Intersimple Sequence Repeats (ISSR) Molecular Fingerprinting Markers for Authenticating Populations of Dendrobium officinale KIMURA et MIGO

Abstract

(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...

Genetic diversity among Polish landraces of common oat (Avena sativa L.)

“…The primers are usually between 16 and 25 bp long and could either be anchored at 3 0 or 5 0 end or also unanchored (Gupta et al 1994;Meyer et al 1993;Wu et al 1994;Zietkiewicz et al 1994). The main advantages of ISSR are: no need for any prior knowledge of genome sequence, low operational cost, low labor-intensity, high stability and reproducibility, and abundance of genomic information (Moreno et al 1998;Shen et al 2006). The main drawback is that ISSRs are inherited mostly as dominant genetic markers in a simple Mendelian fashion and high probability exists that the similar sized fragments are not homologous (Gupta et al 1994;Tsumura et al 1996).…”

Identification of Dendrobium Species Used for Herbal Medicines Based on Ribosomal DNA Internal Transcribed Spacer Sequence

“…[16][17][18][19][20][21][22][23][24][25][26][27] Unfortunately, these methods have only been applied to a limited number of Dendrobium species, and molecular markers that can be applied more universally to Dendrobium have not been developed. To recognize herbal medicines accurately at the species level, a comprehensive DNA sequence database that covers Dendrobium species potentially used for medicine is desired, and to create such a database, the following conditions are required: (1) precise identification of the plant species, (2) comprehensive data banking of reference sequences for the relevant plant group at the species level, and (3) employment of sequence markers that can discriminate interspecific differences.…”

SNP, ARMS and SSH Authentication of Medicinal Dendrobium officinale KIMURA et MIGO and Application for Identification of Fengdou Drugs

“…2,5) Several populations of D. officinale have ever been authenticated by intersimple sequence repeat (ISSR) markers, 6) but this technique maybe have some disadvantages such as lower stability than sequences etc., new methods for population authentication should be developed for selection of germplasm resources during the process of good agriculture practice (GAP) and identification of botanical origins of drugs during crude drug quality control.…”