The Screening Of Growth-related Molecular Marker In Trionyx Sinensis

The Chinese soft-shell turtle, Trionyx sinensis belongs to Reptilia, Testudinata, Trionychidae, Trionyx. It is widely distributed in CHINA, except Tibet, Qinghai and Sinkiang, and found mostly in Yangtze River Valley and South China, it also distributed in Japan, Korea, Vietnam and other Southeast Asian countries. The Chinese soft-shell turtle of the breeding industry developing rapidly due to high demand for edible and medicinal in recent years, the annual output reached 340,000 tons, and the mainly breeding methods are mix culture, pond culture and industrial aquacultures and so on. In order to the economic profit,a lot of aquaculture farm breeding is not standard, especially lead to the spotty quality of the Chinese soft-shell turtle seedling,bring out germplasm characters degradation and decreased the growth speed,all of this block the sustainable development of the Chinese soft-shell turtle industry, therefore, we carrying out the work hope to select to fast growth breeds of the Chinese soft-shell turtle to promote the industrial development and greatly meet of hunman needs, and also hope to provide basic data to germplasm conservation and fine seeds selection of the Chinese soft-shell turtle, thus to promote T. sinensis germplasm identification,conservation and prosperous development of well breeds.In this research, designed SSR primers from genome and transcriptome, widely developed molecular marker and the genetic diversity of three populations of T. sinensis, total 72 individuals, was evaluated. In order to obtain more SSR molecule markers linked to growth traits of Chinese soft-shell turtle, microsatellite marker and molecular biology technique were used to analyze the genetic structure of 120 individuals in the Lvka group. In addition, genetic cloning and sequence polymorphism were used to the functional gene IGF1 to breeding research that hope to find the SNP molecular marker which related to the growth of Chinese soft-shell turtle, and selected to fast growth and high feed nutrient gain ratio of new quality variety applied to production of aquaculture and demonstration promotion.1. Microsatellite markers development and genetic diversity analysis in three populations of T. sinensisT. sinensis is a common freshwater soft-shell turtle in China, and is an important aquaculture species. In this study, 20 polymorphic microsatellite primers were developed from transcriptome. The genetic diversity of three populations of T. sinensis, total 72 individuals, was evaluated. The number of alleles per locus ranged from 3 to 26. The observed and expected heterozygosities varied from 0.208 to 0.958 and from 0.302 to 0.963, respectively. The polymorphic information content varied from 0.283 to 0.953. No significant linkage disequilibrium was detected. These markers will be useful for in future population genetic studies and molecular breeding of T. sinensis.2. Correlation analysis of the microsatellite DNA markers with growth traits of T. sinensisIn order to obtain more molecule markers linked to growth traits of Chinese soft shell turtle, 25 microsatellite DNA markers were selected to analyze the genetic structure of 120 individuals, then compared these markers with min groups and max groups in aspects of Body weight, Carapace length, Carapace width, Body height, Shell length and Shell width. The results indicated that 3 microsatellite loci showed significant or highly significant influences on these growth traits(P<0.05 or P<0.01). LTF1 is significantly correlated with Body weight, Carapace length, Carapace width, Body height, Shell length and Shell width, and LTR87 is significantly correlated with Body weight, Carapace length and Shell width, while LT8 is significantly correlated with body weight, Body height, Shell length and Shell width. Multiple comparison analysis of genotypes related to growth traits showed that BE genotypes of LTF1, BB genotypes of LTR87, and CF, BC, AC and CC genotypes of LT8 played positive roles in growth traits respectively, which gives effective basis to the study of Chinese soft shell turtle marker-assisted breeding.3. Cloning and polymorphic analysis of IGF1 gene and Screening of SNPs associated with growth trait in T. sinensis.The c DNA sequence of IGF1 was obtained. The c DNA sequence of IGF1 was 710bp, and an open reading frame(ORF, 460bp) coding 153 amino acids. The c DNA sequence of IGF1 of T. sinensis was compared and analyzed with blastn through the database of NCBI, the result showed that the IGF1 gene was highly conserved and similarity was high with other animals, the sinilarities were about 85-98%.The phylogenetic tree of the IGF1 gene of aminoacid sequence were established using MEGA5.0 software and it indicate that T. sinensis had the nearest evolutionary relationship with Chelonia mydas, they clustered with Chrysemys Picta bellii in the phylogenetic tree, and displayed a far evolutionary relationship with Gallus gallus, Alligator sinensis, Mus musculus, Homo sapiens.Five SNPs locus S1, S2, S3, S4 and S5 were found in IGF1 gene of min groups and max groups of Chinese soft-shell turtle, which were located in the ORF region of the gene. The results of association analysis between the genotypes of SNPs and the growth traits of the offspring of Chinese soft-shell turtle showed that the growth traits in the genotype GG in S1 and the genotype TT in S3 are higher than the other genotype in the same site. The 5 SNPs sites composed 9 diplotypes(D1-D9). The dominant diplotypes in the max groups were D9 and D5,and in min groups was D1. In conclusion, the genotypes GG in S1 site and TT in S3 site, diplotype D9 of IGF1 gene were closely related to high growth rate in Chinese soft-shell turtle, which can be used as candidate molecular markers for turtle breeding.