目前基于梨属植物基因组开发的SSR引物太少,远不能满足其品种鉴别、遗传多样性分析和分子标记辅助育种等研究与应用的需要。为进一步开发更多的梨SSR引物,本研究从NCBI公共数据库检索到梨的4 338条ESTs和478条基因组序列。通过前处理和聚类去冗余后得到全长为343.81 kb的无冗余的686条ESTs,搜索获得725个SSRs,其平均长度为17.34 bp。在一至九个核苷酸的重复基元中,其主要类型是单核苷酸重复和二核苷酸重复,两者所占的比例分别为84.44%和10.34%。BLASTx同源性比较分析发现,上述686条ESTs中305条在数据库中可以找到同源序列,占ESTs总数的44.46%,功能已知蛋白质中碧桃来源的ESTs所占比例最大,为41.37%。GOA功能分类结果表明,293条ESTs可划分为生物过程、分子功能和细胞成分3大功能类,其中与细胞过程相关的ESTs数量最多,为103条。序列分析结果显示,EST-SSRs与基因组SSRs的平均检出频率分别为2 108.74 loci/Mb和91.11 loci/Mb,两者具有显著的差异。本研究基于梨的EST和基因组序列,应用MISA批量开发技术分别获得了111对EST-SSR引物和291对基因组SSR引物,随机抽取及合成11对引物的验证结果表明,开发得到的SSR引物在梨属植物中具有较好的通用性。
Because of the limit of SSR primers number, the application of SSR marker in genome mapping,genetic diversity, species identification and marker-assisted selection of pears breeding have been greatly delayed in recent years. In order to develop the much more SSR primers in Pyrus L., 4 413 ESTs and 478 genome sequences from the database of NCBI have been downloaded and analyzed, resulting 686 non-redundant ESTs with total length about 343.81 kb. Totally 725 SSRs distributed in 686 ESTs were selected, bioinformatical analysis results showed that the average length of the EST-SSRs were 28.73 bp. Mononucleotide and dinucleotide repeats are the main types, accounting for 84.44% and 10.34% of all the SSRs, respectively. Meanwhile, in the 686EST-SSRs, 305 EST-SSRs can be found in the database through BLASTx homology analysis, accounting for44.46%. In these homologs, proteins with known function from Prunus persica accounting for 41.37%. At the same time, 293 ESTs can be divided into three major functional categories by the analysis of functional classificationfor the gene ontology annotation(GOA), including of biological processes, molecular functions and cellular components. It was found that 103 EST-SSRs were relevant to celluar process. In addition, sequence analysis results indicated there was significant difference on the average SSR rate from ESTs and genomic sequences, and the average SSR rate reached 2 108.74 loci/Mb and 91.11 loci/Mb, respectively. Subsequently, 111 pair EST-SSR and 291 pair genomic SSR primers have already been obtained. At last, 11 pair SSR primers from genomic sequence were randomly selected to detect the transferability of primers, the amplification results indicated that the SSR primers, which developed in this study, were highly transferable.
Molecular Plant Breeding