SCAR-PCR markers for the detection of VAM fungi Gigaspora margarita and Glomus intradices grown using Ri-T DNA root organ culture.

ADHOLEYA ALOK¹, VIJAY GADKAR¹ & S. HIREMATH².

¹ Microbial Biotechnology, Tata Energy Res Inst, Habitat Place, Lodi Rd, New Delhi 1100 03, INDIA, ²Biotechnology Div, USDA-Forest Service, Delaware, Ohio, 43015, USA.

Detection of VAM fungal symbiont in the free living and colonized state, forms an important part of the VAM molecular ecology. Root organ culture is one method which has proved useful for growing these fungi under pure culture conditions.This method is able to provide fungal DNA free from any contamination which is usually associated with field grown VAM fungi.Using the transformed Ri-T DNA of carrot as host we have been able to cultivate G.margarita and G. intradices under in vitro conditions.The fungal DNA was extracted from the spores and RAPD was done using standard 10 mer as PCR primers (60-70% G+C).The polymorphic bands were cloned and sequenced.Primers specific to these genus specific fragments were designed henceforth called SCAR (Sequence Characterized Amplified Region) primers were used for quantitative and qualitative detection of the fungi. The ability of these primers to detect target from complex mixtures (multiplex PCR) and its potential applicability for detection of these two fungi from field samples would be discussed.

Key words: PCR, SCAR, Ri-T DNA, Multiplex PCR