MOLECULAR IDENTIFICATION OF Shorea johorensis IN KETAMBE RESEARCH STATION , GUNUNG LEUSER NATIONAL PARK

Shorea johorensis is one of the red meranti plants in Ketambe Research Station, Gunung Leuser National Park, Aceh Tenggara. Currently, Shorea johorensis also is well known as a major source of valuable commercial timber. This research aims to analyze the phylogenetic of Shorea johorensis based on chloroplast and nuclear DNA in Ketambe Research Station so that it can be known the relationship of Shorea johorensis with other species of Dipterocarpaceae in GenBank database. The research was conducted from July 2015 to August 2016 in Ketambe Research Station and Forestry and Forest Genetics Laboratory of Molecular, Bogor Agricultural University. The method used quadrat sampling technique with purposive sampling and experimental laboratory that consisted of DNA extraction, PCR, electrophoresis, and sequencing. The data analysis was done using BLAST, BioEdit, and MEGA6. The results showed that the phylogenetic tree of Shorea johorensis based on the rbcL and matK showed that Shorea johorensis was closely related with some species of Hopea; but the phylogenetic tree based on psbA-trnH, 5.8S rRNA, ITS2, and 28S rRNA showed that Shorea johorensis was closely related with Shorea robusta.


I INTRODUCTION
Gunung Leuser National Park is one of Nature Conservation Areas in Indonesia covering an area of 1.094.692hectares.This national park is located in Aceh and Sumatera Utara provinces.It is known as one of the World Heritage Sites for the rainforest which has several ecosystems from the coast to the high mountains [1].One of the research stations in Gunung Leuser National Park is Ketambe Research Station.Ketambe Research Station has high and unique biodiversity.It lies in Aceh Tenggara district.A few Dipterocarpaceae species is found growing in Ketambe Research Station [2].Dipterocarpaceae is the most typical family of tropical forest trees in the Malesian region with a geographical distribution that extends to South America and Africa.The family comprises approximately 500 species in 17 genera.It is subdivided into three subfamilies: Dipterocarpoideae, Monotoideae, and Pakaraimoideae.Dipterocarpoideae comprises 470 species in 13 genera [3].The most Dipterocarpaceae are large trees with towering top reaching 70-80 m.Dipterocarpaceae dominate in tropical lowland forest [4].The non-timber products of dipterocarp are used by some wildlife in the forest for their survival therefore, ecologically it is essential.The wood of Dipterocarpaceae is well known as a major source of valuable commercial timber.Currently, the dipterocarps predominate the international tropical timber market and therefore play an important role in the economy of many of the Southeast Asian countries [5].One of the Dipterocarpaceae species is Shorea johorensis.It can be found in Southeast Asian particularly in Borneo, the Peninsular of Thailand and Peninsular Malaysia, and Sumatra.In the timber trade, this Shorea is a type of wood which is grouped into red meranti.Its wood is light, soft, moderately durable, resistant to dry wood borers and fungi but susceptible to termites.Shorea johorensis has a high value of timber trade [6].The chloroplast genome is most widely used as a source of information on the inference of the evolutionary patterns and processes of plants [7] because this genome is thought to evolve slowly, with low mutation rates and maternal inheritance in most angiosperms, along with being a conserved region in structure and gene order.A chloroplast DNA marker that is  [8].On the other hand, the matK gene is a gene encoding the maturase enzyme subunit K.In plant systematics, matK appears as a valuable gene because it has a high phylogenetic signal than another gene [9].The psbA-trnH intergenic spacer is an evolutionary plastid region and employed as a phylogenetic marker [10].
In addition, nuclear DNA is also generally used in evolutionary as well as phylogenetic studies.
Nuclear DNA is transmitted from parent to offspring by nuclear division through sexual or asexual reproduction [11].Since a nuclear genome is biparentally inherited, it is expected to provide more information than a chloroplast or mitochondrial genome on species identity, including hybridization.One of the most useful types of nuclear DNA sequences is the Internal Transcribed Spacer (ITS) region, which contains multiple DNA copies.The ITS region lies between 16S and 28S nuclear ribosomal DNA (nrDNA).Several years ago, ITS regions were often used by experts for molecular phylogenetic analysis on plants in order to understand diversity and answer some issues phylogenetic.This is because the ITS region has superior characteristics namely, has small size (approximately 700 base pairs) and a lot of copying in the nuclear genome [12].These characteristics cause the ITS region to be easy to be isolated, amplified, and analyzed.Therefore, this research aims to analyze the phylogenetic of Shorea johorensis based on chloroplast and nuclear DNA in Ketambe Research Station so that the relationship of Shorea johorensis with some species in Dipterocarpaceae can be determined.

Study Area
The research was conducted in Ketambe Research Station, Gunung Leuser National Park, Aceh Tenggara and Forestry and Forest Genetic Laboratory of Molecular, Bogor Agricultural University.The research was begun on July 2015 to August 2016.The location of the research area is showed from the map (Figure 1).).The ID number of the specimens were written on the outside of the bag using a permanent marker.All packets/bags of the specimen were stored in containers [14].

DNA Extraction
DNA extraction was carried out using Cetyltrimethyl Ammonium Bromide (CTAB) method developed by Doyle and Doyle [15].Young leave of 200 mg was ground in a mortar with liquid nitrogen.The leaf powder was put into 2 mL tubes, 500 extraction buffer solutions and 100 μL polivinilpirodin (PVP) solution were added.The mixture was vortexed and then incubated in a water heater (waterbath) for 60 min at a temperature of 65ºC and every 15 min once reversed.After cooling to room temperature for 15 min, the mixture was added with 500 μL chloroform-isoamyl alcohol (24:1) and centrifuged for 10 min at 10,000 rpm.The upper layer (water phase/supernatant) was separated from the organic phase by using the micropipette into the new tube.Chloroformisoamyl alcohol was added twice.The Supernatant was addes with 500 μL cold isopropanol and NaCl of 300 μL.Samples were incubated overnight in the freezer.The precipitation result was centrifuged at 10.000 rpm for 10 min.The DNA pellet was washed twice using 96% ethanol of 300 μL and dried in a desiccator for 15 min.The dried DNA was added with 50 μL TE buffer (5 M Tris-HCl pH 8.0; 0.5 M EDTA pH 8.0).The DNA then was flicked and centrifuged at 10,000 rpm for 2 min.DNA was stored at -20ºC in the freezer.
The temperature settings of the thermocycler were the initial denaturation at 95°C for 4 min and then 35 cycles with denaturation at 94°C for 30 s, annealing at 58ºC (for rbcL and psbA-trnH) and at 56ºC (for matK) for 1 min, extension at 72 ºC for 1 min and rest at 4ºC [16].For nuclear DNA, amplification segments of DNA were conducted in 20 μL PCR reactions (Green goTaq PCR and PCR MasterMix).All of the PCR components consisted of 10 μL (1X Green goTaq), 1 μL forward primer, 1 μL reserve primer, 3 μL DNA template, and 5 μL Nuclease-Free Water were mixed into one tube.The temperatures for PCR machine were started by initial denaturation at 94ºC for 3 min; 30 cycles 3 stages: (at 94ºC for 30 s), annealing (at 58ºC for 30 s), and extension (at 72ºC for 1 min); and elongation stage at 72ºC for 10 min.The primers in this study were ITS1 and ITS4 (Table 1).For the DNA extraction, gel electrophoresis was done by using electric current with a voltage of 100 volt for 45 min and for the PCR, the process of running electrophoresis was for 30 min with a voltage of 100 volt.The results of running electrophoresis were observed using UV transilluminator.

Sequencing
The nucleotide sequence of the amplicon was identified using the Sanger method carried out by 1st BASE Sequencing INT in Malaysia.The sequencing process was done twice with different directions (forward and reverse).The sequencing data were used for the construction of phylogenetic trees.

Data analysis
The result of sequencing were analyzed with the following stages: (i) Annotation of ITS2 Sequence using ITS2 Database [22], (ii) BLAST (Basic Local Alignment and Search Tool) sequence using GenBank Database, (iii) Sequence alignment using Bioedit program [23], (iv) The result of sequence alignment was used to develop phylogenetic tree by Neighbor Joining (NJ) method with MEGA (Molecular Evolutionary Genetics Analysis) version 6 [24], (v) The reliable test of the tree was done by the bootstrap method 1000 times.

DNA Extraction
DNA extraction is a method of separating DNA from other cell components.The extraction of DNA Dipterocarpaceae was performed to obtain DNA from the genome total of Shorea johorensis which were used as DNA template for PCR amplification process.The most methods for DNA extraction used CTAB buffer solution as cell wall degradation because it has advantages i.e., easy to do and the possibility of DNA degrading enzymes is smaller than other methods [25].Based on the visualization of electrophoresis result in Figure 2, there were DNA bands of the three samples.All of DNA bands look thick, so the process of DNA extraction was successful.The DNA bands of Shorea johorensis were between the 500-750bp (base pair) for rbcL, matK, and ITS region.However for psbA-trnH, the DNA bands of Shorea johorensis were 300 bp.For rbcL and ITS region, all of the three samples were

B. BLAST Analysis
The Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between sequences.The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches.BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families [26].The result of BLAST Analysis of Shorea johorensis can be seen in Table 2.The results of BLAST analysis of Shorea johorensis based on rbcL showed that this Shorea had similarity with some species of Shorea and Hopea.Shorea johorensis had the highest similarity with Hopea ponga.Shorea johorensis had the maximum identity value 99%, the query coverage 97%, and the E-value 0.0 with Hopea ponga.According to Ref.
[27], maximum identity is the highest value of the percentage of identity or compatibility between a query sequence (sequence research) with sequence from GenBank database.Query coverage is the percentage of the nucleotides length aligned with the existing database on BLAST.According to Ref. [28] the value of an E-value is an alleged value that gives statistically significant sizes to the second sequence.The higher value of E-value indicates the lower homology between sequences, while the lower E-value indicates the higher homology between sequences.An E-value of 0 indicates the both of sequences are identical.For matK, Shorea johorensis had similarity with some species of Shorea and Hopea.Shorea johorensis had the highest similarity with Hopea nervosa.Shorea johorensis had the maximum identity value 99%, the query coverage 98%, and the E-value 0.0 with Hopea nervosa.Shorea Johorensis based on psbA-trnH had the highest similarity with Shorea robusta.Shorea johorensis had the maximum identity value 95%, the query coverage 80%, and the E-value 2e-111 (far enough from 0) with Shorea robusta.The E-value between of these sequences 3e-08.The result of BLAST analysis based on 28S rRNA showed the highest similarity with the same species in 5.8S rRNA and ITS2 (Shorea robusta), but it had the difference of E-value with them.Overall, Sequence data from Shorea johorensis based on rbcL, matK, psbA-trnH, 5.8S rRNA, ITS2, and 28S rRNA have not been found in the NCBI database, so this Shorea had the highest similarity to other Dipterocarpaceae species.In addition, chloroplast DNA analysis of Dipterocarpaceae showed the difficulty of distinguishing between closely related species in the genera level primarily to indicate the species of Shorea.In fact, several types in the same tribe have identical sequences [29].

C. PHYLOGENETIC TREE Phylogenetic Tree of Shorea johorensis based on rbcL
The construction of phylogenetic tree was conducted using MEGA 6 program with Neighbor Joining (NJ) method.The construction of phylogenetic tree aims to determine the relationship of among several Dipterocarpaceae species.Based on Figure 4, there were two groups (clades) namely group 1 and group 2. The first group had bootstrap value 75, consisted of Hopea ponga, Shorea brevipetiolaris, Hopea bracteata, Shorea johorensis, and Hopea dryobalanoides.The second group had bootstrap value 90 consisted of Shorea robusta, Parashorea macrophylla, and Shorea parvifolia.Each group formed a monophyletic group.A group of species is a monophyletic if all of the species present in the branches come from one common ancestor [30].In this phylogenetic tree, Shorea johorensis had a closer relationship with Shorea brevipetiolaris and some of Hopea than Shorea parvifolia, Shorea robusta, and Parashorea macrophylla.This is in accordance with the research by Ref. [31], Shorea johorensis formed a separate group with Shorea parvifolia.In addition, chloroplast DNA analysis by Ref. [14 and 32] [38], in the analysis of phylogenetics, outgroup lead to the polarization of characters or characteristics, namely apomorphic and plesiomorphic characters.The apomorphic character is the changed and derived characters which were found in the ingroup (species which were studied), whereas the plesiomorphic character is the primitive character found in the outgroup.The synapomorphic character is a derived character in the monophyletic group.

Phylogenetic Tree of Shorea johorensis based on matK
The pylogenetic tree of Shorea johorensis based on matK is presented in Figure 5.There were two monophyletic groups.The first group had bootstrap value 100, consisted of Shorea johorensis, Hopea bracteata, Hopea dryobalanoides, and Hopea nervosa.The second group had bootstrap value 89, consisted of Parashorea chinensis and Shorea robusta.Shorea johorensis had a closer relationship with some of Hopea than Parashorea chinensis or Shorea robusta.Based on this phylogenetic tree, the matK gene also was not able to separate Shorea, Hopea, and Parashorea to different monophyletic.The phylogenetic analysis using trnL-trnF, trnL, and matK from several species of Dipterocarpoidea also showed that Hopea formed monophyletic with several of Shorea [39].Dipterocarpus costatus was an outgroup because it had a distant relationship with Dipterocarpaceae research samples.7).This is in accordance with the analysis of nuclear DNA conducted by Ref. [36], Shorea and Parashorea were separate and not belonging to a monophyletic group.

Phylogenetic Tree of
Figure 7 The phylogenetic tree of Shorea johorensis based on 5.8S rRNA using the neighbor joining method In addition, Shorea also formed a separate group with Hopea.8).This is in accordance with chloroplast DNA analysis research by Ref. [14 and 33], Parashorea formed a monophyletic group with Shorea.In addition, molecular data from Ref. [34, 35, and 36] also explained that Parashorea was relatively close to several species of Shorea.).This Shorea formed the separate group with Parashorea chinensis.This is in accordance with the nuclear DNA analysis performed by [36], where Parashorea and Shorea were separated and not belonging to a monophyletic group.Trichilia surinamensis was an outgroup.

CONCLUSION
The phylogenetic tree of Shorea johorensis based on the rbcL and matK showed that Shorea johorensis was closely related with some species of Hopea; but the phylogenetic tree based on psbA-trnH, 5.8S rRNA, ITS2, and 28S rRNA showed that Shorea johorensis was closely related with Shorea robusta.

Figure 2
Figure 2 The Results of Gel Electrophoresis of DNA Extraction Shorea johorensis (M = Marker 1 kb DNA Ladder; 1 = Shorea johorensis 1; 2 = Shorea johorensis 2; 3 = Shorea johorensis 3 PCR Amplification PCR Amplification was performed to multiply Shorea johorensis genomic DNA strands with the target of the rbcL, matK, psbA-trnH, and ITS region.The result of electrophoresis is presented in Figure 3.

Figure 4
Figure 4  The phylogenetic tree of Shorea johorensis based on rbcL gene using the neighbor joining method

Figure 5
Figure 5  The phylogenetic tree of Shorea johorensis based on matK gene using the neighbor joining method

Figure 8
Figure 8 The phylogenetic tree of Shorea johorensis based on ITS2 using the neighbor joining method Hopea mengarawan and Hopea dryobalanoides formed a monophyletic group with bootstrap value 100.Based on this phylogenetic tree it appears that Shorea and Parashorea formed a separate group with Hopea.It means that ITS2 was able to separate Shorea with Hopea but not able to separate Shorea with Parashorea.Trichilia surinamensis was an outgroup because it had a distant relationship with other samples.

Figure 9
Figure 9 The phylogenetic tree of Shorea johorensis based on 28S rRNA gene using the neighbor joining method Phylogenetic Analysis Based on 28S rRNA Based on Figure 9, all of the three individuals of Shorea johorensis formed a monophyletic group with Shorea robusta (bootstrap value 62).This Shorea formed the separate group with Parashorea chinensis.This is in accordance with the nuclear DNA analysis performed by[36], where Parashorea and Shorea were separated and not belonging to a monophyletic group.Trichilia surinamensis was an outgroup.

Table 2
Molecular Identification of Shorea johorensis In Ketambe Research Station, Gunung … (Nir Fathiya, Essy Harnelly, Zairin Thomy and Iqbar) The result of BLAST Analysis of Shorea johorensis _________________________________________________________________________________________________ 60 also explained that Shorea johorensis and Shorea parvifolia each formed the monophyletic group.Based on this phylogenetic tree, rbcL gene was not able to separate Shorea, Hopea, and Parashorea to different monophyletic.This is relevant to the cpDNA analysis by Ref.[14 and 33]

Shorea johorensis based on psbA-trnH
Based on Figure6, Shorea johorensis formed the monophyletic group with Shorea robusta (bootstrap value 100).It means this Shorea had a closer relationship with Shorea robusta than Parashorea chinensis.Based on morphology data, Shorea robusta showed some similarities This is in contrast to rbcL and matK genes where Shorea formed a monophyletic group with Hopea.It means that ITS2 was able to separate Shorea with Parashorea and Hopea but not able to separate Parashorea with Hopea.Trichilia surinamensis was an outgroup because it had a distant relationship with other samples.Molecular Identification of Shorea johorensis In Ketambe Research Station, Gunung … (Nir Fathiya, Essy Harnelly, Zairin Thomy and Iqbar) The three individuals of Shorea johorensis formed a monophyletic group with Shorea robusta and Parashorea chinensis with bootstrap value 81 (Figure _________________________________________________________________________________________________63Phylogenetic Analysis Based on ITS2