| Chinese Soft-shelled turtle (Pelodiscus Sinensis), belong to Reptilia, Testudines,Trionychidae, Trionyx, because of its tender meat, delicious flavor, ability of improvingimmunity and mental tonic effect significantly, has been very popular since ancienttimes by consumers and farmers. In recent years, with the improvement of livingstandards, the consumption of soft-shelled turtle is increasing year by year, graduallyexpanding the scale of their farming. However, in recent decades, with the rapidexpanding scale of artificial culture, managers’ nonstandard breeding operations andlarge influx of foreign smuggling turtle, germplasm traits of soft-shelled turtle havedegraded seriously, many problems such as the genetic mixture, decrease of diversitylever, had greatly hindered the development of germplasm protection and breedingindustry in Chinese soft-shelled turtle, which are important issues to be solved. Thus bythe means of Illumina paired–end sequencing technology, we generate a giant ESTdatabase and discover a number of EST-SSRs. Based on the SSR markers, the geneticstructure and genetic diversity of four different cultured populations of soft-shelledturtle was analyzed, and the loci associated with growth traits of soft-shelled turtle werediscovered, and the molecular structure and function of genes associated with growthtraits were also studied. The major results are as following:1Development of EST-SSR markers in the Chinese soft-shelled turtles(Pelodiscus Sinensis) by RNA-SeqBased on the sequences of the cDNA sequencing library, potential SSR loci werediscovered by the MSATCOMMANDER tool, and the primer pairs were designed usingPRIMER3. We selected and validate100pairs of SSR makers (EST-SSRs) associatedwith economic traits obtained in the three GO level2terms including growth,development and metabolism progress based on the result of GO ontologies respectively.And consequently,15polymorphic EST-SSRs pairs were selected researched the thegenetic structure and genetic diversity of four different cultured populations of soft-shelled turtles. The alleles(Na), alleles frequency(P), effective alleles(Ne),observed(Ho) and expected (He) heterozygosity value and polymorphism informationcontent (PIC) values were all analyzed, in the mean while, the clustering dendrogram,was made based on the results of UPGMAmethods using MEGA3.2software.①In all,15319sequences containing19452SSRs were identified from85246consensus sequences (non-redundant or unigenes), the frequency and the distributiondensity were22.82%and177per Mb separately in the soft-shelled transcriptom, and atotal of11801(61%) SSR motifs were designed with primers successfully. The selected100pairs of EST-SSRs associated with economic traits obtained in the three GO levels2terms were validated and consequently23polymorphic SSRs were obtained.②There were totally354alleles were detected across the120soft-shelled turtles of4populations and the number of effective alleles (Ne) were between2and7, theaverage alleles (Na) and effective alleles (Ne) values of each loci were2.95,2.46respectively; the average observed (Ho) and expected (He) heterozygosity values were0.40and0.55respectively, and the mean polymorphism information content (PIC)values was0.48, which has reached to the middle polymorphism lever.(0.5>P>0.25）.③The genetic distances between Yellow River and Japan Pelodiscus sinensispopulations was the largest, which reached to0.157,while the genetic similarity index ofit was the smallest, which indicated that the genetic relationship between these twopopulations was more distant. Otherwise, the genetic distances values and geneticsimilarity index between Yellow River and Huai River were0.0841,0.9194，respectively，suggesting that the genetic relationship between these two populations wasmore closer. The UPGMAdendrogram based on Nei’s genetic distance indicated that theYellow River and Huai River populations are grouped in one cluster, which is thenclustered with Taihu Lake populations, and joined with Japan populations finally.2Correlation analyses of EST-SSR markers with growth traits in Chinesesoft-shelled turtles (Pelodiscus Sinensis)15polymorphic microsatellites were selected to analyze the linkage analysis offour growth traits among60individuals, finding that5loci of which were associatedwith the growth traits in all. The result indicated that: the locus C8387had a significantimpact on body weight, body length, carapace length and a extremely significant difference in side width (P<0.01), and CC at C8387was favorable genotype for thefour growth traits while AB was disadvantage genotype;locus C211showed aextremely significant impact on body weight, carapace length (P<0.01) and asignificant impact on side width (P<0.05), among them allele C was negativelyassociated with the body weight and genotype CC was disadvantage genotype ofcarapace length and side width, while BB and AB were favorable genotypes of sidewidth and body weight respectively; locus C5670had a extremely significant impact onbody weight (P<0.01) and a significant impact on carapace length (P<0.05), in whichBB was the dominant genotype of body weight and carapace length while the AA wasthe negative genotype;the locus C1312and C13038had a extremely significantcorrelation of body weight,carapace length, respectively (P<0.01),in which BC of locusC1312and BB of C13038was dominant genotype of body weight and carapacelength,respectively.3Molecular Cloning and gene expression analysis of MSTN cDNA in Chinesesoft-shelled turtles (Pelodiscus Sinensis)The primers were designed and produced according to soft-shelled turtles MSTNgene cDNA sequence to obtain the whole sequence of it through RACE (rapidamplication of cDNA end) method of5’and3’end. And the structure of MSTN genecDNA sequence and the MSTN protein were predicted by bioinformatical software andmethods, additionally, the expression pattern of MSTN gene was also investigated indifferent ages and tissues using RT-PCR. The results showed that:①the full length of cDNA is2301bp,with an open read frame(ORF）of1134bpencoding375amino acids, having69bp5’-UTR (untranslated region) and1084bp3’-UTR. There were obvious termination codon TGA, polyadenylation signalsAATAAA and poly A tail in the3’-UTR. And a conserved proteolysis processingsite(RARR）and bioactive carboxy terminal region including9cysteine residues wasalso found in the C’ terminal of protein. In addition，this protein owns onetransmembrane domain as well as a signal peptide, and the secondary structure ofprotein contained6α-Helix，16β-Turn，which consisted with the feature of TGF-βsuper family using homology modeling technique. Comparing soft-shelled turtles MSTNwith those of western painted turtle, green turtle, turkey, chicken short-tailed opossum and human，the results revealed that the amino acid homology was between80%~99%.②MSTN gene of the soft-shelled turtle has a certain amount of expression in eachtissue and had formed a significant difference between tissues(P<0.05）,in which therelative expression quality of leg muscles is0.6064, higher than other tissuesobviously(P<0.05), followed by the heart and liver，were0.146and0.1196respectively；while the kidney and spleen had lower relative expression quantity, were0.0346and0.0289，respectively，and there was no significant differences (P>0.05）between otherfour tissues except the leg muscle and heart.③The relative expression quantity of MSTN gene occurred in every age of thesoft-shelled turtle, increasing firstly and then decreased with ageing. Among them,soft-shelled turtle of age3owned the highest levels of relative expression quantity of0.2635, significantly higher than that of the shelled turtle of4-age (P<0.05), whoserelatively expression quantity was0.0844only; followed by the soft-shelled turtles of1-age and2-age,were0.1403and0.1819, repectively，both higher than that of4-age,while there was no significant differences formed among them (P>0.05). |
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