Int'l Conf. on Advances in Science, Engg., Technology & Natural Resources (ICASETNR-15) Aug. 27-28, 2015 Kota Kinabalu (Malaysia)

Evaluation of the Mutagenic Effect of Ethyl Methanesulfonate on Hexokinase nuclear DNA Locus of Solanum lycopersicum

Aswaneeza Binti Khairul Aswandy1, Nur Rawaidah2, Kenneth Francis Rodrigues3, and Cheong Bo Eng4

 Solanaceae family [2]. Tomato has been widely used for Abstract— Tomato (Solanum lycopersicum) is an ideal model genetic studies linking tomato genes to their functions. The plant for assessing the effects of on specific genes due to the direct approach in determining gene function is by analyzing availability of its complete genome, 950 megabases of DNA which the variation that occurs within the organism itself due to aligned to 12 tomato chromosomes with 34,727 coding proteins that mutation in specific gene. The function is first identified from facilitates the task of designing locus specific primers for genetic the mutant organism followed by the identification of specific analysis. A total of 78 seeds were exposed to 0.5% concentration of Ethyl methanesulfonate (EMS). 50 mutant plants were subject to controlling gene [3]. Reverse approach is another Inter Simple Sequence Repeat – Polymerase Chain Reaction (ISSR- alternative to this classic forward genetic approach. PCR) for the purpose of screening for genotypic variation. The Targeting Induced Local Lesions in Genomes (TILLING) presences frequency of occurrence of Single [4], [5] is an alternative technology without the use of genetic Polymorphism (SNPs) were further validated through DNA transformation or gene insertion. This reverse genetic sequencing with the use of gene specific markers which were approach requires gene nucleotide sequences for the screening designated as NHEX and designed by referencing data from the SOL of induced point that managed to alter the mutant genomic database. Phenotypic variation was assessed in terms of organism in terms of their phenotype through the treatment of sugar level in fruits (BRIX Index), flower morphology, leaf chemical [6] Targeting Induced Local Lesions in morphology, fruit morphology, plant’s heights, Cotyledons number and stress responds of the mutant plants. Genomic variation was Genomes (TILLING) technology detects mutation through the observed at the Hexokinase coding gene, site number 147 and 158. screening of mismatch paring in the nucleotide sequence of Sugar level of both control and mutant tomato fruits were also mutants [7]. Full sequence of thousands of individual for determined using a refractometer through refractometric detection. mutation screening could be replace by sequence capture The relatively low sugar level of mutant fruits compared to wild type through TILLING, restricting the size of DNA to be sequence indicates that hexose kinase might involve in carbon catabolite and focuses only on the coding segment of genome [8]. The set repression. Phenotypic variation was also observed with reference to up tomato TILLING platform in this research, enabled the flower morphology, leaf morphology and fruit morphology. The next screening of on specific locus through the phase of this study will involve the culture of the second generation of mutant population (M2) followed by assessment of genetic exposure of chemical mutagen. variation at the target loci. The findings of this study will provide an Ethyl Methanesulfonate (EMS) is effectively proven in insight into the fundamental mechanism of chemical mutagenesis in inducing random point mutation [9]. The alkylating agent, 6 this model plant. EMS results in the build-up of O -ethylguanine on (G) residue which will miss-pairing with Thymine (T) instead of Keywords— Ethyl methanesulfonate, Solanum lycopersicum, Cytosine (C). The miss-pair of Adenine (A)/Thymine (T) will Single Nucleotide Polymorphism, Specific marker occur through continues DNA repair in replacement to Guanine (G)/Cytosine (C) pairing [10]. Occurrence of 70%- I. INTRODUCTION 99% of C/G to A/T change in among mutant OMATO (Solanum lycopersicum) is one of the commercial organism through the treatment of EMS were reported [11], vegetable plants which has a small diploid genome size of [12] Detected mutants were analyzed for variation in specific T950MB across 12 tomato chromosomes [1] compared to HXK gene locus. the other crops within Solanaceae species, which makes Hexokinase are located in multiple intracellular locations. tomato a reference species for sequencing the genome of Hexose monophosphate formed as the end product of the phosphorylation of hexose by hexokinase [13], an important

1 pathway for glucose signaling [14]. Phosphorylation of hexose Aswaneeza Khairul Aswandy is with the Universiti Malaysia Sabah, is catalyzed by three main enzymes, hexokinase Hxk1 and 88400 Sabah Malaysia Nur Rawaidah Mohd Shobri2 is with the Universiti Malaysia Sabah, Hxk2 and glucokinase Glk1. This gene acts as a sensor for 88400 Sabah Malaysia glucose which similar to those regulated in yeast, Kenneth Francis Rodrigues3 is with the Universiti Malaysia Sabah, 88400 Saccharomyces cerevisiae [22]. Hexokinase enzyme is an Sabah Malaysia essential protein which acts as a sugar sensor for genes that Cheong Bo Eng4 is with the Universiti Malaysia Sabah, 88400 Sabah Malaysia take part in both photosynthesis [25] and glyoxylate cycle of

http://dx.doi.org/10.15242/IICBE.C0815048 88 Int'l Conf. on Advances in Science, Engg., Technology & Natural Resources (ICASETNR-15) Aug. 27-28, 2015 Kota Kinabalu (Malaysia) plants [26] in modulating the growth, maturity and plant conducted for one initial step of denaturation at 95°C for 3 responds to stress [27]. In this study, the assessment of total minutes followed by 30 cycles of 95°C for 20 s, 55°C for 40 s, soluble solid (TSS) were measured through an optical 72°C for 60 s: and a final elongation at 72°C for 10 minutes. refractometer. This protocol was optimized for ISSR-1 primer with the use of Hexokinase present in many locations throughout the plant 50 ng of template genomic DNA. DNA of the matured tomato especially in fruits and leaves of Solanum lycopersicum, in this leaves were analyzed for the presence of Single Nucleotide paper we focuses on the Hexokinase gene in the chromosome Polymorphism (SNPs) through DNA sequencing using specific 6, nuclear DNA of tomato in order to analyst the gene function gene marker for HXK locus. of HXK through the development of mutant population using E. Analyzing genotypic variation chemical mutagen Ethyl Methanesulfonate. One mutation was found on the protein level giving variance among mutants and Mutation on the specific locus of HXK were identified control determinant hybrid through the analysis of their sugar through the analysis of the gene sequence of mutant plant in content and genotypic assessment. comparison to the gene sequence of the control determinant hybrid and reference gene from National Center for II. MATERIALS AND METHODS Biotechnology information (NCBI database). The analyzing of chromatogram and nucleotide sequence of the amplified A. Plants Material fragments was performed with EditSeq and SeqMan II Seeds of Yates F1 Hybrid were obtained from Australia and (http://www.dnastar.com/). DNA Baser (www.dnabaser.com) were grown as determinant control hybrid and for the was used to identify trusted and untrusted sequence as high development of mutant population using chemical mutagen, quality of sequence are needed for the screening of SNPs. Ethyl Methanesulfonate (EMS) (Sigma-Aldrich). MEGA 6 (http://megasoftware.net/) was used to screen for variation among sequence of DNA samples of mutants through B. EMS treatment and kill curve sequence alignment. Prior in choosing the optimum concentration of mutagen for F. Brix Index treatment of seeds in developing the mutant population, two concentration of EMS were used. Seeds were sterilized prior Tomato fruits were harvested at 45 days after flowering to treatment. Concentration of 0.5% and 1% EMS (v/v) were (DAF), ripening (breaker) stage. The total soluble solid (TSS) used with the exposure of EMS to seeds for 3 hours, 6 hours, content in fruits (Brix) of tomato fruits were measured by 12 hours and 24 hours. For each of the types of treatment, 50 optical Brix refractometer (N-1 α, alpha Refractometer). 100µl seeds were used. Untreated control seeds were soaked at the of tomato juice were used to cover the prism and the solution same period of time using Mili-Q water as a substitution to undergoes temperature stability of 30 seconds before the EMS. Chemical mutagen, EMS was washed or removed reading of Brix index were recorded. completely with 0.5% ethyl acetate prepared in 0.1M of phosphate buffer of pH 7.0 for 45 minutes and seeds were III. RESULTS AND DISCUSSION continuously washed with Mili-Q water for five times. Treated EMS Mutagenesis on Seeds Germination and untreated seeds were sowed in small pots and grown in a greenhouse or containment facility with maintained optimum For the development of mutant population, the dosage of parameter for the growth of tomato trees. Ethyl Methanesulfonate (EMS) was tested for 0.5% and 1.0% concentration of EMS on 3 hours, 6 hours, 12 hours and 24 C. TILLING hours of treatment. The concentration of EMS can be prepared Mutation at the specific HXK nuclear DNA loci were in a higher amount to induce more mutation but the pitfall of identified by amplifying the DNA template of identified high dosage will contribute to sterility and fertility [15]. matured mutant tomato trees using primer NHEX: 5’- Germination rate of 0.5% and 1.0% of EMS showed reduction AGGACTGATGGCGACAAATC-3’ (forward) and 5’- in seed germination by 15% and 50% respectively. Lethal CCAATGCCAGAACCATCATT-3’ (reverse). PCR reaction Dose, LD15 of 0.5% EMS concentration with 24 hours of EMS were performed at optimized temperature or cycle of 96°C for treatment was used to proceed in developing mutant 3 minutes; 30 cycles of 94°C for 20 s, 60°C for 40 s, and 72°C population as compared to LD50 of 1.0% EMS concentration for 60 s: and a final elongation step at 72°C for 5 minutes. which gave low amount of viable tomato trees in order to analyze the mutagenic effect of EMS. Lethal Dose was similar D. Mutation Screening with the previously reported LD15 for S. lycopersicum cv. M82 From the germinated seeds, 50 mutant tomato trees were [16] and LD20 for the chosen 0.5% EMS treatment on Micro- screened for mutation in terms of their phenotypic and tom seeds [17]. The selected EMS dose in this research genotypic variation in comparison to their control determinant showed 14% decreased in fertility of M1 mutant population in hybrid. Preliminary screening of mutants were analyzed with comparison to 91% of fertile control tomato plants, Table I. six ISSR primers ISSR-1 (5’-GGG(TTC)6-3’), ISSR-2 (5’- The decrease in the number of fertile mutant trees proves the GGG(TTC)4-3’), ISSR-3 (5’-GGG(AAC)6-3’), ISSR-4 (5’- mutagenic effect of EMS in inducing saturated mutation in CCC(AAG)6-3’), ISSR-9 (5’-CTC TCT CTC TCT CTC TAC- treated organisms. 3’), ISSR-10 (5’-CTC TCT CTC TCT CTC TGC- 3’) and ISSR-PCR Amplification focuses on one degenerate primer ISSR-1. ISSR-PCR were A total of 6 random Inter Simple Sequence Repeats (ISSR)

http://dx.doi.org/10.15242/IICBE.C0815048 89 Int'l Conf. on Advances in Science, Engg., Technology & Natural Resources (ICASETNR-15) Aug. 27-28, 2015 Kota Kinabalu (Malaysia) primers were tested as a preliminary tool of mutation screening produce significantly more or reduced amount of amplified where ISSR-1 primer able to generate the most number of bands with the use of ISSR primers, indicating change in the polymorphic bands which was chosen to further analyze number of repeats present in the DNA genome of mutants in mutant DNA samples, Fig. 1. ISSR primer shows variation of comparison to its control determinant hybrid. ISSR primers repeats among the mutant DNA samples except for the number able to identify different sites within the genome of an of bands amplified using genomic DNA of control determinant organism and detecting the difference in polymorphism among hybrid. Thus, confirming the use of ISSR primers as a tool to populations [27]. The primers are reliable in providing evaluate genotypic variation among organisms [18]. Results repeatable results, eliminating the use of Random Amplified were made in repeats and the same number of amplified bands Polymorphic DNA (RAPD) that often gives false positive was obtained, proving the reliability of ISSR primers in results [28] and Restriction Fragment Length Polymorphism providing rapid and accurate results for identification of (RFLP) technology that have limitation in the screening of mutant organisms [19]. Only mutant line affected by EMS will mutation [29].

TABLE I MUTAGENIC EFFECT OF EMS IN M1 GENERATION. Amount of Month and Year Treatment of Ethyl Amount of Planting Condition seeds Lethal Dose (LD) Fertility (%) of Sowing Methanesulfonate, EMS germinated seeds treated November, 2014 Containment Mili-Q water, 24 hours 25 23 Control 21 (91%) facility (constant parameter) November, 2014 Containment 0.5% Ethyl 78 66 15 57 (86%) facility (constant Methanesulfonate, 24 hours parameter)

Analysis of Gene Sequence on HXK Mutants The screening of genetic variation on Hexokinase (HXK)

gene among M1 population was conducted through DNA sequencing, the most efficient tool of detecting mutation among TILLING population of mutants [30]. The Screening of Single Nucleotide Polymorphism (SNPs) shows two mutations at position 147 and 158 with Transition mutation of Thymine (T) to Cytosine (C) and Transversion mutation of Thymine (T) to Adenine (A) as shown in Fig. 2

http://dx.doi.org/10.15242/IICBE.C0815048 90 Int'l Conf. on Advances in Science, Engg., Technology & Natural Resources (ICASETNR-15) Aug. 27-28, 2015 Kota Kinabalu (Malaysia)

(a) (a)

(b)

(b)

Fig. 2 (a) comparison of gene sequence chromatogram of control determinant hybrid, left and mutant line, right at position 147 and Fig. 2 (b) at position 158.

Fig. 2 shows the mutation at specific HXK nuclear DNA locus indicating the reliability of EMS to induce point mutation for the study of gene expression and function. Changes indicated by arrow on both of the mutant line chromatogram samples in comparison to the control determinant hybrid at site 147 Fig. 2 (a) and Fig. 2 (b) at site number 158. Mutant populations were also screen for variation at protein level with identified one point mutation within the target gene: Y30F. Mutation occurs at chromosome 6 on the specific locus, HXK. Genotypic assessment of the mutant line of determinant hybrid detected C-T mutation, caused Tyrosine (Y) to Phenylalanine (F) amino acid substitution at position 30 of the Hexokinase protein sequence (HXK) as shown in Fig. 3. (b) Open Reading Frame (ORFs) of mutant gene was analyzed by ORF finder for insertion which could interrupt the reading frame of subsequent region and could possibly lead to Fig. 3 (a) Open Reading frame of HXK gene based on gene sequence formation of stop codon. Six Open reading frame were and (b) ClustalW protein alignment of control determinant hybrid detected, Fig. 3(a) not to be interrupted by any insertion or and mutant protein. Yellow highlight: mutation position. mutation in the reading frame. Ethyl Methansulfonate, EMS managed to induce point mutation on HXK gene without Rate of mutation calculated based on the gene sequence of causing frameshift mutation on the gene sequence of mutants. HXK gene is 1/2724bp for 0.5% of EMS concentration. Two mutations were identified on mutant sequence of HXK gene based on screened 5448 base pair of mutant tomato trees.

Brix content To further analyst the mutagenic effect of Ethyl Methanesulfonate on HXK gene, total soluble solid (TSS) or total refined sugar in tomato fruits were determined through refractometer (N-1 α, alpha Refractometer). The reading of Brix index measured on control determinant hybrid indicates a higher amount of sugar compared to the total soluble solid of mutant line. Fruits were harvested at 45DAF for both control and mutant line, eliminating bias reading of Brix index obtained as total sugar in tomato fruits differ by the ripeness of the fruits (day of harvest) particularly on the ripening breaker stage with higher amount of glucose and fructose content but significantly lower in starch and sucrose content [20]. Five

http://dx.doi.org/10.15242/IICBE.C0815048 91 Int'l Conf. on Advances in Science, Engg., Technology & Natural Resources (ICASETNR-15) Aug. 27-28, 2015 Kota Kinabalu (Malaysia) fruits were measured for their sugar content, Fig. 4. REFERENCES [1] C. Matsukura, K. Aoki, N. Fukuda, T. Mizoguchi, E. Asamizu, T. Saito, D. Shibata, H. Ezura, “Comprehensive resources for tomato functional genomics based on the miniature model tomato micro-tom” Current Genomics, 2008 9(7),436-443 http://dx.doi.org/10.2174/138920208786241225 [2] E. Emannuel and A. A. Levy, “Tomato Mutants as tools for functional genomics” Plant Biology, January 2002, 5:112-117. http://dx.doi.org/10.1016/s1369-5266(02)00237-6 [3] Y. Okabe, E. Asamizu, T. Saito, C. Matsukura, T. Ariizumi, B. Cecile’, C. Rothan, T. Mizoguchi, H. Ezura, “Tomato TILLING technology: Development of a reverse genetic tool for the efficient isolation of mutants from micro-tom libraries. 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