Applications and Limitations of 28S rRNA Gene Sequences for Monitoring below-Ground Diversity at Degraded Mine Sites Undergoing Eco-Restoration at Purnapani, Odisha, India.

Spent limestone-dolomite mine dumps located in the state of Odisha, in eastern India were targeted for eco-restoration and regeneration of arable land for use by local peoples since 2005. Current status at the study site consists of a multi-tiered, ecologically productive ecosystem that resembles neighboring forest stands. As part of the eco-restoration program, shifts in below-ground diversity were assessed for microbial populations and soil invertebrates. Clonal libraries based on amplicons of 28S rRNA gene primers flanking the D9-D10 expansion segments (traditionally used to detect arthropod DNAs) were generated, and screened by long-read, Sanger sequencing to generate baseline data. Results indicated presence of rhizospheric fungi, invertebrates, and insect-associated fungi likely involved in soil amelioration and establishment of native vegetation over time. The overall utility of PCR primers based on 28S rRNA genes in monitoring below-ground biodiversity at the study site was confirmed using a quantitative real time PCR approach. Advantages, limitations and future applications of sequencing data based on 28S rRNA gene amplicons is described with reference to changes in rhizospheric communities of native plant species that responded to anthropogenic efforts to improve soil health in degraded mine dumps.