Development Of SSR Markers, GHR Gene Cloning And Expression Of Sander Lucioperca

The pikeperch(Sander lucioperca L.) is one of the most economically important freshwater species. In this study, we presented the pikeperch transcriptome using Illumina sequencing technology and developed a set of SSR markers. Microsatellite markers were used to analyze three geographic populations of pikeperch and to do correlation analysis with growth traits of pikeperch. In addition, molecular cloning and gene expression analysis of growth hormone receptor gene in pikeperch were also carried. These studies surely provide basic knowledge for genetic management of breeding stocks and marker-assisted selective breeding of pikeperch.1 Characterization of pikeperch(Sander lucioperca) transcriptome and development of SSR markersThe pikeperch is one of the most economically important freshwater species and has been recently explored as a potential candidate for aquaculture. To facilitate pikeperch research, we sequenced its transcriptome and developed a set of microsatellite markers. We conducted Illumina RNA-sequencing and obtained over 50 million reads from a pooled cDNA library of different tissues. The clean reads were de novo assembled into 56,746 transcripts, with an average length of 1,474 bp. The annotation analysis demonstrated 37,386 transcripts(65.88%) with homologous sequences in the NCBI nr protein database. Of these annotated transcripts, 18,576 sequences were successfully assigned into Gene Ontology(GO) terms, 23,566 transcripts into the Cluster of Orthologous Groups(COG), and 12,081 transcripts to 322 Kyoto Encyclopedia of Genes and Genomes(KEGG) pathways. 16,368 SSRs(≥10 bp) were detected from 11,921 unigenes. The validation of randomly selected 300 SSR markers demonstrated 87% of the markers can be successfully amplified, suggesting RNA-Seq is an efficient tool for the development of molecular markers. This study provides not only a valuable transcriptomic resource, but also a large set of SSR markers for basic aswell as applied research in pikeperch.2 Analysis of genetic diversity on three populations of pikeperch(Sander lucioperca) by microsatellite markersTo investigate the genetic diversity and genetic structure of pikeperch in China,three populations of pikeperch were collected for research, one wild population from Xinjiang, two cultured populations from Shandong province and Suzhou, Jiangsu province, respectively. 20 microsatellite markers were used for analysis. The results showed that out of 20 microsatellite primers, 18 loci were successfully amplicated, of which 14 loci were polymorphic in all the three populations. The number of alleles per locus ranged from two to six, with an average number of 3.6. Among the three populations, the average number of alleles, average expected heterozygosity(He) and average polymorphic information content(PIC) ranged, respectively, from 2.57 to 3.36,0.51 to 0.56, 0.39 to 0.48, which indacated the genetic diversity of the three populations was in the medium to low level. The order of the genetic diversity in the three populations was Xinjiang>Suzhou>Shandong. The value of Fst revealed existing genetic differentiation among the three populations. The highest genetic distance was between Xinjiang population and Shandong population, while the lowest between Shandong population and Suzhou population, showing the two populations had a close relationship. The research revealed that monitoring genetic background and maintaining a large quantity of parent population are necessary measures for preventing genetic bottleneck and sustainable development of pikeperch culture.3 Correlation analysis of microsatellite DNA marks with growth trait of pikeperch(Sander lucioperca)Total 15 microsatellite DNA markers were selected to analyze the genomic DNA of60 individuals of pikeperch. The results showed that the number of average allele was3.81, the value of mean observed heterozygosity was 0.88, the mean excepted heterozygosity was 0.66 and the mean polymorphism information content(PIC) was0.58. The SPASS 16.0 was used to analyze the correlation between microsatellite DNA marks and growth traits of pikeperch. Results uncovered 8 SSR markers had significant on no less than one growth trait, in which 5 SSRs had significant on all selected growth traits.4 Molecular cloning and gene expression analysis of growth hormone receptor in pikeperch(Sander lucioperca)An open reading frame(ORF) clone of the growth receptor(GHR) was isolated from the liver of pikeperch by 5’ and 3’ rapid-amplification of cDNA ends(RACE). The ORF of pikeperch GHR is 1995 bp, which encodes a transmembrane protein of 664 amino acids(aa). The GHR protein of pikeperch possesses the same characteristic motifs and architectural design as GHRs of other fish species. When compared to the other fish GHRs, it is most homologous to the Epinephelus coioides, where the aa identity is 88%. But the GHR sequence of pikeperch is more remotely related to the species of the Cyprinidae species, such as Labeo calbasu(51%) and Cyprinus carpio(49%). Real-time polymerase chain reaction(RT-PCR) using a pair of the GHR gene-specific primers revealed the expression of GHR in various tissues and in different growth stocks of pikeperch. GHR was expressed in all tissues tested, of which expression of GHR was highest in liver, lowest in heart and no significant difference was observed in each tissue between fast-growing stock and low-growing stock,excluding in epidermis.