Genetic Analysis of Itchy Leaves ( Laportea , sp ) in Papua for Herbal Medicinal Products as Development of Studying Economics Value

Papua is rich in biodiversity. The indigenous Papuans utilize most of endemic plants as food, board and medicines. One of the plants used as medicine is Laportea, sp, that locally known as Daun Gatal. The plant however, has not been developed commercially; this might due to lack of scientific providence of medicine content in this plant. The aims of this research is to (1) explore the types of Laportea growing in Papua, (2) conduct bio molecular character, which observes phylogeny relationship or genetic morphology in order to obtain data on the number of species found in Manokwari and Nabire. Bio molecular analysis is performed by PCR. Genetic analysis in this study was conducted through several phases, as follow: (a) Isolation of genomic total, (b) amplification of genes that encoded Rbcl, (c) sequencing the nucleotide sequence of the gene Rbcl of Laportea, (d) phylogeny analysis. The results showed that the DNA quality was good enough to do the next analysis. Of the four samples, two obtained haplo types are Haplo Type 1 (DT.NBX.H, DT.NBX.U and DR.MKW.H) and Haplo Type 2 (DR.NBX.L). The results of the analysis of phylogeny trees showed samples at 1 in the same haplo type, clade sequences of the genus Dendrocnide spp, Discocnide, and Laportea, while the sample at 2 in the same haplotype clade with Laportea interupta, Laportea ruderalis and Urera sp.


Introduction
Indonesia is a country that is extremely rich in biodiversity of medicinal plants 20% -22% plants were cultivated, while around 78% was obtained through direct retrieval (exploration) of forest [1].
Papua is part of east Indonesia. It obtains a huge number of biological state, which is about 20.000 -25.000 species of plants and 142 species of them are endemic Papua [2]. The indigenous people of Papua utilize these plants as food, board and medicines. One of the plants used as medicine is Laportea, sp, the Papuan people called daun gatal and scientifically known as Laportea, sp.
The results showed that laportea Decumana (Roxb) Wed was positive for alkaloids, glycocides, steroids/triterpenoids and negative for saponis, flavonoids and tannins [3]. Then formic acid was influencing the regardless pore dilation thus stimulating blood circulation to relieve aches. In addition to the tribe, some of Laportea are also used to relieve nausea or dizziness pat on the head, and relief pain during child birth.

Sample Collection
The leaves were collected from Manokwari (West Papua Province) and Nabire (Papua Province). Fresh leaves were preserved and dried quickly by using silica gel.

Genomic DNA Isolation
Total genome of the sample leaf was isolated with Genomic DNA mini kit (plant), Geneaid, by using standard protocol of the product. Shortly, the DNA isolation was done by taking approximately 50 -100 mg of sample and grinding with sterile mortar. The grinded sample was added to the lysis buffer GPX1 (400 µL).
To degrade the RNA, RNA-se was added to the solution and incubated at 65˚C for five minutes. Before filtration, 100 µL of GP2 buffer was added then leaved on ice for three minutes. Filtration on filter column with centrifuge at 3500 rpm for 1 minutes. Supernatant of the filtrate was transferred to new micro centrifuge tube 1.5 mL. Then, buffer GP3 (1.5 times volume of supernatant) was mixed with the solution by vortex. The mixture was transferred to the GD column and centrifuge at 15,000 rpm for two minute. The GD column was washed with W1 buffer (400 µL) and wash buffer (600 µL) and each step was centrifuged at 15,000 rpm for 30 seconds. To dry the column, it was centrifuge at 15,000 rpm for three minutes. DNA genome that attached to the column was eluted with heated elution buffer (100 µL). The column and elution buffer were incubated at room temperature for three minutes to let the elution buffer interact with the DNA, then centrifuged at 15,000 rpm for 30 seconds. The extract DNA was kept in freezer.

Amplification of Ribulose 1,5 Difosfat Carboxylase Large Subunit Gene (rbcL)
Amplification of rbcL gen from gatal leaf was done with polymerase chain reaction (PCR) by using thermal cycler machine MJ mini thermocycler, model Promega. PCR reaction was conducted in total volume 50 µL, that consist of 18 µL ddH 2 O, 25 µL Go-Taq-Green master mix, 2.5 µL of primer rbcLaF, 2.5 µL of primer rbcLaR, and 1 µL of DNA genomic extract. The temperature profile of the PCR machine was 80˚C for 10 seconds, 94˚C for 2 minutes, then followed by 35 cycle of 3 steps which were (94˚C for 30 seconds, 50˚C for 30 seconds and 72˚C for 1 minute), the final extension at 72˚C for 5 minute, then closed with 37˚C for one minute.

Electrophoresis
The visualization of PCR product was done by electrophoresis gel agarose 1% with sodium boric buffer and voltage 100 V for 30 minutes. Each well consist of 4 µL PCR product and 1 µL of loading dye. As the marker, Generuller 1 Kb DNA ladder (Thermoscientific) was used. The dying of DNA was conducted by immersing the gel on ethidium bromide solution for 20 minutes and visualize with UV-transilluminator. The gel was documented by digital camera.

Sequencing
The sequencing of rbcL genes is conducted by dideoxy termination sanger. The process was done by company service, PT. Genetika Science Indonesia, Jakarta.
The electropherogram in AB1 format sent by email.

Data Analysis
The sequence of rbcL gene was proof read with the electropherogram with MEGA 5 software [7]. Basic local search alignment tools (BLAST) from NCBI was used to compare our data with the GenBank data (NCBI). Phylogenetic tree analysis (neighbor joining and maximum likelihood) was made with MEGA 5 software.
The bootstrap support in each branch was calculated by bootstrap methods with 1000 replication.

The Spread Pattern of Laportea Spin Nabire and Manokwari Regency
Based on the results of research conducted in the regency of Manokwari and Nabire result that daungatal or by the Latin name is Laportea sp, there is widespread in lowland up to the highland, the deployment begins at a height of 50 meters above sea level to 1500 meters above sea in the Manokwari regency. Laportea plants live in secondary forest and primary forest. In the primary forest, these plants live under stands of large trees with wet environmental conditions but dry land and have plenty of humus. This is consistent with the statement of the WHO [8] that this Laportea plant habitats in the shade and grows well in wet areas but with the dry land. In addition, this plant has been cultivated by some local communities in Manokwari in the yard of the house, where in addition to the needs of family medicine this plant is also traded in the market for profit. Similarly, in the Nabire regency, based on the exploration results obtained plants Laportea were living in primary forests and secondary forests that spread from the lowlands to the highlands of the area Wanggar-Lagari until the Topo-Dogiyai, which ranges at an altitude of 65 masl to 1300 masl. This plant in Nabire is not cultivated by the community because this plant grows wild and naturally so that the beneficiaries are taken directly from nature. From the results of exploration in Manokwari and Nabire obtained some kind of Laportea. Based on altitude, in the district of Manokwari in the lowlands, three plant species of Laportea are found by the local name Consist thin daungatal, red and the Laportea pig. While in the highlands also found that living plants of the Laportea that leaves itchy scratchy thin and red leaves. Based on the exploration results in Nabire, in the lowlands found three types of laportea living is based on local names gatel leaves, leaf green and Laportea scratchy purple, while living on the plateau that leaves itchy scratchy green and purple leaves. Leaf morphology itchy red and green leaves and flowers are shown in Figure 1.

Genomic DNA of Laportea, Isp
A total of six samples of Laportea from Nabire and Manokwari (Table 1), has isolated its genome DNA by using genomic geneaid isolation kit (plant). The success of the isolation of genomic DNA is shown with their band on a 1% agarose gel electrophoresis. Sightings DNA bands of the six samples are presented in Figure   2.
Based on the results of electrophoresis in Figure 2, seen all the DNA samples

Amplicon Gene Ribulose-1,5-Bisphosphate-Carboxylase (Rbcl)
Gen Rbcl of Laportea in the leaf samples in vitro amplification using polymerase chain reaction (PCR). Primary used is Rbcl-Ar [9] and Rbcl-Af [10]. Electrophoresis was performed using 1% agarose gel. As the marker (marker) used DNA marker 1 kb. Long amplicons obtained had a size of about 400 bp. With DNA quality was very good with high levels of brightness, brighter in the DNA bands in DNA marker which has a concentration of 70 ng/uL for most bright ribbon of DNA markers. This means that the DNA amplicon concentration higher than 70 ng/uL. This concentration is enough to be used in the sequencing stage. The results of electrophoresis of products PCR can be seen in Figure 3.
Electrophoresis results showed that there were four samples were successfully

Phylogenetic Analysis
Sequencing of the four samples of Laportea, is derived nucleotide sequences that encode genes Rbcl. The sequence is checked and adjusted (proof reading) with elektroferogram obtained, to ensure the correct sequence. The process of proof reading is based on the forward and reverse sequences of each sample. The quality of the sequences obtained was excellent, as indicated by the high peaks and clean from electroferogram.
The results alignment and the primary cutting edge, obtained along the 552 nucleotide base pairs. Of the four samples of Laportea, 17 points polymorphism obtained. The point of polymorphism is a nucleotide sequence that differs from one sequence to another. Based on the polymorphism, four samples can be classified into two haplotypes, a DNA sequence is said to be different haplotype when  Table 2.
Analysis of the phylo genetic tree created using MEGA software 5 [7]. Based on morphological analysis, obtained the basic information that Laportea samples in this study belong to the genus of Laportea. Therefore, as a reference in the manufacture of the phylogenetic tree, use some sequences of genes Rbcl from NCBI.
According to Wu et al. [11], uriticaceae family members can be grouped into three major clades. One clade of Laportea genus in which there is a third clade, in which there are genus Nanocnide,Uritica, Girardinia, Dendrocnide, Discocnide, Poikilospermum, Urera, Touchardia, Obetia and Laportea. Therefore, the phylo genetic tree made using gene Rbcl of genus-the genus are available at NCBI. A total of 420 base pairs of nucleotides that encode genes Rbcl of 89 sequences (4 samples of leaves itchy, one sequence out group, and 83 sequences from the family Uriticaceae selected from NCBI). The phylo genetic tree was made by the method of maxi-   Ts  Ts  Ts  Ts  Ts  Ts  Ts  Tv mum likelihood parameter K2P + G (Kimura 2 parameter and Gamma distribution). The meter is chosen from the test results the appropriate evolutionary model by using model test [10] in the MEGA 5. Bayesian Inference model with the smallest value is selected as the best model [12]. Bootstrapt 1000 replication method used to test the phylo genetic trees were created. Making the results presented in the phylo genetic tree in Figure 4,  also are politomi, so the kinship of all three cannot be distinguished by using gene Rbcl. Based on morphological analysis, it appears that this sample is a sample of the species Laportea interupta. With the results of phylogenetic tree analysis shows that this sample are in a clade with L. interupta, so it can be sure that the species is L. intruptadarisampel, although in the clade there are other species. Therefore, it is necessary to do further research that examines the kinship and systematics of the group leaves from Papua itch by using some other loci, both chloroplast and nuclear DNA locus.

Conclusions
1) The nucleotide sequence of the gene Rbcl Laportea origin Nabire and Manokwari successfully determined the sequence with a total length reaching 552 base pairs. 3) The results of the phylo genetic tree analysis show samples at 1 in the same haplo type and clade sequences of the genus Dendrocnidespp, Discocnide, and Laportea, while the sample at 2 in the same haplotype clade with Laportea interupta, Laportea ruderalisand Urera sp. 4) Gen Rbcl of the results of this study cannot explain the systematic relationships of these samples to the extent of the species. Multigene analyses or multilocus necessary are able to explain the systematics of the group leaves itchy.