International Research Symposium on Pure and Applied Sciences (IRSPAS)

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Author: search.filters.author.Herath, H. M.

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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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    Production of certain extracellular enzymes by some bacteria and amplification of cellulase gene from Bacillus species
    (4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Jayasinghe, J. A. S. M.; Medhavi, P. I. H. R.; Magana-Arachchi, D. N.; Wanigatunge, R. P.; Herath, H. M.
    Bacteria have received attention, due to their ability to produce extracellular enzymes beneficial in various industries. In the present study, extracellular enzyme production by two thermophilic bacteria (Meiothermus ruber, Tepidimonas ignava) and eight other bacterial isolates (Bacillus thuringiensis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus aryabhattai, Pseudomonas stutzeri, Pseudomonas aeruginosa, Sphingomonas sp., Burkholderia lata) was investigated. Extracellular amylase, protease, pectinase and cellulase production was studied in vitro in media containing starch, skimmed milk, citric pectin and carboxymethylcellulose respectively, at 28 °C, 35 °C, 45 °C and 55 °C. Hydrolyzing Capacity Index (HCI) at day seven was calculated to identify the isolates, which hydrolyzed a substrate with minimal colony formation. Such isolates would have a higher potential in industrial applications. HCI values were analyzed using one-way ANOVA and Tukey’s multiple comparison tests. All isolates, except thermophilic M. ruber, produced at least one extracellular enzyme within 1-3 days. T. ignava, B. thuringiensis and P. aeruginosa produced amylases. All isolates except B. aryabhattai and M. ruber produced proteases. B. thuringiensis, Sphingomonas sp., B. amyloliquefaciens and P. stutzeri produced cellulases. Pectinases were produced only by B. lata. Thermophilic T. ignava produced amylases and proteases at 28 oC and 35 oC but did not produce any enzyme at 55 °C, the temperature of the Maha Oya hot springs from which it was isolated. B. amyloliquefaciens, P. stutzeri, P. aeruginosa, B. pumilus, Sphingomonas sp. and B. lata produced proteases, which were stable at higher temperatures; 45 °C and 55 °C. It was the only enzyme to be produced at those temperatures. According to the HCI values, B. thuringiensis and P. stutzeri were the most efficient degraders of starch and cellulose, respectively. P. stutzeri, Sphingomonas sp. and B. lata were the best protein degraders. A gene coding for glycoside hydrolase (a cellulase) was amplified from bacteria by PCR using primers designed for Bacillus licheniformis ATCC 14580. Although expected amplicon size was ~1683 bp, amplicons of apporiximately 500 bp, 600 bp and 1000 bp were generated from cellulase producing B. thuringiensis. According to the information available in NCBI, B. thuringiensis has glycoside hydrolase gene of 738 bp suggesting that those amplicons could also be some glycoside hydrolase genes of different lengths. This should be confirmed by DNA sequencing. PCR product was generated by the same primers for B. aryabhattai as well, although it did not produce cellulases in vitro. It could be due to non-expression of the particular gene at the experimental conditions used in this study. These Bacillus species are perceived as sources of purified cellulases and the particular genes would be useful also in transformation of other organisms for industrial purposes