基于高通量测序的红豆杉EST-SSRs标记研究

Identification of EST-SSRs in Taxus cuspidata based on high-throughput sequencing

目的:利用高通量测序,对红豆杉Taxus cuspidata转录组进行EST-SSRs发掘和研究。方法:提取红豆杉叶片cDNA,应用454 GS FLX Titanium测序,采用Newbler Assembler Software软件对序列(high-quality sequences)进行从头拼接,获得一致性序列(unique sequences)。用Simple Sequence Repeat Identification Tool(Perl Script)对一致性序列进行分析,检索SSRs模体;采用PRIMER3设计扩增引物。结果:高通量测序获得红豆杉81 148条ESTs,经拼接得到20 557条unique se-quences,从中发现了753个EST-SSRs。依据其邻近序列设计引物,共有519条EST-SSRs获得了扩增引物。在红豆杉EST-SSRs中随机挑选序列构建小样本,克隆测序结果表明87.5%的序列与Sanger测序结果一致。结论:首次获得了红豆杉属EST-SSRs,为红豆杉的种质资源培育、功能基因及基因组差异研究奠定了基础。

Objective： Taxus species are highly valued for the production of taxol. Based on high-throughput sequenceing, EST-SSRs were explored and studied in the transcriptome of Taxus cuspidata. Method： T cuspidata leaf cDNA was extracted and sequenced by 454 GS FLX Titanium. High-quality sequences were assembled using Newbler Assembler Software, which produced unique sequences. SSRs motif was explored using simple sequence repeat identification tool （Perl Script）. Primers were designed using PRIMER3. Result： A total of 81 148 high-quality reads from the needles of T. cuspidata were produced using the Roche GS FLX Titanium system. A total of 20 557 unique sequences were obtained. There were 753 simple sequence repeat motifs identified. Primers of PCR were obtained for 519 EST-SSRs, randomly selected cloning sequencing revealed that 87.5% of ESTs were the same as the results of Sanger sequencing. Conclusion： The results provide the first EST-SSRs collection in Taxus and are essential for future efforts of gene discovery, functional genomics, and genome annotation in related species.