DNA Methods

DNA-LSU



In this section, which is being developed as time permits, methods used at INVAM to carefully select spores, extract DNA from individual spores, amplify a target gene region, and clone and sequence amplicons to individuate sequence variants are discussed. Only two genes have been sequenced at INVAM, with the majority pertaining to an rRNA gene.

1. The full length beta-tubulin gene (TUB2) was characterized by Msiska and Morton (2009a) and an approximate 1000 bp region was selectively amplified for phylogenetic analysis which contains three highly variable introns (Msiska and Morton 2009b). No further work was done with this gene after Msiska left the lab because fungi many clades contained more than one copy (paralogs) which would have required much greater sampling of transformant clones to sort these from orthologs that gave a truer picture of gene evolution. There are 103 sequences representing 47 species (all described) in the INVAM library (stored in a FileMaker Pro database module).

2. A 750-800 bp region near the 5’ end of the 28S rRNA gene (LSU) which spans two highly variable regions, D1 and D2. A fairly extensive library had been developed before Krueger et al. (2012) developed a set of primers to amplify a region of the 18S rRNA gene (SSU), ITS, 5.8S, ITS2, and a similar length of the LSU. Because of that database, cost considerations, and our attention mostly on species-level differences, we continue to select the LSU fragment. Our analysis of its power of resolution relative to concatenation of other rRNA gene repeats can be viewed on this page, which shows only minor differences in tree topology at the genus and species levels. There are 328 sequences representing 78 species (14 of which are undescribed) in the INVAM library as of July, 2013. We are using these sequences internally to better define reference strains and the boundaries between species, especially when morphological or genetic polymorphisms are apparent.

References

* Krüger M, C. Krüger, C. Walker, H. Stockinger, and A. Schüßler A. 2012. Phylogenetic reference data for systematics and phylotaxonomy of arbuscular mycorrhizal fungi from phylum to species level. New Phytologist 193:970-984.
* Msiska, Z. and J.Morton. 2009. Isolation and sequence analyses of a beta-tubulin gene from arbuscular mycorrhizal fungi. Mycorrhiza 19:501-513.
* Msiska, Z. and J. Morton. 2009. Phylogenetic analysis of the Glomeromycota by a partial beta-tubulin gene. Mycorrhiza 19:247-254.