[Identification and analysis of Corydalis boweri, Meconopsis horridula and their close related species of the same genus by using ITS2 DNA barcode].

The study is aimed to ensure the quality and safety of medicinal plants by using ITS2 DNA barcode technology to identify Corydalis boweri, Meconopsis horridula and their close related species. The DNA of 13 herb samples including C. boweri and M. horridula from Lhasa of Tibet was extracted, ITS PCR were amplified and sequenced. Both assembled and web downloaded 71 ITS2 sequences were removed of 5. 8S and 28S. Multiple sequence alignment was completed and the intraspecific and interspecific genetic distances were calculated by MEGA 5.0, while the neighbor-joining phylogenetic trees were constructed. We also predicted the ITS2 secondary structure of C. boweri, M. horridula and their close related species. The results showed that ITS2 as DNA barcode was able to identify C. boweri, M. horridula as well as well as their close related species effectively. The established based on ITS2 barcode method provides the regular and safe detection technology for identification of C. boweri, M. horridula and their close related species, adulterants and counterfeits, in order to ensure their quality control, safe medication, reasonable development and utilization.

[Study on mitochondrial DNA genetic diversity of Tibetan antelope].

With noninvasive DNA genotyping technology, we investigated genetic diversity of Tibetan antelope by analyzing mitochondrial DNA variationìthose samples are collected from Hoh Xil National Nature Reserve, Qinghai province, China. A total of 444-446 bp of the mitochondrial control region was sequenced form 10 individuals. The results showed that A% + T%(61.8%) was higher than C%+G% (38.2%) obviously. Ten haplotypes were identified in the 10 samples. The result showed 48 polymorphic sites after comparisons of the 10 haplotype samples, among them, including 44 transitions, 1 transversion, 1 insertion and 2 deletions. The average genetic distance of haplotypes is 0.031. The haplotypic diversity is 1.000. The nucleotide diversity is 0.0296. The average number of nucleotide differences is 13.127. It showed that the Tibetan antelope population has high variation in mitochondrial D-loop sequence.

[The community and structure of nitrogen-fixing microorganism in Sanjiangyuan natural reserve].

Research on the diversity of microorganism community in natural environment has been concerned hot spot using the newly molecular biotechnology in the world now. To understand the composition and structure of nitrogen-fixing bacteria communities in the Qingzang plateau, the molecular diversity and phylogenetic of nifH genes of Sangjiangyuan natural reserve were examined by using the PCR-RFLP based cloning approach. The 3 samples were come from different sites and different plant types, and their biogeochemical parameters were diverse. DNA was directly extracted from the soil microorganism and amplified the nifH gene fragment using PCR by the primers of nifH-34F 5'-AAAGG(C/T)GG(A/T) ATCGG(C/T)AA(A/G) TCCACCAC-3' and nifH-491R 5'-TFGTT(G/C)GC(G/C)GC(A/G)TACAT(G/C)GCCATCAT-3'. For the nifH gene segment, diverse PCR products were characterized by cloning, restriction fragment length polymorphism (RFLP) analysis and sequencing. A total of 233 clones and 99 operational taxonomic units (OTUs) which were digested the clones by the restriction enzymes MspI and RsaI were obtained from all samples. YS-1 had 63 clones and 24 OTUs, ZD-1 had 75 clones and 28 OTUs, and NQ-1 had 95 clones and 47 OTUs, respectively. They were found 1-2 significant domain groups of clones and shared 4 OTUs in all samples. A wide range of sequence divergence was observed in the 26 nifH clones that were sequenced from all samples. Sequence comparison showed that the nifH clones were 66% to 98% similar. The phylogenetic tree was constructed by the Clustal W and Mega software. 26 sequences could be subdivided into 4 clusters in the phylogenetic tree, and some of them had the closely similar to Proteobacteria, but The majority of the clones were not closely related to any known cultivated nitrogen-fixing bacteria, Therefore, most of them are unique and may represent novel sequences of nitrogen-fixing bacteria.

[Molecular diversity and phylogenetic analysis of nitrogen-fixing (nifH) genes in alp prairie soil of Sanjiangyuan natural reserve].

Research on the diversity of microorganism community in natural environment has been concerned hot spot using the newly molecular biotechnology in the world now. This was the first description of the molecular diversity and phylogenetic analysis of nitrogen-fixing (nifH) genes in alp prairie soil of Sanjiangyuan natural reserve. DNA was directly extracted from the soil microorganism and amplified the nifH gene fragment using PCR by the primers of nifH-34F 5'-AAAGG(C/T)GG(A/T) ATCGG(C/T)AA(A/G) TCCACCAC-3' and nifH-491R 5'-TYGTT(G/C)GC(G/C)GC(A/G)TACAT(G/C)G CCATCAT-3'. For the gene fragment, diverse PCR products were characterized by cloning, restriction fragment length polymorphism (RFLP) analysis and sequencing. 143 clones and 35 different RFLP patterns were received in two samples by the restriction enzymes MspI and RsaI digested. ZD sample had 82 clones and 21 different RFLP patterns, and YS sample had 61 clones and 19 different RFLP patterns. There were shared 5 RFLP patterns in two samples. The analysis result found a significant dominant group of clones occurring in both samples which account for 29.3% and 32.8%, respectively, and several minor groups were also detected. 21 clones were sequenced, and their levels of nucleotide identity were from 71% to 98%. None of the sequenced nifH gene was completely identical to any deposited in the data banks, and therefore each of them belong to a noncharacterized bacterium. Finally, the phylogenetic tree was constructed by the Clustal W and Mega software. 21 sequences can be subdivided into 4 clusters in the phylogenetic tree, and most of them had the closely similar toalpha- , beta-, and gamma-Proteobacteria . The significant dominant group in YS sample and ZD sample had the closely related with Rhodobacter sphaeroides and Delftia tsuruhatensis, respectively. The YS-nifH-11 was the only sequence which had highly similar to Cyanobacteria .

[Genetic diversity of microsatellite loci in captive Amur tigers].

The tiger is one of the most threatened wildlife species since the abundance and distribution of tiger have decreased dramatically in the last century. The wild Amur tiger (Panthera tigris altaica) only distributed in northeast China, the far east area of Russia and the north Korea and its size of wild population is about 450 in the world and 20 in China. Several hundred captive populations of Amur tigers are the main source to protect gene library of tiger and the source of recovering the wild populations. The Breeding Center for Felidae at Hengdaohezi and Haoerbin Tiger Park in Heilongjiang Province is the biggest captive breeding base in China. How to make clear the genetic pedigree and establish reasonable breeding system is the urgent issues. So we use the microsatellite DNA markers and non-invasive technology to research on the genetic diversity of captive Amur tiger in this study. Ten microsatellite loci (Fca005, Fca075, Fca094, Fca152, Fca161, Fca294, Pti002, Pti003, Pti007 and Pti010), highly variable nuclear markers, were studied their genetic diversity in 113 captive Amur tigers. The PCR amplified products of microsatellite loci were detected by non-denatured polyacrylamide gel electrophoresis. Allele numbers, allelic frequency, gene heterozygosity(H(e)), polymorphism information content(PIC) and effective number of allele(N(e)) were calculated. 41 alleles were found and their size were ranged from 110bp to 250bp in ten microsatellite loci, Fca152 had 6 alleles, Fca075, Fca094 and Fca294 had 5 alleles, Fca005 and Pti002 had 4 alleles and the others had 3 alleles in all tiger samples, respectively. The allelic frequencies were from 0.009 to 0.767; The He ranged from 0.385 to 0.707, and Fca294 and Pti010 locus had the highest and lowest value; the PIC were from 0.353 to 0.658, Fca294 and Pti010 locus had the highest and lowest value; and N(e) were from 1.626 to 3.409, Fca294 and Pti010 locus had the highest and lowest value, which showed the ten microsatellie loci had high or medium polymorphism in these Amur tigers and had high genetic diversity. At the same time, we only found even bases variability which showed the even bases repeat sequence (CA/GT) maybe the basic unit for length variability of microsatellite in all loci. In this study, the samples were made up of 75 hair specimens, 23 blood specimens and 15 tissue specimens, we obtained the genome DNA from hairs using the non-invasive DNA technology and demonstrated that DNA derived from hair samples is as good as that obtained from blood samples for the analysis of microsatellite polymorphism. These results imply that microsatellite DNA markers and non-invasive DNA technology can help study the genetic diversity of Amur tiger. This method could be used in the captive management of other endangered species.