Molecular Identification of Malaysian Pineapple Cultivar based on Internal Transcribed Spacer Region

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1 Available online at APCBEE Procedia 4 (2012 ) Molecular Identification of Malaysian Pineapple Cultivar based on Internal Transcribed Spacer Region Topik Hidayat a,b, Farah Izana Abdullah a, Chandrika Kuppusamy a, Azman Abdul Samad a and Alina Wagiran a a ICAAA 2012: July 23-24, 2012, Singapore Department of Biological Sciences, Faculty Bioscience and Bioengineering, Universiti Teknologi Malaysia (UTM), 81310, Skudai, Johor Bahru Malaysia b Department of Biology Education, Indonesia University of Education (UPI), Jalan Dr Setiabudi 229 Bandung 40154, Indonesia Abstract Identification of pineapple cultivar solely based upon traditional method which only based on morphology. However, this approach could results in inaccurate cultivar identification and inconsistent. In this study, we conducted phenetic analysis of 9 Malaysian pineapple cultivars based on DNA sequences of the internal transcribed spacer (ITS) region to evaluate utility of the region as a barcode in identification of pineapple cultivars and to determine phenetic relationships among cultivar. Genomic DNA was directly extracted, and ITS region was amplified and sequenced. Phenetic analysis revealed that the pineapple cultivars used in this study were classified into three groups with high sequence similarity among them. This clearly showed that the DNA barcode from ITS region have good discrimination power to distinguish the pineapple cultivar. Since the tree distinctly separated into three group of cultivar, consensus sequences served as DNA barcode for Malaysian pineapple cultivar can be constructed Published by by Elsevier B.V. B.V. Selection Selection and/or and/or peer review peer review under responsibility under responsibility of Asia-Pacific of Asia-Pacific Chemical, Biological Chemical, & Biological Environmental & Environmental Engineering Society Engineering Society Keywords: ANANAS COMOSUS, DNA BARCODE, ITS REGION, MALAYSIAN CULTIVAR, PHENETIC ANALYSIS 1. Introduction Pineapple (Ananas comosus (L.) Merr.; Family Bromeliaceae) is the third most important tropical fruit Corresponding author. Tel.: +(6) ; fax: (6) address: topik@fbb.utm.my Published by Elsevier B.V. Selection and/or peer review under responsibility of Asia-Pacific Chemical, Biological & Environmental Engineering Society doi: /j.apcbee

2 Topik Hidayat et al. / APCBEE Procedia 4 ( 2012 ) after banana and citrus in the world production [1, 2], without exception in Malaysia [2]. There are many pineapple cultivars available in Malaysia including Maspine, Morris Bentanggur, MD2, Morris Gajah, Gandul, Sarawak Green Local, Josapine, N36, Yankee and even Malaysia Pineapple Industry Board released MD2 variety product of tissue culture technique in to the market. Each of the cultivar has their own uniqueness and economic importance. However the identification to distinguish one from the other cultivars remains the main constraint to date [2].Currently there are no established identification tools at molecular level for Malaysian pineapple. Existed traditional approach that has been used by the growers to identify the cultivars is solely based upon morphological characters, which is extremely inconsistent and time consuming. DNA barcoding is a technique that use standard gene region of DNA or known as DNA Barcode for identification of organism [3]. It plays vital role in managing species diversity and identification of biological specimen (Lahaye et al., 2008). The gene region that usually been used for DNA barcoding standard is the Cyctochrome C Oxidase 1 gene (CO1). This gene is derived from mitochondrial DNA which effectively works in identifying species of animal [4]. Based on conventional method to study about species, identification is only based on morphology which could result in incorrect species recognition. However, by DNA barcoding it allows us to combine morphology and gene sequence together for better and precise identification [5]. The use of the CO1 in plant has been not effective due to its low nucleotide substitution in plant mitochondrial genome [4]. Thus, it is proposed the use of the ITS region as the barcode for plant [6]. The region also proved to be highly effective in distinguishing closely related species in numerous genera [7, 8, 9]. In this research, DNA barcode obtained from the ITS region was used as an alternative approach for identification the grapevine cultivars. The ITS region is the most commonly sequenced locus used as a barcode at the species level [10, 11]. However, in this research, the region was used at cultivar level. 2. Materials and Method 2.1. Cultivar selection and sampling A total of 9 cultivars of pineapple were collected. Selection of this cultivar was made based on information given by Malaysian Pineapple Industry Board. The cultivar were Josapine, Gandul, Yankee, MD2, MD2 (Tissue Culture), Morris Bentanggur, Morris Gajah, N36 and Sarawak Green Local. According to them, these 9 pineapple cultivars have their own commercial value. Young and fresh leaf were selected, labelled and kept in proper storage bag DNA analysis Genomic DNA was extracted from fresh material using QIAGEN Dneasy Mini Plant Kit with slight modification. The DNA obtained was stored in -20 o C for long term usage. The amplification of the ITS region was carried out using primer pairs AB101 and AB102, forward and reverse primer respectively as shown in Figure 1. Each reaction included 5 μl 10x Standard Taq Reaction Buffer, 1 μl of each 10 μm forward and reverse primers, 1 μl of dntps, μl sterile deionized water, 0.25 μl Taq DNA polymerase and 2 μl template genomic DNA. The PCR profile includes 1 cycle predenaturation at 94 o C for 2 minutes, 30 cycles of denaturation at 94 o C for 50 seconds, annealing 50 o C for 1 minutes, extension 72 o C for 7 minutes and final extension at 72 o C for 7 minutes. PCR product was visualized by 0.8% of agarose gel electrophoresis. PCR product were cloned into pgem-t Easy (Promega) and transformed into NEB5 competent cell. Plasmid extraction was conducted by QIAmp Spin Miniprep following manufacturer s instruction. Restriction enzyme digestion was carried by using EcoRI. Digestion mixture was prepared before addition of restriction

3 148 Topik Hidayat et al. / APCBEE Procedia 4 ( 2012 ) enzyme. Mixtures were finally incubated at 37 o C for 4h. The 9 pineapple samples were sent to 1st Base for sequencing by using SP6 and T7 primer pairs Phenetic analysis DNA sequence obtained from the ITS region were edited using CodonCode Aligner ( Multiple alignments were conducted with Clustal X [12]. The 9 aligned sequences were used to construct the phenetic tree with MEGA version 4 [13]. DNA barcode sequence was constructed by using GENEIOUS software [14] after construction of phenetic tree. 3. Results The aligned ITS region consists of 908 characters. Phenetic analysis using Unweighted Pair Group Method with Arithmetic Mean (UPGMA) resulted in single tree as shown in Figure 2. All cultivars were grouped together at sequence similarity of 20%. At 30% similarity index, the tree separated into two clusters. However, at 75% similarity index and above, the tree further separated into three subcluster. The first major subgroup consists of five cultivars which are Gandul, Yankee, MD2, Josapine and MD2/T which exhibited low genetic dissimilarity among the cultivars. Gandul and Yankee were 100% similar, MD2 were 99%, Josapine were 98% and MD2/T were 97% similar based on their nucleotide sequence similarity. The second subgroup contains two cultivars MR Gajah and MR Bentanggur also shows high similarity among them. MR Gajah and MR Bentanggur were 99% similar in their nucleotide sequence. The third subgroup contains two cultivars which are N36 and Sarawak. This group shares 75% sequence similarity among them. Fig. 1. Organization of ITS region of nrdna with location of primer used in this study. Information for primer is also provided. The terms DNA Barcode can be compared with other term such DNA sequence marker which highly applicable for classification of pineapple cultivar in a rapid and cost effective manner. As the tree clearly separated in to two cluster from 0% to 74% similarity index, DNA barcode can be constructed for Cluster A and Cluster B which is derived from consensus sequences. 4. Discussion Phenetic analysis reveals relation among pineapple cultivars based upon overall nucleotide sequence similarity. By phenetic relationship, cultivar with similar characteristic will group together. Three groups of pineapple cultivar obtained and have low dissimilarity index. This shows that this cultivar was closely related.

4 Topik Hidayat et al. / APCBEE Procedia 4 ( 2012 ) Several morphological characters shared by among the first group include presence of spine along leaf margin, linear leaf shape and subulate leaf apex. Most of cultivar in this group has cylindrical fruit shape except for Yankee which has tapered-shape fruit. Both of MR Bentanggur and MR Gajah form second group with 99% nucleotide sequence similarity. Morphologically, this two cultivar shares most of common characteristic such leaf with rough, long and closely arranged spine along their margin, subulate leaf shape with rough leaf surface and cylindrical fruit shape with projected eye. Group 3 represented by N36 and Sarawak. N36 was the product of hybrid between Gandul and Sarawak. This cultivar shares 75% sequence similarity with Sarawak. This result supported by morphological data which shows N36 cultivar much more resembles Sarawak. They shares majority of morphological characteristic such smooth and shiny leaf surface, presence of spines on their leaf and green colour of upper surface and whitish green on lower surface of their leaf. N36 and Sarawak also has cylindrical fruit shape [15]. Classification and identification of pineapple cultivar are important for record and documentation purposes. Until today, morphological-based taxonomy was still main basis for identification. However this method requires expertise with adequate experience for identification of cultivar correctly and longer time required [16]. Moreover, this method usually provides inconsistent data since phenotype of the cultivar were highly influenced by the environmental factor [17]. Thus, molecular approach becomes a promising method for pineapple cultivar identification. By directly looks into genetic materials which are the DNA, influence of external factor such as environment can be avoided. This genetic material has been passed by ancestral to their offspring. Surely, the DNA inherited by the offspring will reflect their ancestor. Cultivar which is highly similar in their morphology and difficult to be identified also can be distinguished by molecular method due to presence of variation in DNA sequence among species [18]. With advancement in PCR and sequencing technology, identification of pineapple cultivar could be done in rapid, effective and cost-effective manner. Some of molecular study also already carried on pineapple such isozymes, RAPD, AFLP, RFLP and cpdna PCR-RFLP [19]. Fig. 2. Phenogram resulted from overall nucleotide sequence similarity obtained by UPGMA software. DNA barcoding is the used of short, standardized DNA region for species identification. The standardized region used known as DNA barcode [20]. There are three genomes in plant that are candidate barcode

5 150 Topik Hidayat et al. / APCBEE Procedia 4 ( 2012 ) includes chloroplast, mitochondrion and nucleus genome [21]. In this study, we developed barcode of ITS region originated from nucleus genome for classification of pineapple cultivar in Malaysia. There are several criteria need to meet by DNA barcode candidate includes high success rate during PCR amplification and adequate DNA sequence polymorphism to allow species characterization [22] and the ITS possess all the characteristic required. To classify pineapple cultivar based on morphology alone is difficult. Thus by using DNA barcode, classification of pineapple cultivar can be done in rapid and accurate manner. DNA barcoding technique works with all stage of life where it can identify species from various life stages includes eggs, larvae and adults. In this case, identification of pineapple cultivar could be done during their juvenile without having to wait until it ripens. DNA sequence which obtained from part of pineapple tree such as their leaf, crown and flower also can be used for identification via DNA barcoding technique. By using this phenetic analysis, we can develop DNA barcode for the nine pineapple cultivar. Since molecular identification method enable rapid and accurate identification of morphologically indefinite alien species [23], our valuable pineapple cultivar can be distinguished from any other pineapple cultivar which might be originated from other pineapple producing country just by comparing DNA barcode that were created, foreign cultivar can be separated from our cultivar. As a conclusion, relation among pineapple cultivar in terms of similarity in nucleotide sequence can be studied from phenetic tree constructed. From the topology of tree which formed of ITS region, universal barcode can be created for pineapple at cultivar level. We confirmed that DNA barcoding is an efficient identification method for Malaysian pineapple cultivar and could be applied to ensure biosecurity of our cultivar. DNA barcoding also can be as supplement to traditional morphology based classification. This result considered being preliminary; with more number of samples and genetic marker compared; more robust tree can be produced. Acknowledgements We express sincere thanks to Haji Sahdan bin Salim, Chief Director of Malaysian Pineapple Industry Board and their staff for providing us with pineapple samples. This research was funded by Research University Grant from the Universiti Teknologi Malaysia (Vote No: Q.J J32), which is gratefully acknowledge. References [1] Botella JR, Smith M. Genomics of Pineapple, Crowning the King of Tropical Fruit. Genomics of Tropical Crop Plants. (Moore, P.H. and Ming, R., Eds.). Springer, Australia 1979; [2] [3] Hebert PDN, Stoeckle MY, Zemlak TS, Francis CM. Identification of Birds through DNA barcodes. PLoS Biol 2004; 2: e312. [4] Hollingsworth PM, Graham SW & Little DP. Choosing and Using a Plant DNA Barcode. PLoS ONE 2011; 6(5): e [5] Kress WJ. Paper floras: how long will they last? a review of flowering plants of the neotropics. Amer J Bot 2004; 91: [6] Yao H, Song J, Liu C, Luo K, Han J, Li Y, Pang X, Xu H, Zhu Y, Xiao. Use of ITS2 Region as the Universal DNA Barcode for Plants and Animals. PLoS ONE 2010; 5(10): e [7] Alvarez I, Wendle JF. Ribosomal ITS sequences and plant phylogenetic inference. Mol Phylogen Evol 2003; 29: [8] Tian B, Liu TL, Liu JQ. Ostryopsis intermedia, a new species of Betulaceae from Yunnan, China. Bot Studies 2010; 51: [9] Liu J, Muller M, Gao LM, Zhnang DQ, Li DZ. DNA barcoding or the discrimination of Eurasian yews (Taxus L., Taxaceae), and the discovery of cryptic species. Mol Ecol Res 2011; 11:

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