| Chinese soft-shelled turtle (Pelodiscus sinensis) is one of the most important economically freshwater cultured species in China, and it is favorable for medicinal value. However, due to inter-specific hybridization, the genetic resource of the turtle has been degrading. To date, few studies were on the breeding for the turtle. In the present study, RNASeq technology was applied in male and female turtle to produce a validated transcriptome database. Analysis of morphometric attributes in female and male turtle was taken and genetic diversity of four cultured populations was assesement by SSR and AFLP. The results are as follows:1. De novo assembly and transcriptomic analysis for male and female P. sinensis was conducted. Total 24 765 075 and 26 707 504 reads were obtained in female and male turtle, respectively.85 781 unigenes were assembled from reads, and mean length was 899 bp. The unigenes were blast by Nt, Swiss-Prot, GO, KEGG, Nr, COG and TrEMBL database, and 25 380 unigenes were annotated. Furthermore,2 445 different expressed genes were obtained among the unigenes which distributed in 193 KEGG signaling pathway. Cytokine-cytokine receptor interaction pathway was the most abundant DGE pathway which including 39 DEGs. Abundant genes and pathway on growth was observed, including genes in GH/IGF axi, TGF signal pathway and MAPK signal pathways etc. The transcriptome sequencing could provided support for functional genes research and molecular breeding in the turtle.2. A correlation coefficient matrix was constructed, in which the body weight was used as the dependent variable and other traits as independent variables. Path coefficients and determination coefficients indicated that plastron length showed predominant direct effect on body weight for 100-day-old turtles, and body height showed predominant direct effect on body weight for 300-day-old turtles. Furthermore, results indicated the morphometric attributes showed predominant direct effect on body weight of 100-day-old female and male turtles were carapace length and plastron length, respectively. Carapace length showed predominant direct effect on body weight of 300-day-old female and male turtles.3. Total 7 278 unigenes contained SSR locus in the transcriptome of turtle, including 4 900 mono-nucleotide SSRs,1 398 di-nucleotide SSRs,1 004 tri-nucleotide SSRs,70 tetra-nucleotide SSRs and 6 penta-nucleotide SSRs. 17 polymorphic SSR markers were isolated and characterized which produced 65 locus, and 12 markers were used for investigating the genetic structure in four cultured population of P. sinensis. Effective alleles, observed heterozygosity, expected heterozygosity and PIC value in four cultured turtles were 1.71～2.92,0.14～0.39, 0.28～0.50 and 0.24～0.46, respectively. Furthormore, the genetic diversity of four cultured populations of turtle was analyzed by amplified fragment length polymorphisms (AFLP). For the assessment, total 372 bands were generated from four populations using 5 different primer combinations,117 of which were polymorphic. The proportion of polymorphic loci among four populations varied from 14.33% to 48.35%. Nei’s genetic diversity and Shannon genetic diversity were ranged from 0.032 to 0.107 and 0.050 to 0.161, respectively. Results showed TW population has the highest Nei’s genetic diversity and Shannon diversity index, and RB population has the lowest Net’s genetic diversity and Shannon diversity index.4. Encoding amino acid cDNA of GHR and GHITM was obtained by PCR. The full length of GHR cDNA was 2000 bp, including an open reading frame (ORF) of 1 698 bp encdong a polypeptide of 566 amino acids. GHR encoding polypeptide contains signal polypeptide, extracellular domain, trasnmembrane domain and intracellular region, and FGEFS motif exists in intracellular region. The GHITM cDNA length was 2 537 bp, including an ORF of 1 050 bp encdong a polypeptide of 350 amino acids. GHITM encoding polypeptide contains extracellular domain, trasmembrane domain and intracellular region. There is a functional domain in intracellular region which was consisted by four transmembrane domains. The results of RT-PCR implied that GHR and GHITM showed higher expression levels in the liver and musscule. The 300-old-day turtles showed higher expression levels of GHR and GHITM genes, and male turtles showed higher expression levels of GHR and GHITM genes... |
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