Population Genetic Structure in Glyphosate-Resistant and -Susceptible Palmer Amaranth (Amaranthus palmeri) Populations Using Genotyping-by-sequencing (GBS)

Abstract

Palmer amaranth (Amaranthus palmeri) is a major weed in United States cotton and soybean production systems. Originally native to the Southwest, the species has spread throughout the country. In 2004 a population of A. palmeri was identified with resistance to glyphosate, a herbicide heavily relied on in modern no-tillage and transgenic glyphosate-resistant (GR) crop systems. This project aims to determine the degree of genetic relatedness among eight different populations of GR and glyphosate-susceptible (GS) A. palmeri from various geographic regions in the United States by analyzing patterns of phylogeography and diversity to ascertain whether resistance evolved independently or spread from outside to an Arizona locality (AZ-R). Shikimic acid accumulation and EPSPS genomic copy assays confirmed resistance or susceptibility. With a set of 1,351 single nucleotide polymorphisms (SNPs), discovered by genotyping-by-sequencing (GBS), UPGMA phylogenetic analysis, principal component analysis, Bayesian model-based clustering, and pairwise comparisons of genetic distances were conducted. A GR population from Tennessee and two GS populations from Georgia and Arizona were identified as genetically distinct while the remaining GS populations from Kansas, Arizona, and Nebraska clustered together with two GR populations from Arizona and Georgia. Within the latter group, AZ-R was most closely related to the GS populations from Kansas and Arizona followed by the GR population from Georgia. GR populations from Georgia and Tennessee were genetically distinct from each other. No isolation by distance was detected and A. palmeri was revealed to be a species with high genetic diversity. The data suggest the following two possible scenarios: either glyphosate resistance was introduced to the Arizona locality from the east, or resistance evolved independently in Arizona. Glyphosate resistance in the Georgia and Tennessee localities most likely evolved separately. Thus, modern farmers need to continue to diversify weed management practices and prevent seed dispersal to mitigate herbicide resistance evolution in A. palmeri.

Keywords: Palmer amaranth; SNP molecular markers; genetic relatedness; glyphosate; herbicide resistance; phylogeography; population genetics.

FIGURE 1

Correlation of shikimate accumulation and EPSPS genomic copy number in all individuals of each of the glyphosate-resistant and -susceptible A. palmeri populations. Shikimate accumulation was measured after incubation in 500 μm glyphosate in an in vivo leaf disk assay Increase in genomic copy number of EPSPS is relative to ALS as measured using qPCR on genomic DNA (A). EPSPS genomic copy number by population (B).

FIGURE 2

Unrooted UPGMA consensus tree after 1,000 bootstrap replications depicting the relationships of A. palmeri individuals from eight populations. Bootstrap values > 70% at nodes are indicated.

FIGURE 3

Clustering of A. palmeri populations based on principal component analysis (PCA) using the filtered and pruned whole dataset of 1,351 SNPs. Analysis was done on all eight populations (A) and on a subset of populations removing the three outlier groups AZ-S2, GA-S, and TN-R (B). Each point represents an individual colored according to the collection site. Glyphosate-resistant individuals are marked by filled symbols and susceptible individuals are marked by empty symbols. Individuals from the same U.S. state have the same symbols.

FIGURE 4

Population structure analysis with K = 4, K = 6, and K = 8 based on 1,351 SNPs of eight A. palmeri populations. Each individual is represented by a vertical bar that is divided by K colored segments representing the likelihood of a membership to each cluster.

FIGURE 5

Clustering of A. palmeri populations based on principal component analysis (PCA) for SNPs found in all eight populations in the chloroplast genome (A) and the mitochondrial genome (B) using the filtered and pruned whole dataset. Each point represents an individual colored according to the collection site. Glyphosate-resistant individuals are marked by filled symbols and susceptible individuals are marked by empty symbols. Individuals from the same U.S. state have the same symbols.

FIGURE 6

Population structure analysis with K = 4, K = 5, and K = 8 based on SNPs from the chloroplast genome for eight A. palmeri populations (A) and K = 5, and K = 8 based on SNPs from the mitochondrial genome for eight A. palmeri populations (B). Each individual is represented by a vertical bar that is divided by K colored segments representing the likelihood of a membership to each cluster.