Dendrobium officinale is a critically endangered perennial herb endemic to China. Determining the levels of genetic diversity and patterns of population genetic structure of this species would assist in its conservation and management. Data of 12 populations were used to assess its genetic diversity and population structure, employing the method of amplified fragment length polymorphism (AFLP). A high level of genetic diversity was detected (H (E) = 0.269) with POPGENE. As revealed by AMOVA analysis, there was moderate variation between pairs of populations with Phi(ST) values ranging from 0.047 to 0.578 and on average 26.97% of the genetic variation occurred among populations. Three main clusters were shown in UPGMA dendrogram using TFPGA, which is consistent with the result of principal coordinate ananlysis (PCO) using NTSYS. Keeping a stable environment is critical for the in situ conservation and management of this rare and endangered plant, and for ex situ conservation it is important to design an integrated germplasm bank.

(previous name: Dendrobium candidum WALL. ex LINDL. 2)) is an endangered species in China and ranked "the first of the nine Chinese fairy herbs." The stems of D. officinale have been used as a traditional Chinese tonic medicine called Tiepi Fengdou 3) for hundreds of years. It can benefit human health in many aspects, such as nourishing yin and clearing away unhealthy heat, benefiting the stomach, promoting the production of body fluid, resisting cancer and prolonging life 1,3) This treasured herb is in severe shortage, making it expensive; the highest grade costs ¥12000-30000 per kg. 4)In our previous studies, rDNA ITS regions of D. officinale and other four adulterant species were sequenced. A pair of allele-specific diagnostic primers was designed to authenticate D. officinale from the other species based on rDNA ITS sequences of D. officinale and the other 37 species of Dendrobium.5,6) Recently, we have focused on the authentication of D. officinale populations using DNA molecular markers, which will lay the foundation for the selection of high-quality population resources.Intersimple sequence repeats (ISSR) have become a good DNA molecular marker for research on populations of the same species, a technique established based on PCR by Zietkiewicz et al. in 1994. 7) The main advantages of ISSR are: no need for DNA sequence information prior to amplification, low cost, simple operation, high stability, and abundance of genomic information. Because of these reasons, ISSR are being used for population authentication and population molecular ecology studies. [8][9][10][11][12][13][14][15][16] The present study was undertaken with the objective of establishing specific molecular markers using ISSR for authenticating eight wild D. officinale populations. MATERIALS AND METHODS Plant MaterialsAll materials used in this study were collected in China. Voucher samples were identified by Dr. Xiaoyu Ding and preserved by means of tissue culture in the College of Life Sciences, Nanjing Normal University. Wherever possible, three to five individuals were sampled from each population. The details are shown in Table 1.DNA Extraction Complete genomic DNA samples were extracted from fresh leaves using Dneasy Plant Mini Kits (QIAGEN), the concentrations of the DNA samples were measured spectrophotometrically, and each sample was diluted to 20 ng ml Ϫ1 with TE buffer for PCR amplification. Selection of Primers To select suitable primers for the study of populations of D. officinale, 76 ISSR primers, which were all purchased from Sangon Co., Ltd., China, were screened using two DNA samples. From the preliminary screening, 32 primers that could amplify visible bands were selected for further examination. Eventually, 10 ISSR primers that produced clear and reproducible bands were selected for the amplification of all DNA samples (Table 2).ISSR Profiling ISSR amplifications were carried out in a 25 ml volume containing Tris-HCl 10 mM, KCl 10 mM, (NH 4 ) 2 SO 4 8 mM (pH 9.0), MgCl 2 1.2-1.8 mM, Taq DNA polymerase1.5 U, dNTP 80 mM, pri...

As a widely used medicinal plant, Alisma orientale is always a possible target for fraudulent labeling. The internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (nrDNA) of the six species of genus Alisma were sequenced, and two variant sites were found to be specific for A. orientale. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis and amplification refractory mutation system (ARMS) analysis were applied to the ITS region for the identification of A. orientale. A restriction site for PSTI useful for PCR-RFLP analysis was detected and a pair of diagnostic primers DFZX-JB02S and DFZX-JB02X were designed for ARMS.

As a widely used medicinal plant, Dendrobium loddigesii Rolfe is always a possible target for fraudulent labeling. The identification of D. loddigesii is generally difficult from its morphological and chemical appearance only. In order to develop a convenient and efficient identification method for D. loddigesii, six pairs of diagnostic ARMS primers were designed based on nrDNA ITS sequences of D. loddigesii and eleven adulterants. The results showed that one diagnostic primer pair (FJB-04-forward, FJB-04-reverse) could be used to authenticate D. loddigesii by generating a fragment of 353 bp at annealing temperatures from 48 degrees C to 62 degrees C while the other diagnostic primer pair (FJB-03-forward, FJB-03-reverse) took on the same effect at annealing temperatures from 49 degrees C to 55 degrees C. This points out the potential of ARMS analysis for authentication of D. loddigesii.

Homozygous alpha-thalassemia [alpha-thal-1], with loss of all four alpha-globin genes, causes lethal hydrops fetalis. The most common mutation producing this syndrome is the Southeast Asian (--SEA) double alpha-globin gene deletion. Erythrocytes from adults heterozygous for the (--SEA) deletion have minute amounts of embryonic zeta-globin chains detectable by anti-zeta-globin monoclonal antibodies. Among 225 peripheral blood samples tested by a simple anti-zeta-immunobinding tetrazolium dye test, 81 were positive and 144 were negative. The majority of subjects were of Filipino, Chinese, or Laotian ancestry. All 81 positive samples were confirmed by Bam HI digests and a zeta-cDNA probe to have the (--SEA) mutation. The (--SEA) double alpha-deletion was the only abnormality in 58. In the others, it was combined with alpha-globin or beta-globin mutations, or coincidental iron deficiency. Four other samples from (--SEA) heterozygotes were negative by this immunologic assay. Anti-zeta negative samples included 78 deletions of the total alpha-globin region, (--Tot), 23 single alpha-globin deletions, and a variety of beta-globin mutations; 16 normocytic samples with normal alpha-genes were also negative. Ten anti-zeta positive and 25 anti-zeta negative samples had benign triplicated zeta-globin genes. In this population, the sensitivity of this test was 95%; and specificity for the (--SEA) mutation was 100%. Anti-zeta immunobinding testing provides rapid, simple, and reliable screening for the (--SEA) double alpha-globin deletion, although it does not detect the (--Tot) total alpha-deletions.