Phylogenetic Relationship of Indonesian Water Yam (Dioscorea alata L.) Cultivars Based on DNA Marker Using ITS-rDNA Analysis

Research on genetic diversity and intra-species classification of Indonesian Dioscorea alata L. based on morphological characters has been done, and the result shows that there are 4 sub-groups of green cultivars group, and 5 sub-groups of purplish-red cultivars group. The objectives of this study are to determine the phylogenetic relationship cultivars of D. alata Indonesia compared to D. bulbifera as nearest species as well as 3 cultivars from GenBank. The young leaves of 18 water yam cultivar accessions were collected from Java, Madura, South Sumatera, South Kalimantan, Centre Celebes (Sulawesi), Ternate, West Papua, and Nusa Tenggara islands of Indonesia. DNA Isolation was conducted using Phytopure reagent. DNA amplification was conducted using thermocycler, predenaturation at 95 oC 5 minutes, denaturation at 95 oC 1 minutes, annealing at 60 oC 3 minutes, and elongation at 72 oC 2 minutes along with 30 cycles. The PCR products were electrophoresed in 1.5% agarose and visualized under UV transiluminator. Fifty-five µl of PCR products with positive targeted band between 700-800 bp were sent to 1st Base Singapore for purification and sequencing of 18S, ITS-1, 5.8S, ITS-2, and 28S rDNA. The DNA sequences were compared and aligned by BioEdit program (version 7.0.5.2) and MEGA programs. Comparison of entire sequences of the tested samples were aligned by software ClustalW (version 1.83). Phylogenetic trees were based on hierarchical clustering of the alignments of 18S, ITS-1, 5.8S, ITS-2, and 28S rDNA and produced by Neigbor-Joining using MEGA 5 software of the bootstrap values (1000 replicates). The study result shows that D. alata cultivars have high genetic variability on ITS-1, 5.8S, and ITS-2 rDNA region. Groups of green and purplish red cultivars formed based on morphological characters are not formed based on ITS-rDNA markers. Sub-groups were formed based on ITS-rDNA molecular markers derived from both the green and purplish-red cultivar groups. This result revealed that two cultivar groups are not similar with RAPD and morphological characters.