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Fouad Et Al., Authentication of Cordia Dentata Poir See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/333457757 Authentication of Cordia dentata Poir. Growing in Egypt using ISSR and DNA barcoding Article · May 2019 CITATIONS READS 0 30 3 authors: Ahmed S. Fouad Rehab Hafez Cairo University Cairo University 6 PUBLICATIONS 11 CITATIONS 8 PUBLICATIONS 25 CITATIONS SEE PROFILE SEE PROFILE Rim Hamdy Cairo University 24 PUBLICATIONS 70 CITATIONS SEE PROFILE Some of the authors of this publication are also working on these related projects: thesis View project All content following this page was uploaded by Ahmed S. Fouad on 29 May 2019. The user has requested enhancement of the downloaded file. Available online freely at www.isisn.org Bioscience Research Print ISSN: 1811-9506 Online ISSN: 2218-3973 Journal by Innovative Scientific Information & Services Network RESEARCH ARTICLE BIOSCIENCE RESEARCH, 2019 16(2): 1474-1484. OPEN ACCESS Authentication of Cordia dentata Poir. Growing in Egypt using ISSR and DNA barcoding Ahmed Sayed Fouad*, Rehab Mahmoud Hafez and Rim Hamdy Botany and Microbiology Department, Faculty of Science, Cairo University, 12613 Cairo, Egypt. *Correspondence: [email protected] Accepted: 25 April. 2019 Published online: 19 May 2019 Cordia dentate was introduced to Egypt as ornamental and timber trees in the beginnings of the 19th Century. Urbanization is responsible for disappearance of many plant species including C. dentata that are represented with only two trees exhibiting different morphological characteristics. The present study aimed to authenticate these trees using rbcl- and matk-based DNA barcoding as well as ISSR markers. Results reflected that matk and rbcl sequences for both trees were 100% identical and showed 100% similarities with corresponding sequences recorded for C. dentate in BOLD System and Gene Bank. Nine ISSR primers, out of ten, reflected polymorphism between the two trees. Thus it is recommended to use DNA barcoding in species identification then ISSR for further intraspecific resolution. Keywords: Cordia dentate, biodiversity, rbcl, matk, ISSR INTRODUCTION 2009). Biodiversity is a general term used to describe Along with another seven Cordia species, C. the sum of all life’s varieties in a defined location dentata was introduced to Egyptian gardens in the or even across the whole planet. It occurs at beginnings of the 19th Century as ornamental and ecosystem, species and genetic levels (Glowka et timber trees (Ascherson and Schweinfurth, 1887; al., 1994). Plant biodiversity is a major source for Delchevalerie, 1899; Bircher and Bircher, 2000; food and drug and constitutes a natural reservoir Diwan et al., 2004; Hamdy, 2010). C. dentate is for genetic raw material essential for breeding also rich in valuable compounds (eg: Rosmarinic programs of many important crops (Rao, 2004). acid, Quercetin, 3-o-rutinoside and Rutin) However, the human activities associated with important for treatment of many human diseases over-usage of plant resources in parallel with the (Thirupathi et al., 2008; Hossan et al., 2014; over-production of pollutants exaggerate the rate Wang et al., 2015; Ganeshpurkar and Saluja, of plant extinction reaching one species per day 2017). The growing urban activities have (Hilton-Taylor, 2000). demolished many gardens and are responsible for The Egyptian territories host 2088 species disappearance of many plant species. belonging to 742 genera of 120 families (Khedr et Consequently, C. dentata in Egypt is represented al., 2002). Cordia L. (family Cordiaceae) is a large with only two individuals growing in zoological pantropical genus including about 300 species of garden; the first is typical C. dentata while the trees and shrubs, distributed in Africa, South Asia second is C. dentata form (Amer et al., 2016). and tropical America (Mabberley, 2008). In the Identification and characterization of Egyptian flora, Cordia was monospecific genus endangered plant species is a prerequisite to represented by C. sinensis (El Hadidy and Boulos, maintain biodiversity (Bapat et al., 2012). Fouad et al., Authentication of Cordia dentata Poir. growing in Egypt using ISSR and DNA barcoding Traditional approaches employing morphological potential. Conversely, matk gives better resolution features require taxonomic expertise and usually but with some amplification concerns (Laiou et al. suffer from subjective biases (Costion et al., 2013). It recommended to use a combination of 2011). Chromatographic profiles also have some these two markers for better results (Ganie et al., limitations being affected with plant age, tissue 2015). source, physiological conditions and The aim of this study is to authenticate the C. environmental factors (Joshi et al. 2004; Zhang et dentata trees growing in Egypt and characterize al., 2007). On the other hand, DNA based the differences between typical C. dentata and C. markers can be used to characterize biodiversity dentata form using ISSR markers along with DNA without fear from the previous sources of error barcoding using rbcl and matk sequences. (Bafeel et al., 2012). An arsenal of non-sequence based molecular MATERIALS AND METHODS markers are available for biodiversity Total genomic DNA was extracted from documentation, the most common of which are about 20 mg liquid nitrogen powdered leaf tissues fragment length polymorphisms (RFLP), amplified collected from each of typical C. dentata and C. fragment length polymorphism (AFLP), simple dentata form trees with aid of Qiagen DNeasy kit sequence repeats (SSR), inter simple sequence (Valencia, California, USA), following the repeats (ISSR) and randomly amplified manufacturer’s protocols. polymorphic DNA (RAPD) (Ganie et al., 2015 for PCR amplifications were carried out using 17- review). RAPD and ISSR are free of many 19 base primers (Table 1) selected based on their limitations facing other markers; they are time, ability to produce clear reproducible banding labor and cost effective and do not necessitate pattern. The reaction mixture comprised of 25 μl prior information about sequences of the target containing one unit Taq polymerase (Promega, organism genome (Muzila et al., 2014). However, WI, USA), 30 pmol of primer, 0.5 μl dNTPs (10 the longer primers used in ISSR, compared with mM), 30 ng template DNA and 1.5 μl MgCl2 (25 RAPD ones, make it more specific with higher mM). The amplification protocol was initial stringent amplifications (Wolfe et al., 1998). In denaturation of 2 min at 94°C; 40 cycles of 30 Sec addition, the abundance of target sequences with denaturation at 94°C, 30 Sec annealing at 50°C high evolution rate for ISSR primers helps in and 2 min extension at 72°C; and final elongation revealing more polymorphic loci, compared with step at 72°C for 7 min. PCR products were RAPD (Ansari et al., 2012). resolved in 1.5% (m/v) agarose gel and visualized DNA barcoding provides another arsenal of under UV light. Band size was determined using molecular markers that are now regularly used for Gel-Doc XR (Bio-Rad) based on 100 bp DNA biodiversity inventories (Costion et al., 2011; de ladder. Only bands appeared in three PCR Vere et al., 2012). It can be defined as employing amplifications were scored. of short uniform nuclear or organelle DNA For matk, and rbcl PCR amplifications were sequences (400-800bp) for the identification of conducted following CBOL Plant Working Group different taxa (Ganie et al., 2015). The slow (2009) employing specific primers (Table 1) in a substitution rates and intramolecular total volume of 50 µl containing about 50 ng recombination exhibited by plant mitochondrial genomic DNA, 1 µl of each primer and 25 µl PCR DNA (Mower et al., 2007) in addition to the Master Mix (Bioline). The amplification protocol for numerous recorded cases of incomplete rbcl was 95℃ for 2 min followed by 34 cycles of concerted evolution of the internal transcribed 94℃ for 1 min, 55℃ for 30 sec and 72℃ for 1 min, spacers (ITS) in plants (Chase et al., 2005; Kress then final extension for 7 min at 72℃. matk et al., 2005) put plastid sequences as front protocol started with 5 min at 94 ℃ then 26 cycle runners in plant DNA barcoding (Hollingsworth, of 94℃ for 1 min, 48℃ for 30 sec and 72℃ for 1 2008). min. The final extension step lasted for 7 min at Considering sequence quality, recoverability 72 ℃. and levels of species discrimination, the Amplicons for rbcl and matk were subjected to Consortium for the Barcode of Life (CBOL) plant purification step employing the QIAquick PCR working group recommended employing rbcl Purification Kit (Qiagen, Hilden, Germany) before and/or matK in barcoding of land plants (CBOL sequencing using Big-dye terminator chemistry in Plant Working Group, 2009). rbcl is considered as 3130xl Genetic Analyzer (Life Technologies, universal barcode due to its high amplification California, USA) according to the standard success rate but it has low discriminatory manufacturer's protocol. Bioscience Research, 2019 volume 16(2): 1474-1484 1475 Fouad et al., Authentication of Cordia dentata Poir. growing in Egypt using ISSR and DNA barcoding Table 1. Primer sequences Maker Primer Sequence ISSR- 1 5'-AGAGAGAGAGAGAGAGYC-3' ISSR- 2 5'-AGAGAGAGAGAGAGAGYG-3' ISSR- 3 5'-ACACACACACACACACYT-3' ISSR- 4 5'-ACACACACACACACACYG-3' ISSR- 5 5'-GTGTGTGTGTGTGTGTYG-3' ISSR ISSR- 6 5'-CGCGATAGATAGATAGATA-3' ISSR- 8 5'-AGACAGACAGACAGACGC-3' ISSR- 9 5'-GATAGATAGATAGATAGC-3' ISSR- 10 5'-GACAGACAGACAGACAAT-3' ISSR- 11 5'-ACACACACACACACACYA-3' 1f 5'-ATGTCACCACAAACAGAAAC-3' rbcl 724r
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