Borneo Ironwood, consists of two species, i.e. Eusideroxylon zwageri and Potoxylon melagangai, is the most well known durable tropical hardwood timber tree of Borneo. Both species are morphologically similar, except

the wood characteristics. This has resulted in taxonomic difference between them. We used PCR with M13 universal primer to determine the genetic relatedness and SCAR marker to distinguish both species. Cluster analysis

grouped both species into two major clusters, suggesting that there are two genetically distantly related species. One diagnostic band (locus M13-16) was successfully converted to SCAR marker. A single fragment (720 bp)

was amplified in all E. zwageri individuals but

completely absent in P. melagangai. Differentiating the species by SCAR marker is very useful for screening large numbers of Borneo Ironwood seedlings collected from

natural populations. The identification of individuals at species level constitutes one of the first basics in any effective conservation programme. In fact, the Borneo Ironwood has been counted as one of the endangered species in Sarawak

Cheksum, Tawan

Ho Wei, Seng

Isa, Ipor

Julaihi, Abdullah

Lee Shiat, Mei

Pang Shek, Ling

Yii Ai, Siew

Unimas Institutional Repository

  1 2 Acta Biologica Malaysiana (2014) 3(1): 10-15 http://dx.doi.org/10.7593/abm/3.1.10  3 4 5 6 7 8 9    DNA Genotyping of Borneo Ironwood using M13 Universal Primer and SCAR Marker Development in Eusideroxylon  zwageri  10 11 12 13 14 15 16 Ho Wei Seng • Yii Ai Siew • Lee Shiat Mei • Cheksum Tawan • Isa Ipor • Pang Shek Ling •    Julaihi Abdullah      17 18 19 20 21 Received: 22 November 2013 / Accepted: 24 July 2014 © Acta Biologica Malaysiana 2014 22 23 24 25 26 27 28 29 30 31 32 Abstract  Borneo Ironwood, consists of two species, i.e. Eusideroxylon zwageri and Potoxylon melagangai, is the most well known durable tropical hardwood timber tree of Borneo. Both species are morphologically similar, except the wood characteristics. This has resulted in taxonomic difference between them. We used PCR with M13 universal primer to determine the genetic relatedness and SCAR marker to distinguish    both    species.    Cluster    analysis  58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92   93 grouped both species into two major clusters, suggesting that there are two genetically distantly related species. One diagnostic band (locus M13-16) was successfully converted to SCAR marker. A single fragment (720 bp) was amplified in all E. zwageri individuals but completely absent in P. melagangai. Differentiating the species by SCAR marker is very useful for screening large numbers of Borneo Ironwood seedlings collected from natural populations. The identification of individuals at species level constitutes one of the first basics in any effective conservation programme. In fact, the Borneo Ironwood has been counted as one of the endangered species in Sarawak.  Keywords  Borneo Ironwood • genotyping • M13 • universal primer • diagnostic band • SCAR   Introductions  Belian or its popular name, Borneo Ironwood is the most famous and well-known durable tropical hardwood timber tree of Borneo, which comes from Lauraceae family. It is officially announced as the State Tree of Sarawak on 20th March 1984. There are two species of Borneo Ironwood, namely Eusideroxylon zwageri and Eusideroxylon melagangai (De Wit, 1949). Both species are not easy to be distinguished in the field because of similar morphological characteristics, except the wood structure (Browne,  1955;  Soerjanegara  and  Lemmens, 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57  Ho W. S., Yii A. S., Lee S. M. Forest Genomics and Informatics Laboratory (fGiL), Department of Molecular Biology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300, Kota Samarahan, Sarawak  Tawan C., Ipor I. Department of Plant Science and Environmental Ecology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300, Kota Samarahan, Sarawak  Pang S. L., Abdullah J. Applied Forest Science and Industry Development Unit, Sarawak Forestry Corporation, 93250 Kuching, Sarawak  Ho W. S.  (      ) Forest Genomics and Informatics Laboratory (fGiL), Department of Molecular Biology, Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300, Kota Samarahan, Sarawak Email: wsho@frst.unimas.my   1 11                                                                                                                       Acta Biologica Malaysiana (2014) 3(1): 10-15 2  3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 1994). E. melagangai has lighter colour of wood, lower durability and natural strength, lower height rays, reddish colour, rather fine texture and longer bands of soft tissue linking up the pores compared to E. zwageri. Besides, it has lighter density (0.70 – 0.80 g/cm³) compared to E. zwageri (0.88 – 1.19 g/cm³) that enabled it to float on the surface of water. The variation in wood characteristics has resulted in taxonomic difference between both species. In 1979, Kostermans finally referred E. melagangai to a new monotypic genus as Potoxylon and named as Potoxylon melagangai Kosterm., which is synonym to E. melagangai Sym. (Soerjanegara and Lemmens, 1994). However, the results could be inconclusive due to developmental and environmental influences on the morphology, problems with low juvenile-mature correlation and the close phenotype resemblance among clones from selection response for similar characteristics (Chong et al. 1995). Thus, it would be best to use PCR-based genetic markers coupled with morphological characteristics to differentiate Borneo Ironwood. PCR-based genetic markers are developmentally stable, governed by complex genetic interactions and not affected by the environment. M13 universal primer is one of the marker systems that have been widely recognized in DNA fingerprinting of plants, animals and microorganisms (Chong et al. 1995; Ho et al. 2006). It is a dominant marker derived from protein III gene of M13 bacteriophage that later proved useful in generating multiple banding patterns in all plant species and potentially an individual-specific banding pattern. Sequence characterized amplified region (SCAR) markers have been developed recently to produce more specific and reproducible results that could be used for species or cultivar identification (Scheef et al. 2003). Reliable SCAR markers have been successfully developed for several crops and woody species, including common bean (Adam-Blondon et al. 1994), rice (Nagvi and Chattoo, 1996) and apple (Evans and James, 2003). To date, there is no published report on the development of species-specific markers in Borneo Ironwood. Hence, the objectives of this study were (1) to genotype Borneo Ironwood by using M13 universal primer and (2) to convert M13 amplified diagnostic band to species-specific sequence characterized amplified region (SCAR) marker in E. zwageri for Borneo Ironwood typing. 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 Materials and Methods  A total of 16 individuals of E. zwageri and P. melagangai maintained in the green house were selected for the present study. E. zwageri were collected from Samunsam Wildlife Sanctuary, Lundu meanwhile P. melagangai were collected from Mulu National Park, Miri, Sarawak on August 2000. The total genomic DNAs of E. zwageri and P. melagangai were isolated using a modified CTAB method from Doyle and Doyle (1987). DNA concentration was determined spectrophotometrically and its integrity and concentration were further determined by 0.8% agarose gel electrophoresis. PCR amplification reaction was performed in volumes of 25 l containing 1X PCR buffer [200 mM Tris-HCl (pH 8.4), 500 mM KCl], 3.0 mM MgCI2, 0.2 mM each of dNTP, 0.4 M M13 universal primer, 1 unit of Taq DNA polymerase (Promega) and 10 ng of genomic DNA. The amplifications were done in a GeneAmp PCR System Model 2400 (Perkin-Elmer) thermocycler, programmed for 35 cycles of 1 minute of denaturing at 94oC, 1 minute of annealing at 48oC and 2 minutes of extension at 72 oC after an initial 2 minutes denaturing at 94oC. The program was then followed by a final extension step at 72 oC for 10 minutes. The PCR products were separated on a 1.2% (w/v) agarose gel for 3 hours at 80 V in 1X TBE buffer and stained with Gelstar nucleic acid gel stain (FMC Bioproducts). The PCR bands were named after the primer and a hyphenated number corresponding to the order of their migrations, e.g., the PCR amplified bands were designated as M13-01, M13-02, and etc. The presence of a band was scored as 1 and absence was scored as 0, based on the following criteria: (1) each locus was assumed as independent or non-allelic, (2) there was no bias in scoring monomorphic fragments versus polymorphic fragments and (3) only fragments in the range of 300 bp to 2.1 kb were considered in order to increase the data reliability. Pairwise genetic similarity was calculated based on the similarity index, Jij = Cij / (ni + nj – Cij) (Jaccard, 1908). The data was then subjected to cluster analysis by the Unweighted Pair-Group Method with Arithmetical Averages (UPGMA) method as described by Sneath and Sokal (1973) using NTSYS-pc software (Rohlf, 1998).   1 Acta Biologica Malaysiana (2014) 3(1): 10-15                                                                                                                       12  2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44  Table 1 Jaccard similarity matrix generated by M13 universal primer (M: Potoxylon melagangai; Z: Eusideroxylon zwageri)               Figure 1: DNA profiles of P. melagangai (lane 2 – 9) and E. zwageri (lane 10 – 17) generated by using M13 universal primer. Arrow (locus M13-16) indicates species-specific, strong and reproducible band that was amplified in E. zwageri and converted into species-specific SCAR marker  45 46 47 48 49 50 51 52 53 54 55 56 57 The diagnostic fragments were identified from the DNA profiles of E. zwageri and P. melagangai individuals generated by using M13 universal primer. Those diagnostic fragments were extracted from the agarose gel and purified using QIAquick Gel Extraction Kit (QIAGEN) and subsequently, cloned into the pGem-T Easy Vector (Promega). Three clones were selected for plasmid extraction using Wizard® Plus SV Minipreps DNA Purification System (Promega) sequenced by an automated ABI 3130xl Genetic Analyzer using Big Dye (dRhodamine) terminator  cycle  sequencing-ready  reaction  kit 58 59 60 61 62 63 64 65 66 67 68 69 70  (Applied Biosystems). Based on the nucleotide sequences of the cloned diagnostic fragments, pairs of oligonucleotide primers ranging from 17 to 30 bases in length were designed using the Primer Premier 5.0 software. The newly designed primers were validated using eight E. zwageri and one P. melagangai samples. Reaction components were as described above, with the following exception: 20 ng of template DNA and the annealing temperature was increased to 65.5oC. This assay was replicated three times to test for the reproducibility of the amplified    diagnostic    fragment.   Amplified   1 13                                                                                                                       Acta Biologica Malaysiana (2014) 3(1): 10-15 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46                   Figure 2: Dendrogram represents the cluster analysis of Jaccard similarities among the Belian individuals, i.e. P. melagangai (Cluster 1) and E. zwageri (Cluster 2)                           Figure 3: Banding pattern of SCAR primer pair amplifying a single and reproducible band specifically for E. zwageri. M is 100 bp ladder and Z1, Z6, Z10, Z11, Z12, Z16, Z17, and Z18 are E. zwageri samples. M10 is P. melagangai sample 47 48 49 50 51 52 53 54 55 56 57 products were resolved using 1.5% agarose gel electrophoresis.   Results and Discussion  The total genomic DNA of all 16 samples of E. zwageri and P. melagangai were isolated using the modified CTAB extraction method (Doyle and Doyle, 1987). The DNA quality of P. melagangai   was    relatively    better    than    E. 58 59 60 61 62 63 64 65 66 67 68 zwageri due to less polysaccharides and secondary metabolites. The presence of these contaminants will cause the DNA solution become very viscous and difficult to extract, especially in E. zwageri samples. The A260/A280 absorbance ratios were 1.28 - 2.08 with DNA concentrations ranged from 10ng/µl to 3µg/µl in both species. Figure 1 shows the multiple banding patterns (25 bands) generated by M13 universal primer in P. melagangai and E. zwageri. The constructed dendrogram based on       1000bp           700bp        M       Z1      Z6      Z10   Z11    Z12    Z16    Z17    Z18   M10   1 Acta Biologica Malaysiana (2014) 3(1): 10-15                                                                                                                       14  2  3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Jaccard similarity matrix (Table 1) had grouped Borneo Ironwood into 2 major clusters that composed of P. melagangai (Cluster 1) and E. zwageri (Cluster 2) (Figure 2). The mean genetic similarity within individuals in Cluster 1 was 0.78 and 0.91 for Cluster 2, meanwhile the mean genetic similarity between Cluster 1 and Cluster 2 was 0.45. This suggests that P. melagangai and E. zwageri are two genetically distantly related species. Two common bands were also found in all the individuals of E. zwageri and P. melagangai (Figure 1), indicating that both E. zwageri and P. melagangai were derived from the same ancestral but had evolved over the years for survival. This may explain why both E. zwageri and P. melagangai have a very close similarity in their vegetative characters (Browne, 1955).  One distinct and highly reproducible diagnostic band was identified from the 25 loci generated by M13 universal primer, i.e. locus M13-16 from E. zwageri. This diagnostic band was then cloned and sent for sequencing (Figure 1). Sequence data from this diagnostic band was submitted to GenBank (accession number FJ624400) and no significant sequence similarities were found with NCBI GenBank non-redundant database. Analysis of sequence data was performed using BLAST search. SCAR marker was created by sequencing a single diagnostic band and designing a specific primer pair to amplify the band of specific size. The SCAR primer pair is designated as Zg16-F (5’-AGGGTCGCTATTGGTGGA- 3’) and Zg16-R (5’–AGCCTCTTTCAGTCGGTCA- 3’). A single and distinct band at approximately 720 bp was observed in all the eight E. zwageri samples while no band was detected in P. melagangai sample (Figure 3). Differentiating this species by SCAR markers is very useful for screening large numbers of Borneo Ironwood seedlings collected from natural populations. To the best of our knowledge, this is the first report on the development of SCAR marker in determining Borneo Ironwood using species specific marker developed from M13 universal primer. We hope this approach could pave the way for the unambiguous identification of Borneo Ironwood. However, more samples need to be used to reconfirm the validity of the designed SCAR marker. Once the confirmation is done, the other three selected diagnostic bands also can be converted to SCAR markers and subsequently, multiplex PCR can be developed using these markers. Multiplex PCR would  save 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 time and money by using only one reaction per sample instead of several reactions, one for each primer (Scheef et al. 2003). The identification of individuals at species level constitutes one of the first basics in any effective conservation programme. In fact, the Borneo Ironwood has been counted as one of the endangered species in Sarawak.    Acknowledgements The authors would like to thank all the lab assistants and foresters involved in this project for their excellent field assistance in species identification and seed collection. This study is supported by UNIMAS Fundamental grant (01(134)/531/2005(30)).   References  Adam-Blondon, A.F., Sevignac, M., Bannerot, H., & Dron, M. (1994). RAPD and RFLP markers linked to a dominant gene (Are) conferring resistance to anthracnose in common bean. Theor Appl Genet, 88, 865-870. Browne, F.G. (1955). Forest Trees of Sarawak and Brunei and their products. Government Printing Office, Sarawak.  Chong, D.K.X., Yeh, F.C., Aravanopoulos, F.A., & Zsuffa, L. (1995). DNA Fingerprinting    Willows (Salix L.) using polymerase chain reaction with the M13 universal primer. Scan      J For Res, 10, 27-31. De Wit, H.C.D. (1949). Spicilegium Malaianum. Bulletin of the Botanical Gardens Buitenzorg. III, 18, 181-212. Doyle, J.J., & Doyle, J.L. (1987). A rapid DNA     isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull, 19, 11-15.  Evans, K.M., & James, C.M. (2003). Identification of SCAR markers linked to Pl-w mildew resistance in apple. Theor Appl Genet, 106, 1178-1183. Ho, W.S., Wickneswari, R., Mahani, M.C., & Shukor, M.N. (2006). Comparative genetic diversity (polymorphisms) studies of Shorea curtisii Dyer ex King (Dipterocarpaceae) using SSR and DAMD markers. J Trop For Sci, 18(1), 557-565. Jaccard, P. (1908). Nouvelles rescherches sur la distribution florale. Bulletin de la Société             Vaudoise des Sciences Naturelles, 44, 223-            270. Kostermans, A.J.G.H. (1979). Potoxylon. A new Borneon genus of Lauraceae. Mal Nat J, 32, 143-148.  Nagvi, N.I., & Chattoo, B.B. (1996). Development of a sequence-characterized amplified region (SCAR) based indirect selection method for a dominant blast resistance gene in rice. Genome, 39, 26-30. Rohlf, F.J. (1998) NTSYSpc: Numerical Taxonomy and    1 15                                                                                                                       Acta Biologica Malaysiana (2014) 3(1): 10-15 2  3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 Multivariate Analysis System. Version 2.0. Applied Biostatistic Inc. New York: Setauket.  Scheef, E.A., Casler, M.D., & Jung, G. (2003). Development of species-specific SCAR markers in bentgrass. Crop Sci, 43, 345-349.                 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 Sneath, P.H.A., & Sokal, R.R. (1973) Numerical taxonomy. San Francisco: W. H. Freeman & Co. Soerjanegara, & Lemmens, R.H.M.J. (1994) PROSEA 5 (1) Timber Trees: Major Commercial timbers. PROSEA Foundation, Bogor, Indonesia.                                                          

DNA Genotyping of Borneo Ironwood using M13 Universal Primer and SCAR Marker Development in Eusideroxylon zwageri

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DNA Genotyping of Borneo Ironwood using M13 Universal Primer and SCAR Marker Development in Eusideroxylon zwageri

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