Indian Journal of Biotechnology Vol 10, January 2011, pp 90-96 Genetic diversity of coal mine spoil by metagenomes using random amplified polymorphic DNA (RAPD) marker Amiya Kumar Patel* and Niranjan Behera School of Life Sciences, Sambalpur University, PO Jyoti Vihar, Burla, Sambalpur 768 019, India Received 13 October 2009; revised 6 April 2010; accepted 15 June 2010 Pit-scarred-landscape with huge heaps of coal mine spoil is the usual scenario of an open cast coal mine area. To assess the status of microbial ecosystem, genetic characterization among 18 metagenomes, sampled within six different age series coal mine spoil overburdens, were analyzed using RAPD. Among studied populations, the genetic diversity was high, having 66.21% polymorphic bands, and it showed Shannon’s information index value 0.3414. Analysis of molecular variance (AMOVA) indicated that 90% of the genetic diversity among populations was attributed to different age series overburdens, while 10% was attributed to different habitats. The existence of genetic variation, as revealed by RAPD, was coupled with bacterial colony forming units (CFUs; r =0.945), and 81.49% variability in CFU can be explained due to genetic diversity (r = 0.902). Further, the relationship between polymorphic loci and Nei’s genetic diversity index (H) was observed to be significant (r =0.876), indicating gradual enrichment in the existing genetic composition due to reclamation process with the increase in age, and mine spoil gradually starts supporting greater genetic diversity. Keywords: Coal mine spoil, diversity index, genetic diversity, RAPD Introduction Soil ecosystem destruction by open cast coal mining activities often results in removal of biologically rich top soil and subsequently subsoil, which combines with coal residues and rock fragments to constitute ‘coal mine spoil’. Coal mine spoil ecosystem is a stressed ecosystem, where open cast mining stress disrupts the original land shape and, consequently, the landscape that emerges out of mining activity is devoid of supportive and nutritive capacity for biomass development. Extremities of conditions in a fresh coal mine spoil allow the existence of only specific group, such as, chemolithotrophic 1,2 , acidophilic 3,4 and thermophilic 5,6 group of bacteria. Being the initial colonizers, their presence and activity is very important for subsequent colonization of other microbial groups in the coal mine spoil. These activities on coal mine habitat led to reclamation of the spoil to soil through function of time (evolution) and space (spatial adaptation) and enrichment of microbial diversity. Genetic diversity of an ecosystem is used as an indicator to measure the cumulative impact of multiple stresses on population(s) and its adaptation to the habitat. Analysis of diversity is particularly important when the soil ecosystems respond to changing environmental conditions and such changes in the composition of the soil microflora can be crucial for the functional integrity of the soil 7 . Hence, assessment of the microbial diversity of coal mine spoil can provide an insight into the ecological well being of the habitat 8,9 and the successional changes in microbial communities during the process of reclamation. Direct cultivation or indirect molecular approaches can be used to explore and exploit the microbial diversity present in a coal mine spoil. But the cultivation methods cover only 0.1 to 1.0% of the diversity, leaving large segment of diversity unexplored due to the heterogeneity of the microbial communities present in various ecosystems. Hence, metagenomic approach was used to assess coal mine spoil microbial diversity. Molecular markers have been potentially used to survey and explore the diversity of soil metagenome, bacterial taxonomy and phylogeny 10,11 . Randomly amplified polymorphic DNA (RAPD) based detection of genetic polymorphism 12 has been successfully utilized on —————— *Author for correspondence: Mobile: 91-9338686900 E-mail: amiya_gene@yahoo.com Present Address: Division of Biotechnology, Majhighariani Institute of Technology and Science (MITS), At- Sriram Vihar, Bhujbala, PO Kolnara, Rayagada, 765 017