Genomic-based epidemiology using EPSPS copy number and associated duplication markers
Genomic DNA from 113 individuals representing 27 of the populations evaluated for glyphosate resistance above, along with 36 individuals representing 11 populations used in Gaines et al. (2016) and 58 individuals representing 18 populations collected in Montana, was used for real time PCR to measure the relative copy number of genomic EPSPS , as well as the longer type I (56.1 kb) and shorter type II (32.7 kb) segments associated with EPSPSduplication, and the mobile genetic element (MGE) Fhy3/FAR1 (Patterson et al., 2019). Primer sequences for these features as well as the normalization gene Acetolactate Synthase (ALS ) were 1) EPSPS, For (5’-CGCTATATGTTGGATGCTCTAAG-3’), Rev (5’-CACTCCTATTCTCTTTACCAGC-3’); 2) Type I (56.1 kb), For (5’-GACGGAAATACCCTCAATATAGACA-3’), Rev (5’-ACGCCCAAGATGTACATTGATA-3’); 3) Type II (32.7 kb), For (5’-GACGGAAATACCCTCAATATAGACA-3’), Rev (5’-CATGCCTTTGATGTCCAAGTTT-3’); 4) MGE, For (5’-GAAGATAGCGAGACGTTTGAG-3’), Rev (5’-CGGCTTGATCGGTTAAGATAC-3’); and 5) ALS, For (5’-CCAGAAAAGGCTGCGATG-3’), Rev (5’-CTGACTCGCTCTGATTCCA-3’). A GR control (population M32) with high EPSPS copy number, presence of both type I and type II EPSPS duplication segments, and an increased copy number of MGE was included along with a susceptible control (7710) containing a single copy of EPSPS , no copies of type I or II markers, and a low copy number of MGE. The qPCR protocol of Patterson et al. (2019) was used and relative copy number was calculated using the ∆Ct method (Schmittgen & Livak, 2008). The type I and type IIEPSPS duplication qPCR primers have a forward primer in the MGE and a reverse primer in either the type I or type II sequence, respectively, enabling amplification only when the MGE is located next to the type I or type II repeat segment. Using these markers, we quantified the number of type I (56.1 kb) or type II (32.7 kb) repeats in individuals from multiple populations. Three haplotypes were defined based on the four qPCR markers, using EPSPS >1.4 to define increased EPSPS , type I and type II >0 to define presence of the two markers of EPSPS gene duplication described in Patterson et al. (2019), and MGE <10 defined as ‘normal’ and ≥10 defined as ‘increased’ MGE. Populations and their haplotypes and geographic locations were plotted in R using ggplot and sf packages (R, 2019).