One of the most devastating emerging pathogens of wildlife is the chytrid fungus, Batrachochytrium dendrobatidis (Bd), which affects hundreds of amphibian species around the world. Genomic data from pure Bd cultures has advanced our understanding of Bd phylogenetics, genomic architecture, and mechanisms of virulence. However pure cultures are laborious to obtain and whole genome sequencing is comparatively expensive, so relatively few isolates have been genetically characterized. Thus we still know little about the genetic diversity of Bd in natural systems. The most common non-invasive method of sampling Bd from natural populations is to swab amphibian skin. Hundreds of thousands of swabs have been collected from amphibians around the world, but Bd DNA collected via swabs is often low in quality and/or quantity. In this study, we developed a custom Bd genotyping assay using the Fluidigm Access Array platform to amplify 192 carefully-selected regions of the Bd genome. We obtained robust sequence data for pure Bd cultures and field-collected skin swabs. This new assay has the power to accurately discriminate among the major Bd clades, recovering the basic tree topology previously revealed using whole genome data. Additionally, we established a critical value for initial Bd load for swab samples (150 Bd genomic equivalents) above which our assay performs well. By leveraging advances in microfluidic multiplex PCR technology and the globally distributed resource of amphibian swab samples, non-invasive skin swabs can now be used to address critical spatial and temporal questions about Bd and its effects on declining amphibian populations.
- microfluidic multiplex PCR
- Batrachochytrium dendrobatidis
- Disease Biology
- Ecological Genetics