A total 26 accessions of endemic orchids were collected by local people from the jungle of Papua, Indonesia territorial. Phenotypic of Papua’s endemic orchid (PEO) were used as plant materials in this study it sown in the Figure 1. The young leaf samples were preserved by using silica gel granules in zip lock plastic according to previous reported procedures [ 19 ]. The dried young leafs were grinded directly without using liquid nitrogen by using mortar and pestle until the tissue become fine powder. Isolation and extraction of genomic DNA from the dried leaf samples were performed by using procedures as described in Qiagen DNA extraction kit. The genomic DNA was achieved its keep in −20˚C in the freezer until ready to use.

Ten decamer RAPD primers were employed in this research as follows: OPA1 (5’-CAGGCCC TTC-3’), OPA4 (5’-AATCGGGCTG-3’), OPA12 (5’-TCTGTGCTGG-3’), OPU3 (5’-CTATGCCGAC-3’), OPU8 (5’-GGCGAAGGTT-3’), OPAW-05 (5’-CTGCTTCGAG-3’), OPAE-16 (5’-TCCGTGCTGA-3’), OPD08 (5’-GTGTGCCCCA-3’), OPD11 (5’-AGCGCCATTG-3’), and OPU8 (5’-AGCGCCATTG-3’). The decamer random primers were provided original sequences by Operon Technologies, Alameda, USA and synthesized by Integrated DNA Technologies. The PCR reagents were mixed in a 25 µl volume containing 10 ng genomic DNA, 2.0 µl of 50 pM primer, 2.5 µl of 10× polymerase buffer, 2.5 µl of 2.5 mM dNTPs, 1.0 U AmpliTaq Gold, 2.0 µl of 3 mM MgCl₂, and Milli Q Water.

Amplification was performed in a programmable Thermal Cycler (Takara) for an initial denaturation at 94˚C for 2 min, 40 cycles denaturation at 94 for 1 min, annealing at 37˚C, extension at 72˚C for 2 min, final extension at 72˚C for 5 min, and holding at 4˚C. The Amplified DNA fragments were resolved in 2.0% agarose gel, electrophoresis at 100 V using 1× TAE buffer, standard molecular weight marker using a 100 bp DNA ladder (Takara), staining with Ethidium bromide (1 µl/l) for 30 min, visualization and documentation by using UV illuminator equipment and digital camera. The PCR reaction was repeated more than one times per sample for each primer to ensure the reproducibility of the amplified bands. Faint bands were not considered for scoring and calculation.

RAPD fragments were scored as present (1) and absent (0). The scorer band in the formed of a binary data were analyzed the genetic variation, and genetic relationships among Papua’s endemic orchids. The dissimilarity matrix was calculated by using distance coefficient. The dissimilarity was employed for clustering of the Papua’s endemic orchids by the Unweighted Pair-Group Method Arithmetic Average (UPGMA), using the Sequential Agglomerative Hierarchical Nested Cluster Analysis (SAHN-clustering) [ 20 ] and TREE program from NTSYS-pc, version 2.02 packages [ 21 ]. Bootstrap analysis by 1000 permutation times were performed by using software Tools for Genetic Analysis (TFPGA 1.3); [ 22 ]. The average polymorphic information contents (PIC) of RAPD primers were calculated by using [ 23 ] formula (PIC_i = 1 − ∑pi², pi is the frequency of the i bands).

Amplification fragments by using ten RAPD primers and performed in the PCR tools were resulted 54 numbers of polymorphic fragments and no monomorphic band. The number of polymorphic bands per primer ranged from 4 (OPU8) to 7 (OPU3) with averaged 5.4 bands per assay unit. The number of polymorphic band that were observed in this work is lower than the number of bands were reported in the species of Phalaenopsis [ 15 ] and in the genus of Catasetum [ 24 ]. The fragment size range among 100 and 1400 bp, and the number of genotypes range from 9 (OPA12) to 17 (OPAW-05) with average 13.5 genotypes (Table 1).

The PIC value was performed to estimate the power of discriminatory the markers. A large proportion of PIC value has a high discrimination power. The PIC values were calculated range from 0.71 to 0.92. In the previous studies were reported that the PIC of within species of Phalaenopsis reached 0.99 [ 15 ]. Primer OPA1 and OPD11 have the highest PIC value (0.92), therefore both these primers have the highest discrimination power comparing overall primer used in this research. Otherwise, the lowest PIC value was observed at primer OPA12 with 0.71 PIC values (Table 1). An example of amplification pattern for molecular characterization generated by RAPD markers is shown in Figure 2.

The genetic dissimilarities (GDs) among examined orchids were range 0.10 to 0.94 based on [ 25 ] unbiased coefficients. The highest GDs values were found between A. calepsum (Ac25) and P. amibilis (Ac21) with 0.94 GDs value then followed by pairwise differences of A. Calepsum and C. asperata with 0.90 GDs value and the smallest GDs values among genera were found between D. aries (Ac11) and A. Calepsum (Ac25) with 0.23 GDs value (Table 2). Genetic distances of PEO were found in this study, it showed a

wide range of genetic distances. In the previous studies were reported that the genetic variation of orchid species also shows high variation [ 26 - 28 ]. The range of high genetic distance values based on the results of Nei’s unbiased distance analysis is reflected from the highly variable phenotype of orchid flowers (Figure 2).

The GDs among species of Dendrobium were range 0.10 to 0.90 based on Nei’s unbiased coefficient. The highest GDs values were found between D. Stratiotes (Ac08) and D. anosnum (Ac14) with 0.90 GDs value then followed by pairwise differences of D. Stratiotes (Ac08) and Dendrobium sp3 with 0.77 GDs value and the smallest GDs value were found between D. Lineale type1 (2) and Dendrobium sp1 (Ac03) with 0.10 GDs value. The calculation results of genetic variation among orchid species in this investigation resemble those of Cajanus cajan, Cajanus albicans, and Cajanus liniatus which have a wide range of genetic differences based on RAPD markers [ 29 ].

Dendrogram construction of PEOs is based on 54 polymorphic fragments of 10 RAPD primers as markers by using UPGMA method of Nei’s unbiased distance [ 24 ]. Dendrogram construction showed that PEO samples different from another and separated to form group by their self at the 0.40 coefficient value. This phenomenon correlated with different morphological phenotypes of flower. In the previous studies were reported that a lot of species of plant showed us their phenotypic traits correlated with molecular assessment [ 30 - 33 ]. Conformities of morphological traits and genetic characteristics by using molecular marker were reported also at sago palm species [ 34 , 35 ]. At the 0.6 coefficient value indicates that PEO sample is divided into nine groups (Figure 3) i.e. each sample at the genera level was separated into their own groups. Group 1 consists of two individuals namely Ascoglossum calepsum and Dendrobium sp2; Group 2 consists of four individuals namely Bulbophylum phalaenopsis, Dendrobium anosnum, Dendrobium Aaries, and Dendrobium digibum; Group 3 consists of seven individuals namely Dendrobium nindii, Dendrobium capituliflorum, Bulbophylum patens, Dendrobium mirbelianum type 2, Dendrobium antenatum type 1, Dendrobiums spectabile, and Dendrobium mirbelianum type 1; Group 4 consist Collagen asperata only; Group 5 consist Dendrobium simillieae only; Group 6 consists of two individuals

namely Dendrobium antenatum and Dendrobium macrophyllum; Group 7 consists of Dendrobium affine and Dendrobium sculery; Group 8 consists of Dendrobium lineale type 2, Dendrobium gouldii, and Phalaenopsis amibilis; and Group 9 consists of Grammatophyllum scriptum, Dendrobium sp1, Dendrobium discolor, and Dendrobium lineale type 1.