Alternative splice variants and differential relative abundance patterns of vasa mRNAs during gonadal development in the Chinese mitten crab Eriocheir sinensis.

Gonadal development usually involves alternative splicing of sex-related genes. Vasa, a highly conserved ATP-dependent RNA helicase present mainly in germ cells, has an important function in gonadal development. As an important sex-related gene, recent evidence indicates that different splice variants of vasa exist in many species. In this study, there was identification of two types of vasa splice variants in the Chinese mitten crab Eriocheir sinensis, termed Esvasa-l and Esvasa-s, respectively. Furthermore, splice variants of Esvasa-s were sub-divided into Esvasa-s1, Esvasa-s2, Esvasa-s3, Esvasa-s4, and Esvasa-s5, based on differing numbers of TGG repeats. Results from genomic structure analyses indicated that these forms are alternatively spliced transcripts from a single vasa gene. Results from tissue distribution assessments indicate the vasa splice variants were exclusively expressed in the gonads of male and female adult crabs. In situ hybridization results indicate Esvasa mRNA was mainly present in the cytoplasm of previtellogenic oocytes. As oocyte size increased, relative abundance of Esvasa mRNA decreased and became distributed near the cellular membrane. The Esvasa mRNA was not detectable in mature oocytes. In testis, Esvasa mRNA was detected in spermatids and spermatozoa, but not in spermatogonia and spermatocytes. Notably, results from qPCR analysis of Esvasa-l and Esvasa-s indicate there are different relative proportions during gametogenesis, implying that splice variants of the Esvasa gene may have different biological functions during crab gonadal development.

Identification and profiling of microRNAs during gonadal development in the giant freshwater prawn Macrobrachium rosenbergii.

As post-transcriptional regulators, microRNAs (miRNAs) play an important role in growth and reproductive processes. So far, there is limited information regarding crustacean miRNAs. To explore the potential role of miRNAs in the gonadal development of the prawn Macrobrachium rosenbergii, we constructed seven small RNA libraries from ovarian and testicular tissues at various stages using somatic tissue as the control. A total of 1,954 known and 129 novel miRNAs were retrieved. By comparing differentially expressed miRNAs (DEMs) between testes and ovaries, forty-one miRNAs were identified with sex-biased expression patterns, including 17 ovary-biased and 24 testis-biased patterns. Furthermore, the putative target genes of the sex-biased miRNAs, such as cyclin L1, mitogen-activated protein kinase 7 (MAPK 7), heat shock protein (HSP), and zinc finger protein, were significantly enriched in many reproduction-related pathways including the Gonadotropin-releasing hormone (GnRH) pathway, glycolysis, gluconeogenesis pathway, ovarian steroidogenesis, estrogen signaling pathway, MAPK pathway, Wnt pathway, and insulin signaling pathway, implicating potential regulatory roles of miRNAs in reproduction. These data aid in the further investigation of the mechanism of gonadal development and reproductive regulation mediated by miRNA in M. rosenbergii.

A second generation SNP and SSR integrated linkage map and QTL mapping for the Chinese mitten crab Eriocheir sinensis.

The Chinese mitten crab Eriocheir sinensis is the most economically important cultivated crab species in China, and its genome has a high number of chromosomes (2n = 146). To obtain sufficient markers for construction of a dense genetic map for this species, we employed the recently developed specific-locus amplified fragment sequencing (SLAF-seq) method for large-scale SNPs screening and genotyping in a F1 full-sib family of 149 individuals. SLAF-seq generated 127,677 polymorphic SNP markers, of which 20,803 valid markers were assigned into five segregation types and were used together with previous SSR markers for linkage map construction. The final integrated genetic map included 17,680 SNP and 629 SSR markers on the 73 linkage groups (LG), and spanned 14,894.9 cM with an average marker interval of 0.81 cM. QTL mapping localized three significant growth-related QTL to a 1.2 cM region in LG53 as well as 146 sex-linked markers in LG48. Genome-wide QTL-association analysis further identified four growth-related QTL genes named LNX2, PAK2, FMRFamide and octopamine receptors. These genes are involved in a variety of different signaling pathways including cell proliferation and growth. The map and SNP markers described here will be a valuable resource for the E. sinensis genome project and selective breeding programs.

[Genetic structure of wild Macrobrachium nipponense populations in Taihu Lake based on microsatellite analysis].

By using eight highly polymorphic microsatellite DNA loci, this paper analyzed the genetic structure of wild Macrobrachium nipponense populations in Taihu Lake. For the 15 M. nipponense populations in the Lake, there were at least three of the loci presenting heterozygosity deficiency and obvious deviation from Hardy-Weinberg equilibrium after Bonferroni correction. The observed heterozygosity values of the 15 populations were all above 0. 683, displaying a high genetic diversity, but the diversity varied obviously with site. For example, the genetic diversity of the eastern and southern populations at Dukou and Luxiang was higher than that of the western and northern populations at Huazhuang and Yangzhu. For the 15 populations, parts of the loci showed heterozygote excess and departure from mutation-drift equilibrium, suggesting that the population structure had experienced bottleneck effect and the population amount had declined. The AMOVA analysis across all the populations and loci showed that the genetic divergence among the 15 populations was at a lower level (F(ST) = 0.011 ). 98.9% of the genetic variation came from intra-population, and 1.1% came from inter-population, suggesting that all the M. nipponense populations in the Lake could be protected and managed as a single unit in genetic resource. However, the genetic distance between Huazhuang and Wutangmen populations reached 0.206, being close to the delimitation of species identification. Further studies would be needed for the sustainable utilization of the genetic resource of M. nipponense in Taihu Lake.

[Microsatellite analysis of genetic variation of the oriental river prawn Macrobrachium nipponense in Qiantang River].

Genetic diversity and genetic structure of 7 wild stocks of oriental river prawn Macrobrachium nipponense in Qiantang River, i.e. Wen-yan, Fu-yang, Chang-kou, Tong-lu, Xin-an-jiang, She-xian and Xiu-ning, were investigated using 10 microsatellite DNA markers. The result showed that all the 10 loci were highly polymorphic. There was a trend that the level of genetic diversity of wild stocks in downstream and midstream were higher than the upstream ones'. Sign test and Wilcoxon sign rank test results showed that the stocks in Qiantang River had no bottleneck effect, and the number of stocks had not declined recently. F(ST) among stocks ranged from 0.0201 to 0.1069. Analysis of molecular variance (AMOVA) revealed that a higher portion (93.48%) of variations existed within individuals, while lower portion (6.52%) existed among stocks. F(ST) and AMOVA analysis across all stocks and loci indicated the medium level of divergence among the stocks. The NJ clustering tree based on D(A) genetic distance demonstrated that the stocks of adjacent geographical position clustered together. 413 individuals obtained from six wild stocks could be divided into two potential populations based on the genetic structure. This study demonstrated that genetic diversity and genetic structure of M. nipponense stocks were relevant to geographical position where they survived.