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

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.