APPLICATION OF INTERNAL TRANSCRIBED SPACER OF NUCLEAR RIBOSOMAL DNA FOR IDENTIFICATION OF ECHINOPS MANDAVILLEI KIT TAN

The present study explored the use of internal transcribed spacers (ITS) sequences (ITS1-5.8S-ITS2) of nuclear ribosomal DNA (nrDNA) for identification of Echinops mandavillei Kit Tan, an endemic species to Saudi Arabia. The sequence similarity search using Basic Local Alignment Search Tool (BLAST) and phylogenetic analyses of the ITS sequence of E. mandavillei Kit Tan showed high level of sequence similarity (98%) with E. glaberrimus DC. (section Ritropsis). The novel primary sequence and the secondary structure of ITS2 of E. mandavillei could have a potential use for molecular genotyping. Introduction The genus Echinops L. belonging to the subtribe Echinopsinae of Cynareae, of the family Asteraceae comprise about 120 species (Vidović, 2011), and distributed in tropical Africa, the Mediterranean basin, temperate regions of Eurasia, Central Asia, Mongolia and North-eastern China, with the maximum number of species occurring in the Caucasus and the Middle East (Susanna and Garcia-Jacas, 2007). The genus received considerable interest for establishing natural groups with infrageneric classification (Sánchez-Jiménez et al., 2010). Morphological characters, like the pappus, which is a key taxonomic character of Cynareae, the type and density of indumentum on stems, leaf shapes and phyllaries are considered least significance in dissemination of Echinops species (Mozaffarian, 2006; Sánchez-Jiménez et al., 2010). In Saudi Arabia, there are nine Echinops species, viz. E. abuzinadianus Chaudhary, E. erinaceus Kit Tan, E. glaberrimus DC., E. hystrichoides Kit Tan, E. macrochaetus Fresen., E. mandavillei Kit Tan, E. sheilae Kit Tan, E. viscosus DC. and E. yemenicus Kit Tan. Of them, E. abuzinadianus, E. mandavillei and E. sheilae are endemic to Saudi Arabia, while remaining species have been reported from different geographic locations of Arabian Peninsula. E. mandavillei was reported to occur in Dahna, Summan and Nafud sands (Chaudhary, 2000). The DNA sequence technology provides series of new data for molecular phylogeny and DNA barcoding which has now-a-days changed the paradigm of species identification (Ali and Choudhary, 2011; Ali et al., 2014). From the first report of the utility of the internal transcribed spacers (ITS) sequence of nuclear ribosomal DNA (nrDNA) in plants (Baldwin, 1992), it has been Corresponding author. E-mail: majmalali@rediffmail.com International Biological Material Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305 806, South Korea Institute of Genetics and Biotechnology, St. István University, Gödöllo H-2103, Hungary Department of Botany, University of Delhi, Delhi 110007, India


Introduction
The genus Echinops L. belonging to the subtribe Echinopsinae of Cynareae, of the family Asteraceae comprise about 120 species (Vidović, 2011), and distributed in tropical Africa, the Mediterranean basin, temperate regions of Eurasia, Central Asia, Mongolia and North-eastern China, with the maximum number of species occurring in the Caucasus and the Middle East (Susanna and Garcia-Jacas, 2007).The genus received considerable interest for establishing natural groups with infrageneric classification (Sánchez-Jiménez et al., 2010).Morphological characters, like the pappus, which is a key taxonomic character of Cynareae, the type and density of indumentum on stems, leaf shapes and phyllaries are considered least significance in dissemination of Echinops species (Mozaffarian, 2006;Sánchez-Jiménez et al., 2010).In Saudi Arabia, there are nine Echinops species, viz.E. abuzinadianus Chaudhary, E. erinaceus Kit Tan, E. glaberrimus DC., E. hystrichoides Kit Tan, E. macrochaetus Fresen., E. mandavillei Kit Tan, E. sheilae Kit Tan, E. viscosus DC. and E. yemenicus Kit Tan.Of them, E. abuzinadianus, E. mandavillei and E. sheilae are endemic to Saudi Arabia, while remaining species have been reported from different geographic locations of Arabian Peninsula.E. mandavillei was reported to occur in Dahna, Summan and Nafud sands (Chaudhary, 2000).
The DNA sequence technology provides series of new data for molecular phylogeny and DNA barcoding which has now-a-days changed the paradigm of species identification (Ali and Choudhary, 2011;Ali et al., 2014).From the first report of the utility of the internal transcribed spacers (ITS) sequence of nuclear ribosomal DNA (nrDNA) in plants (Baldwin, 1992), it has been extensively used to distinguish even very closely related species (Chen et al., 2010;Yao et al., 2010).Moreover, in the last two decades, the ITS sequence technology has gained much attention, along with the smartest genes available for the molecular phylogeny and taxonomy (Ali et al., 2013).
The ITS sequence technology has been used for molecular phylogeny of Echinops (Garnatje et al., 2005), and series of other genera of Cynareae (Susanna et al., 1999;Vilatersana et al., 2000;Wang et al., 2005Wang et al., , 2007;;Hidalgo et al., 2006); however, these studies did not include systematics of Echinops species occurring in Saudi Arabia.Hence, the present study aims to establish molecular signature of Echinops mandavillei Kit Tan based on ITS sequence of nrDNA.

Plant materials:
The leaf material of Echinops mandavillei Kit Tan was collected from herbarium specimen (Saudi Arabia, Al-Nafud, 29.4'N, 39.58'E, 5 May 1985, H.O. Al-Hassan 195) housed at National Herbarium and Genebank, National Agriculture and Animal Resources Research Centre, Riyadh, Saudi Arabia (RIY).The taxonomic identification of specimen was confirmed with the aid of Flora of Saudi Arabia (Chaudhary, 2000).

DNA isolation and amplification:
Genomic DNA was extracted from 10 mg silica gel-dried leaves using the protocol of DNeasy Plant Mini kit (QIAGEN, Valencia, CA, USA).The ITS regions were amplified using the primers ITS1 and ITS4 as described by White et al. (1990).Double-stranded polymerase chain reaction (PCR) products were produced through 35 cycles of 95°C for 1 min, 48°C for 1 min and 72°C for 1 min, with a 10 min final extension cycle at 72°C.PCR products were purified with SolGent PCR Purification kit-Ultra (SolGent, Daejeon, South Korea), and forwarded to sequencing using the same primers, 2L BigDye, 1µl primer (20 pM), template DNA and purified water to reach a 10µl reaction volume.Cycle sequencing used was 25 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min.

DNA sequencing and data analysis:
DNA sequencing was performed by ABI Prism 377 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA).Each sample was sequenced in the sense and anti-sense direction.The nucleotide sequences of both DNA strands were obtained and analyzed by Sequence Navigator (Perkin-Elmer/Applied Biosystems) to ensure accuracy of the base pair sequences.The sequence was submitted to GenBank (accession number KJ187107).

Results and Discussion
The ITS region (ITS1-5.8S-ITS2) of Echinops mandavillei Kit Tan sequenced in the present study was found 634 bp, where ITS1 region 252 bp (GC content 54%), 5.8S gene 164 bp (GC content 53%), and ITS2 region 218 bp (GC content 50%).The BLAST search of ITS sequence of E. mandavillei Kit Tan showed maximum identity (98%) with E. glaberrimus DC.Parsimony analysis of the entire ITS region resulted in 431 maximally parsimonious trees with consistency index of 0.691, homoplasy index of 0.459, and retention index of 0.763.The phylogenetic tree constructed by the present analyses shows Echinops to be monophyletic (bootstrap support 100%; Fig. 1).The tree also provides a clear resolution at the sectional level and the result confirms an earlier report (Sánchez-Jiménez et al., 2010), and E. mandavillei Kit Tan nested within the clade of the section Ritropsis (Fig. 1). Figure 2 illustrates specific nucleotide differences between E. mandavillei Kit Tan and E. glaberrimus DC., in total seven SNPs (four nucleotides in ITS1 region, i.e. at the alignment position 11, 81, 226 and 234, and three nucleotides in ITS2 region, i.e. at the alignment position 4, 58 and 165) were observed.
The secondary structures of ITS2 region of E. mandavillei Kit Tan and E. glaberrimus DC. were constructed and compared (Fig. 3 A-B), which contained a central ring (primary ring) and four helices.However, the two structures differed in the four helical regions, in stem loop numbers, sizes, position, and screw angle.On the basis of the ITS2 secondary structure, E. mandavillei Kit Tan could be discriminative from other species of the genus.The morphological identification depends on sufficient experience and can easily be affected by the geographical environment and biocoenosis (Marcon et al., 2005;Rai et al., 2012).In contrast, DNA sequence is hardly influenced by environmental characteristics and developmental stages (Liu et al., 2011); and therefore, the DNA barcoding may be an effective supplement to traditional/classical morphological methods (see Hebert et al., 2003).The species identification using DNA barcodes has been successfully used across the algae, fungi, plants, and animals, hence; the DNA barcoding has now been proven useful in biodiversity assessment, biomonitoring, forensics, illegal trade of endangered species and their products, ecology, medicinal and poisonous plants and conservation genetics (see Hebert et al., 2003;Fišer Pečnikar and Buzan, 2014;Ali et al., 2014).
DNA barcoding efforts worldwide have resulted in the formation of the Consortium for the Barcode of Life (CBOL), and the Barcode of Life Database (BOLD), which contain more than 2.7 million records, with 2 million barcodes belonging to over 170,000 species (Ratnasingham and Hebert, 2007;BOLD Systems, 2013).The China Plant BOL Group has proposed that ITS1/ITS2 should be incorporated into the core of barcode for seed plants (Li et al., 2011).In the present study, we supplied the ITS barcode of E. mandavillei Kit Tan which is new for GenBank databases.An increasing number of studies also suggest that DNA secondary structures are crucial for genomic stability and cellular processes, such as transcription (Bochman et al., 2012;Salvi and Mariottini, 2012), and our study has also provided new data of E. mandavillei Kit Tan for this purposes.

Fig. 1 .
Fig. 1.Neighbour joining tree of Echinops species including E. mandavillei inferred from ITS sequences of nrDNA.Bootstrap values greater than 50% in 1,000 bootstrap replicates are shown above lines.

Fig. 2A .
Fig. 2A.Alignments of ITS1 sequences of E. mandavillei compared to E. glaberrimus, B. Alignments of ITS2 sequences of E. mandavillei compared to E. glaberrimus.Gaps in clustal line indicate nucleotide differences.