Two A. tuberculatus were the originating populations in this study. This species is an obligate outcrosser so inbred lines could not be developed to establish parents for the inheritance studies. The seeds of the R population were collected in 2014 from a field in Platte County, NE, where mesotrione resistance was confirmed (Oliveira et al., 2017a). The S population seeds were collected from Dixon County, NE; this population was known to have high sensitivity to mesotrione. For both populations, seeds were collected from at least 20 plants. The seeds were cleaned and stored separately (R and S populations) at 5 C until used in the greenhouse study in 2015 and 2016 at the University of Nebraska-Lincoln. Seeds were planted in 1200 cm³ plastic pots for pairwise crosses containing peat:soil:sand:vermiculite (4:2:2:2) potting mix. In addition, seeds were planted in 164 cm³ cone-tainers (Ray Leach “Cone-tainer” SC10®, Stuewe and Sons Inc, Tangent, OR, USA) for dose-response and segregation analysis with the same potting mix described. Plants were supplied with adequate water and kept under greenhouse conditions at 28/20 C day/night temperature with 80% relative humidity. In addition, twice per week, plants were fertilized with 3 mg of NPK (20-10-20 Peters® Professional, JR Peters Inc., Allentown, PA, USA) for each 100 cm³ of the potting mix until plants were 8–10 cm tall. Artificial lighting was provided using metal halide lamps (600-μmol photons m⁻² s⁻¹) to ensure a 15-h photoperiod.

The R population was grown from seeds in a greenhouse and selected with a POST application of the recommended rate of 105 g ai ha⁻¹ of mesotrione (Callisto®, Syngenta Crop Protection, Research Triangle Park, NC, USA) when R seedlings were 10–15 cm tall. The recommended mesotrione rate caused low injury (<30%) on R plants. Therefore, the R plants were kept for individual pairwise crosses with the S plants. Single S and R plants growing in pots were paired according to floral synchrony and enclosed with pollination bags (100 by 185-cm) to exclude external pollen. Male plant inflorescences were gently shaken in the morning during pollination period (~3 weeks) to ensure cross-fertilization between S and R individuals. At maturity, seeds were collected from each female plant, cleaned, and bagged separately, then designated as F1 families (Figure 1).

The F1 families were derived from 20 parental crosses. Four F1 families were advanced to the next generation of crosses (Table S1), including three S × R and one R × S (female × male). The F1 plants were termed RS/F1-5, SR/F1-8, SR/F1-9, and SR/F1-13 (Table S1). The R parent of the SR/F1-13 family was derived from an R × R cross of the R population seeds, and the R × R cross was performed under greenhouse conditions, similarly as described for the S × R cross. The remaining F1 families were derived from field collected R and S seeds. The F1 plants were treated with 105 g ai ha⁻¹ of mesotrione when plants were 10 to 15 cm tall. The F1 plants survived with variable mesotrione injury (data not shown) confirming that they were crosses between R and S. A. tuberculatus is dioecious, preventing F1 self-pollination to produce true F2 plants (Figure 1). Therefore, the F1 family SR/F1-9 and SR/F1-13 individuals were separately cross-pollinated using the procedures described above to make pseudo-F2 plants (hereinafter referred as F2). The F2 plants were designed F2-9 (from SR/F1-9) and F2-13 (from SR/F1-13). In addition, F1 male plants were also allowed to pollinate the parental susceptible female plants (S) to produce backcross susceptible (BC/S) families (Figure 1). Three BC/S families were made and designed BC-8/S (from SR/F1-8), BC-9/S (from SR/F1-9), and BC-13/S (from SR/F1-13; Table S1).

The greenhouse experiments were set in completely randomized design with four replications and repeated twice in 2016 at the University of Nebraska-Lincoln. Mesotrione dose-response studies were conducted separately with R and S (parent); and F1 families, including RS/F1-5, SR/F1-9, and SR/F1-13.

The mesotrione dose was 0, 0.125×, 0.25×, 0.5×, 1×, 2×, 4×, 8×, 16×, where 1× represents 105 g ai ha⁻¹ (labeled use rate of mesotrione). Mesotrione was mixed with 1% (v/v) crop oil concentrate (Agri-Dex®, Helena Chemical Co, Collierville, TN, USA) and 20.5 g L⁻¹ of ammonium sulfate (DSM Chemicals North America Inc., Augusta, GA, USA). Herbicide treatments were applied with a single tip chamber sprayer (DeVries Manufacturing Corp, Hollandale, MN, USA) fitted with an 8001 E nozzle (Spraying Systems Co., North Avenue, Wheaton, IL 60139), calibrated to deliver 140 L ha⁻¹ spray volume at 210 kPa at a speed of 3.7 km h⁻¹. Control was assessed visually 21 d after treatment (DAT) using a scale of 0 to 100% (where 0 indicates no injury and 100 indicates plant death). Control ratings were based on symptoms such as bleaching, necrosis, and stunting of plants compared to non-treated control plants. Aboveground biomass was harvested at 21 DAT from each experimental unit and oven-dried at 65 C until reaching constant weight before the weight of biomass (g plant⁻¹) was recorded.

The effective mesotrione doses needed to control and reduce biomass by 50% (ED₅₀) and 90% (ED₉₀) for the parental and F1 families were determined using the three-parameter log-logistic curve of the drc package of the R statistical software (Knezevic et al., 2007).

In this model, Y is the control (%) or biomass reduction (g plant⁻¹), d is the upper limit, and e represents the ED₅₀ value relative to d. The parameter b is the relative slope around the parameter e, and x is the mesotrione dose in g ai ha⁻¹.

The resistance level was calculated by dividing the effective dose for providing 50% control or 50% biomass reduction (ED₅₀) of the R or F1 populations by the ED₅₀ of the S population. The resistance level indices for the respective ED₅₀ between the R or F1, and the S were compared using the EDcomp function of package drc in R software (Ritz and Streibig, 2005). The EDcomp function compares the ratio of ED₅₀ using t-statistics, where P-value < 0.05 indicates that herbicide ED₅₀ values are different between the R or F1 and the S.

The degree of dominance (D) was calculated following the formula

Where D is the degree of dominance and X₁, X₂, and X₃ are the log(ED₅₀) of R, F1, and S population, respectively. When D = 0, the F1 has a resistance level that is the mean of the R and S (additive). When D = 1 there is an indication of complete dominance, 0 < D < 1 supports the model of incomplete dominance, −1 < D < 0 of incomplete recessive, and D = −1 of complete recessive (Stone, 1968).

The R and S parent, SR/F1 (SR/F1-8, SR/F1-9, and SR/F1-13), F2 (F2-9 and F2-13) and BC/S (BC-8/S, BC-9/S, and BC-13/S) families were treated with three mesotrione doses attempting to discriminate S from R plants. These doses were 26, 105, and 420 g ai ha⁻¹, which represent 0.25×, 1×, and 4× the recommended rate, respectively. The 0.25× dose was chosen to assess segregation at a below-label dose. The 1× dose was chosen to evaluate segregation at recommended label dose at which plants are selected under field conditions. The higher dose (4×) was selected to likely allow mesotrione resistance segregation to occur with minimal interference of minor resistance loci (Busi et al., 2014). There were two runs (repetitions in time) of this experiment for each mesotrione dose, and each run included BC/S and F2 populations represented by 29 to 98, and 56 to 98 plants, respectively. In addition, parent (R and S) populations were represented by 8 to 17 plants, and F1 populations were represented by 8 to 24 plants. A total of 2,908 A. tuberculatus plants were screened with mesotrione in this study. Herbicide application was the same as described above in dose-response studies. At 21 DAT, all populations were visually evaluated and assessed as dead or alive. Alive plants were separated into different injury level groups, including low (<40%), medium (41 to 79%), high (80 to 98%), and dead (>98%; Figure S1). Plant aboveground biomass was harvested, oven-dried until constant weight, and biomass (g plant⁻¹) was recorded. The plant biomass and injury level for parental, SR/F1, F2, and BC/S families were represented in violin plots with a rotated kernel density plot using package ggplot2 in R statistical software (Wickham, 2009).

The experimental null hypothesis was that mesotrione resistance segregates as controlled by a single major gene (one locus). The segregation analysis in F2 and BC/S families was based on the observed survival ratio (alive/total treated plants) compared to expected survival assuming one gene locus segregation (Table S2). For the F2 and BC/S families, the expected number of surviving plants was described by the following equations (Tabashnik, 1991; Busi et al., 2013; Han et al., 2014):

F2: One gene locus model (1R:2F1:1S),

BC/S: One gene locus model (1F1:1S),

where R, F1, and S are the number of observed surviving plants of the R, F1, and S families at each mesotrione dose (26, 105, and 420 g ai ha⁻¹). Thus, for each dose, the expected number of survivors for F2 and BC/S populations was calculated with the total number of treated plants multiplied by the theoretical one locus segregation ratio for F2 and BC/S families.

A chi-square goodness-of-fit test (χ²) was used to compare the observed and expected plant survival based on a one locus segregation model. The P-values were obtained to indicate the probability in rejecting the null hypothesis for F2 and BC/S families at one locus segragation. For example, for one locus segregation, the null hypothesis (H₀) is that the BC segregates as 0.5F1:0.5S. The significant level is α = 0.05 and if P-value < 0.05, the null hypothesis is rejected.

The R population displayed a high level of resistance whereas S was susceptible (Figures 2A,B). The level of resistance of R compared to S was 19-fold based on ED₅₀ values (Table 1). The generated F1 families provided an intermediate and relatively lower level of resistance (3-4-fold) based on ED₅₀ values, except the SR/F1-13 family (11-fold). The biomass (g plant⁻¹) of parental and F1 families corroborated with visual control, resulting in similar ED₅₀, ED₉₀, and resistance levels (Table 2).

The F1 families expressed a variable degree of dominance (D). For example, based on control, the degree of dominance of SR/F1-13 family was 0.62 (Table 3). In contrast, the SR/F1-9 family had a negative degree of dominance (D = −0.22). The RS/F1-5 family had a degree of dominance close to zero (D = −0.04). The pooled-F1 family resulted in D = 0.11. Similar degree of dominance trend was also observed with plant response to mesotrione based on biomass (g plant⁻¹) of the F1 families (Table 3).

The segregation at below-label mesotrione dose (26 g ai ha⁻¹) resulted in 77% survival of S plants when averaging both runs, and survived S plants showed high injury (>80%; Figure 2A). In addition, the 26 g ai ha⁻¹ resulted in high survival rate (≥87%) of the SR/F1, F2, and BC/S families across runs. Therefore, this dose was not the best to describe a segregation analysis for this R population. Nonetheless, a single major gene model would not fit (P-value < 0.05) the majority of F2 and BC/S families (Table 4).

The recommended (105 g ai ha⁻¹; Table 5) and a high mesotrione rate (420 g ai ha⁻¹; Table 6) effectively distinguished R and S plant survival. At these two rates, R plants survived with low injury (< 40%), and S plants died (Figures 3C,E). However, F1 family survival and level of injury at 105 and 420 g ai ha⁻¹ varied within families (Figures 3D,F). At mesotrione rate of 105 g ai ha⁻¹, segregation in each family deviated from the corrected 1R:2F1:S (F₂) and 1F1:1S (BC) ratios expected for one major gene in the two experimental runs (Table 5). Two and three loci segregation model tested fitted for most of F2 and BC/S, families (data not shown). For 420 g ai ha⁻¹, only F2-13 (first run) and BC-8/S (second run) families did not deviate from the expected one major locus (Table 6). However, BC-8/S (first and second run), F2-13 (second run), and BC-13/S (first run) also did not deviate from two and three loci segregation (data not shown). In general, at 105 and 420 g ai ha⁻¹ doses, the plant mortality was variable but higher than expected.

The plant biomass (g) and injury levels (%) of the segregation analysis at different mesotrione doses on A. tuberculatus families was illustrated with combined (Figures 3A,C,E) or separated (Figures 3B,D,F) violin plots. The plant biomass of the generated families overlapped the R and S parent phenotypic ranges. The violin plots showed either continuous or bell-shaped distribution of biomass (g) across families and mesotrione rates (Figures 3A–F). For example, at 105 g ai ha⁻¹ of mesotrione, the F2-9 family showed a majority of plants with low injury and highly variable biomass (Figure 3D). The F2-13 family had a medium to high injury with relatively low uniform biomass. Nonetheless, the F2 families resulted in high plant survival (Table 5). In contrast, the BC/S families resulted in a uniform relatively low biomass across families and mesotrione doses (Figures 3B,D,F). In addition, the shape of the phenotype was more continuous and thinner as the mesotrione dose increased (Figures 3A–F).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.