Assiut J. Agric. Sci., (47) No. (5) 2016 (72-82) ISSN: 1110-0486 Website: http://www.aun.edu.eg/faculty_agriculture E-mail: [email protected] Authentication of Euphorbia peplus L. Family Euphorbiaceae Growing in Egypt Using Finger Printing Mohamed, G.I.A. 1; A. M. Zaher2; A. A. Ali2 ; Hanaa M. Saeyd2 and Sabrin R. Mohamed2 1 Genetic Department, Faculty of Agriculture, Assiut University, Assiut-Egypt. 2 Pharmacognosy Department, Faculty of Pharmacy, Assiut University, Assiut-Egypt. Received on: 8/8/2016 Accepted for publication on: 21/8/2016 Abstract RAPD-PCR was performed using six random primers to identify the ge- netic diversity among six plant samples belong to two genera (Euphorbia and Ricinus). The dendrogram, based on genetic distance, depict the relationship among the investigated plant samples, separate clearly the six samples. The clos- est relationship was observed between E. geniculata and E. aphylla; and E. pul- cherrima and E. peplus, while this relationship was quite separated between these four samples and the other two samples E. cactus and R. communis. Fragments generated by the six primers show a polymorphism ratio of 88.9%. Bands 3500 and 750 bp generated by primer OP-Z13, and also bands 2000, 1500, 1400, 1200, 1000, 720 and 550 bp generated by primer OP-A09 existing only in the plant samples of E. geniculata and E. aphylla, which suggest that these bands can be used as a positive molecular marker to identify these plant samples. Bands 2500, 1720, 1650, 1300, 950 and 250 bp generated by primer OP-A09, and band 1200 bp generated by primer OP-A20 and band 350 bp generated by primer OP-Z19 and band 250 bp generated by primer OP-Z17 were common in all plant samples of family Euphorbiaceae. Moreover, band 430 bp generated by primer OP-Z17 was characterized for Ricinus communis and absent in other plants of genus Eu- phorbia. Also, band 2700 bp generated by primer OP-A20 and band 210 bp gen- erated by primer OP-Z19 existing only in Euphorbia peplus. This study high- lights the usefulness of RAPD assay for determining genetic variation in different plant genera and for estimating genetic distances between different plant sam- ples. Moreover, knowledge of genetic distance among genera and species, and genetic diversity/structure within genera could be useful for conservation of ge- netic resources. Data presented here are the first report in Egypt of genetic varia- tion inside genera Euphorbia and Ricinus described at the molecular level. We consider this work as a first step in molecular characterization of genera Euphor- bia and Ricinus, thus, it is recommended to extend the panel of samples and primers in the future. Keywords: Fingerprinting, RAPD-PCR, Genetic marker, Euphorbia, Ricinus. Introduction the best method for the detection of Authentication of medicinal contaminants and could be an excel- plants is a critical issue (Core, 1962; lent method for plant identification Lawrence, 1968 and Kirticar and (Jassbi, 2006; Ria et al., 2009 and Basu, 1975). Macroscopic and micro- Uchida et al., 2010). Molecular biol- scopic studies can be used, as they ogy offers an assortment of tech- are rapid and inexpensive identifica- niques that can be very useful for au- tion techniques. Chemical analysis is thentication of medicinal plants, Mohamed, et al., 2016 DNA base-pair sequences guides the and Ricinus growing in Egypt has not production of proteins and enzymes. been sufficiently studied. This pro- These in turn decide features such as voked us to carry out this study to leaf shape and flower color, as well provide information at the molecular as direct synthesis of a wide range of level about the authentication and ge- phytochemicals in plants. DNA fin- netic diversity of these genera, em- gerprint can distinguish plants from ploying RAPD-PCR molecular mark- different families, genera and species. ers. DNA fingerprinting refer to the use Materials and Methods of techniques based polymerase chain Six plant samples belong to tow reaction (PCR), a system for the am- genera, Euphorbia (1= E. geniculate, plification of DNA, reveal the spe- 2= E. pulcherrima, 3= E. peplus, 4= cific profile for a particular organism E. aphylla and 5= E. cactus) and which is a unique as a fingerprint Ricinus (6= R. communis) collected (Powledge, 1995 and Rosso and Phil- from Assiut University Campus, were ippe, 2001). Medicinal plants have subjected to Random Amplified Po- always an important place in the lymorphic DNA (RAPD) technique therapeutic system. The use of natural using six decamer random primers, products in the treatment of various obtained from Operon Technologies diseases has played an important role Inc.: USA. in medical therapy for many years Extraction and Purification of and plants of the genus Euphorbia are Genomic DNA: Total genomic DNA known to possess considerable me- was extracted using the Qiagen dicinal and economic importance DNeasy (Qiagen Santa Clara, CA). components (Tian et al., 2010 and This was performed following the Chaabi et al., 2007). Euphorbiaceae manufacturer's instructions. DNA is one of the largest families of higher concentration was determined by di- plants comprising about 283 genera luting the DNA (1:5) in dH2O. The and 7500 species (Core, 1962; Law- DNA samples were electrophoresed rence, 1968; Tackholm, 1974; Kirti- in 1% agarose gel against 10µg of a car and Basu, 1975; Watt and Breyer- DNA size marker. This marker cov- Brandwijk, 1962; Boulos, 1980 and ers a range of concentration between Benson, 1957) that are further charac- 95ng to 11ng. Samples concentration terized by the frequent occurrence of was adjusted at 25 ng/µl using TE milky sap. Genus Euphorbia is the buffer pH 8.0. one of the largest six genera of flow- PCR conditions for RAPD analysis: ering plants with approximately 1600 PCR for RAPD analysis was species (Boulos, 1980 and Chopra, done using 25 ng genomic DNA of 1973). Economically, important six plant samples. The PCR mixture products including foods, drugs and and amplification conditions were rubber (Watt and Breyer-Brandwijk, prepared according to Williams et al., 1962 and Boulos, 1980) are obtained (1990) with minor modifications. A from certain Euphorbia species. Re- set of six primers RAPD (OP-A09, viewing the available literature, ge- OP-A20, OP-B03, OP-Z13, OP-Z17 netic diversity of genera Euphorbia and OP-Z19) were used. The amplifi- 73 Assiut J. Agric. Sci., (47) No. (5) 2016 (72-82) ISSN: 1110-0486 Website: http://www.aun.edu.eg/faculty_agriculture E-mail: [email protected] cation reaction was carried out in 25 (0.5 µg/ml) in 1X TBE buffer at 95 µl reaction volume. The contents of volts. PCR products were visualized on PCR mixture are shown in Table 1. UV Transilluminator and photographed. PCR amplification cycles were per- RAPD-PCR profiles were analyzed us- formed in a Perkin-Elmer/GeneAmp® ing Gene profiler 3.1 software. The PCR system as follows: Pre- banding profiles were scored (using 1kb o DNA Ladder RTU, promega) and the Denaturation (one cycle) at 94 C for presence or absence of each size class 5 min, followed by 40 cycles of: De- o was scored as (+) or (-), respectively. naturation step at 94 C for 1 min, The scored bands were analyzed by un- o Annealing step at 36 C for 1 min, weighted pair-group method based on o and an elongation step at 72 C for arithmetic mean (UPGMA) to estimate 1.5 min, then final extension at 72 oC similarity, genetic distances and recon- for 7 min. struct the dendogram. Results Table 1. contents of PCR mixture for Genetic diversity and relationship RAPD analysis between six species belong to two gen- 0.2 mM dNTPs 2.5 µl era (Euphorbia and Ricinus) were stud- 1X reaction buffer + MgCl2 2.5 µl ied using Random Amplified Polymor- 1 µM primer 3.0 µl phic DNA (RAPD) technique. Six oli- 25 ng template DNA 1.0 µl godecamers arbitrarily primers used in 1 units Taq (super thermal) 1.0 µl the present investigation to generate RAPD profiles from the six plant sam- H2O 15.0 µl Total volume 25.0 µl ples. All primers were amplified suc- cessfully on the genomic DNA from taken samples yielding distinct RAPD The amplification products were patterns. revolved by electrophoresis in 1.5% aga- rose gel containing ethidium bromide (MFigures 11, 2 and 2 3 and 3 Table 4 2 ). The 5 number 6 of 1 2 3 4 5 6 M OP A09 OP Z13 Figure 1. Agarose-gel electrophoresis of RAPD products generated by primer OP-A09 and OP-Z13 in the examined samples, M = DNA marker. 74 Mohamed, et al., 2016 M 1 2 3 4 5 6 1 2 3 4 5 6 OP A20 OP B03 Figure 2: Agarose-gel electrophoresis of RAPD products generated by primer OP-A20 and OP-B03 in the examined samples, M = DNA marker. M 1 2 3 4 5 6 1 2 3 4 5 6 OP OP Z19 Z17 Figure 3. Agarose-gel electrophoresis of RAPD products generated by primer OP-Z17 and OP-Z19 in the examined samples, M = DNA marker. 75 Assiut J. Agric. Sci., (47) No. (5) 2016 (72-82) ISSN: 1110-0486 Website: http://www.aun.edu.eg/faculty_agriculture E-mail: [email protected] Table 2. Summary of all fragments generated by the assay of the six primers, and their molecular size (bp) in all six samples of Euphorbia and Ricinus where (+) means presence and (-) means absence. Primer M.W. Samples Primer M.W. Samples code (bp) 1 2 3 4 5 6 code (bp) 1 2 3 4 5 6 OPZ13 3500 + - - + - - OPA09 950 + + + + + + OPA20 3200 + + + + - - OPZ17 900 + + + - - - OPB03 3200 + + - + - - OPA20 850 + + + - - - OPZ13 3000 - + - + - - OPZ19 850 + + + + - + OPZ17 3000 + + + - - - OPA09 750 - + + + + + OPA20 2800 + + - + - - OPA20 750 + - - - - OPZ13 2800 - - - - + + OPB03 750 + + + + + - OPA20