Browsing by Author "Ranatunga, M. A. B."

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Comparison of different RNA extraction protocols: An optimized RNA extraction protocol for tea leaves [Camellia sinensis (L.) O. Kuntze]
(4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Keerthika, V.; Ranatunga, M. A. B.; Perera, D.; Herath, H.
Extracting high-quality RNA is critical for downstream applications such as quantitative real time PCR (qRT PCR), RNA sequence based transcriptomics and a prerequisite for ensuring representation of all expressed genes in a cDNA library. Tea is a popular non- alcoholic beverage crop and tissues contain abundant polysaccharides, phenolic compounds and other metabolites, which hinder isolation of high-quality RNA. Tender two leaves and a bud harvested is the economically important tissues of tea. A large number of extraction protocols have been exploited or modified for tea, viz commercial kits, CTAB-based methods and SDS-based methods. However; difficulties were encountered in terms of purity and quantity of isolated RNA, while some of the methods were time-consuming. Hence, the present study was aimed to optimize protocol/s for extracting good quality RNA suitable for downstream applications from two leaves and a bud tissue of tea. Two different RNA extractions protocols based on CTAB and hot borate with modifications along with three commercial RNA extraction kits were used to extract RNA from two tea cultivars. Out of which, a CTAB-based protocol optimized for pomegranate plant tissues with minor modifications resulted in the highest RNA yield, varying from 524 ± 4 to 776 ±16 μg/ml for cultivars TRI 2023 and TRI 2025 and high integrity as confirmed by Gel electrophoresis (distinct and visible 28S rRNA and 18S rRNA bands). The extracted RNA was further used for cDNA synthesis, expression of 18s house keeping gene through Realtime-polymerase chain reaction (qRT-PCR). Results showed that the yield and quality of total RNA extracted were suitable for qRT-PCR, depicting the quality of RNA for the downstream applications. Commercially available extraction kits despite of giving sufficient amount of RNA, had lesser yield and quality when compared to the CTAB based method. The protocol optimized for pomegranate was modified with omitting washing the RNA pellet with LiCl and replacing the extraction buffer (CTAB, PVP, NaCl, EDTA, Tris HCl, Spermidine, β-mercaptoethanol) with extraction buffer used for grapevines. This protocol enables successful isolation of RNA from two leaves and a bud tissues of tea within two days without the use of toxic and expensive chemicals such as phenol, guanidium isothiocyanate and guanidium hydrochloride. The protocol is efficient, simple, and reproducible and is therefore recommended for RNA extraction from plants with high polyphenol contents
Morpho-molecular genetic diversity and population structure analysis to enrich core collections in tea [Camellia sinensis (L.) O. Kuntze] germplasm of Sri Lanka and India
(Genetic Resources and Crop Evolution, 2023) Kottawa-Arachchi, J. D.; Ranatunga, M. A. B.; Sharma, R. K.; Chaudhary, H. K.; Attanayake, R. N.; Amarakoon, A. M. T.; Gunasekare, M. T. K.; Sharma, B.; Kumar, N.; Sood, V. K.
Despite tea [Camellia sinensis (L.) O. Kuntze] is one of the top non-alcoholic beverages consumed around the world; its genetic and phenotypic diversity is less understood compared to other plantation crops. The study’s aims were to undertake phenotypic and genotypic characterization of Sri Lankan and Indian tea germplasm and to identify diverse accessions to accelerate tea breeding programmes in both countries. A total of 171 tea accessions, comprising 94 Sri Lankan and 77 Indian accessions were used. All the accessions were subjected to phenotyping and genotyping using 28 polymorphic simple sequence repeat (SSR) markers. Based on 16 morphological characters, the first three principal components explained 57.61% and 58.43% of the total variability of Sri Lankan and Indian accessions, respectively. Young shoot pubescence, young shoot pigmentation, serration of leaf margin, and mature leaf colour contributed positively to the grouping of accessions. Based on Neighbor-joining analysis, all Sri Lankan accessions grouped in a single cluster, whereas Indian accessions grouped in two distinct clusters. The Gower’s distance method was the most appropriate than other methods for developing core subsets. Among 21 Sri Lankan core accessions selected, 11 accessions belong to introductions, five TRI-developed cultivars and five estate selections. Among 18 Indian core accessions selected, 11 belong to China types, two Assam types and five Indian recommended cultivars. The current study is the first study to compare tea germplasm of both countries and the results are useful for tea crop improvement programme, conservation and utilization of tea germplasm in India and Sri Lanka in the future.