Application of the ITS2 Region for Barcoding Medicinal Plants of Selaginellaceae in Pteridophyta

Background Selaginellaceae is a family of nonseed plants with special evolutionary significance. Plants of the family Selaginellaceae are similarly shaped and easily confused, complicating identification via traditional methods. This study explored, for the first time, the use of the DNA barcode ITS2 to identify medicinal plants of the Selaginellaceae family. Methodology/Principal Findings In our study, 103 samples were collected from the main distribution areas in China; these samples represented 34 species and contained almost all of the medicinal plants of Selaginellaceae. The ITS2 region of the genome was amplified from these samples and sequenced using universal primers and reaction conditions. The success rates of the PCR amplification and sequencing were 100%. There was significant divergence between the interspecific and intraspecific genetic distances of the ITS2 regions, while the presence of a barcoding gap was obvious. Using the BLAST1 and nearest distance methods, our results proved that the ITS2 regions could successfully identify the species of all Selaginellaceae samples examined. In addition, the secondary structures of ITS2 in the helical regions displayed clear differences in stem loop number, size, position, and screw angle among the medicinal plants of Selaginellaceae. Furthermore, cluster analysis using the ITS2 barcode supported the relationship between the species of Selaginellaceae established by traditional morphological methods. Conclusion The ITS2 barcode can effectively identify medicinal plants of Selaginellaceae. The results provide a scientific basis for the precise identification of plants of the family Selaginellaceae and the reasonable development of these resources. This study may broaden the application of DNA barcoding in the medicinal plant field and benefit phylogenetic investigations.

[1]  Dong Liang,et al.  PTIGS-IdIt, a system for species identification by DNA sequences of the psbA-trnH intergenic spacer region , 2011, BMC Bioinformatics.

[2]  D. Smith.  Unparalleled GC content in the plastid DNA of Selaginella , 2009, Plant Molecular Biology.

[3]  P. Hollingsworth.  Refining the DNA barcode for land plants , 2011, Proceedings of the National Academy of Sciences.

[4]  C. Rothfels,et al.  rbcL and matK Earn Two Thumbs Up as the Core DNA Barcode for Ferns , 2011, PloS one.

[5]  E. Shakirov,et al.  Selaginella moellendorffii telomeres: conserved and unique features in an ancient land plant lineage , 2012, Front. Plant Sci..

[6]  J. Starr,et al.  Plant DNA barcodes and species resolution in sedges (Carex, Cyperaceae) , 2009, Molecular ecology resources.

[7]  J. John,et al.  Protection of immunocompromised mice from fungal infection with a thymus growth-stimulatory component from Selaginella involvens, a fern , 2011, Immunopharmacology and immunotoxicology.

[8]  Yue Shi,et al.  Structural characterization and identification of biflavones in Selaginella tamariscina by liquid chromatography-diode-array detection/electrospray ionization tandem mass spectrometry. , 2011, Rapid communications in mass spectrometry : RCM.

[9]  P. Mariottini,et al.  Molecular phylogenetics in 2D: ITS2 rRNA evolution and sequence-structure barcode from Veneridae to Bivalvia. , 2012, Molecular phylogenetics and evolution.

[10]  C. China Pharmacopoeia,et al.  Pharmacopoeia of the People's Republic of China , 2010 .

[11]  M. Nei,et al.  MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. , 2007, Molecular biology and evolution.

[12]  De‐Zhu Li,et al.  Comparative analysis of a large dataset indicates that internal transcribed spacer (ITS) should be incorporated into the core barcode for seed plants , 2011, Proceedings of the National Academy of Sciences.

[13]  Hong-Mei Zhang,et al.  Isolation and Cytotoxic Activity of Selaginellin Derivatives and Biflavonoids from Selaginella tamariscina , 2012, Planta medica.

[14]  W. John Kress,et al.  A DNA barcode for land plants , 2009, Proceedings of the National Academy of Sciences.

[15]  M. Guerra,et al.  Variation in chromosome numbers, CMA bands and 45S rDNA sites in species of Selaginella (Pteridophyta). , 2005, Annals of botany.

[16]  Zhenzhong Wang,et al.  Testing the potential of proposed DNA barcodes for species identification of Zingiberaceae , 2011 .

[17]  Petra Korall,et al.  The phylogenetic history of Selaginellaceae based on DNA sequences from the plastid and nucleus: extreme substitution rates and rate heterogeneity. , 2004, Molecular phylogenetics and evolution.

[18]  C. Yang,et al.  Bioactive selaginellins from Selaginella tamariscina (Beauv.) Spring , 2012, Beilstein journal of organic chemistry.

[19]  N. Pathak,et al.  In vitro and in vivo evaluation of the anticarcinogenic and cancer chemopreventive potential of a flavonoid-rich fraction from a traditional Indian herb Selaginella bryopteris , 2011, British Journal of Nutrition.

[20]  J. Ryals,et al.  Comparative metabolic profiling between desiccation-sensitive and desiccation-tolerant species of Selaginella reveals insights into the resurrection trait. , 2012, The Plant journal : for cell and molecular biology.

[21]  Ting Gao,et al.  Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species , 2010, PloS one.

[22]  J. Gershenzon,et al.  Nonseed plant Selaginella moellendorffii has both seed plant and microbial types of terpene synthases , 2012, Proceedings of the National Academy of Sciences.

[23]  Katrin Paeschke,et al.  DNA secondary structures: stability and function of G-quadruplex structures , 2012, Nature Reviews Genetics.

[24]  G. Petersen,et al.  How Many Loci Does it Take to DNA Barcode a Crocus? , 2009, PloS one.

[25]  A. Ebihara,et al.  Molecular Species Identification with Rich Floristic Sampling: DNA Barcoding the Pteridophyte Flora of Japan , 2010, PloS one.

[26]  Shilin Chen,et al.  Using DNA Barcoding to Identify Species within Euphorbiaceae , 2010, Planta medica.

[27]  A. Galimberti,et al.  DNA barcoding to analyse taxonomically complex groups in plants: the case of Thymus (Lamiaceae) , 2013 .

[28]  De‐Zhu Li,et al.  Plant DNA barcoding in China , 2011 .

[29]  Michael S. Barker,et al.  The Selaginella Genome Identifies Genetic Changes Associated with the Evolution of Vascular Plants , 2011, Science.

[30]  Thomas Dandekar,et al.  5.8S-28S rRNA interaction and HMM-based ITS2 annotation. , 2009, Gene.

[31]  Y. Li,et al.  Authentication of the family Polygonaceae in Chinese pharmacopoeia by DNA barcoding technique. , 2009, Journal of ethnopharmacology.

[32]  G. Zeng,et al.  Bioactive flavones and biflavones from Selaginella moellendorffii Hieron. , 2010, Fitoterapia.

[33]  W. Feng,et al.  Antihyperglycemic activity of Selaginella tamariscina (Beauv.) Spring. , 2011, Journal of ethnopharmacology.

[34]  G. Zeng,et al.  Antimicrobial selaginellin derivatives from Selaginella pulvinata. , 2010, Bioorganic & medicinal chemistry letters.

[35]  P. Hebert,et al.  Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species , 2003, Proceedings of the Royal Society of London. Series B: Biological Sciences.

[36]  Youping Deng,et al.  Extensive Pyrosequencing Reveals Frequent Intra-Genomic Variations of Internal Transcribed Spacer Regions of Nuclear Ribosomal DNA , 2012, PloS one.

[37]  Jingyuan Song,et al.  Species identification of medicinal pteridophytes by a DNA barcode marker, the chloroplast psbA-trnH intergenic region. , 2010, Biological & pharmaceutical bulletin.

[38]  Chun-lin Long,et al.  Pyrrolidinoindoline alkaloids from Selaginella moellendorfii. , 2009, Journal of natural products.

[39]  Ke‐long Huang,et al.  Target-guided isolation and purification of antioxidants from Selaginella sinensis by offline coupling of DPPH-HPLC and HSCCC experiments. , 2011, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[40]  Petra Korall,et al.  Phylogenetic relationships in Selaginellaceae based on RBCL sequences. , 2002, American journal of botany.

[41]  Thomas Hackl,et al.  ITS2 database IV: interactive taxon sampling for internal transcribed spacer 2 based phylogenies. , 2012, Molecular phylogenetics and evolution.

[42]  Ting Gao,et al.  Identification of medicinal plants in the family Fabaceae using a potential DNA barcode ITS2. , 2010, Journal of ethnopharmacology.

[43]  L. Quintans-Júnior,et al.  Antinociceptive effect of ethanolic extract of Selaginella convoluta in mice , 2012, BMC Complementary and Alternative Medicine.

[44]  J. Banks.  Selaginella and 400 million years of separation. , 2009, Annual review of plant biology.

[45]  Jingyuan Song,et al.  Use of ITS2 Region as the Universal DNA Barcode for Plants and Animals , 2010, PloS one.