DNA Barcoding of Two Narrow Endemic Plants; Astragalus Argaeus and Astragalus Stenosemioides from Mount Erciyes, Turkey


 The genetic resources and biological diversity of countries are very important. Biodiversity and genetic resources should be protected, especially as endemic species. In this concept, DNA barcoding studies are an effective way to identify an unknown taxon and protected the biodiversity of a country. Astragalus argaeus and A. stenosemioides are narrow endemic species from Mt. Erciyes, Turkey. To determine its phylogenetic relationships and DNA barcoding, sequence data from the chloroplast DNA (matK region) were analyzed by parsimony, maximum likelihood, and Bayesian methods. In this study, A. argaeus, A. stenosemioides samples, and 23 sequences from GenBank, including a closely related species were performed. The phylogenetic analysis showed that the matK gene region could clearly distinguish A. argaeus and A. stenosemioides from its closely related species. DNA barcoding surveys can contribute to taxonomic and biodiversity research, various molecular ecology, and population genetics studies. Also, it is possible to determine the species by separating the matK DNA gene region, which is one of the molecular characters, and A. argaeus and A. stenosemioides have been successfully barcoded for the first time; therefore, it has been shown that this gene region can be used for barcoding.


Introduction
Many molecular markers are used for plant and animal genetic studies. Recently, the most commonly used methods to determine the genetic variation of DNA-based methods such as DNA barcoding, phylogenetic studies (Carew et al. 2005). It is very important for taxonomy and biodiversity studies by using DNA barcode methods to fast and correctly make taxa identi cation. In particular, DNA barcoding has been provided a great utility in biodiversity surveys. Moreover, the Conservation of biodiversity and DNA barcoding is a useful tool to quickly and accurately identify species. Also, DNA barcoding has the potential to prompt the discovery of new species.
Turkey has a signi cant and rich ora because of its geographical location and its paleogeography and ecological features. Turkey is the only country covered almost entirely by three of the world's 34 global biodiversity hotspots (the Caucasus, Iran-Anatolian, and the Mediterranean) ( Vural & Şapcı 2012). In this study, the ability of DNA barcoding to con rm the identities of Critically Endangered endemic plant taxon (Astragalus argaeus) in Mountain Erciyes was assessed using a DNA barcode (matK).

Taxon collection
Research materials were having gathered from Mount Erciyes, Kayseri, Turkey, and deposited at Herbarium of Erciyes University, Turkey (ERCH).
DNA isolation, ampli cation, and PCR product sequencing Genomic DNA (gDNA) from leaf tissues was extracted by using a commercial DNA extraction kit (DNeasy Plant Mini Kit, Qiagen), following the manufacturer's protocol. Agarose gel electrophoresis of 1.5% was used to detect the presence of gDNA with ethidium bromide. The obtained genomic DNA was used in PCR for the ampli cation of the matK gene region using speci c primer pairs 1R_KIM and 3F_KIM (Fazekas et al. 2008).
The PCR products were sequenced in both directions (Forward and Reverse) using an automatic sequencer (ABI 3100 Genetic Analyzer, RefGen Biotechnology, Ankara, Turkey). Reaction mixture used for the PCR ampli cations contained a volume of 50 µl; 10 × Taq

Phylogenetic analyses
Sequences were aligned used for the multiple sequence alignment with Geneious Software 6R (available from http://www.geneious.com). Phylogenetic analyses were performed by using the sequences of matK gene regions obtained from GenBank (NCBI: The National Center for Biotechnology Information) and this study ( Table 1). The related matK sequences of Astragalus species (23) (Drummond et al. 2006) and also selected to Yule process for speciation as tree prior (Yule 1925). Checking of the Quality analysis was done by using likelihood parameters and values, which were estimated by using different runs in Tracer V.1.5 (Rambaut & Drummond 2009

Results And Discussion
In this study, narrow endemic A. argaeus and A. stenosemioides were evaluated in the molecular data. Sequences of plastid DNA (matK gene) were successfully ampli ed from A. argaeus and A. stenosemioides. These sequences were analyzed together with sequences from GenBank and constructed phylogenetic tree. The phylogenetic tree was obtained by the Bayesian method using the BEAST program (Fig. 1). The phylogenetic tree constructed from matK sequences was clustered into the two main groups. Astragalus caragnae and A. dactylocarpus were grouped in the rst lineage. Other species were classi ed in the second lineage. When we evaluated the DNA barcode-based tree, it shows that consistent with classical taxonomic classi cation. In this concept, the matK gene region sequences of the genus were useful to solve taxonomic problems. DNA barcoding involves sequencing a standard region of DNA as a tool for species identi cation. However, there has been no agreement on which region(s) should be used for barcoding land plants. An ideal DNA barcode should be routinely retrievable with a single primer pair. Additionally, it should be amenable to bidirectional sequencing with little requirement for manual editing of sequence traces, and also provide maximal discrimination among species. In this concept, there are several candidate barcode regions. One of these candidate barcode gene regions is the matK gene region (Ford et al. 2009  Phylogenetic tree using BEAST Bayesian inference based on cp DNA (matK gene region).