Development And Application Of SSR And Cloning Of Growth-related Gene HDAC1in Tegillarca Granosa

According to sequences originated from magnetic beads enrichment library andEST library, Genomic-SSRs and EST-SSRs were developed respectively. Comparativestudy was also carried out to detect the characterization of distribution of EST-SSRs inTegillarca granosa and the difference of genomic-SSR and EST-SSR while applied inpopulation genetic analysis.14SSRs were used to determine genetic structure betweenhybrid populations and purebred populations so as to provide biological information inthe further breeding programs. HDAC1cDNA was cloned by SMART RACE (RapidAmplification of cDNA Ends) technique and its expression status in different tissueswere also analyzed.1. Development and comparative study of Genomic-SSR and EST-SSR in T.granosa:47pairs of primer were designed according to flanking sequence frommagnetic bead enrichment library,17of them amplified expected fragments while17(100%) were polymorphic, the average number of alleles (Na), observed heterozygosity(Ho), expected heterozygosity (He) and polymorphism information content (PIC) were6.8,0.468,0.785,0.733.1683SSRs were detected by screening11344ESTs, theaverage density of SSRs was1per3.86kb of EST. Of120pairs of primer designed,62pairs amplified expected fragments steadily while29(46.77%) were polymorphic aftertested in30individuals of T. granosa. The average Na, Ho, He, PIC were4.2,0.372,0.554,0.500. The result of bivariate correlation analysis processed by SPSS showed:there was highly significant positive correlation between the length of SSR coresequence and Na, Ho, He, PIC at the0.01level, Spearman correlation coefficient were0.632,0.387,0.657,0.664, respectively.2. Genetic analysis of the purebred and crossbred populations originated from two different geographic stocks of T. granosa: As an important commercial marine bivalve,cultured bloody calm (T. granosa) populations are confronted with problems such asdecrease of genetic diversity and character degeneration. To study the probability ofimproving germplasm resource by hybridization,4populations were produced by adiallel mating of2different geographic stocks. And14polymorphic microsatellite lociwere used to analyze the genetic structure of4offspring populations. The resultrevealed that the mean He and PIC of the purebred populations (ZZ and KK) werehigher than that of reciprocal cross populations (ZK and KZ). However the average Hoof ZK and KZ was higher than that of ZZ and KK. The largest value (0.067) of geneticdifferentiation coefficient (Fst) was between ZK and KZ, and the smallest value (0.020)was between ZZ and KK. The value of Nei’s (1978) unbiased genetic distance was inline with Fstvalue, reciprocal cross populations showed the largest genetic distance,meanwhile purebred populations showed the smallest genetic distance. Our studyrevealed crossbred populations of the bloody calm presented relatively highheterozygosity and genetic variation, which may probably benefit the breeding work inthe further study.3. Cloning of HDAC1gene and analysis of expression in different tissues T.granosa:HDAC1is in charge of deacetylation of histone, so as to adjust gene expression,and also play an important role in cell differentiation. Using RACE technology, we getthe full length of HDAC1cDNA, which is2275bp long including26bp5’-UTR,1587bp ORF,662bp3’-UTR. The result analyzed by biological software showed HDAC1gene could encode528amino acids, molecular measure is59.91kDa, theoreticalisoelectric point is5.65, no significant signal peptide sequence and transmembraneregion were found, and HDAC1protein showed hydrophilism primarily. By RT-PCR，HDAC1were found in all tissues, and foot showed highest expression, which indicatesthat HDAC1might have functions of promoting muscle cell differentiation.