.1%) and tetra-(4.7%). EST-SSR markers based on the 1822 SSR-containing contigs were developed, of which 1582 contigs could design primer pairs. Of the 35 primer pairs designed, 29 produced clear PCR fragment patterns with one or two bands. Polymorphic genotypes were obtained for 28 loci (80%) with the number of alleles per locus ranging from 3 to 12 for the 16 studied individuals. The average PIC value was 0.573, which indicates that the identified EST-SSR markers have a high degree of polymorphism. Principal Coordinates Analysis (PCA) showed that these EST-SSR loci can be used for identifying the provenances, even individuals of Chinese fir. [Conclusion] Combined SSRs mining and multiplex-PCR methods, we established the flow chart of EST-SSR markers development from transcriptome sequences of Chinese fir, and developed 28 polymorphic EST-SSR loci. These markers have been used in our ongoing analysis of genetic diversity in Chinese fir. Compared with traditional methods of SSR markers development, our method significantly improved PCR efficiency and dramatically reduced project costs. The new technologies will promote molecular genetics studies in Chinese fir, and also provide a basis for SSR marker development in other species.

Key words: Cunninghamia lanceolata, microsatellite markers, EST-SSR, transcriptome sequences, de novo assembly

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