聚丙烯酰胺凝胶与毛细管电泳检测龙须菜SSR位点的比较研究

Comparison on Two SSR Detection Methods of Polyacrylamide Gel Electrophoresis and Capillary Electrophoresis in Gracilariopsis lemaneiformis

以12个野生型龙须菜个体为材料,选择7个SSR位点,分别使用PAGE电泳银染检测和毛细管电泳荧光检测每个位点的多态性信息,并对这两种方法的检测结果进行了比较。PAGE与毛细管电泳分型结果相一致的4个位点包括全部的五碱基和六碱基重复:H88、S96、H97和H98。而分型结果不一致的3个位点则均为三碱基重复:H30、S25和S28。经测序检验,PAGE结果均准确,而毛细管电泳分型结果出现误差。在样本量少,SSR位点的变异小时,使用PAGE检测SSR位点更为准确。

Twelve wild individuals of Gracilariopsis lemaneiformiswere used to detect polymorphism of 7 SSR loci by polyacrylamide gel electrophoresis （PAGE） and capillary electrophoresis, for the purpose of comparing the detection efficiency of the two methods and exploring an accurate SSR detection system. Genotyping results of capillary electrophoresis in 4 SSR loci was consistent with that of PAGE, including all penta and hexa nucleotide repeats： H88, S96, H97 and H98, while all tri nucleotide repeats： H30, S25 and S28 showed inconsistent results in two methods. At SSR locus H30, amplified bands of 12 individuals in PAGE analysis had no size difference, which indicated that there was no polymorphism. However, the size of target fragments obtained by capillary electrophoresis fluorescent detection system were compared among 12 samples, giving a result that size differences existed at this locus. Through the analysis of fluorescent peak, size difference of 1 base pair could be caused by inaccurate selection of target peak, while size difference of 3 base pairs was equal to one repeat unit of tri nucleotide repeats, which were identified as size polymorphism. Therefore, 2 alleles were detected by capillary electrophoresis at locus H30. At SSR loci S28 and S30, the genotyping results of capillary electrophoresis both showed that there was no size difference existed among 12 sample, however, amplified bands in PAGE gel displayed separation among individuals and were identified as 2 alleles, which was the evidence of polymorphism. PAGE results of the three SSR locus was further verified by gel extraction and sequencing. SSR sequences of 12 samples obtained by sequencing displayed the true polymorphic information, which were fully consistent with PAGE results. The errors existing in capillary electrophoresis detection were mainly caused by peak selection and fragment size reading, especially when repeat unit length of SSR locus was short, size difference of target fragments was small and the number of sample was small. In contrast with capillary electrophoresis, the genotyping result of PAGE was more accurate and credible. Therefore, PAGE detection was suggested to be suitable for identifying polymorphic SSR loci with small sample size and low polymorphism.