杨树锈菌表达序列微卫星分析及EST-SSR标记开发

通过对64 498条杨树锈菌EST进行拼接,得到1 998个拼接片段(contigs),发现了604个SSR位点。在拼接片段中SSR平均密度为每736.6 bp含有1个SSR。在SSR中,三核苷酸重复基元的SSR类型最多,占总数的44.70%。在二碱基重复中,最主要的优势重复单元是AC和AT,三碱基中AGT和AAG为优势重复单元,四碱基、五碱基重复类型中,(AAAN)n和(AAAAN)n为对应的优势重复单元,这些优势重复单元中富含碱基A和T,研究还发现杨树锈菌表达序列中微卫星丰度很高。杨树锈菌突变频率很高,由试验结果推测杨树锈菌基因中含有的微卫星序列是杨树锈菌基因变异的一个重要驱动力。杨树锈菌EST序列中高度变异的微卫星(长度大于20 bp)约占15.07%。针对检测到的微卫星位点,共设计了455对引物,并选取了30个SSR引物对对杨树锈菌基因组DNA进行PCR扩增,其中有27个引物对扩增成功,占90.0%。扩增产物电泳结果显示,有21个引物对扩增出的谱带在预期片段大小范围之内,有8个引物对在杨树锈菌DNA中检测到了多态性,多态性引物对占26.7%。

Analysis of Microsatellites Containing in ESTs and Development of EST-SSR Markers for Poplar Leaf Rust Fungi

In this study,64498 ESTs of poplar leaf rust fungus Melampsora was assembled into 1998 contigs,and 604 microsatellites(SSRs) were detected on these contigs,with 736.6bp containing one microsatellite on average.Among these SSRs,trinucleotide repeats were the most abundant repeats(44.70%).As for the composition of microsatellites,AC and AT repeats were the richest motif in dinucleotide repeats,and AGT,AAG repeats were the most frequent motifs in trinucleotide repeats,whereas(AAAN)n and(AAAAN)n repeats were dominant in tetra-and pentanucleotide repeats,respectively.All the dominant repeat motifs of different types of SSRs were rich in A and T.In EST sequences of Melampsora genome,microsatellites longer than 20bp accounted for about 15.07%.It is noticeable that microsatellites were found to be highly rich in the expressed sequences of Melampsora genome,which implies that SSRs play a significant role in triggering the gene mutation in Melampsora genome.A total of 455 SSR primers were designed for the detected microsatellites using Primer 5.0 and Oligo 6.0,and 30 primer pairs were randomly selected for amplification test.Among these primer pairs,27 primer pairs succeed in amplification,with a successful rate of 90%.Eight primer pairs generated polymorphic fingerprints in leaf rust fungus collected from different poplar genotypes,accounting for 26.7% of the tested primer pairs.