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).