Detection of Glyphosate Resistance Gene EPSPS in Different Fodders

ZANCO Journal of Pure and Applied Sciences The official scientific journal of Salahaddin University-Erbil ZJPAS (2017), 29 (s4); s157-s168 http://dx.doi.org/10.21271/ZJPAS.29.s4.19 Detection of Glyphosate Resistance Gene EPSPS in Different Fodders Shatha A. Yousif1, Samia Khalel2, Tagreed A. Saeed1 and Abdul Kareem Q. Mohammed1 1 Ministry of Science &Technology, P.O. Box 765 Baghdad- Iraq. 2 Al Nahreen University, Baghdad- Iraq. ARTICLE INFO ABSTRACT Article History: Received: 01/06/2017 Accepted: 05/08/2017 Published: 20/12/2017 Keywords: Despite the controversy over GM food and feed, these products are still on the market. Iraqi Biosafety Law had not yet come into force and no law has so far been enacted on the labeling of genetically modified food and feed. To our knowledge there is no available quantitative data on the prevalence of GM crops in feeds in Iraq. The aim of the present study was to detect the presence of GMO feeds by PCR based method. Five fodder kinds from various markets in Iraq were chosen as materials. Feed1 (seed mixture; including safflower, flax, millet with colorful protein supplements), feed 2 which feed crushed, feed 3 (seeds mixture; including wheat, corn, sorghum and sunflower), feed 4 which is grain mixture with main barley seeds and feed 5 which is a fodder pellet. The screening of all samples was performed using the primers for Chloroplast rbcL (internal control), CaMV35S promoter, NOS terminator, Cry1Ac and EPSPS genes. The study proved the existence of genetic modification in all samples. For confirmation procedures the amplified fragments corresponding to the EPSPS were verified by sequencing. GMO Detection, Fodder; PCR, DNA sequencing. *Corresponding Author: Shatha A. Yousif yousifshatha@yahoo.com 1. INTRODUCTION Genetically modified organisms (GMO) are unique, mankind created forceful modification of their genome through gene technology. As revealed by International Service for the Acquisition of Agri-Biotech Application (ISAAA), The global area of GM crops increased from 1.7 million hectares in 1996 to 179.7 million hectares in 2015 by 28 countries (James, 2015). From 1994, the first GM plant approved for consumption by Food and Drug Administration (FDA) in USA was the Calgene's Flavr Savr tomato (Dale, 2015). The first crop approved for food production was Roundup Ready (RRTM) soybean in 1996, it was developed by Monsanto and confers tolerance to Roundup herbicide (Rott et al., 2004). According to scientific data collected until today there were no records of negative effects of GM food which could be harmful for human and animal health, except its possible allergenicity of its protein product, so many scientists agree that this should not be a reason against GMO (Bachas-Daunert and Deo, 2008) but from other hand the usage of GM plants raised many controversies and public concerns about the possible its impact on human health, the environment and socio-economic systems (Daniell, 1999, Romeis et al., 2013). In consequence, low public acceptance of GM products has resulted in increasing the regulatory requirements for safety assessment 158 Yousif, Sh. et al. /ZJPAS: 2017, 29 (s4): s157-s168 of food and feed containing GMOs and different countries have established their own biosafety laws and regulations regarding their production, import and risk assessments (Ahmed, 2002, De Jong, 2010, Premanandh et al., 2012, Stewart and Knight, 2005). To meet these regulative demands it is necessary to continuously analyze the presence of GM event in food/feed. Two classical approaches, based on proteins such as Enzyme-Linked Immunosorbent Assay (ELISA) and DNA, have been used in order to reveal the presence of GMOs (Al-Salameen et al., 2012). Polymerase chain reaction (PCR) is preferred by many analytical laboratories interested in the detection of GM organisms because of its high sensitivity and reliability (Ahmed, 2002, Anklam et al., 2002, Holst-Jensen et al., 2003). PCR-based detection can be evaluated in at least four categories related to the level of specificity namely screen-specific, genespecific, construct-specific, and event-specific evaluation. The first category include common DNA elements in GMOs, such as promoters and terminators that are present in many different GMOs (Holst-Jensen, 2009, Miraglia et al., 2004). Sometimes, marker genes are also used as screening targets, such as gene encoding resistance to ampicillin (bla) and neomycin/kanamycin (nptII) antibiotics used in selection. In the second category, which detect a part of the trait gene associated with the specific genetic modification such as CryIAb and CryIAc (Bt) or EPSPS genes. The third level includes the junctions between the promoter sequence used to drive the transgene and the transgene itself and in category 4 detect the sequences which are event-specific, such as junction between the gene and its integration site are targeted for PCR amplification (HolstJensen et al., 2003). Glyphosate (Monsanto RoundUp) is a broad spectrum herbicide that kills plants by binding to an essential enzyme (5Enolpyruvylshikimate-3-phospate synthaseEPSPS). Herbicide resistance GM crops have been developed through the insertion of EPSPS gene (James, 2015, Randhawaand Firk, 2006) and thus allows transgenic plants to survive herbicide treatment. Another example of genetic modification is the development of crop resistant to specific groups of insects, these crops contain a gene from the soil born bacterium Bacillus thuringiensis coding for insecticidal toxin CRY (Mehrotra et al., 2011). Herbicide tolerant GM and insects resistant have occupied most of the total area of global GM crops. Iraqi Biosafety Law had not yet come into force, to our knowledge there is no available quantitative data on the prevalence of GM crops in feeds in Iraq. The aim of the present study was to detect GMO from feeds using PCR technique and to monitor the presence of EPSPS and Bt gene commercially available in feeds in the Iraqi market. 2. MATERİAL AND METHODS Five fodder products (kinds) from various markets in Iraq were chosen as materials. Feed1 (seed mixture; including safflower, flax, millet with colorful protein supplements), feed 2 which feed crushed, feed 3 (seeds mixture; including wheat, corn, sorghum and sunflower), feed 4 which is grain mixture with main barley seeds and feed 5 which is a fodder pellet (Figure 1A-E). One kilogram of each feed products was homogenized in grinder individually. 1 gm of each sample from the same kind of feed products was pooled together to form representative sample for further analysis. All feeds DNAs were extracted from 100 mg samples by the cetyltrimethylammonium bromide (CTAB) method (Meriç et al., 2014). The DNA isolation method was repeated three times; in addition, water instead of the sample was used in one 159 Yousif, Sh. et al. /ZJPAS: 2017, 29 (s4): s157-s168 tube in each set, in case of possible contamination risks that may be caused by the environment during the process. DNA yield and purity were determined by a NanoDrop 2000c UV/Vis spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and DNA integrity was assessed by electrophoretic methods; agarose gel electrophoresis (1% agarose gel stained with 0.5 μg/ml of ethidium bromide in 1 x TBE buffer, voltage at 90 V). The gels were photographed using Gel Documentation System (AE-9000 E-graph ; ATTO, Japan). GMO scanning of the investigated feed products was performed to determine whether or not they contained GMO products. Screening of all samples was performed using primers for CaMV35S promoter which amplifies a product size of 123bp, primers for NOS terminator which amplifies a product size of 118bp (Lipp et al. 2001), Chloroplast rbcL (internal control) which amplifies a product size of 500bp (Kress, et al., 2009, Levin et al., 2003), primers for Cry1Ac (Mehrotra et al., 2011) which amplifies a product size of 155bp and primers for EPSPS gene. The PCR reactions were carried out using AccuPower®PCR PreMix (Bioneer, Korea) containing 250 μM of each deoxyribonucleoside triphosphate (dNTPs), 30 mM of KCl, 10 mM of Tris- HCl (pH 9.0), 1.5 mM of MgCl2, 1 Unit of Top DNA polymerase and tracking dye. PCR was performed in a final volume of 20 μl of PCR mix containing 100 ng each of the primers and 200 ng DNA. The PCR amplification was carried out in a 20 μl reaction mix on a MyGenieTM Thermal Block (Bioneer, Korea), PCR reaction was performed in duplicate. Negative controls (without template DNA) were also run along with the DNA samples of interest to avoid the detection false positive. The program consisted of a single cycle of DNA polymerase activation for 10 min at 95°C followed by the thermal step cycle programme included an initial denaturation at 94 °C for 3 min followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at either 55 °C (P-35S and T NOS), 56 °C (Cry1Ac) and 60 °C (EPSPS gene) for 30 s and extension at 72 °C for 30s. A final extension step was performed at 72 °C for 5 min. The amplified DNA fragments were separated by electrophoresis in a 1% agarose gel (supplemented with 0.5 μg/ml of ethidium bromide) in 1x TBE buffer at 90 V and visualized using Gel Documentation System. Visible bands of the expected size of EPSPS gene were purified using a Wizard® SV Gel and PCR Clean-Up System kit. Samples were sent to Macrogen (Korea) for sequences. Sequence manipulations were conducted in BioEdit Sequence Alignment Editor v.7.0.5.3 (http://www.mbio.ncsu.edu/bioedit/page2.html) (Hall, 1999). Each sequence was queried in NCBI using Nucleotide BLAST search. 3. RESULTS AND DİSCUSSİON Many methods in GMO analysis are based on precipitation of the DNA using CTAB extraction buffer. CTAB extraction method has been widely applied in molecular genetics and is considered efficient for a wide range of foods and feeds products (Anklam, et al, 2002, Gryson et al., 2004) from other hand the costs are lower compare with commercial kits (Gryson et al., 2004, Sönmezoğlu and Keskin, 2015). The highest average DNA amounts were obtained from feed1 (124 ng/μl), feed2 (533ng/μl), feed3 (247 ng/μl), feed4 (305 ng/μl) and feed5 (538 ng/μl) and the average DNA purity (A260/A280 ratio) of all samples ranged reached 2. Also this method produced a clear DNA band on the agarose gel. The 500 bp band size expected as a result of amplification of Chloroplast rbcL (internal 160 Yousif, Sh. et al. /ZJPAS: 2017, 29 (s4): s157-s168 control) was found in all samples (Table 1). This gene has to be specific for plant species and use it as a control to evaluate the DNA quality and PCR efficacy is necessary to exclude possibility of false negative results due to possible inhibitor presence or inappropriate DNA quality (Zdjelar et al., 2013). Based on the gel electrophoresis results, analysis of fodders (Table1) showed the presence of CaMV 35S promoter sequence in feed 1, feed 2 and feed 4. In addition to that, all samples except feed1 had NOS terminator sequence. Most GMOs contain CaMV35S promoter and NOS terminator are commonly used in genetic engineering as regulator sequences and are used as universal molecular markers for analysis of most GM plants, food and feed (Mandaci et al., 2014) and a positive result of these markers confirmed the transgene sequence is present. In such cases further PCR tests should be performed with gene-specific or construct specific primers designed to amplify the specific transgenic DNA (Zdjelar et al., 2014). To address this possibility, all the samples that had tested were also tested for herbicide and insect resistant. Herbicide resistance gene (EPSPS) and gene for insecticide protein (Cry1 Ac) were found in all samples (Table 1, Figure 2 and 3). This analysis gave an evident that all samples had multiple GM events. The presence of the GM materials in feeds were similar to the result of Meriç et al. (2014) who found that all 11 feed samples were positive for the NOS terminator, 35S promoter and EPSPS. In another study, demonstrated the presence of glyphosateresistant GM plants under the control of P35S promoter and NOS terminator in feed products that collected from the Malaysian and Vietnamian local markets (Tung Nguyen et al., 2008). GM plants are not cultivated in Iraq, but the distributions of GMO indicated that this materials originated from ambiguous imported sources. Although GM plants are not produced in many countries e.g Jordan (Herzallah, 2012), Lebanon (Sakr et al., 2014) and Malaysia (Tung Nguyen et al., 2008) but a related studies found many feed samples which are not labeled were positive for GMO regulatory elements and they emphasized the need for controlling of all of the imported feed products by the authorities responsible for GM monitoring. In order to validate the GMO detection method, selected bands (280 pb) for EPSPS gene have been sent for sequencing. EPSPS sequences for feeds were compared to those in the Genbank database managed (NCBI). Table 2 showed high similarity for all feed samples to construct pTLE8 vector (GenBank Acc. JX434028.1 ) after BLAST analysis. pTLE8 vector is used in genetic modification to transfer the gene for herbicide resistance; EPSPS gene). Expected (E) value ranged from 1e-132 to 9e-129 that means these E values fulfilled the requirements of significant matches. The sequences for all samples were aligned with pTLE8 vector using ClustalW (Figure 4) through alignment in BioEdit program. All feed sequences partially aligned to vector, start from 2707bp and up to 2970 bp of the vector structure and this site is a special zone EPSPS gene in this vector which begins from 2481 bp and ending at 3656 pb. The results of this analysis confirmed our screening results. EPSPS gene sequences showed some mutations caused by single nucleotide insertion, delete or change. This result was in agreement with previous research of Tung Nguyen et al. (2008) who concluded that those mutations might have resulted from the mismatches of Taq polymerase during PCR amplification and the troubleshooting of DNA sequencing rather than the permanent changes themselves. 161 Yousif, Sh. et al. /ZJPAS: 2017, 29 (s4): s157-s168 CONCLUSIONS In the present study, genetically modified feeds gathered from Iraqi market, was detected. The results in this study demonstrated the presence of Cry1Ac and EPSPS genes under the control of P35S promoter and/or NOS terminator. In order to validate the GMO detection method, selected bands for EPSPS gene have been sequenced and the analysis confirmed our screening results. Polymerase Chain Reaction based method and DNA sequencing is useful tool for investigation or screening GM events in feeds. This is the first documented study on the presence of genetically modified feeds in Iraqi markets. The interpretation of the presence of GM fodder may be explained that this materials may be originated from ambiguous imported sources, so monitoring will be necessary to control the distribution of unlabelled GM containing feeds. Yousif, Sh. et al. /ZJPAS: 2017, 29 (s4): s157-s168 162 A B C D FIGURE 1. Fodder kinds, A; feed 1 (seed mixture; including safflower, flax, millet with colorful protein supplements), B; feed 2 which feed crushed, C; feed 3 (seeds mixture; including wheat, corn, sorghum and sunflower), D; feed 4 which is grain mixture with main barley seeds and E; feed 5 which is a fodder Pellet. E Yousif, Sh. et al. /ZJPAS: 2017, 29 (s4): s157-s168 163 TABLE 1. Screening of various fodder samples for chloroplast rbcL and transgenic content. chloroplast rbcL Feed1 + Feed2 + Feed3 + Feed4 + Feed5 + (+) = possitive; (-) = negative. Fodders CaMV 35S promoter + + + - NOS terminator + + + + EPSPS gene Cry1 Ac conclusion + + + + + + + + + + + + + + + 164 Yousif, Sh. et al. /ZJPAS: 2017, 29 (s4): s157-s168 FIGURE 2: Agarose gel electrophoresis (1%) of FIGURE 3: Agarose gel electrophoresis (1%) of amplification products of Cry1Ac from different amplification products of EPSPS from different fodder samples. From left to right Lane 1; 100 bp fodder samples. From left to right Lane 1; 100 DNA ladder (Bioneer), Lane 2; feed1, Lane 3; bp DNA ladder (Bioneer), Lane 2; feed1, Lane feed2, Lane 4; feed3, Lane 5; feed 4, Lane 6; feed5, 3; feed2, Lane 4; feed3, Lane 5; feed 4, Lane 6; Lane 7; negative control. feed5. Yousif, Sh. et al. /ZJPAS: 2017, 29 (s4): s157-s168 165 TABLE2. Similarities between fodder samples and pTLE8 vector. Fodders Feed1 Feed2 Feed3 Feed4 Feed5 Identity 263/264(99%) 260/263(99%) 252/263(96%) 264/264(100%) 260/263(99%) E value 1e-132 9e-129 2e-115 2e-134 9e-129 Gaps 0/264(0%) 0/263(0%) 0/263(0%) 0/264(0%) 0/263(0%) 166 Yousif, Sh. et al. /ZJPAS: 2017, 29 (s4): s157-s168 ....|....| ....|....| ....|....| ....|....| ....|....| 2710 2720 2730 2740 2750 vector pTLE8 GCCTCGTCGG GGTCTACGAT TTCGACAGCA CCTTCATCGG CGACGCCTCG feed1 ------TCGG GGTCTACGAT TTCGACAGCA CCTTCATCGG CGACGCCTCG feed2 ------TCGG GGTCTACGAT TTCGACAGCA CCTTCATCGG CGACGCCTCG feed3 ------TCGG GGTCTACGAT TTCGACAGCA CCTTCATCGG CGACGCCTCG feed4 ------TCGG GGTCTACGAT TTCGACAGCA CCTTCATCGG CGACGCCTCG feed5 ------TCGG GGTCTACGAT TTCGACAGCA CCTTCATCGG CGACGCCTCG ....|....| ....|....| ....|....| ....|....| ....|....| 2760 2770 2780 2790 2800 vector pTLE8 CTCACAAAGC GCCCGATGGG CCGCGTGTTG AACCCGCTGC GCGAAATGGG feed1 CTCACAAAGC GCCCGATGGG CCGCGTGTTG AACCCGCTGC GCGAAATGGG feed2 CTCACATAGC GCCCGATGGG CCGCGTGTTG AACCCGCTGC GCGAAATGGG feed3 CTCACTTACC GCCCGCTGGG CCGCGTGTTG AACCCGCTGC GCAAAATGGG feed4 CTCACAAAGC GCCCGATGGG CCGCGTGTTG AACCCGCTGC GCGAAATGGG feed5 CTCACAAAGC GCCCGATGGG CCGCGTGTTG AACCCGCTGC GCGAAATGGG ....|....| ....|....| ....|....| ....|....| ....|....| 2810 2820 2830 2840 2850 vector pTLE8 CGTGCAGGTG AAATCGGAAG ACGGTGACCG TCTTCCCGTT ACCTTGCGCG feed1 CGTGCAGGTG AAATCGGAAG ACGGTGACCG TCTTCCCGTT ACCTTGCGCG feed2 CGTGCAGGTG AAATCGGAAG ACGGTGACCG TCTTCCCGTT ACCTTGCGCG feed3 CGTGCAGGTG AAATCGGAAG ACGGTGACCG TCTTCCCGTT ACCTTGCGCG feed4 CGTGCAGGTG AAATCGGAAG ACGGTGACCG TCTTCCCGTT ACCTTGCGCG feed5 CGTGCAGGTG AAATCGGAAG ACGGTGACCG TCTTCCCGTT ACCTTGCGCG ....|....| ....|....| ....|....| ....|....| ....|....| 2860 2870 2880 2890 2900 vector pTLE8 GGCCGAAGAC GCCGACGCCG ATCACCTACC GCGTGCCGAT GGCCTCCGCA feed1 GGCCGAAGAC GCCGACGCCG ATCACCTACC GCGTGCCGAT GGCCTCCGCA feed2 GGCCGAAGAC GCCGACGCCG ATCACCTACC GCGTGCCGAT GGCCTCCGCA feed3 GGCCGAAGAC GCCGACGCCG ATCACCTACC GCGTGCCGAT GGCCTCCGCA feed4 GGCCGAAGAC GCCGACGCCG ATCACCTACC GCGTGCCGAT GGCCTCCGCA feed5 GGCCGAAGAC GCCGACGCCG ATCACCTACC GCGTGCCGAT GGCCTCCGCA ....|....| ....|....| ....|....| ....|....| ....|....| 2910 2920 2930 2940 2950 vector pTLE8 CAGGTGAAGT CCGCCGTGCT GCTCGCCGGC CTCAACACGC CCGGCATCAC feed1 CAGGTGAAGT CCGCCGTGCT GCTCGCCGGC CTCAACACGC CCGGCATCAC feed2 CAGGTGAAGT CCGCCGTGCT GCTCGCCGGC CTCAACACGC CCGGCATCAC feed3 CAGGTGAAGT CCGCCGTGCT GCTCGCCGGC CTCAACACGC CCGGCATCAC feed4 CAGGTGAAGT CCGCCGTGCT GCTCGCCGGC CTCAACACGC CCGGCATCAC feed5 CAGGTGAAGT CCGCCGTGCT GCTCGCCGGC CTCAACACGC CCGGCATCAC ....|....| ....|....| ....|....| ....|....| ....|....| 2960 2970 2980 2990 3000 vector pTLE8 GACGGTCATC GAGCCGATCA TGACGCGCGA TCATACGGAA AAGATGCTGC feed1 GACGGTCATC GAGCCGATCA ---------- ---------- ---------feed2 GACGGTCATC GAGCCGATC- ---------- ---------- ---------feed3 GACGGTCATC GAGCCGATC- ---------- ---------- ---------feed4 GACGGTCATC GAGCCGATCA ---------- ---------- ---------feed5 GACGGTCATC GAGCCGATC- ---------- ---------- ---------- FIGURE 4. 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