Genomics and metagenomics technologies to recover ribosomal DNA and single-copy genes from old fruit-body and ectomycorrhiza specimens

Abstract

Citation: Tedersoo L, Liiv I, Kivistik PA, Anslan S, Kõljalg U, Bahram M (2016) Genomics and metagenomics technologies to recover ribosomal DNA and single-copy genes from old fruit-body and ectomycorrhiza specimens.MycoKeys 13: 1-20. doi: 10.3897/mycokeys.13.8140 Abstract High-throughput sequencing (HTS) has become a standard technique for genomics, metagenomics and taxonomy, but these analyses typically require large amounts of high-quality DNA that is difficult to obtain from uncultivable organisms including fungi with no living culture or fruit-body representatives. By using 1 ng DNA and low coverage Illumina HiSeq HTS, we evaluated the usefulness of genomics and metagenomics tools to recover fungal barcoding genes from old and problematic specimens of fruit-bodies and ectomycorrhizal (EcM) root tips. Ribosomal DNA and single-copy genes were successfully recovered from both fruit-body and EcM specimens typically <10 years old (maximum, 17 years). Samples with maximum obtained DNA concentration <0.2 ng µl-1 were sequenced poorly. Fungal rDNA molecules assembled from complex mock community and soil revealed a large proportion of chimeras and artefactual consensus sequences of closely related taxa. Genomics and metagenomics tools enable recovery of fungal genomes from very low initial amounts of DNA from fruit-bodies and ectomycorrhizas, but these genomes include a large proportion of prokaryote and other eukaryote DNA. Nonetheless, the recovered scaffolds provide an important source for phylogenetic and phylogenomic analyses and mining of functional genes.

Novel soil-inhabiting clades fill gaps in the fungal tree of life

“…Both fruiting bodies and cultures form an excellent basis for genomic analysis to understand the functional capacities of undescribed taxa and improve phylogenetic resolution [16, 32, 33]. Metagenomics and single-cell genomic analyses offer promising tools for taxonomic and functional characterization of bacteria [17] and aquatic microeukaryotes [34] in their intimate environment, and these methods may provide satisfactory results also for unicellular zoosporic fungi [20].…”

Fungal identification biases in microbiome projects

“…However, due to greater PCR efficiency, short amplicons are less prone to amplification biases, and probably yield more accurate quantitative insights (Ihrmark et al ., ). We advocate that metagenomic approaches are unlikely to replace amplicon‐based surveys of eukaryotes in the near future, because of their low biomass and problems with sequence assignment to OTUs and reference taxa (Tedersoo et al ., ,b; b).…”

Multigene phylogeny of Endogonales, an early diverging lineage offungi associated with plants

“…For the other 26 specimens, three (12 specimens), two (13 specimens) or one (1 specimen) gene sequences were successfully generated (Supplementary Table 1). Most missing gene sequence data were of the ITS-28S rRNA region, which is known to be problematic for Endogonales (Tedersoo et al 2016). The RPB2 gene also proved challenging to amplify and sequence.…”

“…Even though they are distributed across temperate and tropical habitats in the Northern and Southern Hemispheres, sporophores of most of these fungi are rarely collected and molecular data are limited or not available. Further, there are idiosyncratic challenges when working with Endogonales rDNA, because ITS rDNA does not amplify or sequence well, or is degenerate (Tedersoo et al 2016). Consequently, Endogonales are often conspicuously underrepresented in environmental molecular surveys and databases (e.g.…”