Development And Application Of Snp Markers In Genome Of Shrimp (fenneropenaeus Chinensis)

Fenneropenaeus chinensis is a commercially important farmed species in China since the 1970s. Since the development of molecular biology techniques in the early 1990s, DNA markers have been widely applied to the structural and functional analysis of important genes, the analysis of population genetic structure, development of genetic linkage map, marker-assisted selection and QTL analysis. The purposes of this study were to identify the facticity of putative SNPs with amplification refractory mutation system (ARMS)-PCR method. Tetra-primer amplification refractory mutation system PCR (Tetra-primer ARMS PCR) technique is one of the better methods for SNP genotyping because of the lower cost and convenience.1. Tetra-primer ARMA-PCR is introduced to investigate single nucleotide polymorphisms (SNPs) genotyping in Chinese shrimp (Fenneropenaeus chinensis) with 80 putative SNPs locus. For a SNP locus, 2 allele specific inner primers which their 3 ends matched to the two alleles and 2 flanking outer primers were designed, respectively. Primers were designed to amplify fragments of variying sizes for each allele band in order for them to be easily resolved using agarose gel electrophoresis. The SNP was successfully genotyped as PCR were conducted with Mg²⁺ 1.5mmol/L, dNTP 0.2mmol/L, Taq polymerase 0.5U, inner/outer primers 4/1 at annealing temperature in Ta and following touchdown profiles. 20 out of the 80 tetra-primer ARMA-PCR primer sets were validated and the outer and the expected inner bands were amplified. Homozygous and heterozygous were detected by agarose gel and the genotypes were obtained.Twenty SNP tetra-primer ARMA-PCR primer sets were validated and used to investigate the genetic structure of six selected families of marine shrimp F. chinensis. The effective number of alleles ranged from 1.127 to 1.993, with an average value of 1.600. The average values of expected and observed heterozygosities of the SNPs ranged from 0.505⁰.609 and 0.373⁰.487, respectively. Polymorphism information content (PIC) values ranged from 0.145 to 0.373. Among 120 population-locus cases (six populations×twenty loci), only 8 (6.7%) showed significant deviation (P<0.05), while the other 112 (93.3%) were in Hardy-Weinberg equilibrium (HWE) (P>0.05). The frequencies of minor alleles ranged from 0.378 to 0.497.2. Three hundred and ten SNP tetra-primer ARMA-PCR primer sets were validated of eight hundred primers and the polymorphic SNP primers were two hundred sets between the parents. The polymorphic markers presented in this study provide a useful tool for population genetics, pedigree analysis, linkage map construction and marker-assisted selection (MAS) of Fenneropenaeus chinensis. The genetic linkage map of Fenneropenaeus chinensis was constructed using 200 SNP markers. Linkage map was performed using one F₁ family, and a composite linkage map was generated by incorporating map information from the male and female map. The composite linkage map contained 180 markers covering 899.3cM with an average spacing of 5.2cM. The number of linkage groups in the intergrated linkage map was 16.One hundred and eighty SNP tetra-primer ARMA-PCR primer sets were validated and used to investigate the genetic structure of F₁ family. The effective number of alleles ranged from 1.041 to 1.962. The values of expected and observed heterozygosities of the SNPs ranged from 0.067⁰.772 and 0.169⁰.657, respectively. Polymorphism information content (PIC) values ranged from 0.135 to 0.481. The MAF ranged from 0.021 to 0.492.3,A GLM procedure was used to analyze the correlation between the 180 SNPs and body weight. Results uncovered that C2904-168 and C12871-235 had a significant impact on body weight. The detection and location of QTL were performed based on map. One putative QTL (LOD≥2.0) associated with the body weight was detected and located in the male map LG16. The location of QTLs with the growth would be very useful for molecular-assisted selection and map-based cloning of functional genes.The results indicated that tetra-primer ARMA-PCR is a simple, rapid and efficient method for SNP genotyping which make it useful in a broad aspects of Fenneropenaeus chinensis genetic and breeding studies. The genetic linkage map of Fenneropenaeus chinensis was constructed using SNP markers. In summary, the SNPs study of Fenneropenaeus.chinensis is encouraging and suggesting that SNP markers have been useful for genetic and breeding studies in Fenneropenaeus. chinensis.