Allele frequency analysis of Chinese chestnut (Castanea mollissima) populations using fluorescent simple sequence repeats (SSR) analysis

The aim of this study was to establish a method for allele frequency detection in bulk samples. The abundance of polymerase chain reaction (PCR) products in bulk leaf samples was detected using fluorescent labeled Simple sequence repeat (SSR) primers and an Applied biosystems (AB) 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, an average of 4.6 alleles per locus, were detected among 17 chestnut populations with the primer CmTCR10 (NED) and a total of 41 alleles, an average of 2.4 alleles per locus, were detected with the primer CmTCR24 (6-FAM). Multiplexing the PCR reaction by combining the primer pairs of CmTCR10 and CmTCR24, using different fluorescent dyes for different primers, showed that the alleles could be discriminated and the sizes of the amplified segments were similar. Furthermore, the exact sizes of the amplified fragments and the abundance of the PCR products were determined by fluorescent SSR. After data analysis with GeneScan software and allele calling and output with Genotyper software, allele frequencies were calculated for equal pooled samples in each population using the FREQS-R module in the R statistical computing language. The results indicate that it is feasible to determine allele frequencies in bulked samples based on the detection of SSR-PCR products. The advantages and additional applications of this method are also discussed. The abundance of the PCR products can be used to determine the allele frequencies in bulk samples of chestnut populations.