A rapid and cost‐effective quantitative microsatellite genotyping protocol to estimate intraspecific competition in protist microcosm experiments

Abstract

Summary1. High levels of intraspecific variation are commonly observed in natural microbial populations, yet the consequences of this variation for ecological and evolutionary processes remain poorly understood. Protists are excellent experimental models for investigating fundamental and applied questions in ecology and evolution, but studying intraspecific variation remains a challenge due to a lack of molecular resources to aid in quantifying and distinguishing strains during experiments. 2. Here we present a molecular method, quantitative microsatellite genotyping, to accurately quantify strain-specific frequencies from microcosm experiments of the marine flagellate Oxyrrhis marina, both between many pairs of strains and between strains in a multistrain mixture. 3. We find that for pairs of strains, the method is effective for relative frequencies as low as 0Á02 and with around 99% accuracy. The method is able to quantify four strains reasonably well, though less accurate than for pairs (range 92-97% accuracy). 4. This makes accessible a cheap and easy-to-implement method for quantifying strain (or allele) frequencies and is suitable for use in a broad range of single-celled eukaryotes (protists) where copy number should correlate well with number of individuals (i.e. cells). This opens up the possibility of examining the role of intraspecific variation using experimental protist microcosms.

Microsatellite markers for the dinoflagellate Gambierdiscus caribaeus from high-throughput sequencing data

“…Annealing temperatures ranged from 52 to 60°C in the different PCR reactions. Similar numbers of microsatellites were developed for other dinoflagellate species in different laboratories (Frommlet and Iglesias-Rodriguez 2008;Lundholm et al 2014;Minter et al 2015) with the exception of higher numbers of markers for the genus Alexandrium (Nagai et al 2004(Nagai et al , 2006(Nagai et al , 2014Sehein et al 2016). Fur- thermore, the ratio of applicable microsatellites to the amount of tested markers in our study (partial genome 7.8%, transcriptome 23%) is comparable to the success rate of other studies that use high-throughput sequencing (Nagai et al 2014;Minter et al 2015).…”

“…Similar numbers of microsatellites were developed for other dinoflagellate species in different laboratories (Frommlet and Iglesias-Rodriguez 2008;Lundholm et al 2014;Minter et al 2015) with the exception of higher numbers of markers for the genus Alexandrium (Nagai et al 2004(Nagai et al , 2006(Nagai et al , 2014Sehein et al 2016). Fur- thermore, the ratio of applicable microsatellites to the amount of tested markers in our study (partial genome 7.8%, transcriptome 23%) is comparable to the success rate of other studies that use high-throughput sequencing (Nagai et al 2014;Minter et al 2015). Thus, this study shows that screening of high-throughput sequencing data represents an efficient alternative to traditional methods for microsatellite development such as dual-suppression PCR and compound SSR PCR, which are usually very labor intensive and time consuming (Nagai et al 2014).…”

Application of high resolution melting assay (HRM) to study temperature-dependent intraspecific competition in a pathogenic bacterium

“…However, due to technical limitations, such as tracking individual isolates in co-cultures, relatively few studies have empirically examined how joint changes in intraspecific interactions and temperature affect ecological responses to increasing temperatures 12 . Based on a diagnostic SNP, we successfully established a method based on real-time PCR and HRM analysis for the rapid differentiation and quantification of two closely related genotypes of F .…”

Company matters: The presence of other genotypes alters traits and intraspecific selection in an Arctic diatom under climate change

“…Previously, this method has only been used to quantify the relative abundance of pairs of genotypes (John et al, 2015;Minter, Lowe, Brockhurst, & Watts, 2015;Sildever, Sefbom, Lips, & Godhe, 2016). Previously, this method has only been used to quantify the relative abundance of pairs of genotypes (John et al, 2015;Minter, Lowe, Brockhurst, & Watts, 2015;Sildever, Sefbom, Lips, & Godhe, 2016).…”

“…Through asqPCR using our microsatellites, we followed the dynamics of change in relative strain abundances in the multistrain populations using filter samples taken at three time-points (t1, t2, tfin). Previously, this method has only been used to quantify the relative abundance of pairs of genotypes (John et al, 2015;Minter, Lowe, Brockhurst, & Watts, 2015;Sildever, Sefbom, Lips, & Godhe, 2016). Here, we extended this method to monitor the relative abundances of six genotypes in artificial assemblages.…”