Genetic variation in Canada wildrye endophytes as assessed with High-Resolution Melt analysis

Background/Question/Methods

Endophytic fungi are common endosymbionts of cool season grasses. Canada wildrye (Elymus canadensis) hosts a variety of endophyte species from the genus Epichloë, including an asexual, vertically transmitted interspecific hybrid and a sexual, potentially horizontally transmitted species. The differing reproductive strategies within a single host species provides a system where hypotheses related to the evolution of mutualisms and parasitisms can be tested.

One hypothesis is that the variation in reproductive strategies will create variation in the geographical distribution of endophyte genotypes, which in turn may lead to variation in their interactions with local Canada wildrye genotypes. Asexual, vertically transmitted endophytes are expected to have lower migration rates and experience higher genetic drift than their sexual, horizontally transmitted counterparts, and therefore exhibit more genetic variation between populations.

To assess genetic variation in Canada wild-rye endophytes, we developed a genotyping assay utilizing high-resolution melt (HRM) analysis. HRM is based on quantifying the melting temperatures of PCR products on a very fine scale. As a result, changes in melting temperature caused by single base differences are detectable.  Additionally, this technique is sensitive enough to allow for the identification of heterozygous individuals or individuals harboring more than one copy of a gene.

Results/Conclusions

We were able to identify genetic variation, including hybrid status, in E. canadensis endophytes from natural populations ranging from Texas to Minnesota with HRM analysis using several genetic markers. Specifically, HRM analysis was able to identify single nucleotide polymorphisms (SNPs) in b-tubulin (tubB) and translation elongation factor 1-a (tef-1a). The SNP variation was confirmed by sequencing representative samples from each variant group and comparing them to known sequences in GENBANK.

HRM analysis was found to be able to distinguish unique genotypes of Canada wild-rye endophytes, as well as to identify hybrid individuals. The genotypes found in this study correspond with previously described endophyte species harbored by Canada wildrye. Genotypes of tubB, specifically, are very similar to versions of tubB found in the recently described Epichloë x canadensis, an interspecific hybrid endophyte found in Canada wildrye.