The Screen And Correlation Analysis Of Pou1f1, PSS Gene SNPs With Growth Traits Of Largemouth Bass

The largemouth bass (Micropterus salmoides L.), common name Californian bass, was subordinate to Perciformes, Porcoidei, Cehtrachidae, Micropterus. It's one of important cultivated fish in China. In recent twenty years, production units do not observe the rules of breeding, picking the small and early mature individuals as the parent, along with the small founder population and not introduce protospecies regularly, result in low genetic diversity and production performance degradation which have seriously affected the benefit of largemouth bass culture. So it's necessary to carry out the fish breed improvement programme. Marker assistant selection (MAS) which has the high stability and the information content, is influenced slightly by the environmental condition and is an effective way to selective breeding. In the research, we selecte the SNPs site in the POU1F1 and PSSⅢpromoters and make correlation analysis aimed to apply in MAS programme.1. POU1F1 cDNA and promoter cloneThe POU1F1 (pituitary specific transcription factor1) protein play a crucially positive role in the embryo development of prehypophysis and the expression of GH. We got largemouth bass POU1F1 cDNA and promoter sequence using 3′Race, RT-PCR and GenomeWalker methord. POU1F1 of largemouth bass consisted of 7 exons and 6 introns, the cDNA sequence length is 3141bp, the length of 5′UTR is 620bp, the length of 3'UTR is 1384bp, CDS 1128bp , coding 375 aa and highly similarily to Acanthopagrus schlegelii (88%) and Sparus aurata (88%), in the next place is Rainbow trout (75%), which is unanimous to the existing classification. The promoter sequence of largemouth bass POU1F1 was also cloned according Genomewalk which is 1629bp in length, predicted the existence of four octamer transcription factor 1 (Oct-1) binding sites, one homeobox transcription factors sites, two cAMP-responsive element binding proteins sites and a TATA box.2.The SNPs screening and correlation analysis of largemouth bass POU1F1 promoterUsing directly sequencing, two nucleotides mutations were identified which located at the -18 and -183 sites, and utilizing restriction enzymeonly AluⅠand BsrBⅠtwo haplotypes were identified (haplotype A and haplotype B). The different genotype individuals and the mutant alleles in stochastic population (126 individuals) were examined. The frequency of A haplotype and B haplotype was 0.246 and 0.754 respectely. The population correlation analysis showed that the AA and AB genotype has significant correlated with individuals' weight, body length, full length, body depth and body width (P<0.05), but there is no significant correlation between AA genotype individuals and AB genotype individuals.The growth correlation of POU1F1 genotype was further verified in stochastic population composed of 293 individuals. Population association analysis showed that the individual of the genotype AA and AB type was significantly higher than that of BB genotype in weight, head length, caudal peduncle depth (P<0.05), it also has the tendency in body length, full length, body depth and caudal peduncle length that the AA and AB genotype individuals precede than BB genotype individuals. And there was no significant difference between the AA genotype of type AB type individuals in growth traits .In order to eliminate the effect brought by the genetic differences in background, selected the sire and dam both belong to the POU1F1 gene promoter genotype AB, established AB×AB full sibs. Randomly choose 67 offsprings to make correlation analysis, finding that the BB genotype individuals had a lower weight, full length and body length. The transcription level of the different POU1F1 genotype generations was studied using real-time quantitative PCR using different genotype individuals. No significant difference in transcription was found between POU1F1 gene in different genotypes individuals, but the difference in GH gene transcription volume was significant (P<0.05), the BB genotype individuals' GH gene transcription volume was the highest, significantly higher than AA and AB genotype individuals. We conjecture that the haplotye is only a molecular marker of the growth traits of largemouth bass.The correlation analysis in the three groups showed that the haplotype in largemouth bass POU1F1 promoter has intimate relationship with the growth traits, so its adhibition in molecular marker assisted breeding is significant.3. The PSS cDNA and genome sequence clone and sequence analysis Somatostatin take part in pituitary secretion. All of PSSⅠ,PSSⅡand PSSⅢcan code somatostatin. Both of PSSⅠand PSSⅢgene consisted of 2 exons and 1 intron. The cDNA and genome sequences of PSSⅠand PSSⅢwre cloned using RT-PCR, GenomeWalker and 3′Race. PSSⅠDNA sequence was cloned which is 1626bp in length; the second exon was located in 1402-1626, 225bp in length; the 1-1401bp may the intron, the first exon was not cloned; the 3'UTR was 221bp in length got according 3' Race. The length of the PSSⅢDNA sequence was 2439bp, among that the length of the promoter sequence was 1751bp, the first exon lacated in 1752-1857 was 106bp, the first intron was 355bp in 1858-2212, the second exon in 2213-2439 was 227bp, the 3′UTR was 335bp. By comparing the PSSⅢcDNA sequence with the epinephelus coioides, we knew that the 5'UTR of the largemouth bass was 197bp which was located in 1555-1751.4. The SNPs screening of PSSⅢpromoter and correlation analysis Five SNPs had been found in PSSⅢpromoter: A→C mutation in -870bp, C→T mutation in -448bp, A→G mutation in -101bp, C→T mutation in -54bp and C→T mutation in -24bp. The genotype frequency was between 0 and 93.5%. The correlation analysis showed that -870bp, -448bp, -54bp and -24bp four SNPs had no significant difference between the different genotype individuals in growth traits, but the differences in individuals' weight, body length, full length, caudal peduncle length and caudal peduncle depth were significant in -101 site. From -102bp to -98bp is the Pax2 binding site when the -101bp is nucleotide A.