Molecular analysis of grass carp (Ctenopharyngodon idella) by SRAP and SCAR molecular markers

The sequence-related amplified polymorphism (SRAP) technique was used to analyze the gene differentiation between two cultured populations [Freshwater Fisheries Research Center (FFRC) and Qianzhou populations] and one wild population (Hanjian population) of grass carp (Ctenopharyngodon idella). Some loci showed quite different genetic frequencies, attributable to artificial selection, which imply that these fragments are putative markers of germplasm identification. We developed a simple and effective method to further characterize these SRAP fragments. Specific SRAP bands were cut directly from polyacrylamide gels, re-amplified, cloned, and sequenced. Twenty-one putative genetic markers were sequenced, ranging from 137 to 357 bp. The sequences were submitted to the database of the Genome Sequence Survey. A BLAST analysis showed that eight SRAP fragments were highly similar to functional genes, whereas the other 13 had no similarity, indicating that these markers are tightly linked to the germ identification trait although only eight are functional genes. Three primers were designed according to this sequence information and used for PCR amplification of the three populations. A sequence-characterized amplified region (SCAR1) was positively amplified in the artificially cultured populations but not in the wild population. The frequency of the SCAR3 marker in the cultured populations was 87% (26/174), whereas it was only 6% (6/100) in the wild population. A specific band was isolated from all individuals in the wild population with the SCAR3 primers, whereas the specific band was amplified from only seven individuals in the FFRC population and from none of the Qianzhou population. The frequency of SCAR2 in the artificially cultured populations was 96.5%. These results indicate that SCAR1 could be used as a specific molecular marker for population identification. The SCAR markers used in this study offer a powerful, easy, and rapid method for genetic analysis and the discrimination of different populations.