Enhanced rates of herbicide metabolism in low herbicide‐dose selected resistant Lolium rigidum

Lolium rigidum is an obligately cross‐pollinated, genetically diverse species and an economically important herbicide resistance‐prone weed. Our previous work has demonstrated that recurrent selection of initially susceptible L. rigidum populations with low herbicide rates results in rapid herbicide resistance evolution. Here we report on the mechanisms endowing low‐dose‐selected diclofop‐methyl resistance in L. rigidum. Results showed that resistance was not due to target‐site ACCase mutations or overproduction, or differential herbicide leaf uptake and translocation. The in vivo de‐esterification of diclofop‐methyl into phytotoxic diclofop acid was rapid and similar in resistant versus susceptible populations. However, further metabolism of diclofop acid into non‐toxic metabolites was always faster in resistant plants than susceptible plants, resulting in up to 2.6‐fold lower level of diclofop acid in resistant plants. This corresponded well with up to twofold higher level of diclofop acid metabolites in resistant plants. The major polar metabolites of diclofop acid chromatographically resembled those of wheat, a naturally tolerant species. Clearly, recurrent selection at reduced herbicide rates selected for non‐target‐site‐based enhanced rates of herbicide metabolism, likely involving cytochrome P450 monooxygenases.     

Cytochrome P450 CYP81A10v7 in Lolium rigidum confers metabolic resistance to herbicides across at least five modes of action

Rapid and widespread evolution of multiple herbicide resistance in global weed species endowed by increased capacity to metabolize (degrade) herbicides (metabolic resistance) is a great threat to herbicide sustainability and global food production. Metabolic resistance in the economically damaging crop weed species Lolium rigidum is well known but a molecular understanding has been lacking. We purified a metabolic resistant (R) subset from a field evolved R L. rigidum population. The R, the herbicide susceptible (S) and derived F₂ populations were used for candidate herbicide resistance gene discovery by RNA sequencing. A P450 gene CYP81A10v7 was identified with higher expression in R vs. S plants. Transgenic rice overexpressing this Lolium CYP81A10v7 gene became highly resistant to acetyl-coenzyme A carboxylase- and acetolactate synthase-inhibiting herbicides (diclofop-methyl, tralkoxydim, chlorsulfuron) and moderately resistant to hydroxyphenylpyruvate dioxygenase-inhibiting herbicide (mesotrione), photosystem II-inhibiting herbicides (atrazine and chlorotoluron) and the tubulin-inhibiting herbicide trifluralin. This wide cross-resistance profile to many dissimilar herbicides in CYP81A10v7 transgenic rice generally reflects what is evident in the R L. rigidum. This report clearly showed that a single P450 gene in a cross-pollinated weed species L. rigidum confers resistance to herbicides of at least five modes of action across seven herbicide chemistries.     

Distinct non-target site mechanisms endow resistance to glyphosate,ACCase and ALS-inhibiting herbicides in multiple herbicide-resistant <Emphasis Type="Italic">Lolium rigidum</Emphasis>

This study investigates mechanisms of multiple resistance to glyphosate, acetyl-coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS)-inhibiting herbicides in two Lolium rigidum populations from Australia. When treated with glyphosate, susceptible (S) plants accumulated 4- to 6-fold more shikimic acid than resistant (R) plants. The resistant plants did not have the known glyphosate resistance endowing mutation of 5-enolpyruvylshikimate-3 phosphate synthase (EPSPS) at Pro-106, nor was there over-expression of EPSPS in either of the R populations. However, [¹⁴C]-glyphosate translocation experiments showed that the R plants in both populations have altered glyphosate translocation patterns compared to the S plants. The R plants showed much less glyphosate translocation to untreated young leaves, but more to the treated leaf tip, than did the S plants. Sequencing of the carboxyl transferase domain of the plastidic ACCase gene revealed no resistance endowing amino acid substitutions in the two R populations, and the ALS in vitro inhibition assay demonstrated herbicide-sensitive ALS in the ALS R population (WALR70). By using the cytochrome P450 inhibitor malathion and amitrole with ALS and ACCase herbicides, respectively, we showed that malathion reverses chlorsulfuron resistance and amitrole reverses diclofop resistance in the R population examined. Therefore, we conclude that multiple glyphosate, ACCase and ALS herbicide resistance in the two R populations is due to the presence of distinct non-target site based resistance mechanisms for each herbicide. Glyphosate resistance is due to reduced rates of glyphosate translocation, and resistance to ACCase and ALS herbicides is likely due to enhanced herbicide metabolism involving different cytochrome P450 enzymes.     

Biochemical characterization of metabolism‐based atrazine resistance in Amaranthus tuberculatus and identification of an expressed GST associated with resistance

Rapid detoxification of atrazine in naturally tolerant crops such as maize (Zea mays) and grain sorghum (Sorghum bicolor) results from glutathione S‐transferase (GST) activity. In previous research, two atrazine‐resistant waterhemp (Amaranthus tuberculatus) populations from Illinois, U.S.A. (designated ACR and MCR), displayed rapid formation of atrazine‐glutathione (GSH) conjugates, implicating elevated rates of metabolism as the resistance mechanism. Our main objective was to utilize protein purification combined with qualitative proteomics to investigate the hypothesis that enhanced atrazine detoxification, catalysed by distinct GSTs, confers resistance in ACR and MCR. Additionally, candidate AtuGST expression was analysed in an F₂ population segregating for atrazine resistance. ACR and MCR showed higher specific activities towards atrazine in partially purified ammonium sulphate and GSH affinity‐purified fractions compared to an atrazine‐sensitive population (WCS). One‐dimensional electrophoresis of these fractions displayed an approximate 26‐kDa band, typical of GST subunits. Several phi‐ and tau‐class GSTs were identified by LC‐MS/MS from each population, based on peptide similarity with GSTs from Arabidopsis. Elevated constitutive expression of one phi‐class GST, named AtuGSTF2, correlated strongly with atrazine resistance in ACR and MCR and segregating F₂ population. These results indicate that AtuGSTF2 may be linked to a metabolic mechanism that confers atrazine resistance in ACR and MCR.     

Glyphosate,paraquat and ACCase multiple herbicide resistance evolved in a <Emphasis Type="Italic">Lolium rigidum</Emphasis> biotype

Glyphosate is the world’s most widely used herbicide. A potential substitute for glyphosate in some use patterns is the herbicide paraquat. Following many years of successful use, neither glyphosate nor paraquat could control a biotype of the widespread annual ryegrass (Lolium rigidum), and here the world’s first case of multiple resistance to glyphosate and paraquat is confirmed. Dose–response experiments established that the glyphosate rate causing 50% mortality (LD₅₀) for the resistant (R) biotype is 14 times greater than for the susceptible (S) biotype. Similarly, the paraquat LD₅₀ for the R biotype is 32 times greater than for the S biotype. Thus, based on the LD₅₀ R/S ratio, this R biotype of L. rigidum is 14-fold resistant to glyphosate and 32-fold resistant to paraquat. This R biotype also has evolved resistance to the acetyl-coenzyme A carboxylase (ACCase) inhibiting herbicides. The mechanism of paraquat resistance in this biotype was determined as restricted paraquat translocation. Resistance to ACCase-inhibiting herbicides was determined as due to an insensitive ACCase. Two mechanisms endowing glyphosate resistance were established: firstly, a point mutation in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, resulting in an amino acid substitution of proline to alanine at position 106; secondly, reduced glyphosate translocation was found in this R biotype, indicating a co-occurrence of two distinct glyphosate resistance mechanisms within the R population. In total, this R biotype displays at least four co-existing resistance mechanisms, endowing multiple resistance to glyphosate, paraquat and ACCase herbicides. This alarming case in the history of herbicide resistance evolution represents a serious challenge for the sustainable use of the precious agrochemical resources such as glyphosate and paraquat.     

Precision genome editing in plants via gene targeting and piggyBac‐mediated marker excision

Precise genome engineering via homologous recombination (HR)‐mediated gene targeting (GT) has become an essential tool in molecular breeding as well as in basic plant science. As HR‐mediated GT is an extremely rare event, positive–negative selection has been used extensively in flowering plants to isolate cells in which GT has occurred. In order to utilize GT as a methodology for precision mutagenesis, the positive selectable marker gene should be completely eliminated from the GT locus. Here, we introduce targeted point mutations conferring resistance to herbicide into the rice acetolactate synthase (ALS) gene via GT with subsequent marker excision by piggyBac transposition. Almost all regenerated plants expressing piggyBac transposase contained exclusively targeted point mutations without concomitant re‐integration of the transposon, resulting in these progeny showing a herbicide bispyribac sodium (BS)‐tolerant phenotype. This approach was also applied successfully to the editing of a microRNA targeting site in the rice cleistogamy 1 gene. Therefore, our approach provides a general strategy for the targeted modification of endogenous genes in plants.     

No evidence for early fitness penalty in glyphosate‐resistant biotypes of Conyza canadensis: Common garden experiments in the absence of glyphosate

Strong selection from herbicides has led to the rapid evolution of herbicide‐resistant weeds, greatly complicating weed management efforts worldwide. In particular, overreliance on glyphosate, the active ingredient in RoundUp^®, has spurred the evolution of resistance to this herbicide in ≥40 species. Previously, we reported that Conyza canadensis (horseweed) has evolved extreme resistance to glyphosate, surviving at 40× the original 1× effective dosage. Here, we tested for underlying fitness effects of glyphosate resistance to better understand whether resistance could persist indefinitely in this self‐pollinating, annual weed. We sampled seeds from a single maternal plant (“biotype”) at each of 26 horseweed populations in Iowa, representing nine susceptible biotypes (S), eight with low‐level resistance (LR), and nine with extreme resistance (ER). In 2016 and 2017, we compared early growth rates and bolting dates of these biotypes in common garden experiments at two sites near Ames, Iowa. Nested ANOVAs showed that, as a group, ER biotypes attained similar or larger rosette size after 6 weeks compared to S or LR biotypes, which were similar to each other in size. Also, ER biotypes bolted 1–2 weeks earlier than S or LR biotypes. These fitness‐related traits also varied among biotypes within the same resistance category, and time to bolting was inversely correlated with rosette size across all biotypes. Disease symptoms affected 40% of all plants in 2016 and 78% in 2017, so we did not attempt to measure lifetime fecundity. In both years, the frequency of disease symptoms was greatest in S biotypes and similar in LR versus ER biotypes. Overall, our findings indicate there are no early growth penalty and possibly no lifetime fitness penalty associated with glyphosate resistance, including extremely strong resistance. We conclude that glyphosate resistance is likely to persist in horseweed populations, with or without continued selection pressure from exposure to glyphosate.     

Genetic control of a cytochrome P450 metabolism-based herbicide resistance mechanism in Lolium rigidum

The dynamics of herbicide resistance evolution in plants are influenced by many factors, especially the biochemical and genetic basis of resistance. Herbicide resistance can be endowed by enhanced rates of herbicide metabolism because of the activity of cytochrome P450 enzymes, although in weedy plants the genetic control of cytochrome P450-endowed herbicide resistance is poorly understood. In this study we have examined the genetic control of P450 metabolism-based herbicide resistance in a well-characterized Lolium rigidum biotype. The phenotypic resistance segregation in herbicide resistant and susceptible parents, F1, F2 and backcross (BC) families was analyzed as plant survival following treatment with the chemically unrelated herbicides diclofop-methyl or chlorsulfuron. Dominance and nuclear gene inheritance was observed in F1 families when treated at the recommended field doses of both herbicides. The segregation values of P450 herbicide resistance phenotypic traits observed in F2 and BC families was consistent with resistance endowed by two additive genes in most cases. In obligate out-crossing species such as L. rigidum, herbicide selection can easily result in accumulation of resistance genes within individuals.     

Inheritance of evolved glyphosate resistance in Lolium rigidum (Gaud.)

Resistance to the non-selective herbicide, glyphosate, has evolved recently in several populations of Lolium rigidum (Gaud.). Based upon the observed pattern of inheritance, glyphosate resistant and susceptible populations are most probably homozygous for glyphosate resistance and susceptibility, respectively. When these populations were crossed and the F₁ progeny treated with glyphosate, the dose response behavior was intermediate to that of the parental populations. This observation, coupled with an absence of a difference between reciprocal F₁ populations, suggests that glyphosate resistance is inherited as an incompletely dominant nuclear-encoded trait. The segregation of resistance in F₁×S backcrosses suggests that the major part of the observed resistance is conferred by a single gene, although at low glyphosate treatments other genes may also contribute to plant survival. It appears from this study that a single nuclear gene confers resistance to glyphosate in one population of L. rigidum. Received: 17 May 2000 / Accepted: 1 September 2000     

Base editing with high efficiency in allotetraploid oilseed rape by A3A‐PBE system

CRISPR/Cas‐base editing is an emerging technology that could convert a nucleotide to another type at the target site. In this study, A3A‐PBE system consisting of human A3A cytidine deaminase fused with a Cas9 nickase and uracil glycosylase inhibitor was established and developed in allotetraploid Brassica napus. We designed three sgRNAs to target ALS, RGA and IAA7 genes, respectively. Base‐editing efficiency was demonstrated to be more than 20% for all the three target genes. Target sequencing results revealed that the editing window ranged from C1 to C10 of the PAM sequence. Base‐edited plants of ALS conferred high herbicide resistance, while base‐edited plants of RGA or IAA7 exhibited decreased plant height. All the base editing could be genetically inherited from T₀ to T₁ generation. Several Indel mutations were confirmed at the target sites for all the three sgRNAs. Furthermore, though no C to T substitution was detected at the most potential off‐target sites, large‐scale SNP variations were determined through whole‐genome sequencing between some base‐edited and wild‐type plants. These results revealed that A3A‐PBE base‐editing system could effectively convert C to T substitution with high‐editing efficiency and broadened editing window in oilseed rape. Mutants for ALS, IAA7 and RGA genes could be potentially applied to confer herbicide resistance for weed control or with better plant architecture suitable for mechanic harvesting.     

Inheritance of resistance to anti-microtubule dinitroaniline herbicides in an “intermediate” resistant biotype of Eleusine indica (Poaceae)

Inheritance of resistance to the anti-microtubule dinitroaniline herbicides was investigated in a goosegrass biotype displaying an intermediate level of resistance (I). Reciprocal crosses were made between the I biotype and previously characterized susceptible (S) or resistant (R) biotypes. Eight F₁ hybrids were identified, and F₂ populations were produced by selfing. The dinitroaniline-herbicide response phenotype (DRP) of F₁ plants, and F₂ seedlings was determined using a root-growth bioassay. The DRP of F₁ plants of S × I was “susceptible” (i.e., identical to the S parental plants), and the DRP of F₁ plants of I × R was “intermediate” (i.e., identical to the I parental plants). Nonparental phenotypes were not observed in F₁ plants. Results indicated susceptibility to be dominant over intermediate resistance and intermediate resistance to be dominant over high resistance. Analysis of reciprocal crosses ruled out any role for cytoplasmic inheritance. When treated at the discriminating concentration (e.g., 0.28 ppm oryzalin), F₂ seedlings of S × I were classified as either S or I phenotype, and F₂ seedlings of I × R were classified as either I or R phenotype. Again, nonparental phenotypes were not observed. The 3:1 (S:I or I:R) segregation ratios in F₂ seedlings were consistent across all eight F₂ families. The results show that dinitroaniline herbicide resistance in the I biotype of goosegrass is inherited as a single, nuclear gene. Furthermore, it suggests that dinitroaniline resistance in goosegrass is controlled by three alleles at a single locus (i.e., Drp-S, Drp-i, and Drp-r).     

Investigating the genomic basis of discrete phenotypes using a Pool‐Seq‐only approach: New insights into the genetics underlying colour variation in diverse taxa

While large‐scale genomic approaches are increasingly revealing the genetic basis of polymorphic phenotypes such as colour morphs, such approaches are almost exclusively conducted in species with high‐quality genomes and annotations. Here, we use Pool‐Seq data for both genome assembly and SNP frequency estimation, followed by scanning for F_ST outliers to identify divergent genomic regions. Using paired‐end, short‐read sequencing data from two groups of individuals expressing divergent phenotypes, we generate a de novo rough‐draft genome, identify SNPs and calculate genomewide F_ST differences between phenotypic groups. As genomes generated by Pool‐Seq data are highly fragmented, we also present an approach for super‐scaffolding contigs using existing protein‐coding data sets. Using this approach, we reanalysed genomic data from two recent studies of birds and butterflies investigating colour pattern variation and replicated their core findings, demonstrating the accuracy and power of a Pool‐Seq‐only approach. Additionally, we discovered new regions of high divergence and new annotations that together suggest novel parallels between birds and butterflies in the origins of their colour pattern variation.     

Population genomic analyses from low‐coverage RAD‐Seq data: a case study on the non‐model cucurbit bottle gourd

Restriction site‐associated DNA sequencing (RAD‐Seq), a next‐generation sequencing‐based genome ‘complexity reduction’ protocol, has been useful in population genomics in species with a reference genome. However, the application of this protocol to natural populations of genomically underinvestigated species, particularly under low‐to‐medium sequencing depth, has not been well justified. In this study, a Bayesian method was developed for calling genotypes from an F₂ population of bottle gourd [Lagenaria siceraria (Mol.) Standl.] to construct a high‐density genetic map. Low‐depth genome shotgun sequencing allowed the assembly of scaffolds/contigs comprising approximately 50% of the estimated genome, of which 922 were anchored for identifying syntenic regions between species. RAD‐Seq genotyping of a natural population comprising 80 accessions identified 3226 single nuclear polymorphisms (SNPs), based on which two sub‐gene pools were suggested for association with fruit shape. The two sub‐gene pools were moderately differentiated, as reflected by the Hudson's F_ST value of 0.14, and they represent regions on LG7 with strikingly elevated F_ST values. Seven‐fold reduction in heterozygosity and two times increase in LD (r²) were observed in the same region for the round‐fruited sub‐gene pool. Outlier test suggested the locus LX3405 on LG7 to be a candidate site under selection. Comparative genomic analysis revealed that the cucumber genome region syntenic to the high F_ST island on LG7 harbors an ortholog of the tomato fruit shape gene OVATE. Our results point to a bright future of applying RAD‐Seq to population genomic studies for non‐model species even under low‐to‐medium sequencing efforts. The genomic resources provide valuable information for cucurbit genome research.     

A mechanism of chlorotoluron resistance in Lolium rigidum

Many biotypes of Lolium rigidum Gaud, (annual ryegrass) have developed resistance to herbicides; however, few have developed resistance to phenylurea herbicides. Two biotypes with different histories of herbicide selection pressure were six to eight times less sensitive to the phenylurea herbicide, chlorotoluron, than a susceptible biotype. Resistance was not due to differences in the herbicide target site as oxygen evolution by thylakoids isolated from resistant and susceptible biotypes was similarly inhibited by diuron and chlorotoluron. There was no difference in the uptake and distribution of chlorotoluron into resistant and susceptible plants. There was a twofold greater rate of chlorotoluron detoxification in resistant plants with N-demethylation being a major detoxification reaction. Resistant plants treated with a 3-h pulse of 120 M chlorotoluron recovered net carbon fixation after 42 h, half the time taken by susceptible plants. The mixed-function oxidase inhibitor 1-aminobenzotriazole (70 M) intensified the effects of chlorotoluron in resistant plants when applied in combination with the herbicide for 7 d. 1-Aminobenzotriazole also inhibited the metabolism of chlorotoluron in both resistant and susceptible plants. The cytochrome P-₄₅₀ inhibitor, piperonyl butoxide piperonyl butoxide, interacted with chlorotoluron when applied to plants growing in soil. Chlorotoluron applied with reduced plant dry weight to a greater extent than chlorotoluron alone. It appears, therefore, that enhanced detoxification is the major mechanism of resistance to chlorotoluron in the resistant biotypes studied.Abbreviations ABT 1-aminobenzotriazole - VLR1 Victorian L. rigidum biotype 1 — herbicide susceptible - VLR69 Victorian L. rigidum biotype 69 — herbicide resistant - WLR2 Western Australian L. rigidum biotype 2 — herbicide resistant M.W.M.B, was supported by an Australian Postgraduate Research Award and a supplementary scholarship from the Grains Research and Development Corporation. We are very grateful to Dr. E. Ebert, Ciba Geigy, Basal, Switzerland for providing [¹⁴C]chlorotoluron and standards of chlorotoluron metabolites. We express our gratitude to Dr. John Huppatz of the CSIRO Division of Plant Industry for providing ABT. We also thank Ciba Geigy Australia for providing technical-grade chlorotoluron and formulated phenylurea herbicides.     

Occurrence of a herbicide-resistant acetyl-coenzyme A carboxylase mutant in annual ryegrass (Lolium rigidum) selected by sethoxydim

The spectrum of herbicide resistance was determined in an annual ryegrass (Lolium rigidum Gaud.) biotype (SLR 3) that had been exposed to the grass herbicide sethoxydim, an inhibitor of the plastidic enzyme acetylcoenzyme A carboxylase (ACCase, EC 6.4.1.2), for three consecutive years. This biotype has an 18-fold resistance to sethoxydim and enhanced resistance to other cyclohexanedione herbicides compared with a susceptible biotype (VLR 1). The resistant biotype also has a 47- to >300-fold cross-resistance to the aryloxyphenoxypropanoate herbicides which share ACCase as a target site. No resistance is evident to herbicide with a target site different from ACCase. The absorption of [4-¹⁴C]sethoxydim, the rate of metabolic degradation and the nature of the herbicide metabolites are similar in the resistant and susceptible biotypes. While the total activity of the herbicide target enzyme ACCase is similar in extracts from the two biotypes, the kinetics of herbicide inhibition differ. The concentrations of sethoxydim and tralkoxydim required to inhibit the activity of ACCase by 50% are 7.8 and >9.5 times higher, respectively, in the resistant biotype. The activity of ACCase from the resistant biotype was also less sensitive to aryloxyphenoxypropanode herbicides than the susceptible biotype. The spectrum of resistance at the whole-plant level is correlated with resistance at the ACCase level and confirms that a less sensitive form of the target enzyme endows resistance in biotype SLR 3.Abbreviations ACCase acetyl-coenzyme A carboxylase - AOPP aryloxyphenoxypropanoate - CHD cyclohexanedione - GR₅₀ dose giving 50% reduction of growth - IG₅₀ dose giving 50% reduction of germination - LD₅₀ lethal dose 50 This work was partially supported by The Grains Research and Development Corporation of Australia through a grant to Dr. R. Knight, Department of Plant Science, Waite Agricultural Research Institute. The encouragement and generous support of Dr. R. Knight is gratefully acknowledged.     

ZFN‐mediated gene targeting of the Arabidopsis protoporphyrinogen oxidase gene through Agrobacterium‐mediated floral dip transformation

Previously, we showed that ZFN‐mediated induction of double‐strand breaks (DSBs) at the intended recombination site enhanced the frequency of gene targeting (GT) at an artificial target locus using Agrobacterium‐mediated floral dip transformation. Here, we designed zinc finger nucleases (ZFNs) for induction of DSBs in the natural protoporphyrinogen oxidase (PPO) gene, which can be conveniently utilized for GT experiments. Wild‐type Arabidopsis plants and plants expressing the ZFNs were transformed via floral dip transformation with a repair T‐DNA with an incomplete PPO gene, missing the 5′ coding region but containing two mutations rendering the enzyme insensitive to the herbicide butafenacil as well as an extra KpnI site for molecular analysis of GT events. Selection on butafenacil yielded 2 GT events for the wild type with a frequency of 0.8 × 10^?3 per transformation event and 8 GT events for the ZFNs expressing plant line with a frequency of 3.1 × 10^?3 per transformation event. Molecular analysis using PCR and Southern blot analysis showed that 9 of the GT events were so‐called true GT events, repaired via homologous recombination (HR) at the 5′ and the 3′ end of the gene. One plant line contained a PPO gene repaired only at the 5′ end via HR. Most plant lines contained extra randomly integrated T‐DNA copies. Two plant lines did not contain extra T‐DNAs, and the repaired PPO genes in these lines were transmitted to the next generation in a Mendelian fashion.     

Inheritance of fenoxaprop-P-ethyl resistance in a blackgrass (Alopecurus myosuroides Huds.) population

A blackgrass population has developed resistance to fenoxaprop-P-ethyl following field selection with the herbicide for 6 consecutive years. Within this population, 95% of the individuals are also resistant to flupyrsulfuron. Both the inheritance(s) and the mechanism(s) of resistances were investigated by making crosses between the resistant and a susceptible biotype. The inheritance was followed through the F₁ and F₂ generations either by spraying the herbicide on seedlings at the three-leaf stage or using a seedling bioassay, based on coleoptile length. No maternal effects were evident in the fenoxaprop-P-ethyl responses of the F₁ plants, suggesting that the inheritance was nuclear. Some F₁ families treated with fenoxaprop-P-ethyl segregated in a 3:1 (resistant:susceptible) ratio, indicating that the resistance was conferred by two dominant and independent nuclear genes. This was confirmed by the 15:1 (R:S) ratio observed in the F₂ generation treated with fenoxaprop- P-ethyl. The use of selective inhibitors of herbicide de-toxifying enzymes (aminobenzotriazole, pyperonylbutoxide, malathion and tridiphane) with the F₂ plants suggested that each of the two genes may govern two different mechanisms of fenoxaprop-P-ethyl resistance: the ACCase mutation previously postulated and an enhanced herbicide metabolism, mediated by cytochrome P 450 mono-oxygenases (P 450) susceptible to malathion. The P 450 activity may also confer resistance to flupyrsulfuron. This study clearly indicates that two distinct mechanisms of resistance may co-exist in the same plant. Received: 18 August 2000 / Accepted: 6 December 2000     

QST–FST comparisons with unbalanced half‐sib designs

Q_ST, a measure of quantitative genetic differentiation among populations, is an index that can suggest local adaptation if Q_ST for a trait is sufficiently larger than the mean F_ST of neutral genetic markers. A previous method by Whitlock and Guillaume derived a simulation resampling approach to statistically test for a difference between Q_ST and F_ST, but that method is limited to balanced data sets with offspring related as half‐sibs through shared fathers. We extend this approach (i) to allow for a model more suitable for some plant populations or breeding designs in which offspring are related through mothers (assuming independent fathers for each offspring; half‐sibs by dam); and (ii) by explicitly allowing for unbalanced data sets. The resulting approach is made available through the R package QstFstComp.