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1.
Genome Biol Evol ; 11(10): 2927-2940, 2019 10 01.
Article in English | MEDLINE | ID: mdl-31518388

ABSTRACT

Increased copy number of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene confers resistance to glyphosate, the world's most-used herbicide. There are typically three to eight EPSPS copies arranged in tandem in glyphosate-resistant populations of the weed kochia (Kochia scoparia). Here, we report a draft genome assembly from a glyphosate-susceptible kochia individual. Additionally, we assembled the EPSPS locus from a glyphosate-resistant kochia plant by sequencing select bacterial artificial chromosomes from a kochia bacterial artificial chromosome library. Comparing the resistant and susceptible EPSPS locus allowed us to reconstruct the history of duplication in the structurally complex EPSPS locus and uncover the genes that are coduplicated with EPSPS, several of which have a corresponding change in transcription. The comparison between the susceptible and resistant assemblies revealed two dominant repeat types. Additionally, we discovered a mobile genetic element with a FHY3/FAR1-like gene predicted in its sequence that is associated with the duplicated EPSPS gene copies in the resistant line. We present a hypothetical model based on unequal crossing over that implicates this mobile element as responsible for the origin of the EPSPS gene duplication event and the evolution of herbicide resistance in this system. These findings add to our understanding of stress resistance evolution and provide an example of rapid resistance evolution to high levels of environmental stress.


Subject(s)
3-Phosphoshikimate 1-Carboxyvinyltransferase/genetics , Bassia scoparia/genetics , Gene Duplication , Glycine/analogs & derivatives , Herbicides , Bassia scoparia/drug effects , Bassia scoparia/enzymology , DNA Transposable Elements , Drug Resistance/genetics , Evolution, Molecular , Gene Expression , Glyphosate
2.
Pest Manag Sci ; 74(10): 2306-2315, 2018 Oct.
Article in English | MEDLINE | ID: mdl-29083527

ABSTRACT

BACKGROUND: Resistance to the synthetic auxin herbicide dicamba is increasingly problematic in Kochia scoparia. The resistance mechanism in an inbred dicamba-resistant K. scoparia line (9425R) was investigated using physiological and transcriptomics (RNA-Seq) approaches. RESULTS: No differences were found in dicamba absorption or metabolism between 9425R and a dicamba-susceptible line, but 9425R was found to have significantly reduced dicamba translocation. Known auxin-responsive genes ACC synthase (ACS) and indole-3-acetic acid amino synthetase (GH3) were transcriptionally induced following dicamba treatment in dicamba-susceptible K. scoparia but not in 9425R. Chalcone synthase (CHS), the gene regulating synthesis of the flavonols quertecin and kaemperfol, was found to have twofold higher transcription in 9425R both without and 12 h after dicamba treatment. Increased CHS transcription co-segregated with dicamba resistance in a forward genetics screen using an F2 population. CONCLUSION: Prior work has shown that the flavonols quertecin and kaemperfol compete with auxin for intercellular movement and vascular loading via ATP-binding cassette subfamily B (ABCB) membrane transporters. The results of this study support a model in which constitutively increased CHS expression in the meristem produces more flavonols that would compete with dicamba for intercellular transport by ABCB transporters, resulting in reduced dicamba translocation. © 2017 Society of Chemical Industry.


Subject(s)
Acyltransferases/genetics , Bassia scoparia/drug effects , Dicamba/pharmacology , Herbicide Resistance/genetics , Herbicides/pharmacology , Plant Proteins/genetics , Acyltransferases/metabolism , Bassia scoparia/enzymology , Bassia scoparia/genetics , Gene Expression Regulation, Plant/drug effects , Plant Proteins/metabolism , Plant Weeds/drug effects , Plant Weeds/enzymology , Plant Weeds/genetics
3.
Plant Sci ; 261: 69-79, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28554695

ABSTRACT

Glyphosate is considered the world's most important herbicide, but widespread and continual use has resulted in the evolution of resistance. Kochia scoparia (kochia) has evolved resistance via tandem gene amplification of glyphosate's target, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and resistant populations have been reported from the Canadian Prairies and the Northern Great Plains. Here, we evaluated the fitness costs of EPSPS amplification in kochia by comparing susceptible and resistant full siblings from segregating F2 populations generated from within six populations. Kochia was expected to be highly diverse because of strong gene flow; however, six of the seven field-collected parents with higher EPSPS copy number were homozygous. Under competitive greenhouse conditions, the EPSPS type of the line's maternal parent showed persistent effects: delayed emergence, delayed flowering, and reductions in viable seed count and weight overall. High EPSPS copy number individuals had reduced seed count and weight, reduced competitive ability, and reduced final height in mixed stands, but better germination of the F3. However, all characteristics were highly variable and fitness costs were not constant across genetic backgrounds. In the absence of selection from glyphosate, kochia with increased EPSPS copy number will be at a competitive disadvantage in some genetic backgrounds.


Subject(s)
Bassia scoparia/drug effects , Glycine/analogs & derivatives , Herbicide Resistance , Herbicides/pharmacology , 3-Phosphoshikimate 1-Carboxyvinyltransferase/genetics , 3-Phosphoshikimate 1-Carboxyvinyltransferase/metabolism , Bassia scoparia/enzymology , Bassia scoparia/genetics , Bassia scoparia/physiology , DNA Copy Number Variations/genetics , DNA Copy Number Variations/physiology , Glycine/pharmacology , Herbicide Resistance/genetics , Herbicide Resistance/physiology , Plant Breeding , Glyphosate
4.
Planta ; 241(2): 463-74, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25366557

ABSTRACT

MAIN CONCLUSION: Field-evolved resistance to the herbicide glyphosate is due to amplification of one of two EPSPS alleles, increasing transcription and protein with no splice variants or effects on other pathway genes. The widely used herbicide glyphosate inhibits the shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Globally, the intensive use of glyphosate for weed control has selected for glyphosate resistance in 31 weed species. Populations of suspected glyphosate-resistant Kochia scoparia were collected from fields located in the US central Great Plains. Glyphosate dose response verified glyphosate resistance in nine populations. The mechanism of resistance to glyphosate was investigated using targeted sequencing, quantitative PCR, immunoblotting, and whole transcriptome de novo sequencing to characterize the sequence and expression of EPSPS. Sequence analysis showed no mutation of the EPSPS Pro106 codon in glyphosate-resistant K. scoparia, whereas EPSPS genomic copy number and transcript abundance were elevated three- to ten-fold in resistant individuals relative to susceptible individuals. Glyphosate-resistant individuals with increased relative EPSPS copy numbers had consistently lower shikimate accumulation in leaf disks treated with 100 µM glyphosate and EPSPS protein levels were higher in glyphosate-resistant individuals with increased gene copy number compared to glyphosate-susceptible individuals. RNA sequence analysis revealed seven nucleotide positions with two different expressed alleles in glyphosate-susceptible reads. However, one nucleotide at the seven positions was predominant in glyphosate-resistant sequences, suggesting that only one of two EPSPS alleles was amplified in glyphosate-resistant individuals. No alternatively spliced EPSPS transcripts were detected. Expression of five other genes in the chorismate pathway was unaffected in glyphosate-resistant individuals with increased EPSPS expression. These results indicate increased EPSPS expression is a mechanism for glyphosate resistance in these K. scoparia populations.


Subject(s)
3-Phosphoshikimate 1-Carboxyvinyltransferase/metabolism , Bassia scoparia/drug effects , Bassia scoparia/enzymology , Bassia scoparia/metabolism , Gene Amplification/physiology , Glycine/analogs & derivatives , Herbicides/pharmacology , 3-Phosphoshikimate 1-Carboxyvinyltransferase/genetics , Bassia scoparia/genetics , Gene Amplification/genetics , Gene Expression Regulation, Plant , Glycine/pharmacology , Herbicide Resistance/genetics , Glyphosate
5.
Plant Physiol ; 166(3): 1200-7, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25037215

ABSTRACT

Recent rapid evolution and spread of resistance to the most extensively used herbicide, glyphosate, is a major threat to global crop production. Genetic mechanisms by which weeds evolve resistance to herbicides largely determine the level of resistance and the rate of evolution of resistance. In a previous study, we determined that glyphosate resistance in Kochia scoparia is due to the amplification of the 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) gene, the enzyme target of glyphosate. Here, we investigated the genomic organization of the amplified EPSPS copies using fluorescence in situ hybridization (FISH) and extended DNA fiber (Fiber FISH) on K. scoparia chromosomes. In both glyphosate-resistant K. scoparia populations tested (GR1 and GR2), FISH results displayed a single and prominent hybridization site of the EPSPS gene localized on the distal end of one pair of homologous metaphase chromosomes compared with a faint hybridization site in glyphosate-susceptible samples (GS1 and GS2). Fiber FISH displayed 10 copies of the EPSPS gene (approximately 5 kb) arranged in tandem configuration approximately 40 to 70 kb apart, with one copy in an inverted orientation in GR2. In agreement with FISH results, segregation of EPSPS copies followed single-locus inheritance in GR1 population. This is the first report of tandem target gene amplification conferring field-evolved herbicide resistance in weed populations.


Subject(s)
3-Phosphoshikimate 1-Carboxyvinyltransferase/genetics , Bassia scoparia/enzymology , Glycine/analogs & derivatives , Herbicide Resistance , Herbicides/pharmacology , 3-Phosphoshikimate 1-Carboxyvinyltransferase/metabolism , Bassia scoparia/genetics , Biological Evolution , Chromosome Mapping , Gene Amplification , Gene Expression Regulation, Plant/drug effects , Glycine/pharmacology , In Situ Hybridization, Fluorescence , Models, Biological , Glyphosate
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