Male-specific Y-chromosomal regions in waterhemp (Amaranthus tuberculatus) and Palmer amaranth (Amaranthus palmeri).

Amaranthus tuberculatus and Amaranthus palmeri are agronomically important weed species, both with stable dioecious reproductive systems. An understanding of the genetic basis of sex determination may lead to new methods of managing these troublesome weeds. Previous research identified genomic sequences associated with maleness in each species. Male-specific sequences were used to identify genomic regions in both species that are believed to contain sex-determining genes, i.e. the male-specific Y (MSY) region. These regions were compared to understand if sex determination is controlled via the same physiological pathway and if dioecy evolved independently. A contiguously assembled candidate MSY region identified in Amaranthus palmeri is approximately 1.3 Mb with 121 predicted gene models. In Amaranthus tuberculatus, several contigs, with combined length of 4.6 Mb and with 147 gene models, were identified as belonging to the MSY region. Synteny was not detected between the two species' candidate MSY regions but they shared two predicted genes. With lists of candidate genes for sex determination containing fewer than 200 in each species, future research can address whether sex determination is controlled via similar physiological pathways and whether dioecy has indeed evolved independently in these species.

Evolution of Glyphosate-Resistant Weeds.

Widespread adoption of glyphosate-resistant crops and concomitant reliance on glyphosate for weed control set an unprecedented stage for the evolution of herbicide-resistant weeds. There are now 48 weed species that have evolved glyphosate resistance. Diverse glyphosate-resistance mechanisms have evolved, including single, double, and triple amino acid substitutions in the target-site gene, duplication of the gene encoding the target site, and others that are rare or nonexistent for evolved resistance to other herbicides. This review summarizes these resistance mechanisms, discusses what is known about their evolution, and concludes with some of the impacts glyphosate-resistant weeds have had on weed management.

Resistance to acetolactate synthase inhibitors is due to a W 574 to L amino acid substitution in the ALS gene of redroot pigweed and tall waterhemp.

Several Amaranthus spp. around the world have evolved resistance (and cross resistance) to various herbicide mechanisms of action. Populations of redroot pigweed (RRPW-R) and tall waterhemp (TW-R) in Mississippi, USA have been suspected to be resistant to one or more acetolactate synthase (ALS) inhibiting herbicides. Whole plant dose-response experiments with multiple ALS inhibitors, ALS enzyme assays with pyrithiobac, and molecular sequence analysis of ALS gene constructs were conducted to confirm and characterize the resistance profile and nature of the mechanism in the RRPW-R and TW-R populations. Two susceptible populations, RRPW-S and TW-S were included for comparison with RRPW-R and TW-R, correspondingly. The resistance index (R/S; the herbicide dose required to reduce plant growth by 50% of resistant population compared to the respective susceptible population) values of the RRPW-R population were 1476, 3500, and 900 for pyrithiobac, imazaquin, and trifloxysulfuron, respectively. The R/S values of the TW-R population for pyrithiobac, imazaquin, and trifloxysulfuron were 51, 950, and 2600, respectively. I50 values of RRPW-S and RRPW-R populations for pyrithiobac were 0.062 and 208.33 µM, indicating that the ALS enzyme of the RRPW-R population is 3360-fold more resistant to pyrithiobac than the RRPW-S population under our experimental conditions. The ALS enzyme of the TW-R population was 1214-fold resistant to pyrithiobac compared to the TW-S population, with the I50 values for pyrithiobac of ALS from TW-R and TW-S populations being 87.4 and 0.072 µM, correspondingly. Sequencing of the ALS gene identified a point mutation at position 574 of the ALS gene leading to substitution of tryptophan (W) residue with a leucine (L) residue in both RRPW-R and TW-R populations. Thus, the RRPW-R and TW-R populations are resistant to several ALS-inhibiting herbicides belonging to different chemical classes due to an altered target site, i.e., ALS. Resistance in Amaranthus spp. to commonly used ALS-inhibiting herbicides warrants an integrated weed management scheme incorporating chemical, mechanical, and cultural strategies by growers.

Coexpression Clusters and Allele-Specific Expression in Metabolism-Based Herbicide Resistance.

In the last decade, Amaranthus tuberculatus has evolved resistance to 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-hydroxyphenylpyruvate dioxygenase inhibitors in multiple states across the midwestern United States. Two populations resistant to both mode-of-action groups, one from Nebraska (NEB) and one from Illinois (CHR), were studied using an RNA-seq approach on F2 mapping populations to identify the genes responsible for resistance. Using both an A. tuberculatus transcriptome assembly and a high-quality grain amaranth (A. hypochondriacus) genome as references, differential transcript and gene expression analyses were conducted to identify genes that were significantly over- or underexpressed in resistant plants. When these differentially expressed genes (DEGs) were mapped on the A. hypochondriacus genome, physical clustering of the DEGs was apparent along several of the 16 A. hypochondriacus scaffolds. Furthermore, single-nucleotide polymorphism calling to look for resistant-specific (R) variants, and subsequent mapping of these variants, also found similar patterns of clustering. Specifically, regions biased toward R alleles overlapped with the DEG clusters. Within one of these clusters, allele-specific expression of cytochrome  P450  81E8 was observed for 2,4-D resistance in both the CHR and NEB populations, and phylogenetic analysis indicated a common evolutionary origin of this R allele in the two populations.

Resistance to clethodim in Italian ryegrass (Lolium perenne ssp. multiflorum) from Mississippi and North Carolina.

BACKGROUND: Clethodim, an acetyl-CoA carboxylase (ACCase)-inhibiting herbicide, is one of the few postemergence chemical control options available to growers of Mississippi to manage glyphosate and/or other herbicide resistant Italian ryegrass populations. Recently, clethodim failed to adequately control Italian ryegrass populations across Mississippi. A sethoxydim, also an ACCase inhibitor, -resistant Italian ryegrass population from North Carolina was cross-resistant to clethodim. This research characterized the magnitude and mechanisms of clethodim resistance in the Mississippi and North Carolina Italian ryegrass populations via whole-plant herbicide dose response, cross resistance, and metabolism studies, and molecular analysis. RESULTS: Two clethodim-resistant biotypes from Mississippi, MS24 and MS37, were 10- and 4-fold resistant, respectively, relative to a susceptible (SUS1) biotype. A North Carolina biotype, NC21, was 40-fold resistant to clethodim compared to SUS1. Two additional biotypes from North Carolina, NC22 and NC 23, recorded shoot dry weight reduction of only 17-30% of nontreated at the highest clethodim dose of 2.17 kg ha-1 , (8×). The NC22 biotype was cross-resistant to sethoxydim, fluazifop, quizalofop, and pinoxaden. Metabolic inhibitors such as piperonyl butoxide and 4-chloro-7-nitrobenzofurazan did not affect resistance of MS37, MS51, and NC22 biotypes to fenoxaprop, clethodim, or pinoxaden. The MS37 biotype had three target site mutations, I2041N, C2088R, and G2096A. Another clethodim-resistant biotype from Mississippi, MS51, had only the C2088R substitution. The NC22 and NC23 biotypes had I1781L, I2041N, and D2078G replacements. CONCLUSION: This study shows that the mechanism of resistance to clethodim in Italian ryegrass from Mississippi and North Carolina is due to target site modifications in the ACCase gene leading to broad cross-resistance to other ACCase-inhibiting herbicides. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.

Optimizing RNA-seq studies to investigate herbicide resistance.

Transcriptomic profiling, specifically via RNA sequencing (RNA-seq), is becoming one of the more commonly used methods for investigating non-target site resistance (NTSR) to herbicides due to its high throughput capabilities and utility in organisms with little to no previous sequence information. A review of the weed science RNA-seq literature revealed some basic principles behind generating quality data from these types of studies. First, studies that included more replicates per biotype and took steps to control for genetic background had significantly better control of false positives and, consequently, shorter lists of potential resistance genes to sift through. Pooling of biological replicates prior to sequencing was successful in some cases, but likely contributed to an overall increase in the false discovery rate. Although the inclusion of herbicide-treated samples was common across most of the studies, it ultimately introduced difficulties in interpretation of the final results due to challenges in capturing the right sampling window after treatment and to the induction of stress responses in the injured herbicide-sensitive plants. RNA-seq is an effective tool for NTSR gene discovery, but careful consideration should be given to finding the most powerful and cost-effective balance between replicate number, sequencing depth and treatment number. © 2017 Society of Chemical Industry.

Two new PPX2 mutations associated with resistance to PPO-inhibiting herbicides in Amaranthus palmeri.

BACKGROUND: Resistance to herbicides that inhibit protoporphyrinogen oxidase (PPO) is a widespread and growing problem for weed managers across the midwestern and midsouthern United States. In Amaranthus spp., this resistance is known to be conferred by a glycine deletion at the 210th amino acid (ΔG210) in PPO2. Preliminary analysis indicated that the ΔG210 mutation did not fully account for observed resistance to PPO inhibitors in two Amaranthus palmeri populations from Tennessee and one from Arkansas. RESULTS: Sequencing PPX2 cDNA from six resistant plants uncovered two new mutations at the R98 site (R98G and R98M), a site previously found to endow PPO-inhibitor resistance in Ambrosia artemisiifolia. Sequencing of this region from additional plants sprayed with 264 g fomesafen ha-1 showed the presence of one or both R98 mutations in a subset of the resistant plants from all three populations. No plants sensitive to fomesafen contained either mutation. A derived cleaved amplified polymorphic sequence (dCAPS) assay to test for the presence of these mutations in A. palmeri was developed. CONCLUSION: Two new mutations of PPX2 (R98G, R98M) likely confer resistance to PPO-inhibitors in A. palmeri, and can be rapidly identified using a dCAPS assay. © 2017 Society of Chemical Industry.