香菇表达序列标签(EST)的SSR分析及EST-SSR引物的筛选

从真菌基因组计划网站(FGP)和NCBI网站数据库下载了总长度为8.1Mb的11150条香菇的EST(包括香菇的10条cDNA)序列，通过SSRhunter 1.3软件结合手工查找，从中发现2.83％即316条EST含有一共469个SSR.，平均每17.3kb出现一个EST-SSR。在所有EST-SSR中，三碱基和六碱基SSR出现最多，分别占EST-SSR总数的38.00％和20.00%，出现较多的基元为(A)n、(T)n、(GA)n、(AG)n、(TGA)n、(GAT)n和(TCTTT)n，占所有EST-SSR的35.39％。利用Oligo 6.0软件设计了51对EST-SSR引物，并选用其它物种的24对引物，对香菇菌株进行了PCR扩增。实验结果表明，设计的51对引物有39对具有明显的扩增产物，选用的其它物种的24对引物均没有清晰可见的特异性扩增产物。部分有扩增产物的EST-SSR引物具有多态性。

Analysis of SSRs in ESTs database of Lentinula edodes and selection of EST-SSR primers

In this study, 11150 ESTs (including 10 cDNAs), about 8.1Mb in length, were downloaded from ESTs databases of Lentinula edodes in Fungal Genomics Project (FGP) website and NCBI website. By searching with SSRhunter 1.3 software and handwork, 469 SSRs were identified in 316 ESTs, approximately 2.83% of total ESTs. The average distance between SSRs was about 17.3kb. Trinucleotide and hexanucleotide ESR-SSRs were found to be the two most aboundent repeats, accounting for 38.00% and 20.00% of the total ESR-SSRs respectively, and (A)n, (T)n, (GA)n, (AG)n, (TGA)n, (GAT)n and (TCTTT)n to be the most familiar motifs, which comprised about 35.94% of all EST-SSRs. Agarose electrophoresis manifested that 39 out of 51 EST-SSRs primer pairs designed by Oligo 6.0 software possessed obvious PCR product in L. edodes, while there were no visible PCR product amplified by the selected primer pairs form other species. Some EST-SSR primer pairs revealed polymorphism in electrophorogram.