Large-subunit rDNA based differentiation of anaerobic rumen fungi using restriction fragment length polymorphism

Abstract Text: The contribution of rumen anaerobic fungi to animal production systems is substantial despite their lower numbers in the rumen. Utilizing both cultivation-based and advanced genomic tools, several reports have described the repertoire of ligno-cellulolytic enzymes secreted by these fungi. However, characterization of anaerobic rumen fungi is a tedious process with difficulties in isolation methods, their pleomorphic nature and lack of appropriate targeted genetic markers to understand fungal diversity.  Previously, we have identified a new marker, D1/D2 domain of 28S rDNA of LSU (Large-subunit; LSU) region which provided a better resolution in species differentiation of genus Orpinomyces than internal transcribed spacer (ITS) region, a preferred genetic marker for most fungal diversity studies. In the current study, we applied the same D1/D2 marker to assess species level distribution of several fungal lineages. For this, nearly 100 isolates of known lineages (Orpinomyces, Neocallimastix, Anaeromyces, Piromyces, Caecomyces, Cyllamyces) and unknown anaerobic fungi were cultivated from the rumen of different herbivorous animals. The isolates were extracted for their genomic DNA using modified method of Cetyl tri-methyl ammonium bromide (CTAB) and also MoBio genomic DNA extraction kit. The genomic DNA samples were PCR-amplified for D1/D2 regions of LSU (≈780 bp) using NL1 and NL4 primers. The amplified PCR products were purified, sequenced and characterized for phylogeny. Based on the phylogenetic analysis, representative sequences from identified fungal lineages were further subjected to in silico restriction digestion which led to the identification of two restriction enzymes, AluI (recognition sequence, AG▼CT) and HinfI (recognition sequence, G▼ANT C). About 34 selected sequences were co-digested by these two restriction enzymes using Restriction Fragment Length Polymorphism (RFLP) assay. The RFLP analysis of electrophoretic runs revealed distinct riboprints for individual fungal lineages that were identified in this study. Sequences annotated to similar fungal lineages showed comparable riboprints which confirmed that targeting D1/D2 region of the LSU gave repeatable results. Our study summarized that RFLP-based differentiation of fungal lineages was better accomplished when D1/D2 region of the LSU was used as a genetic marker.

Keywords: Rumen, Anaerobic fungi, LSU, PCR-RFLP, Co-digestion