title: Large-scale isolation of microsatellites from Chinese Mitten Crab Eriocheir sinensis via a Solexa Genomic Survey.
abstract: Microsatellites are simple sequence repeats with a high degree of polymorphism in the genome; they are used as DNA markers in many molecular genetic studies. Using traditional methods such as the magnetic beads enrichment method, only a few microsatellite markers have been isolated from the Chinese mitten crab Eriocheir sinensis, as the crab genome sequence information is unavailable. Here, we have identified a large number of microsatellites from the Chinese mitten crab by taking advantage of Solexa genomic surveying. A total of 141,737 SSR (simple sequence repeats) motifs were identified via analysis of 883 Mb of the crab genomic DNA information, including mono-, di-, tri-, tetra-, penta- and hexa-nucleotide repeat motifs. The number of di-nucleotide repeat motifs was 82,979, making this the most abundant type of repeat motif (58.54%); the second most abundant were the tri-nucleotide repeats (42,657, 30.11%). Among di-nucleotide repeats, the most frequent repeats were AC motifs, accounting for 67.55% of the total number. AGG motifs were the most frequent (59.32%) of the tri-nucleotide motifs. A total of 15,125 microsatellite loci had a flanking sequence suitable for setting the primer of a polymerase chain reaction (PCR). To verify the identified SSRs, a subset of 100 primer pairs was randomly selected for PCR. Eighty two primer sets (82%) produced strong PCR products matching expected sizes, and 78% were polymorphic. In an analysis of 30 wild individuals from the Yangtze River with 20 primer sets, the number of alleles per locus ranged from 2--14 and the mean allelic richness was 7.4. No linkage disequilibrium was found between any pair of loci, indicating that the markers were independent. The Hardy-Weinberg equilibrium test showed significant deviation in four of the 20 microsatellite loci after sequential Bonferroni corrections. This method is cost- and time-effective in comparison to traditional approaches for the isolation of microsatellites.
authors.affiliation: Key laboratory of Freshwater Aquatic Genetic Resources Certificated by Ministry of Agriculture, College of Life Science, Shanghai Ocean University, 999 Hucheng Huan Road, Shanghai 201306, China. gfqiu@shou.edu.cn.
authors.forename: Liang-Wei
authors.initials: LW
authors.lastname: Xiong
authors.forename: Qun
authors.initials: Q
authors.lastname: Wang
authors.forename: Gao-Feng
authors.initials: GF
authors.lastname: Qiu
mesh_terms: D000818:Animals; D003386:Brachyura / Q000235:genetics*; D002681:China; D003001:Cloning, Molecular / Q000379:methods*; D056726:Genetic Association Studies; D056426:Genetic Loci; D016678:Genome; D018895:Microsatellite Repeats; D017422:Sequence Analysis, DNA / Q000379:methods*