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.

A first generation microsatellite-based linkage map of the Chinese mitten crab Eriocheir sinensis and its application in quantitative trait loci (QTL) detection

“…Compared with EST-SSRs, genomic-SSRs exhibit significantly higher levels of polymorphism and are more suitable for map construction. However, the generation of genomic-SSR markers is expensive and requires intensive labor with conventional methods (Xiong et al, 2012a); moreover, the Chinese mitten crab genome has many chromosomes (2n = 146) (Du et al, 1986). This indicates that considerably more SSR markers and more funds are required to construct the same dense genetic linkage map for the mitten crab compared with other aquatic species.…”

“…This indicates that considerably more SSR markers and more funds are required to construct the same dense genetic linkage map for the mitten crab compared with other aquatic species. Fortunately, our previous study resulted in more than 15,000 genomic-SSR marker candidates via a genome survey using next generation sequencing technology (Xiong et al, 2012a), making it possible to develop a framework linkage map for the crab. In the present study, we constructed a first generation linkage map using 1300 SSR markers genotyped 92 F1 offspring from a F1 full-sib family.…”

“…The SSR markers were derived from two sources: one set of 1225 SSRs was previously developed from scaffolds in the Solexa genomic survey of E. sinensis (Xiong et al, 2012a). The SSR markers were named after the scaffold number.…”

“…Unfortunately, because of a lack of enough SSRs, fully SSR-based linkage maps have not yet been developed in crabs and shrimps, with the exception of a recently reported SNPbased map of the black tiger shrimp Penaeus monodon (Baranski et al, 2014). In our previous study, by taking advantage of the Solexa genomic survey, approximately 15,000 candidate SSR markers were isolated from mitten crabs, and those markers are suitable for construction of a linkage map (Xiong et al, 2012a). Here, we constructed a first generation SSR-based linkage map of the mitten crab.…”

Large‐scale identification of microsatellites for a major invasive weed, Ageratina adenophora, using the Illumina sequencing technology

“…These newly identified microsatellite loci could be considered as the potential loci for development of SSR molecular marker only if there are enough non‐repeated flanking regions around the microsatellite loci to design PCR primers (Toth et al ., ; Xiong et al ., ). To calculate the number of promising candidate SSR marker loci, we further screened the newly identified microsatellite loci to find which of them containing suitable flanking PCR primer sites using the criterion as described in .…”