IRSPAS 2016

Permanent URI for this collectionhttp://repository.kln.ac.lk/handle/123456789/15651

Browse

Now showing 1 - 2 of 2

Optimization of a genomic DNA extraction technique for genetic diversity studies of selected orchid cvs. with ornamental values
(Faculty of Science, University of Kelaniya, Sri Lanka, 2016) Farook, F.; Attanayake, R.N.; Senanayake, S.P.
Orchidaceae is one of the largest and most diverse families of flowering plants and a major export ornamental crop. Cultivar development is the key for the success of ornamental flower industry and therefore, it is vital to identify the genetic diversity of the cultivars. In such an attempt, the first step is to optimize basic molecular biological techniques involved in genetic diversity analysis. However, DNA extraction from orchids is challenging compared to the other plant species since orchids are rich in polysaccharides and secondary metabolites, which can act as inhibitors in downstream applications. Most of the standardized protocols require liquid nitrogen freezing step, which is not an affordable practice in the laboratories of developing countries. Therefore, optimization of a low cost protocol to obtain pure DNA is necessary. The objective of the current study was to optimize extraction of genomic DNA for molecular marker based genetic diversity studies of selected orchid cultivars with ornamental value. Leaf pieces and pestle and mortar were stored at -80°C for at least three days. DNA extraction was done from frozen orchid leaves (50-100 mg young leaves) using Promega Wizard genomic DNA purification kit (Promega Inc. USA), classical CTAB protocol and modified CTAB protocol. Except modified CTAB protocol, none of the other methods produced high quality DNA as determined by spectrophotometry and agarose gel electrophoresis. However, the method was successful only for the Dendrobium cultivars but not for the Phalaenopsis leaves tested. Successful amplification of orchid rDNA-ITS region confirmed that the quality of extracted DNA is suitable for other PCR based molecular marker studies such as Random Amplified Polymorphic DNA (RAPD) and Simple Sequence Repeats (SSRs). The modified method was reliable and reproducible.
Pleurostomophora richardsiae associated with decaying woods in a dry zone forest of Sri Lanka
(Faculty of Science, University of Kelaniya, Sri Lanka, 2016) Bandara, R.H.; Deraniyagala, S.R.A.S.; Attanayake, R.N.
Wood decay is a biological process by which cellulose and lignin are converted into carbon dioxide and water. This process is important for forest sustainability since it uses cellulose and lignin, the two most abundant organic compounds on earth to recycle nutrients. Nearly 83% of the total forest coverage in Sri Lanka is comprised with dry mixed forests and these forests are rich in hard wood bearing plant species such as Diospyros ebenum, Manilkara hexandra and Drypetes sepiaria. Therefore, fungi associated with decaying these hard wood species should also be efficient lignin and cellulose degraders with possible biotechnological importance. The objective of the current study was to identify such decaying wood-associated fungi found in a dry zone forest using DNA barcoding approach and to maintain these cultures for future research. Decaying hard wood pieces were collected from the forest floor of Dimbulagala forest reserve and brought to the laboratory. Wood pieces were surface sterilized, cut into small pieces, plated on selective PDA media. Pure cultures of fungi were obtained and morphological characterization was done. For molecular characterization, fungal mycelia grown in PDB were subjected to total genomic DNA isolation. Fungal rDNA-ITS region was amplified and sequenced from both directions. Sequences were manually edited and BLASTN searches reveled that Trichoderma species and Lasiodiplodia species were among the frequently found species. Species delineation of certain fungal isolates were challenging and therefore, molecular phylogeny based identification was applied. Maximum Likelihood method based on the Kimura 2-parameter model was used and interestingly Pleurostomophora richardsiae (isolate name DDW 05), a possible plant pathogenic fungus, was found to be associated with a decaying hard wood sample. P. richardsiae has been reported to cause grapevine dieback and cankers in Italy and Spain. This finding is interesting since forest dieback has long been an unidentified problem in Sri Lanka and it could be possible that P. richardsiae is among the organisms causing forest dieback and cankers in Sri Lanka. Previous reports also show that this fungus has been isolated from wood, ground wood pulp, sewage and soil in North America, Europe, Africa and several countries in Asia. Therefore, further research is needed to confirm the pathogenicity of the isolate.