| Grass carp(Ctenopharyngodon idella) had been cultured in China for thousands of years. Due to its fast growth and high quality of meat, its scale of aquaculture had been gradually expanded, and it became one of the most important freshwater cultured species in China. However, influenced by the continuous deterioration of environment and the improper artificial releasing, the genetic structure of the wild groups of grass carp had been severely compromised. In addition, with little attentions to the renewal and replacement of broodstock, or even reverse selection and inbreeding coefficient, the economic traits of grass carp had been seriously degraded. Even though the resources research of grass carp had been carried out for more than thirty years, there existed none excellent variety with the advantage traits or deserves for large-scale promotion. By using molecular markers, we can not only have a better understanding of the germplasm resources, but also shorten the process of selection and breeding process of grass carp. In previous researches, most studies on molecular markers were limited to SSR, and there were rarely studies on SNPs. Due to its unique characters, such as widely distribution in genomic and heridity stability, SNP markers had been widely used for the construction of genetic linkage map and molecular marker assisted breeding. In this study, we tried different methods to find the effective and efficient way to develop SNP markers in grass carp. And details were as follows: 1. Isolation and characterization of SNP markers from transcriptome of grass carpIn order to get enough SNP markers rapidly and lay foundation for molecular marker assisted breeding of grass carp, our group attempted to develop and detect SNP markers from a transcriptome. The transcriptome, which was sequenced by Next Seq500 technology, obtained 108452 mutations and 37661 contigs. Among all the mutations, the percentage of transition sites was 65.67% and the ratio of transition and transversion was 3.12. In this research, 60 mutation sites were randomly selected and 39 pairs of primers were designed. Finally, only 30 of the selected mutations were successfully genotyped by using SNa Pshot technology. After prediction of software, the average Ne(number of effective allele), Ho(observed heterozygosity), He(expected heterozygosity) and PIC(polymorphic information content) of the genotyped 39 mutations were 1.66, 0.3346, 0.3758 and 0.2922, respectively. In addition, 23 mutations presented moderate polymorphism, and 7 mutations presented low polymorphism; however, none SNP markers showed high polymorphism. Analyzed by Popgen 32, 10 SNPs showed significantly deviated from Hardy-Weinberg equilibrium(P<0.05). The above results indicated that it was an effective way to develop SNP markers from transcriptome of grass carp, and the polymorphic SNP markers might lay foundation for researches on grass carp, such as genetic structure analysis, linkage map construction and encryption. 2. Polymorphism of MSTN-1 and the association with growth traits and muscle compositions of juvenile grass carpPrevious studies suggested that the myostatin-1(MSTN-1) gene played as an important role in the growth and development of grass carp. In order to analysis whether the polymorphism of grass carp MSTN-1 gene was associated with growth traits and muscle compositions, 192 individuals from Yangtze River were used in this study. Three polymorphic loci were found in the 3824 bp MSTN-1 gene: C1799 T, C1842 T and TGAAGCGCTGGTTCT /2585-, which were named as Locus 1(wild-type EE/mutant EF), Locus 2(wild-type HH/mutant HI) and Locus 3(wild-type BB/deletion type BD), respectively. A general linear model was used to analysis the correlation between those three single nucleotide polymorphisms(SNPs) and grass carp traits. The results showed that two SNPs have a significant influence on grass carp growth traits. Whereas, none of those SNPs have a significant effect on grass carp muscle compositions. Multiple comparisons found that the body length and body weight of individuals with HI genotype were significantly higher than that of wide-type individuals, conversely, mutant individuals with BD genotype were significantly lower than wide-type individuals at body length and body weight in haplotype analysis; the groups with HI or BD genotype also had the similar results in fold type combinations analysis, while the body length of groups with BD genotype were not significantly lower than other groups. Results indicate that HI is a beneficial mutation on growth traits, while BD is a detrimental mutation on grass growth traits. Preliminary indicated the MSTN-1 gene could be a candidate modifier gene in Molecular Maker-assisted Selection(MAS) of grass carp. 3. Polymorphism of IGF1 gene and the association with growth traits in grass carpIn this study, we tried to evaluate the application of the SNP genotyping method, whose primers were designed by the specific of alleles, in grass carp IGF1 gene. Firstly, 7 SNPs were pre-detected by direct sequencing. Then three primers, including one public primer and two specific primers, were designed for each mutation. The length of the two specific primers, whose 3’ terminals were strictly complement with the alleles of each mutation, were deferent at 4bp. However, the 4 groups of primers could not type all the mutations in IGF1 gene. Therefore, we made some improvements on the re-designed 5 groups of primers with artificial introduced mismatches. With the re-designed primers and polymerase chain reaction, we tried to obtain the genotypes of 384 individuals. Statistical analysis found that the average successful rate of the re-designed primers was 85.57%. In view of the successful rate and efficient, we believed that this method can be applied to SNP genotyping researches with a medium scale sample size. In addition, we analyzed the connection between growth traits and SNPs of IGF1 in grass carp, however there was no correlation between them(P>0.05). |
