The Resarch Of SSRs And SNPs Markers On Laboratory Carassius Auratus Red Variety C1HD

OBJECTIVE: To research the Laboratory Carassius auratus Red variety C1HD by microsatellite DNA(SSRs) and Single Nucleotide Polymorphisms(SNPs) molecular markers.METHODS: 1.As the SSRs markers research, selecting six pairs microsatellite primers of common carp randomly, eight samples of laboratory Carassius auratus red variety C1HD and common Carassius auratus red variety were analyzed by PCR. Then the microsatellite DNA marker of laboratory Carassius auratus red variety C1HD was selected after PCR products electrophoresis were analysed respectively. 2.As the SNPs markers research, two pairs primers referring to the SNPs sites of Zebrafish Genome Bank to SNPs of genes were designed and the genome DNA of twelve samples of laboratory Carassius auratus red variety C1HD and common Carassius auratus red variety repectively was amplificatied by single-tube bi-directional allele specific amplification (SB-ASA). Then the SNPs genotype of samples were determined after electrophoresis analysis of amplification products, which were sequenced. Then the SNPs markers of C1HD were selected after analysising the data.RESULTS: 1.As the SSRs markers research, selecting six pairs microsatellite primers of common carp randomly, eight samples of laboratory Carassius auratus red variety C1HD and common Carassius auratus red variety were analyzed by PCR. Results of amplification products after electrophoresis showed that they were only amplified a allele by MFW14, MFW24 and MFW25 primers in two types of Carassius auratus red variety respectively. They were amplified multiple alleles altogether by MFW1 and MFW5 primers in two types of Carassius auratus red variety respectively.Amplification products appeared a allele of approximately 180bp length by primers MFW15 in common Carassius auratus red variety type, but amplification products of laboratory Carassius auratus red variety C1HD type did not appear its.2.As the SNPs markers research, two pairs primers referring to the twenty-four SNPs sites of Zebrafish Genome Bank of twenty genes were designed and the genome DNA of twenty-four samples of Carassius auratus red variety was amplificatied by SB-ASA.Amplification products to electrophoresis analysis and showed that three sites Zk261O7 gene zSNP30112411 (A/G type), zC227H20 gene zSNP24678043 (T/A type) and zC39F23 gene zSNP26474328(C/G type) can been genotyped. It was that different internal reference were amplified fragment length 432bp, 482bp, 365bp. The corresponding characteristic segments length were common Carassius auratus red variety 153bp (on behalf of genotype A) and laboratory Carassius auratus red variety C1HD of 314bp (on behalf of genotype G), common Carassius auratus red variety 228bp (on behalf of genotype T) and laboratory Carassius auratus red variety C1HD of 289bp (on behalf of genotype A), common Carassius auratus red variety 254bp (on behalf of genotype C) and laboratory Carassius auratus red variety C1HD of 141bp (on behalf of genotype G).So characteristic length of the fragments can discriminate laboratory Carassius auratus red variety C1HD and common Carassius auratus red variety.Genotyping results were completely consistent with sequencing. Data analysis showed DNA fragments length were sequenced 10933bp in the twenty-four SNPs sites. There were ninety-two SNPs and seventy haplotypes in total. Thirty-two and sixty-seven SNPs were found respectively in laboratory Carassius auratus red variety C1HD and common Carassius auratus red variety.The SNPs frequencyθwas 0.47, 1.02 SNPs/kb respectively in laboratory Carassius auratus Red variety C1HD and common Carassius auratus red variety,and the SNPs frequencyπwas 0.31×10-3,1.27×10-3 respectively.So differentiation of SNPs exist different regions.Allcle frequencies at sixty-five SNPs in ninety-two SNPs loci showed significiant difference between laboratory Carassius auratus red variety C1HD and common Carassius auratus red variety.Three SNPs of zSNP30124211(G/A type),zSNP24678043(A/T type) and zSNP26474328(C/G tye) at three genomic sites of zK261O7,zC227H20 and zC39F23 can discriminate laboratory Carassius auratus red variety C1HD and common Carassius auratus red variety.Thirty-eight and forty-seven haplotype were detected in laboratory Carassius auratus red variety C1HD and common Carassius auratus red variety, and fifteen common haplotypes.Mean haplotype divergence was 0.14, 0.234 in them respectively. Haplotype differentiation(Gst) was between 0 and 1.CONCLUSION: 1.As the SSRs markers research, the band of 180bp only amplified in common Carassius auratus red variety from primer(MFW15) could be used as a microsatellite DNA marker for discriminating laboratory Carassius auratus red variety C1HD and common Carassius auratus red variety.2.As the SNPs markers research, three conclusions made as follow. (1)Different characteristic lenth fragments to three SNPs sites of zSNP30124211(A/G type), zSNP24678043(T/A type) and zSNP26474328(C/G type) can be used to be SNPs markers to discriminate laboratory Carassius auratus red variety C1HD and common Carassius auratus red variety. (2)SB-ASA method was successfully used to conduct SNPs genotyping of laboratory Carassius auratus red variety. (3)The SNPs diversity of common Carassius auratus red variety was larger than laboratory Carassius auratus red variety C1HD.