International Research Symposium on Pure and Applied Sciences (IRSPAS)

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    Phylogenetic relationships of selected commercial Dendrobium hybrids in Sri Lanka
    (4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Kahagalla, T. H.; Herath, H. M.; Attanayake, R. N.; Senanayake, S. P.
    Nuclear rDNA-ITS regions and chloroplast matK genes are useful in delineating plant species. In this study, genetic relatedness of eight commercial Dendrobium hybrids (A-H) with a range of attractive flower colours was studied using nuclear rDNA-ITS and chloroplast matK sequences. Genomic DNA was extracted from fresh, young leaves using a modified cetyltrimethylammonium bromide based protocol. rDNA-ITS and matK were amplified using PCR in 25 μl reactions containing 1X PCR buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 1.25 U Taq DNA polymerase, 0.4 μM forward and reverse primers and 1.00 μl of genomic DNA. The optimized thermal cycling conditions were initial denaturation at 95 oC for 5 minutes, 35 (rDNA-ITS) and 40 (matK) cycles of denaturation at 95 oC for 40 seconds, annealing at 55 oC (rDNA-ITS) and 48 oC (matK) for 40 seconds, extension at 72 oC for 40 seconds and final extension at 72 oC for 10 minutes. rDNA-ITS and matK PCR products were subjected to Sanger sequencing. Sequences were manually edited using BioEdit 7.0.5.3. and ContigExpress software. Sequences were aligned to the nucleotide database in the National Center for Biotechnology Information using mega BLAST program. Forty-three related sequences were obtained from GenBank and the sequences were aligned using ClustalW implemented in MEGA 7.0.26 software. Phylogenetic analysis was performed by generating trees of ITS, matK and concatenated sequences of ITS and matK. The phylogenetic relationships were analyzed using Maximum Likelihood analysis with 1000 bootstrap replications. Phalaenopsis aphrodite, Liparis kumokiri and Malaxis spicata were used as outgroups. Combined gene-tree was estimated using RAxML-HPC BlackBox tool in CIPRES Science Gateway platform. Resulting trees were viewed using Figtree v1.4.3. In the combined gene tree, selected hybrids were clustered into two distinct groups. Dendrobium hybrids A, B, C, E and F were clustered with Dendrobium bigibbum var bigibbum and Dendrobium phalaenopsis (72% bootstrap). Hybrids G, H and D were clustered with Dendrobium nindii and Dendrobium taurinum (79% bootstrap). In matK gene tree, all the selected hybrids were clustered together with Dendrobium kingianum (90% bootstrap). In rDNA-ITS gene tree, hybrids A, B, C, E and F were clustered with Dendrobium bigibbum var bigibbum and Dendrobium phalaenopsis while hybrids D, G and H were clustered with Dendrobium taurinum and Dendrobium nindii (81% bootstrap). Therefore, though high variation in floral morphology is observed among the selected imported commercial hybrids, they were represented from a narrow genetic background. This is an indicative of genetic bottleneck most likely due to selective breeding and it is important to incorporate more diverse varieties in future breeding programs to maintain a diverse genetic background
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    Diversity of decaying wood associated fungi in Dimbulagala forest of Sri Lanka
    (Research Symposium on Pure and Applied Sciences, 2018 Faculty of Science, University of Kelaniya, Sri Lanka, 2018) Shashikala, M. M. P.; Deraniyagala, A.; Attanayake, R.N.
    Sri Lanka is one of the 34-biodiversity hotspots in the world bearing a tremendous diversity in flora and fauna. Therefore, it should hold true for fungal species diversity as well. In Sri Lanka, tropical wet evergreen rain forest reserves, mainly Sinharaja forest is well studied for the macro and micro fungal diversity. However, studies in the dry zone and intermediate zone forests are neglected though 83% of the country’s forest cover belongs to this category. The current study was initiated to describe the decaying wood associated fungal diversity in a dry mixed evergreen forest reserve. Decaying wood samples were collected from Dimbulagala forest reserve. Decaying hard wood pieces of at least 6 inches length were collected randomly. Fungal strains associated with the decaying woods were isolated into PDA or semi-selective medium and pure cultures were obtained. Macroscopic and microscopic features were observed. Total genomic DNA was isolated from a modified CTAB method and Polymerase Chain Reaction (PCR) was conducted targeting rDNA-ITS region using universal ITS primers and Sanger dideoxy sequencing was carried out to determine the nucleotide sequence of the region. Sequences were manually edited and compared with the GenBank using Basic Local Sequence Alignment Search Tool (BLAST). Phylogenetic relationships among decaying wood associated fungi were determined using MEGA (version 7.0) and according to the phylogenetic analysis well-defined clusters of fungi that belongs to divisions Ascomycota and Bascidiomycota were found. Fungal cultures were maintained at the Department of Botany using dry filter papers and in sterilized distilled water. A total of 55 fungal isolates were obtained from 36 decaying wood pieces and 35 fungal species were successfully identified. Results indicated that Sri Lankan dry mixed evergreen forests are rich in species of Paecilomyces, Daldinia, Trametes, Perenniporia, Phanerochaete, Hypoxylon, Schizophyllum, Lentinus, Fusarium, Coriolopsis, Phlebia, Coprinellus, Gymnopilus, Scytalidium, Trichoderma, Xylogone, Lasiodiplodia, Neoscytalidium and Pleurostoma. Species of Trichoderma and Lasiodiplodia were the most abundant species. T . harzianum T. lixii, T . longibrachiatum, and T . erinaceus were also found. Out of six Lasiodiplodia isolates, three were L . crassispora, and the rest belonged to the species of L . pseudotheobromae and L . theobromae. Some of the isolated fungi were already known plant pathogens and some were well-known biodegraders. The results indicated that the least studied Sri Lankan dry mixed evergreen forests are rich in various fungal species and could serve as another source in finding biotechnologically important fungal species.
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    Molecular identification of selected Dendrobium cultivars.
    (International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Silva, W.E.R.; Attanayake, R.; Senanayake, S.P.
    The family Orchidaceae includes more than 25,000 species, and the genus Dendrobium consists of over 1,450 species around the world. Today many unidentified Orchid cultivars are available in the market and growers use different vernacular names. Authentication of parental materials is important for conservation and selecting cultivars as parental materials in breeding experiments. However, Dendrobiums are well known for their difficulty in identification due to vegetative similarity among different species and morphological dissimilarity among same species. Since DNA barcoding has been proposed to be one of the most promising tools for accurate identification of taxa, this project was initiated with the objectives of identifying selected commercial Dendrobium cultivars and to determine their phylogenetic relatedness. Twelve commercial Dendrobium cultivars were selected based on the flower morphology. Genomic DNA was extracted from young leaves using a modified Cetyltrimethylammonium bromide (CTAB) method. PCR amplification of DNA was performed using universal ITS 1 and ITS 4 primers. PCR products were sequenced at Genetech Pvt. Ltd., Sri Lanka. Sequences were manually edited using BioEdit software version 7.1.9. Out of 12 samples, 9 samples produced non-specific amplification and only 3 samples produced good quality sequences of nearly 700 bp length. BLAST analysis was performed and sequences were deposited in the GenBank (MF535341, MF535342, and MF535343). Sequences of the current study with other 26 sequences from the GenBank were used in maximum likelihood analysis implemented in Mega 6.0 software with 1000 bootstrap replications. Liparis kumokiri (AY907087) was used as the out group for the analysis. Dendrobium cultivar Triple Fantacy (MF535341) resulted 99% similarity to Dendrobium bigibbum var. bigibbum and Dendrobium bigibbum var. superbum (KP142215 and KP142213) in the BLAST analysis. Unidentified Dendrobium cultivar (MF535343) was 94% similar to Dendrobium bigibbum var. bigibbum (KP142215) and Dendrobium bigibbum var. superbum (KP142214). In addition, both Triple Fantacy and unidentified Dendrobium cultivar, were clustered together with Dendrobium bigibbum var. bigibbum and Dendrobium bigibbum var. superbum. Therefore, Dendrobium cultivars, both Triple Fantacy and unidentified Dendrobium cultivar were identified up to species level as Dendrobium bigibbum. Dendrobium cv. Thailand Tommy (MF535342) resulted 99% similarity to Dendrobium nindii (AY239985) and clustered with Dendrobium nindii with 99% bootstrap support. Thus, the identity of Dendrobium cv. Thailand Tommy was confirmed to be Dendrobium nindii. In summary, DNA barcoding with ITS sequence was successfully used in resolving species identity of selected commercial Dendrobium cultivars in Sri Lanka.