MOLECULAR IDENTIFICATION AND PHYLOGENETIC RELA- TIONSHIPS OF Origanum syriacum L. AND Origanum vulgare L. US- ING CHLOROPLAST rbcL AND matK BARCODES GENES M. ZOELFAKAR S. AHMED1*, EL-SHAIMAA S. EL-DEMERDASH1*AND SH. D. IBRAHIM2 1. Genetic Resources Department, Desert Research Center (DRC), 1, Mathaf El-Matariya Street, El- Matariya B.O.P 11753 El-matariya, Cairo, Egypt. 2. Molecular Genetics and Genome Mapping Lab., AGERI, ARC, 9 Gamma ST., Giza, Cairo 12619, Egypt *corresponding authors: [email protected], [email protected] enus Origanum is considered as an major constituent of its essential oil were G important multipurpose aromatic isolated by Baser et al., (2003). It is espe- perennial herb used in folk medicine and cially antimicrobial, economic importance as food additive since ancient times which and specific biological characters (Asdal belongs to the family Lamiaceae or Labi- et al., 2006) and antioxidant properties atae, tribe Mentheae subfamily Nepetoi- (Mastelic et al., 2008). In Egypt, Origa- deae. It is comprises 43 species and 18 num syriacum L. subsp. Sinaicum (Boiss.), hybrids widely distributed in Eurasia and commonly known as 'Syrian marjoram' is North Africa (Ietswaart, 1980; Duman et an aromatic, herbaceous and perennial al., 1998). Ietswaart (1980) identified six plant growing wild in the Sinai desert of subspecies within O. vulgare based on Egypt (Tackholm, 1974). It is endemic differences in indumentums, number of species grow in mountainous areas of sessile glands on leaves, bracts and caly- Saint Katherine, south of Sinai in dry ces, and in size and color of bracts and rocky habitats, also have a vernacular lo- flowers. Lamiaceae family contains about cal name as ''Za'atar Katherine'' or Barda- 236 genera having 6900 to 7200 species qwish and from the conservation point of (Harley et al., 2004). Origanum vulgare view, it is an endangered plant (Tackholm, L. is a perennial aromatic herb, widely 1974; Boulos, 2002). Origanum syriacum naturally distributed all over Euroasia and L. species, as one of wild plant species, is North Africa and is one of the most traded endangered and has limited distribution. and consumed spice (Kokkini, 1997). Ori- There are many challenges affecting both ganum vulgare L. is used as medicinal conservation and the distribution of wild plant because of the essential oils pro- plants in SKP (Saint Katherine Protec- duced in the aerial parts (Skoula and Har- torate) such as; feral donkeys, plant over borne, 2002). Thymol and carvacrol as a collection, tourist intrusions (e.g., tres- ______________________________________ Egypt. J. Genet. Cytol., 49:191-211, July, 2020 Web Site (www.esg.net.eg) 192 M. Z. S. AHMED et al. passing beyond trail borders and collec- (http://barcoding.si.edu/DNABarCoding.h tion of firewood during camping), over- tm). For instance, barcoding strategies grazing, collection for scientific research, have been deployed for the verification of urbanization and settlement expansion and plant products from several medicinal quarries as recorded by Guenther et al., plants (Asahina et al., 2010; Xue and Li, (2005), Assi (2007), Hatab (2009) and 2011). Large data information gathered Khafagi et al., (2013). The taxonomy of from two chloroplast chDNA barcodes Origanum was found to be rather complex (rbcL and matK) at molecular level used a and nearly all of the sections are afflicted universal barcode system far beyond taxo- with some kind of taxonomic uncertainties nomic studies of land plants. Some inves- (Lukas, 2010). tigators have chosen a combination of two regions (matK and rbcL) as a satisfactory For molecular characterization and compromise that best meets the DNA bar- identification of the biological species coding criteria (Hebert, 2003; Chase et al., based on using DNA sequence data were 2005; De Vere et al., 2012 and; Fazekas et widely used for DNA barcoding in the last al., 2008& 2012). Using four candidate decades. Several international organiza- barcode regions (rpoB, rbcL, matK and tions, including iBOL (the International trnH-psbA to evaluate the utility of using Barcode of Life Project) http:// markers plant DNA barcodes in 64 species www.ibol.org/, CBOL (the Consortium specimens, encompassing six different for the Barcode of Life) and ECBOL (the genera (i.e. Mentha, Ocimum, Origanum, European Consortium for the Barcode of Salvia, Thymus and Rosmarinus) to reduce Life), have applied in large-scale DNA cost and time for species identification barcoding projects aiming to identify and (De Mattia et al., 2011 & 2012). Using classify all life on earth. Recently, the one region barcodes from rbcL, matK, Consortium for the Barcode of Life psbA-trnH loci of 14 Labiatae species (CBOL) Plant Working Group (CBOL were identified and analyzed with species 2009 and 2010) proposed two other chlo- identification from Pakistan (Schori and roplast regions, the protein coding rbcL Showalter, 2011). Universal primers were and matK, as a 2-locus combination bar- used for amplification of matK and rbcL code. DNA barcoding techniques play an Loci in 2 different plant species (covering important role in the identification of pol- 14 families) from Saudi Arabia (Bafeel et ymorphic plant species having a problem- al., 2011). To classify between 36 samples atic taxonomic identity for the biodiversity of Thymus spp. the core barcode regions investigation (Ajmal et al., 2014). DNA (matK and rbcL) and the plastid intergenic barcoding technique in plants with high spacer trnH-psbA were compared (Federi- effective and robust conserved regions is a ci et al., 2013). Three cpDNA loci (matK, characterizing using a partial DNA se- rbcL and trnH-psbA) as single region or as quence from a standard and agreedǦupon multi-region barcodes based on CBOL position in the genome of all species were used and analyzed for medicinal Phylogenetic relationships of O. syriacum L. and O. vulgare L. 193 using chloroplast rbcL AND matK barcodes genes plants of the Labiatae (Lamiaceae) family studied species. Phylogenetic relationship (Theodoridis et al., 2012). DNA barcod- analysis and homology modeling of both ing as part from authentication of traded sequences (matK + rbcL genes) using medicinal plants, also finds application in Basic Local Alignment Search Tool biodiversity monitoring, conservation im- (BLASTn) and MEGA 7 software pro- pact assessment, monitoring of illegal gram to apply comparative sequences be- trading, forensic botany, etc. (Nithaniyal tween of them. et al., 2014; Ferri et al., 2015 and Mishra et al., 2016). For more than a decade, oth- MATERIALS AND METHODS er several applications has been widely tested in the DNA barcoding, molecular Plant materials collection systematics, identification at molecular level and community phylogenetic of The two Origanum species availa- some medicinal plants (Vohra and Khera, ble in Egypt; Origanum syriacum L. 2013; Techen et al., 2014; Zhou et al., subsp. sinaicum (Boiss.) (Ecotype wild 2014; Parveen et al., 2016; Bezeng et al., species) and Origanum vulgare L. (culti- 2017 and Chen et al., 2019). vated species) were collected for the pre- sent investigation. The fresh young leaves The importance of Origanum spe- were collected as bulk for each species in cies is due to active ingredients and essen- spring of 2018, transferred into liquid ni- tial oils which confer their medicinal, cul- trogen, and kept frozen at - 80 qC till use. inary and pharmaceutical properties. The Origanum species chosen for the pre- Moreover, threatens such as over- sent study were wild type Origanum syri- collecting of plants and human construc- acum L. subsp. sinaicum (Boiss.) which tions in plants natural habitats which put was collected from the mountains of Saint them in their way to extinction. Therefore, Catherine Protectorate (SKP), South Sinai the current investigation was carried out to Governorate, Egypt and the other is culti- identify and authenticate the endemic wild vated species; Origanum vulgare L. which was collected from private farms at Kir- Origanum species, Origanum syriacum L. dasa region, Giza Governorate, Egypt. subsp. sinaicum (Boiss.) in Egypt and compare them with cultivated species; DNA extraction, primers design and Origanum vulgare L. using two chloro- PCR amplification plast genes (rbcL and matK genes) as the most important DNA barcode at the mo- Genomic DNAs were extracted lecular level. In addition, to distinguish from 100 mg frozen tissue and loaded in between the two Origanum species under 2.0 ml eppendorf tubes with one 5 mm study and with the other available species stainless steel bead. Samples were ground on NCBI database to fulfill a strict con- using Tissue Lyser II (Qiagen, Ltd.) until servation plan and maintenance of the the material became powder (frequency 20 194 M. Z. S. AHMED et al. Hz x 2 x ~ 30 sec) following the proce- GU381797.1 (Origanum rotundifolium), dure described by DNeasy Plant Mini Kit GU381801.1 (Origanum microphyllum), (Qiagen Inc., Cat.no.69104, USA), this GU381800.1 (Origanum dayi), was performed following the manufactur- AY840165.1 (Origanum vulgare), and er's instruction. The concentrations and MG256495.1 (Mentha spicata) were used quality of the genomic DNA samples were for multiple sequences alignment of nu- estimated on spectrophotometer ND-2000 cleotide (BLSTn) to design specific pri- (Nanodrop, USA). Finally, all the ge- mers pair of matK gene. The entire coding nomic DNA samples were diluted to a plastid maturase fragment (matK) was final concentration of 40 ng/ μl with TE amplified ~ 884 bp or less using the pri- buffer (10 mM Tris-HC1, pH 8.0 and 1 m mer pairs matK_466Fwd (5`- GTC CAT M EDTA), then stored at -20qC for further GTG GAA ATC TTG ATT C -3`) and use. DNA fragments were amplified via matK_1349Rev (5`- CGT ACA GTA CTT standard polymerase chain reaction TTG TGT TTA CG -3`) and PCR frag- (PCR). The entire coding plastid rbcLa ment ~ 884 bp or less 850 bp according to (first part of rbcL gene ~ 700bp or less) CBOL Plant Working Group (2009).