Xiaoqing Wang

Microsatellite-centromere mapping in large yellow croaker (Pseudosciaena crocea) using gynogenetic diploid families.

The large yellow croaker (Pseudosciaena crocea) is an economically important marine fish in China. Inheritance of 22 heterozygous microsatellite loci was examined in normal crossed diploid families and meio-gynogenetic families in P. crocea. Two gynogenetic families were produced via inhibition of the second polar body in eggs fertilized with UV-irradiated sperm. The ratio of gynogenesis was proven to be 100% and 96.9% in the two families, respectively. Of the 22 examined loci, 4 showed a segregation distortion in both control and gynogenetic families. Microsatellite–centromere (M–C) map distances were examined using 18 loci with normal Mendelian segregation. Estimated recombination rates ranged between 0 and 1.0 under the assumption of complete interference. High recombinant frequencies between heterozygous markers and the centromere were found in large yellow croaker, as in other teleosts. The average recombination frequency was 0.586. Ten loci showed high M–C recombination with frequency greater than 0.67. M–C distances provide useful information for gene mapping in large yellow croaker.

Identification of Immune-Related Genes and Development of SSR/SNP Markers from the Spleen Transcriptome of Schizothorax prenanti.

Schizothorax prenanti (S. prenanti) is mainly distributed in the upstream regions of the Yangtze River and its tributaries in China. This species is indigenous and commercially important. However, in recent years, wild populations and aquacultures have faced the serious challenges of germplasm variation loss and an increased susceptibility to a range of pathogens. Currently, the genetics and immune mechanisms of S. prenanti are unknown, partly due to a lack of genome and transcriptome information. Here, we sought to identify genes related to immune functions and to identify molecular markers to study the function of these genes and for trait mapping. To this end, the transcriptome from spleen tissues of S. prenanti was analyzed and sequenced. Using paired-end reads from the Illumina Hiseq2500 platform, 48,517 transcripts were isolated from the spleen transcriptome. These transcripts could be clustered into 37,785 unigenes with an N50 length of 2,539 bp. The majority of the unigenes (35,653, 94.4%) were successfully annotated using non-redundant nucleotide sequence analysis (nt), and the non-redundant protein (nr), Swiss-Prot, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. KEGG pathway assignment identified more than 500 immune-related genes. Furthermore, 7,545 putative simple sequence repeats (SSRs), 857,535 single nucleotide polymorphisms (SNPs), and 53,481 insertion/deletion (InDels) were detected from the transcriptome. This is the first reported high-throughput transcriptome analysis of S. prenanti, and it provides valuable genetic resources for the investigation of immune mechanisms, conservation of germplasm, and molecular marker-assisted breeding of S. prenanti.

Characterization of a RacGTPase up-regulated in the large yellow croaker Pseudosciaena crocea immunity☆

The Rac proteins are members of the Rho family of small G proteins and are implicated in the regulation of several pathways, including those leading to cytoskeleton reorganization, gene expression, cell proliferation, cell adhesion and cell migration and survival. In this investigation, a Rac gene (named as LycRac gene) was obtained from the large yellow croaker and it was expressed in Escherichia coli and purified. Subsequently the specific antibody was raised using the purified fusion protein (GST-LycRac). Moreover, the GTP-binding assay showed that the LycRac protein had GTP-binding activity. The LycRac gene was ubiquitously transcribed and expressed in 9 tissues. Quantitative real-time RT-PCR and Western blot analysis revealed the highest expression in gill and the weakest expression in spleen. Time-course analysis revealed that LycRac expression was obviously up-regulated in blood, spleen and liver after immunization with polyinosinic polycytidynic acid (poly I:C), formalin-inactive Gram-negative bacterium Vibrio parahemolyticus and bacterial lipopolysaccharides (LPS). These results suggested that LycRac protein might play an important role in the immune response against microorganisms in large yellow croaker.

Molecular cloning and functional characterization of a RabGTPase in large yellow croaker (Pseudosciaena crocea).

The molecular mechanisms of the immune system against pathogens in large yellow croaker (Pseudosciaena crocea) are not well known, despite its economic importance as an aquaculture species. In this investigation, a Rab gene (named as LycRab gene) was obtained from this fish, which exhibited high homology with Rab8 of other species. It was expressed in Escherichia coli, and the specific antibody was raised using the purified fusion protein (GST-LycRab). The LycRab protein, containing characteristic signatures of Rab proteins with 5 GTP-binding domains, had GTP-binding activity. The LycRab gene was ubiquitously expressed in all analyzed tissues as revealed by Western blot, although expression levels varied from tissue to tissue. Real-time PCR revealed that the LycRab gene was up-regulated after immunization with poly I:C, formalin-inactive Gram-negative bacterium Vibrio parahaemolyticus or bacterial lipopolysaccharides (LPS), suggesting that LycRab protein might play an important role in large yellow croaker defense against pathogens infection. This discovery might contribute better understanding to the molecular events involved in fish immune responses.

Development of SNP markers associated with immune-related genes of Schizothorax prenanti

Schizothorax prenanti (S. prenanti), one of the important endemic commercial fish in China, is mainly distributing in the upstream of the River Yangtze and the tributaries. The wild population is facing serious challenges of germplasm degeneration due to the overfishing, water pollution and construction of hydropower stations; therefore, it is very urgent to develop genetic resource of S. prenanti to protect the wild population. In this study, we used Illumina Hiseq2500 sequencing to develop single nucleotide polymorphism (SNP) markers in S. prenanti. From 37,785 unigenes with functional annotation, 857,535 putative SNPs were identified. Among them, 33 SNPs from immune-related genes were randomly selected and 20 loci exhibited significant polymorphisms in genotyping by Sequenom MassARRAY. As far as our best knowledge, this is the first report about the SNP markers development in S. prenanti based on Illumina RNA sequencing. These SNP markers should not only be useful for population conversation, but also for construction of genetic linkage map and economic performance improvement of S. prenanti.

Development of SNP markers associated with growth-related genes of Pelodiscus sinensis

The Chinese soft-shelled turtle (Pelodiscus sinensis) is one of the most important aquatic animals in China. The low growth rate is the main factor which hinders the development of the turtle’s industry. Therefore, it is urgent to development available molecular marker associated with genetic variation in growth for P. sinensis. The aim of this study was to identify and validate SNPs from growth-related gene of P. sinensis. The Illumina Hiseq4000 sequencing was used to develop single nucleotide polymorphism (SNP) markers in P. sinensis. Among them, 38 SNPs from growth-related genes were randomly selected and 24 loci exhibited significant polymorphism in genotyping by ARMS-PCR. The observed and expected heterozygosity ranged from 0.0625 to 0.5208 and 0.0807 to 0.4974, respectively. Significant deviation from HWE was observed for 3 loci. These findings provide molecular basis and candidate markers for P. sinensis breeding.