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

Abstract

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 o C 5 minutes, denaturation at 95 o C 1 minutes, annealing at 60 o C 3 minutes, and elongation at 72 o C 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 1 st 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.

Cluster analysis of Dioscorea alata accessions of Purwodadi Botanic Gardens (Indonesia) collection based on morphological characteristic and SSR markers

“…(local varieties). That is supported by the results of the study of Purnomo et al (2017), which states that the D. alata kinship tree based on the results of RAPD molecular analysis shows no groups formed based on regional origin.…”

“…Some studies of tuber processing were found, and its tuber could be used for bakery, cookies, flakes, muffins, noddle, vermicelli (Hapsari 2014); flour (Afidin et al 2014); chips (Putri et al 2017); fries (Munawaroh et al 2018), etc. Dioscorea alata in Indonesia showed high morphological diversity, especially in tuber shape, size, skin, and flesh color. Accessions clustering and naming of D. alata used different local names depending on the origin of the plant/tuber, which confused identification (Onwueme and Ganga 1996;Herison et al 2010;Purnomo et al 2017;Kinasih et al 2017). Characterization and clustering of D. alata in Indonesia are needed as base information to make valid accession names and arrange development programs.…”

Genetic variation and phylogenetic relationships of Thelymitra javanica (Orchidaceae: Orchidoideae) in East and Central Java, Indonesia

“…There is a possibility that the ITS-nrDNA in some species of Thelymitra are conservative and indicative of incomplete concerted evolution of nrDNA, as Hribova et al (2011) found in Musa. The utilization of ITS-nrDNA sequence in this study might be insufficient to reveal the genetic variation of T. javanica at the intraspecies level, although Yukawa et al (2013) successfully used ITS-nrDNA to verify the taxonomical ambiguity of Grammatophyllum speciosum, and Purnomo et al (2017) successfully constructed the phylogenetic relationships of water yams (Dioscorea alata L.). Khademi et al (2016) also successfully used ITS-nrDNA to investigate unresolved clades in Acer monspessulanum subspecies in Iran.…”

Genetic variability of Begonia longifolia Blume from Indonesia based on Nuclear DNA internal transcribed spacer (ITS) sequence data

“…Nuclear rDNA consists of ITS 1, located between 18S and 5.8S, and ITS 2, located between 5.8S and 26S (Edger et al 2014). This region is widely used for studies at the intraspecific level because it showed high variability within some species, such as Passiflora (Mäder et al 2010), Withania somnifera (Mir et al 2010) and Dioscorea alata (Purnomo et al 2017). In Begonia, ITS sequences showed significant intra and interspecific differences (96%-100%) in B.…”