基于SSR荧光标记技术的玉米群体混合样本基因频率分析方法

Establishment of Fluorescent SSR Technique on Detecting Allelic Frequency in Maize (Zea mays L.) Populations with Bulk Sampling Strategy

【目的】建立玉米群体混合样本等位基因及其频率的分析方法。【方法】采用ABI全自动遗传分析仪,利用5′端荧光标记的SSR引物,通过混合样本中PCR扩增产物检测峰高与丰度对应,分析基因频率。【结果】与聚丙烯酰胺凝胶电泳银染法相比,ABI全自动遗传分析仪的检测灵敏度高。20个供试玉米群体中,荧光引物Phi072(6-Fam)共检测到82个等位基因位点,平均每个群体4.1个;引物Phi084(Hex)检测到43个等位基因位点,平均每个群体2.15个。多重PCR(multiplex PCR)荧光SSR检测结果显示,可以明确区分不同等位基因。荧光SSR能准确地读出片段大小,定量PCR扩增产物的丰度。用GeneScan获取数据,用Genotyper软件对数据进行设置与输出,使用R软件的FREQS-R模块,初步探讨利用荧光SSR定量化数据,计算供试群体15个单株混合样本的等位基因频率。【结论】可通过荧光SSR产物的丰度来确定混合取样群体的基因频率。

【Objective】The objective was to establish the detecting method on allele frequencies in bulk samples.【Method】The abundance of the PCR products in bulk samples were used to represent the frequencies by using fluorescence labeled SSR primers and based on ABI automatic DNA analyzer.【Result】Compared with the conventional SSR technique based on polyacrylamide gel electrophoresis（PAGE） and silver staining,fluorescent SSR was much more sensitive.A total of 82 alleles,and 4.1 alleles per locus in average,were detected with the primer Phi072（6-Fam） among 20 domestic maize landraces.Moreover,a total of 43 alleles,and 2.15 alleles in average,were detected with the primer Phi084（Hex）.The multiplex PCR combining primer pairs with phi072 and phi084 showed that the alleles could be clearly discriminated even the sizes of the amplification segments were quite similar by using the different fluorescent dyes in different primers.Furthermore,the exact sizes of the amplification segments and the abundance of the products can be determined in fluorescent SSR.After data capturing in GeneScan,data setting and output in Genotyper,allelic frequencies were detected in equally pooled samples with 15 individuals in each population using the FREQS-R module in R program.The results suggested that it was feasible to determine allelic frequencies in bulked samples based on detecting the abundance of SSR-PCR products.The advantages and further applications of this method were also discussed.【Conclusion】The abundance of the PCR products can be used in determining the allele frequencies in bulk samples of maize populations.