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).