中文摘要：

【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CMi B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35 633个contigs,总长度31.5 Mb,其中最小拼接长度155 bp,最大23 794 bp,平均长度884 bp。得到2 156个SSR位点,分布于1 822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1 582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

英文摘要：

【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 CMi B 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 35 633 contigs,the total length was 31. 5 Mb,of which mini- and max-contigs were 155 bp and 23 794 bp,respectively,with an average length of 884 bp. In total,2 156 SSRs were identified distributed in 1 822（ 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 1 822 SSR-containing contigs were developed,of which 1 582 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 for28 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