INTRODUCTION
The origins and spread of herbicide resistance in a landscape context
may result from a single (or few) independent sources followed by
dispersal via gene flow versus multiple, localized, independent
evolutionary events (Baucom, 2019). A summary of previous research
indicates that gene flow often contributes similarly to or more than
independent evolution to the occurrence of resistance among populations
(Beckie, Busi, Bagavathiannan, & Martin, 2019). Use of highly effective
herbicides, such as glyphosate, across large areas generates a stark
selection pressure in which individuals with resistance alleles exhibit
high fitness. This widespread selection intensity increases the
probability that rare resistance mutations that arise independently will
increase in frequency rather than be lost via genetic drift, resulting
in a rapid increase in the frequency of a resistance allele following
movement to a new location via pollen or seed-mediated dispersal (Beckie
et al., 2019). Genomic-based epidemiology enables identification of
unique resistance alleles as well as tracking movement of individuals
across a landscape when the mutations associated with herbicide
resistance are known, providing insight into the origins and spread of
resistance mechanisms (Comont & Neve, 2020). Understanding these
patterns of independent origins and movement will provide insights for
improved management practices, such as focusing area-wide management
approaches to prevent sources of seed introduction to new areas when
gene flow is moving resistance, or managing selection pressure on a
field-by-field basis when resistance is evolving independently and
frequently.
To investigate the question of gene flow and independent evolution for
herbicide resistance using genomic-based epidemiology, we used kochia
[Bassia scoparia (L.) A. J. Scott, synonymous with Kochia
scoparia (L.) Schrad.], an introduced weed that occurs in the
semi-arid arable lands of the western United States and Canada (Friesen,
Beckie, Warwick, & Van Acker, 2009). Kochia collections from around
North America exhibit high levels of genetic diversity in the invaded
range and a lack of true population structure (Kumar, Jha, Jugulam,
Yadav, & Stahlman, 2019; Mengistu & Messersmith, 2002). This lack of
structure is most likely due to several reproductive traits that
emphasize cross pollination as well as long-range dispersal of both
pollen and seed (Beckie, Blackshaw, Hall, & Johnson, 2016). Kochia has
protogynous flowers in which the stigmas emerge first and are receptive
to pollen from other flowers before pollen production within the same
flower occurs, reducing the self-pollination rate (Guttieri, Eberlein,
& Thill, 1995). Additionally, kochia is a well-known tumbleweed
species, with some plants dispersing seeds for dozens or even hundreds
of miles (Kumar et al., 2019). This dispersal mechanism greatly
increases the spread of herbicide-resistance alleles and makes
containment extremely difficult (Beckie et al., 2016; Kumar et al.,
2019; Stallings, Thill, Mallory-Smith, & Shafii, 1995).
Herbicide-resistance mechanisms can be classified as either target-site
(mutations or changes in expression of the gene encoding the protein
inhibited by the herbicide) or non-target-site (mechanisms that reduce
the concentration of active herbicide reaching the target site protein)
(Gaines et al., 2020). Target-site mechanisms can be considered
specialist adaptations while non-target-site mechanisms can be
considered generalist mechanisms, as they can sometimes confer
resistance across different herbicide modes of action (Baucom, 2019).
Evolution of resistance to the herbicide glyphosate has included both
target-site and non-target-site mechanisms, including the parallel
evolution of increased copy number of the gene encoding the glyphosate
target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in
multiple species (Gaines, Patterson, & Neve, 2019; Patterson, Pettinga,
Ravet, Neve, & Gaines, 2018).
The first report of glyphosate-resistant (GR) kochia was from Kansas in
2007 (Waite et al., 2013) and reports have since confirmed glyphosate
resistance in multiple US states and Canadian provinces (Kumar et al.,
2019). The widespread regional evolution of GR kochia has negatively
impacted the sustainability of reduced-tillage weed management and
moisture and soil conservation during fallow periods in the Central
Great Plains of North America (Kumar et al., 2019). The mechanism of
glyphosate resistance has been thoroughly investigated in kochia, in
terms of physiology and fitness penalty as well as the genetic
mechanisms that cause resistance (Beckie et al., 2018; Kumar & Jha,
2015; Martin et al., 2017; Osipitan & Dille, 2017; Wiersma et al.,
2015). Increased copy number of the EPSPS gene has been
identified as the resistance mechanism in all studied kochia populations
to date from across seven US states (Montana, Wyoming, Oregon, Idaho,
Nebraska, Kansas, and Colorado) (Gaines et al., 2016; Godar, Stahlman,
Jugulam, & Dille, 2015; Kumar, Felix, Morishita, & Jha, 2018; Kumar,
Jha, Giacomini, Westra, & Westra, 2015; Wiersma et al., 2015). In
kochia, EPSPS gene duplications are tandem and occur at a single
locus (Jugulam et al., 2014; Patterson et al., 2019).
The EPSPS locus has been sequenced from a single GR kochia
individual using BAC libraries. The EPSPS repeat unit was
variable with two units being most common; 1) a full-length repeat
containing EPSPS and six other flanking genes and 2) a less
frequent form containing EPSPS and only three other flanking
genes (Patterson et al., 2019). A ~15 kb mobile genetic
element (MGE) was found inserted flanking both upstream and downstream
of the entire tandem duplication. Additionally, a copy of the MGE was
found between every repeat unit, indicating the MGE has been
co-duplicated after subsequent crossing over events (Patterson et al.,
2019). Once the structure of the repeat was determined, quantitative PCR
markers that specifically amplify the two types of repeats and the MGE
were developed to confirm the sequence and measure the copy number of
each part of the repeat structure.
Due to long distance gene dispersal in kochia and its prevalence in
cropping systems where it can be carried in/on agricultural equipment,
we tested the hypothesis that a single EPSPS gene duplication
event occurred initially in Kansas and then subsequently spread to
initiate all other GR kochia populations and its alternative, that gene
duplication may have occurred multiple times in independent events in a
case of parallel evolution. In this study, we used two approaches to
address our question. First, we used a genomic-based epidemiology
approach with the qPCR markers developed for measuring the various units
of the EPSPS tandem duplication to investigate whether all GR
plants had the same EPSPS repeat structure. Second, we used a
population genetics approach with simple sequence repeat (SSR) markers
to determine the relatedness of GR and glyphosate-susceptible (GS)
kochia populations from across the Central Great Plains, Northern
Plains, and the Pacific Northwest. Combined, these approaches provide
insight into the origins and spread of glyphosate resistance alleles in
kochia.