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A Journal on Biotechnology and Molecular Biology
Indexed/Abstracted in: EMBASE, Science Citation Index Expanded (SciSearch), Scopus
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Minerva Biotecnologica 2014 September;26(3):143-8
Nuclear DNA content and genome size estimation of Stevia rebaudiana using flow cytometry
Yadav A. K., Singh S., Bhardwaj G.
CSIR-Institute of Himalayan Bioresource Technology, Palampur, India
AIM: Nuclear DNA C-value and genome size are important biodiversity characters. Flow cytometry (FCM) is commonly used to determine nuclear DNA content and plant genome size estimates in many crop plants. This paper describes a simple and reliable method for extracting intact nuclei, and estimation of genome size of Stevia rebaudiana.
METHODS: The method involves preparation of aqueous suspensions of intact nuclei whose DNA is stained using a DNA fluorochrome (propidium iodide). Estimation of nuclear DNA content of Stevia rebaudiana (variety Madhuguna) was done through flow cytometry using different types of buffers. The buffers used were, Tris MgCl2 buffer, Otto buffers I and II, Galbraith buffer, Arumuganthan and Eareles buffer, Marie’s NIB buffer and Woody plant buffer. The nuclei isolated with different buffers were analyzed through flow cytometer (BD FACS Calibur System, USA).
RESULTS: Woody plant buffer with some modifications (Tris HCl 200 mM, MgCL2 4 mM, EDTA Na2 2 mM, NaCl 86 mM, Sodium meta bisulphite 10 mM, 1% PVP 10 and 0.5% triton-X 100 with pH 7.2) gave the best peak as it isolates the maximum number of intact nuclei in comparison to other buffers. Different plants (pea, maize and tomato) were used as internal reference standards. Tomato was found to give best results with stevia for estimation of nuclear DNA content in comparison to pea and maize. The aqueous suspension of intact nuclei of both the samples i.e. stevia variety Madhuguna and tomato (Solanum lycopersicum) variety Stupicke were analyzed in flow cytometer. By using tomato variety Stupicke having nuclear DNA content 1.96 pg per 2 C (provided by the Institute of Experimental Botany, Olomouc, Czech Republic) as an internal reference standard, the relative fluorescence intensity (RFI) of stevia nuclei was calculated. The measurements of RFI of stained nuclei were performed on 10 plants of variety Madhuguna on a linear scale and 20,000 nuclei were analyzed for each sample. The genome size of stevia (2C value) was estimated to be 2.72 pg or 2660 Mbp.
CONCLUSION: Stevia rebaudiana might be a good candidate for genome sequencing due to possession of comparatively smaller genome size (1 C=1.36 pg) or 1330 Mbp. The noted genome size of Stevia rebaudiana will have important implications on structural and functional genomics research and provides information for consideration in genome sequencing undertakings of this economically important medicinal plant and would be utilized in advancing biochemical and evolutionary studies of the genus Stevia.