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Luteinizing hormone (LH), a pituitary gonadotropin, coupled with LH receptor (LHR) is essential for the regulation of the gonadal maturation in vertebrates. Although LH homolog has been detected by immunocytochemical analysis, and its possible role in ovarian maturation was revealed in decapod crustacean, so far there is no molecular evidence for the existence of LHR. In this study, we cloned a novel LHR homolog (named EsLHR) from the Chinese mitten crab Eriocheir sinensis. The complete sequence of the EsLHR cDNA was 2775bp, encoding a protein of 924 amino acids, sharing 71% amino acids identity with the ant Zootermopsis nevadensis LHR. EsLHR expression was found to be high in the ovary, while low in testis, gill, brain, and heart, and no expression in the thoracic ganglion, eye stalk, muscle, and hepatopancreas. Quantitative PCR revealed that the expression level of EsLHR mRNA was significantly higher in the ovaries in previtellogenic (Pvt), late vitellogenic (Lvt), and germinal vesicle breakdown (GVBD) stages than that in the vitellogenic (Mvt) and early vitellogenic (Evt) stages (P < 0.05), and, the highest and the lowest expression were in Lvt, and Evt, respectively. The strong signal was mainly localized in the ooplasm of Pvt oocyte as detected by in situ hybridization. The crab GnRH homolog can significantly induce the expression of EsLHR mRNA at 36 hours post injection in vivo (P < 0.01), suggesting that EsLHR may be involved in regulating ovarian development through GnRH signaling pathway in the mitten crab.

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.