Paddy cultivated Inseptisol Soil rhizosphere Metagenomics

During the rice cultivation season of 2010, Inceptisol soil samples were collected from a rice cultivate farmer field located at Varanasi district of Uttar Pradesh state, India. The soil samples were collected at plow layer rhizosphere 3 -7 inch depth. The samples were analyzed for its texture and physico-chemical properties which includes the organic carbon %, ammonium, nitrate, phosphate content, water holding capacity and pH. The region is classified as a part of Indo-Gangetic plains under the climate of tropical and dry monsoon with annual precipitation average of 670 mm in 2010 which is mostly distributed in rice season (June to December), accounting for 90-95 % of total precipitation in a year. The minimum temperature varied from 8C in January to 29C in June and maximum temperature from 10 to 28C and the maximum between 28 and 42C. The rice-wheat-rice cropping system is under intensive practice in this area. At the time of plowing basal dose of fertilizer (KCl + P2O5 + farmyard manure) was applied at the rate of 60:60:1000 kg/ha. Twenty- one day old seedlings of rice (Oriza sativa) plants were transplanted in the field. The field was submerged before transplanting but it was not entirely flooded. Irrigation was done whenever required. Chemical fertilizer was applied in the form of urea at 30 kg N/ha at the time of tillering and before grain filling stages as per normal former’s practices. META-DNA extraction kit was used and library was prepared by emulsion PCR. All DNA amplicons were gel-purified by gel extraction kit (Qiagen, USA) and concentrations were determined using Bioanalyzer High sensitivity chip (Agilent, USA) and qubit fluorometer (Invitrogen, USA). The amplicons were then pooled together in equimolar ratios into a tube breedwise before sequencing. Samples were further preceded for Roche 454 GS FLX Titanium chemistry pyrosequencing using 454 Roche platform (Roche, USA)