Establishment of effective method for detecting the allelic frequency of chestnut populations

The objective was to establish the method for detecting the allele frequencies in bulk samples.The abundance of the PCR products in bulk samples,which represents the frequencies,was detected using fluorescence labeled SSR primers and based on ABI automatic DNA analyzer.Compared with the conventional SSR technique based on polyacrylamide gel electrophoresis(PAGE) and silver staining,fluorescent SSR was much more sensitive.A total of 78 alleles,and 4.6 alleles per locus in average,were detected with the primer CmTCR10(NED) among 17 chestnut populations.Moreover,a total of 41 alleles,and 2.4 alleles in average,were detected with the primer CmTCR24(6-FAM).The multiplex PCR combining primer pairs with CmTCR10 and CmTCR24 showed that the alleles could be discriminated and the sizes of the amplification segments were quite similar by using the different fluorescent dyes with different primers.Furthermore,the exact sizes of the amplification segments and the abundance of the products can be determined by fluorescent SSR.After data capturing by GeneScan software,data setting and output in Genotyper software,allelic frequencies were detected in equally pooled samples 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 the abundance detection of SSR-PCR products.The advantages and further applications of this method were also discussed.The abundance of the PCR products can be used to determine the allele frequencies in bulk samples of chestnut populations.