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Molecular characterization of Manikaran hot spring microbial community by 16S rRNA and RAPD analysis

Bhupender Sharma, Richa Verma, Kamal Dev, Rampa Thakur

The biodiversity of thermophiles inManikaran hot spring was determined by using 16S rRNA and RAPD based methods. Thermophilic microbes were isolated from the soil and water samples collected from Manikaran hot spring, situated in Kullu district of Himachal Pradesh. The microorganisms were primarily differentiated based on the morphology, colour and texture thus purified by successive streaking on nutrient agar plates. Pure cultureswere further differentiated based on GramÂ’s staining. Seven different microbial strains were analysed by RAPD using four sets of randomprimers. The RAPD data was further analysed by phylogenetic analysis for genetic relatedness. Further, two strains were analyzed by PCR amplification of gene encoding 16S rRNA by using standard primers (27 F/1492 R).Amplified DNAproducts were sequenced on both strands, using 27 F and 1492 R primers and the quality of DNA sequences were analysed using chromatogram 3.2 to remove low quality bases (nucleotides) so as to give trimmed sequences. Trimmed sequences were assembled by using mobyle Portal online merger application to give full length 16S rDNA sequence. Merged DNA sequences were analysed by BLAST (BLASTn) program at NCBI database and identified as Pseudomonas species (MS1-1) and Bacillus licheniformis (MS1-3) with 97%similarity.