Isolation and characterization of single nucleotide polymorphisms in the common carp (Cyprinus carpio L.) genome

Abstract

In this study, we constructed a genomic library to identify a set of new SNPs from the common carp (Cyprinus carpio L.) genome. Putative SNPs were validated by amplification of 41 fragments from the library using unrelated individuals. A total of 211 SNPs, 18 Indels and 6 microsatellites were identified from a total length of 18,244 bp genomic sequences. A/G was the most common substitution, and the ratio of transitions to transversions was 2.3:1. Ten validated SNPs were selected for genetic diversity analysis in three wild populations of common carp, and all loci were polymorphic, with the average observed (H o ) and expected (H e ) heterozygosities were 0.2154 and 0.3321, respectively. These new SNPs would be good resources for population genetics, parentage analysis and conservation genetics in common carp.Keywords Cyprinus carpio L. Á Single nucleotide polymorphisms (SNPs) Á Validation Á Genetic diversityAs an important aquaculture species, common carp (Cyprinus carpio L.) has been domesticated for about 4,000 years, and widely distributed from Asia to Europe with huge number of varieties, strains or breeds. During the past decades, owing to habitat degradation, commercial overexploitation and water pollution, population resources of common carp have declined in China leading to the sharp reduction of natural fisheries in the rivers and lakes. Genetic variations of common carp populations, varieties or strains worldwide have been extensively studied using such molecular markers as mitochondrial DNA, AFLP and microsatellite DNA. Single nucleotide polymorphisms (SNPs) are the most abundant DNA variation in the vertebrate genome, and this type of marker is increasingly important in population genetics, linkage analysis and genome-wide association studies (Elahi et al. 2004). Nowadays, only a limited number of SNPs from expressed sequence tags (ESTs) were identified and utilized in the common carp (Kongchum et al. 2010;Zheng et al. 2011). SNPs discovery on a large scale throughout the entire genome was not reported for this species. In this study, we isolated and characterized a set of new SNPs in common carp for further genetics and conservation studies.An SNP-enriched genomic library was constructed for common carp, primarily following the method described by Xu et al. (2009). DNA from ten unrelated individuals from the Yangtze River was mixed equally as a DNA pool and was digested with MseI. The fragmented DNAs were ligated to specific adapters (5 0 -TACTCAGGACTCAT-3 0 / 5 0 -GACGATGAGTCCTGAG-3 0 ). Ligated fragments were amplified with MseI adaptors pecific primers. Heteroduplex of PCR products, formed by denaturation and annealing, was digested by CEL I nuclease and elongated by Bst. Target DNA containing SNPs were ligated into a pMD18-T vector (TaKaRa) and transformed into E. coli DH5a cells. After PCR confirmation, 50 random positive clones, ranging from 400 to 1,000 bp, were sequenced. Primer Premier 5 was used to design primers for 49 unique (Table S1) for 45 s and 72°C for 1 min, with last el...