Development And Application Of SNP Markers In The Sea Cucumber (Apostichopus Japonicus)

The sea cucumber(Apostichopus japonicus) belongs to Holothuroidea, Echinodermata, Aspidochirotida in taxonomy. The sea cucumber has been an important aquaculture species for high market value. As a promising molecular marker, single nucleotide polymorphism (SNP) is widely used in genetic map construction, QTL analysis, genetic diversity assessment, pedigree identification and molecular breeding for its advantages such as typically bi-allelic, abundance and high-through genotyping. The development of SNP from expressed sequence tag (EST) database is a new and easy way. High resolution melting (HRM) is a kind of highly effective mutation screening and genotyping technology for the advantages of simple, precise operation and acting in closed pipe. In this study, single nucleotide polymorphism loci are developed from EST database of A. japonicas by high resolution melting method, and SNP loci are used to assess the genetic diversity of A. japonicas and analyze the relationship between SNP markers and growth traits in A. japonicas. The results provide new insight of SNPs in the fields of high-density genetic mapping, biodiversity conservation, population structure analysis and marker-assisted breeding.1. The development and characterization of EST-SNPThe alignment of the 7,739 ESTs downloaded from GenBank produced 1878 contigs and 229 putative SNPs were selected for validation. A total of 51 gene-derived SNPs were isolated and characterized by means of HRM analysis,47 EST-SNPs are novel developed. The observed and expected heterozygosity ranged from 0.0345 to 0.5000 and from 0.0345 to 0.5643, with an average of 0.2405 and 0.3055, respectively.2. Comparison of genentic diversity in cultured populations in China and wild populations in JapanThirty-two of SNP loci were applied for estimating the genetic similarity and variation between the five hatchery stocks from China and two wild stocks from Japan. No obvious differences in observed or expected heterozygosity were spotted between the wild and hatchery populations. The pair-wise Fst (ranged from 0.0119 to 0.0236) and the genetic identity (ranged from 0.9802 to 0.99J5) showed no obvious differentiation between the wild and cultured stocks. The analysis of AMOVA indicated the source of variation came from the "level of within the populations". The results indicate that the hatchery sea cucumbers have considerable genetic variation through the process of domestication.3. Correlation analysis of SNP markers with growth traitsForty-six SNP markers are used to analyze the genetic diversity of a full-sib family with high resolution melting method, and examined the correlations of SNP markers with the traits of body length, body width, body weight, body wall weight, dressing percentage. The analysis of correlations suggested that 10 loci of the 46 SNPs were correlated with the growth traits significantly (P<0.05). Of the 10 SNP loci, the genotype of AB at SNP183 locus was correlated with dressing percentage, while the genotype of AAat SNP189 was correlated with body length significantly, respectively. Because the genotype of BB at eight loci (SNP4, SNP146, SNP154, SNP163, SNP164, SNP175, SNP178 and SNP216) was significantly correlated with body length, body width, bodyweight, and body wall weight, respectively, suggesting that the genotype of BB was superiority at these loci.