亚洲棉EST-SNP的挖掘及其在陆地棉中的验证

【目的】随着不同棉种序列数据库的逐步完善以及高通量测序技术的发展,棉花单核苷酸多态性(Single nucleotide polymorphism,SNP)标记开发可利用的公共数据资源逐步增加。【方法】本研究基于陆地棉祖先基因组的现代种亚洲棉表达序列标签(Expressed sequence tag,EST)数据库,利用CAP3对亚洲棉EST数据库进行拼接。拼接获得7 187个重叠群(Contig),再利用Quality SNP软件进行SNP位点分析。【结果】在807条含有4条以上EST序列的Contig中查找到2 690个SNP位点。通过筛选次要等位基因频率大于30%的位点,获得953个可靠度较高的候选SNP,通过电子筛选,最终获得可用于陆地棉分析的SNP 149个,利用位点特异性聚合酶链式反应以及酶切扩增多态序列验证了EST-SNP的准确性。【结论】本研究证实基于亚洲棉EST数据库挖掘用于陆地棉研究的EST-SNP切实可行,并有望将EST-SNP用于陆地棉遗传图谱构建、重要性状的基因定位以及分子标记辅助育种。

Development of EST-SNP Markers in Gossypium arboreum and Their Validation in G.hirsutum

Objective] With the development of the cotton genome sequence database and next-generation high-throughput sequencing techniques,the resources available for generating single nucleotide polymorphism (SNP) markers are gradually expanding.Method] The Gossypium arboreum expressed sequence tags (ESTs) downloaded from the NCBI database were assembled into 7 187 contigs using the CAP3 program.Additionally,the QualitySNP program was used for SNP mining.Result] A total of 2 690 SNPs were obtained from 807 contigs that consisted of more than four ESTs.We obtained 953 highly reliable candidate SNPs by screening for a minor loci frequency of more than 30％.Finally,a total of 149 candidate EST-SNPs that may be used in G.hirsutum were obtained through in silico screening.An allele-specific polymerase chain reaction and cleaved amplified polymorphic sequence molecular markers were used to validate the accuracy of the selected candidate SNPs in G.hirsutum.Conclusion] The EST-SNP markers from G.arboreum may be used to analyze G.hirsutum.The obtained EST-SNP markers will be used to construct genetic maps,map important traits,and complete marker-assisted selection in G.hirsutum.