CFM surveying, specimen collection, and DNA extraction
A comprehensive survey for CFM was conducted throughout the canola-producing regions of Alberta, Saskatchewan, and Manitoba in 2017 and 2018 (Vankosky et al. in prep ). Surveyors visited 546 fields, from the northern limit of canola production to the southern limit of CFM range in Alberta and Saskatchewan. In Manitoba, the survey was mostly limited to the agricultural extent in the northwest of the province, with the exception of a single, additional site in Portage la Prairie. At each site, 100 canola racemes along the edge of each field were examined. All galled flowers found were collected and returned to the laboratory in a refrigerated container. In the laboratory, buds were dissected, and larvae were placed into individual 2 ml tubes and frozen at -80 °C (Vankosky et al. in prep ). From all survey results, we subsampled sites for genetic analysis by selecting the sites that had the highest CFM densities, defined as any location where more than four larvae were sampled. Our genetic sampling also aimed to maximize the geographic scope across the range of CFM.
Genomic DNA was extracted from whole specimens sampled at 16 localities (Table S1) using a QIAamp DNA Micro Kit (QIAGEN). The final DNA concentration of each sample (either with or without WGA, see below) was standardized to 20 ng/µl for library preparation following the two-enzyme genotyping-by-sequencing (GBS) method of Poland et al.(2012).