| As it is delicious and nutritious, Pelodiscus sinensis become delicacies and medicinal dish in China since ancient time. With the improvement of people’s living standard, accelerating the artificial propagation has become an inevitable trend by the reason of market requirement. However, as the non-standard introduction, lacking of breed conservation, the breed of Pelodiscus sinensis become confused and reduce the genetic diversity in recent years. Germplasm resources is the origin of industry and the fundamental guarantee of breeding populations. In order to develop and protect the germplasm resources of Pelodiscus sinensis rationally, the Pelodiscus sinensis of Zhejiang province Ningbo yaojiang river basin was regarded as the main research object, firstly, the chromosome preparation method of Pelodiscus sinensis was explored, following by the analysis of karyotype and G-band, it was aimed at providing the theory basis for the gender studies and artificial breeding of Pelodiscus sinensis. In order to identify different Pelodiscus sinensis geographic population accurately, the genetic diversity of six Pelodiscus sinensis geographical populations was analyzed by the methods of RAPD and DNA barcode. The results are as follows:In the aspect of karyotype analysis, we found that the best chromosome specimens could be gained by the quantity of colchicine 1μg/g(weight), effecting 1h. The karyotype formula of it is 2n=66=4m+4st+34sm+22T+ZZ/ZW, NF=104(male) and 105(female). In 33 pairs of chromosomes, the No. of 1~6 are large chromosomes, and the rest are small chromosomes, the sex chromosome type are ZZ(male) and ZW(female). The G-banding of chromosomes were made by trypsinase method, and the schema chart of it was mapped out.In the analysis of RAPD, RAPD technique was applied to analyze the genetic diversity of six Pelodiscus sinensis geographical populations, 112 individuals, by nine random primers. It has been detected a total of 156 loci, of which 142(91.03%) exhibited polymorphism, only 14 loci appeared to the six Pelodiscus sinensis geographical populations stably. Proportion of polymorphic loci(P), Nei’s gene diversity index(h) and Shannon information index(I) between different Pelodiscus sinensis geographical populations was 39.34~75.00%, 0.1144~0.2643, 0.1144~0.2643, respectively. In the various of genetic diversity indexes, Pelodiscus sinensis Yaojiang River strain had always been the highest level, suggested that it had highest genetic diversity among six Pelodiscus sinensis geographical populations. Yaojiang River strain had the nearest genetic relationship with Qingxi Huabie, and the farthest with Yellow River strain. It could not be found specific markers to distinguish different Pelodiscus sinensis geographical populations by electrophoretic band, however, it could be distinguished in the molecular phylogenetic tree based on individual genetic distance.In terms of DNA barcode identification, according to the existing Pelodiscus sinensis of COI gene sequence fragment, the specific primers were designed to be used for the middle segment, 3’ and 5’RACE reaction, after that, used universal primers, which were designed by the overall length of Pelodiscus sinensis COI gene sequence it got before, for the DNA barcoding analysis of genetic diversity among six Pelodiscus sinensis geographical populations of 111 individuals. From 1534 bp sequence of Pelodiscus sinensis COI gene, it had been detected 928 of conserved sites(C), 606 of variable sites(V), 337 of parsimony informative sites(Pi), 269 of Singleton sites(S), the average content of each nucleotide was A=30.10%, T=30.27%, C=23.41%, G=16.22%, which the content of A+T(60.37%) was obviously higher than C+G(39.63%). The whole group of haplotype diversity(H) was 0.972, the nucleotidy diversity(Pi) was 0.0171, indicated that each strain of Pelodiscus sinensis had a high genetic diversity level. Thr average genetic distance between and within different Pelodiscus sinensis geographical populations was 0.0203~0.0417 and 0.0113~0.0338, respectively. The interspecific and intraspecific genetic distance did not form an effective barcode clearance, therefore, individuals of different Pelodiscus sinensis geographical populations did not cluster together according to the population in the individuals NJ tree. Nevertheless, in the analysis of the Trionychidae between different genera, it had been formed an effective barcode clearance between interspecific and intraspecific genetic distance, different turtle clustered together accordance with the features of the genus respectively, and the nodes with high approval ratings. Consequently, as a DNA barcode, the COI gene could be effectively used to distinguish different genus of Trionychidae, but do not apply to the separate identification of different Pelodiscus sinensis geographical populations. |
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