Zhou Wei, Motohashi Keiichi, Suga Haruhisa, Fukui Hirokazu, Kageyama Koji
The United Graduate School of Agricultural Science, Gifu University, Gifu, Japan.
FEMS Microbiol Lett. 2009 Apr;293(1):85-91. doi: 10.1111/j.1574-6968.2009.01518.x.
A strategy combining dual-suppression PCR and thermal asymmetric interlaced PCR was used to determine sequences flanking microsatellite regions in Pythium helicoides. The primer pairs were designed to amplify loci containing (AC)n, (GA)n, (AGC)n, (CAC)n(CAA)n, (TCA)n and (CTTT)n repeats from the P. helicoides nuclear genome. The PCR products of each primer pair, amplified from three representative isolates collected from different hosts and locations, were cloned and sequenced. Different degrees of polymorphism were detected among these microsatellite markers. The numbers of alleles were 6, 2, 4, 11, 4 and 4 in YL-AC, YL-AGC, YL-CAA, YL-CTTT, YL-GA and YL-TCA, respectively. Allele analysis of 30 P. helicoides isolates showed length polymorphisms in all loci, except for YL-AC, using capillary electrophoresis. Thus, we have developed a simple method for designing PCR primers to amplify microsatellite markers from P. helicoides.
采用双重抑制PCR和热不对称交错PCR相结合的策略来确定盘旋腐霉（Pythium helicoides）微卫星区域侧翼的序列。设计引物对以扩增来自盘旋腐霉核基因组中含有（AC）n、（GA）n、（AGC）n、（CAC）n（CAA）n、（TCA）n和（CTTT）n重复序列的位点。从不同宿主和地点收集的三个代表性分离株扩增得到的每个引物对的PCR产物，进行克隆和测序。在这些微卫星标记中检测到不同程度的多态性。YL-AC、YL-AGC、YL-CAA、YL-CTTT、YL-GA和YL-TCA中的等位基因数分别为6、2、4、11、4和4。对30个盘旋腐霉分离株的等位基因分析表明，除YL-AC外，所有位点均存在长度多态性，采用毛细管电泳进行分析。因此，我们开发了一种简单的方法来设计PCR引物，以从盘旋腐霉中扩增微卫星标记。
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