Analysis Of EST Database For Largemouth Bass(Micropterus Salmoides) And Screening Of Molecular Markers Of Association With Growth Traits

1. Establishs and analysis of EST database for largemouth bassIn this study, Roche454high-throughput technology was used to conducttranscriptome sequencing and establish ESTs database in the largemouth bass northernsubspecies and florida subspecies. The results showed that a total of468671and332322ESTs were generated from northern subspecies and florida subspecies. The average lengthwas306.5bp and304.4bp, respectively. Assembly of the largemouth bass northernsubspecies and florida subspecies’ high quality ESTs resulted in42056and35743contigs.The average length was612.6bp and588.2bp, respectively. EST database of largemouthbass northern subspecies and florida subspecies was merged and then compared withknown protein databases.78938EST sequences in total were annotated. Using the GeneOntology （GO） information, we classified the sequences according to molecularfunctions、 cellular functions and biological processes.By exploring the mergedlargemouth bass EST library, over25469and8547putative SSRs and SNPs wereidentified. Comparison of the northern subspecies and florida subspecies sequence wasmade by randomly selecting75contigs from the EST database. Results showed that ESTsequences of the two subspecies homology were99.2%.2、The development of EST-SSRs in largemouth bassWe selected randomly133SSRs sequenced, and designed these microsatellitesprimers. Fifteen pairs of primers can amplified clear and polymorphic products by methodof PCR. Fifteen pairs of EST-SSRs primers were employed to detect the polymorphism oflargemouth bass northern subspecies. The results show that the allele number ranges from2to5alleles, averaging2.533alleles per locus. The observed and expectedheterozygosities range from0.150to0.857(mean0.480) and from0.262to0.739(mean 0.550), respectively. The average polymorphism information content(PIC) ranged from0.261-0.740(mean0.384). One out of the15loci(Contig44566) tested showed a verysignificant deviation from HWE after Bonferroni correction.3. SNPs markers screening of largemouth bass and polymorohisms of SNPs in APOgene and association analysis with growth traitsIn this study, genomic fragments of metabolism genes such as apolipoprotein,activin and zincfinger protein etc were amplified based on the sequences of many available contigs in the EST-SNPdatabase of largemouth bass. We obtained14SNPs loci. In order to further study apolipoprotein(Apo-A1、Apo-A4and Apo-C1)，we will verify sites of apolipoprotein in another group，the result is：A+489C mutations from Apo-A1gene, A+633T mutations from Apo-A4gene, A+24G�'�A+75Cmutations from Apo-C1gene mutations from activin gene were identified after sequencing the PCRproducts. Regarding the four SNPs, the genotype and gene frequency of159largemouth bass werefurther assayed with the method of SnaPshot. A general linear model was used to do the correlationanalysis among the apolipoprotein and activin gene SNPs and growth traits. In Apo-A1gene, A+489Csite showed significant association at body weight and body high（P<0.05）. In Apo-A4and Apo-C1genes, A+633T、A+24G and A+75C sites showed significant association at body weight, body highand total length（P<0.05）.