Single nucleotide mutation in the barley acetohydroxy acid synthase (AHAS) gene confers resistance to imidazolinone herbicides

Hyejin Lee; Sachin Rustgi; Neeraj Kumar; Ian Burke; Joseph P. Yenish; Kulvinder S. Gill; Diter von Wettstein; Steven E. Ullrich

doi:10.1073/pnas.1105612108

New Research In

Contributed by Diter von Wettstein, April 8, 2011 (sent for review March 25, 2011)

Abstract

Induced mutagenesis can be an effective way to increase variability in self-pollinated crops for a wide variety of agronomically important traits. Crop resistance to a given herbicide can be of practical value to control weeds with efficient chemical use. In some crops (for example, wheat, maize, and canola), resistance to imidazolinone herbicides (IMIs) has been introduced through mutation breeding and is extensively used commercially. However, this production system imposes plant-back restrictions on rotational crops because of herbicide residuals in the soil. In the case of barley, a preferred rotational crop after wheat, a period of 9–18 mo is required. Thus, introduction of barley varieties showing resistance to IMIs will provide greater flexibility as a rotational crop. The objective of the research reported was to identify resistance in barley for IMIs through induced mutagenesis. To achieve this objective, a sodium azide-treated M₂/M₃ population of barley cultivar Bob was screened for resistance against acetohydroxy acid synthase (AHAS)-inhibiting herbicides. The phenotypic screening allowed identification of a mutant line showing resistance against IMIs. Molecular analysis identified a single-point mutation leading to a serine 653 to asparagine amino acid substitution in the herbicide-binding site of the barley AHAS gene. The transcription pattern of the AHAS gene in the mutant (Ser653Asn) and WT has been analyzed, and greater than fourfold difference in transcript abundance was observed. Phenotypic characteristics of the mutant line are promising and provide the base for the release of IMI-resistant barley cultivar(s).

Barley (Hordeum vulgare L.) is an important annual crop in the Pacific Northwest (PNW) for 2- or 3-y rotations with winter wheat (Triticum aestivum), pea (Pisum sativum), lentil (Lens culinaris), or fallow (1). One of the major reasons for the worldwide decline in barley acreage is its sensitivity to commonly used herbicides. Many of the widely used herbicides imposing barley plant-back restrictions belong to the group B herbicides that were first commercialized in 1982 (2). The advantages of these herbicides are low application rates, broad spectrums of weed control, soil residual activity, high margins of crop safety, and low mammalian toxicity (3). This herbicide group consists of five different chemical families: imidazolinones (4), sulfonylureas (5), triazolopyrimidines (6), pyrimidyloxybenzoates (7), and sulfonlyaminocarbonyl-triazolinones (8). These herbicides target acetolactate synthase (ALS; EC 2.2.1.6), also known as acetohydroxyacid synthase (AHAS), which is an octameric enzyme with four catalytic and four regulatory subunits (9). AHAS catalyses two parallel reactions in the synthesis of branched-chain amino acids. The first reaction is condensation of two pyruvate molecules to yield acetolactate, leading to the production of valine and leucine, and the second reaction is the condensation of pyruvate and α-ketobutyrate to yield acetohydroxybutyrate, leading to the production of isoleucine (10). The AHAS-inhibiting herbicides are known to bind at the substrate access channel, blocking the path of substrate to the active site (11 –13). When AHAS is inhibited, deficiency of the amino acids causes a decrease in protein synthesis, which in turn, slows down cell division rate (5, 14). This process eventually kills the plant after showing symptoms in meristematic tissues, where biosynthesis of amino acids primarily takes place (15). Resistant plants, in most cases, depend on reduced sensitivity to these herbicides by an isoform of AHAS, which does not severely affect its catalytic activity. Most AHAS isoenzymes resistant to the herbicides carry substitutions for the amino acid residues Ala122, Pro197, Ala205, Asp376, Trp574, or Ser653 (amino acid numbering refers to the sequence in Arabidopsis thaliana). The amino acid residues Ala122, Pro197, and Ala205 are located at the N-terminal end of the enzyme, whereas Asp376, Trp574, and Ser653 are located at the C-terminal end (16, 17). Amino acid substitutions at Ala122 and Ser653 confer high levels of resistance to imidazolinone herbicides (IMIs), whereas substitutions at Pro197 endow high levels of resistance against sulfonylureas and provide low-level resistance against IMIs and triazolopyrimidine herbicides (18 –22). Substitutions at Trp574 endow high levels of resistance to imidazolinones, sulfonylureas, and triazolopyrimidines (19, 23–26), whereas substitutions at Ala205 provide resistance against all AHAS-inhibiting herbicides (24, 27).

In the case of barley, which is a preferred rotational crop after wheat, no IMI resistance is reported for any of the varieties cultivated in the PNW. Thus, introduction of barley varieties with resistance to IMI will provide greater flexibility as a rotational crop. The objective of the research reported was to identify resistance in barley for IMIs through induced mutagenesis of barley cultivar Bob followed by phenotypic, genetic, and molecular characterization of the identified mutation(s). Development of an IMI-resistant barley cultivar through induced mutagenesis in Bob will have many inherent advantages; for instance, the line is adapted to the PNW, it has excellent feed quality, and it will be nontransgenic in origin. In addition, IMI-resistant barley has less risk of introducing herbicide resistance to the most common monocotyledonous weeds in the barley fields. In contrast, IMI-resistant wheat has a potential risk of transferring herbicide resistance to jointed goat grass (Aegilops cylindrica), which shares its D genome with common wheat (28, 29). Also, ryegrass (Lolium perenne), both as forage crop and weed, has recently been shown to develop IMI resistance because of gene flows from IMI-resistant crops through inter- and/or intraspecies hybridizations (30).

Results and Discussion

Screening Procedure and Selection of Resistant Lines.

The two-row spring feed barley cultivar Bob (31) was mutagenized using sodium azide (NaN₃) using a modified procedure developed by Kueh and Bright (32). Approximately 500 g seeds were presoaked for 16 h at 0 °C followed by 8 h soaking at 20 °C with aeration in a water bath. The seeds were treated with 1 mM NaN₃ (pH 3) for 2 h, rinsed three times, and dried. The mutagenized seeds were planted in isolation at the Spillman Agronomy Farm, Pullman, WA, in the spring of 2007. The plants from these seeds, the M₁ generation, were selfed, and M₂ seeds were bulk harvested at maturity. The M₂ seeds were screened for imidazolinone resistance using a modified procedure developed by Newhouse et al. (33). About 250 seeds were placed on a sterile 10 × 1.5-cm Petri dish and soaked in 25 mL imazethapyr solution [1,120.3 μM; 48.06 g active ingredient (ai)·ha⁻¹] for 48 h. The imazethapyr presoaked seeds were planted at 1–2 cm in 25 × 50-cm flats containing commercial potting mix (Sunshine Mix 1/ LC 1; Sun Gro) in a glasshouse under a 16-h light (22 °C) and 8-h dark (16 °C) cycle. Each flat contained ∼1,000 seeds or four treated Petri dishes, and each flat was watered as needed. Emerged seedlings were visually evaluated after 4 wk. Putative M₂ mutants were transferred to pots and grown under the same glasshouse conditions to maturity. The M₃ seeds from putative M₂ mutants were harvested individually, and 5–10 M₃ seeds from each plant were planted to increase seeds for further validation. The M₄ seeds were used for verification using imazamox. The M₄ seeds from each line were individually placed into the cells of a 25 × 50-cm, 72-celled flat. Two identical setups with barley cultivar Bob (WT) were used as controls: one with imazamox treatment and the other without herbicide treatment. Imazamox was applied over foliage when most plants were at the two-leaf stage in an herbicide chamber. The spray solution included 818 μM (34.7 g ai·ha⁻¹) imazamox with nonionic surfactant (NIS) at 0.25% of the solution volume. A moving nozzle cabinet sprayer with a flat-fan nozzle tip was calibrated to deliver 140 L·ha⁻¹ spray solution at 206 kPa in a single pass. The three flats, one each for treated mutant, treated WT, and nontreated WT plants, were visually evaluated 21 d after imazamox application.

After screening more than 2 million M₂ seeds, one line was confirmed to show imidazolinone resistance, because all 72 M₄ plants derived from this line survived with little symptoms after herbicide application (Fig. 1). This homozygous line showed no difference in aboveground biomass compared with nontreated controls of the susceptible WT Bob (Fig. 2A). The imazamox resistance did not seem to interfere with mutant fitness in general, although a difference in plant height was observed (Fig. 2B). This might be explained by a slightly delayed germination of the mutants as a possible result to additional mutations induced by the mutagen treatment. The mutant barley line was reciprocally backcrossed to its progenitor Bob. Segregation ratios of F₂ progenies confirmed the semidominant nature of the mutation and monogenic inheritance of the IMI resistance (Table S1).

Fig. 1.

Phenotypic confirmation of resistance in the selected M₄ mutant line. Negative control is the Bob without imazamox treatment (rows 1 and 2), positive control is Bob with imazamox treatment (rows 3 and 4), Morex with imazamox treatment is in rows 5 and 6, and mutant plants with imazamox treatment are in rows 7 and 8 (70 g ai·ha⁻¹).

Fig. 2.

Phenotypic observations on the mutant line with positive and negative controls. (A) Above-ground biomass was represented as percentage of nontreated control plants. The values are averages and obtained from plants dried 21 d after imazamox treatment. (B) Plant height was represented as percentage of nontreated control plants. The values are averages and obtained from plants 21 d after imazamox treatment. Bob^trt, Bob with imazamox treatment; Bob^nontrt, Bob without imazamox treatment; mutant, mutant with imazamox treatment. Double asterisks indicate significance at ≤0.01; ns indicates no significant at ≤0.05.

Molecular Characterization of the Mutation.

The molecular nature of the mutant conferring IMI resistance in barley was determined by PCR amplification and subsequent sequencing of an ∼850-bp fragment from the genomic DNA of the mutant and five barley cultivars: Bob, Morex, Steptoe, Champion, and Baronesse. The fragment was amplified using the wheat AHAS-specific primers CM-F and CM-R (Table S2) (34). DNA sequences obtained from five susceptible barley cultivars (including WT Bob) and the mutant line were aligned and searched for any potential mutation in the gene sequence. The sequence analysis revealed a single transition from G to A at around nucleotide 1742 when based on the wheat AHAS gene sequence AY210406 (Fig. S1). The point mutation leads to an amino acid substitution from serine 653 (AGC) to asparagine (AAC) at the herbicide-binding site of the acetohydroxyacid synthase protein. The mutation (Ser653Asn) seems to be identical to the mutation conferring resistance against IMIs in Arabidopsis, tobacco (Nicotiana tobaccum), maize (Zea mays), rice (Oryza sativa), oilseed rape (Brassica napus), and wheat (35). To eliminate the possibility of mutations other then G to A transition in HvAHAS, sequences of 1,845 bp from Bob and 1,798 bp from the mutant were obtained (Fig. S2) and aligned with the barley AHAS reference sequence (AF059600). The sequence alignment revealed no other point mutation(s) between Bob and mutant (Fig. S3). However, two transitions leading to synonymous changes between AF059600 and the sequences obtained from Bob and mutant were observed (Fig. S3). The changes seem to be varietal differences, and they could be used to develop SNP markers. The phylogenetic analysis of the amino acid sequences around the amino acid substitution at Ser653Asn revealed a highly conserved domain of ∼23 aa in the grass lineage (Fig. 3). The amino acid substitution (Ser653Asn) resides in the γ-domain at the C-terminal end of the catalytic subunit of the AHAS enzyme. These subunits aggregate to form a tetramer and complex with another tetramer of four regulatory subunits to build the AHAS apoenzyme (9). The amino acid location of the point mutation detected in the current study was also modeled on the crystal structure of the catalytic subunit of the Arabidopsis AHAS enzyme by matching the partial amino acid sequence of the barley mutant AHAS with the Arabidopsis mutant AHAS sequence (http://www.rcsb.org/pdb/home/home.do) (Fig. 4) (13). A one-step allele-specific assay for the barley AHAS mutation (Fig. 5) allows identification of the mutant in production trials and breeding of resistant cultivars.

Fig. 3.

Comparative sequence alignment of AHAS showing phylogenetic relationships among members of grass lineage and Arabidopsis; the rectangular box indicates the position of the amino acid substitution providing resistance against imidazolinone herbicides. *Sequences obtained from seed mutagenized wheat cv. CDC Teal.

Fig. 4.

Putative location of point mutation conferring resistance against IMIs in barley on the 1D, 2D, and crystal structure of Arabidopsis AHAS enzyme. The 2D topology diagram was modified from Duggleby et al. (9). [Reprinted from Plant Physiology and Biochemistry, 46(3), RG, Duggleby, JA McCourt, LW Guddat, Structure and mechanism of inhibition of plant acetohydroxyacid synthase, 309–324, Copyright (2008), with permission from Elsevier.]

Fig. 5.

(A) One-step allele-specific assay for AHAS mutation using a combination of an allele-specific primer (ALS_M_R) and a locus-specific primer (ALS_O_F). (B) PCR product obtained using locus-specific primers (ALS_O_F and ALS_O_R) used as loading controls. Primers used are described in Table S1, and their putative locations are depicted in Fig. 3. M, size markers (100 bp ladder; New England Biolab); arrowhead, expected product size.

The C-terminal ends of the catalytic subunits form the substrate access channel, and substitutions at 6 (A122, F206, Q207, K256, W574, and S653) of 12 aa residues in the catalytic subunit are known to provide resistance against imidazolinone herbicides (9). Four different substitutions were reported at Ser-653, replacing Ser with Asn, Thr, Phe, or Ile (9, 36), but to date, no negative impact of substitutions at Ser653 on plant performance has been reported, giving these point mutations a great commercial value for the development of IMI-resistant crops (37). The crystal structure of Arabidopsis AHAS with the IMI herbicide imazaquin indicates that a replacement of Ser653 with the larger side chain carrying amino acids Asn or Thr likely obstructs binding of the quinoline ring of imazaquin to the enzyme and thereby, leads to the herbicide resistance in the mutant plants (Fig. S4) (13). Single amino acid substitutions at one of six aa residues (A122, F206, Q207, K256, W574, and S653) are known to be sufficient to convert AHAS from a sensitive to resistant form (17). In addition, a limited initial test of the mutant line with sulfosulfuron [used rate = 534.4 μM (35.2 g ai·ha⁻¹) with 0.25% NIS] indicated that the line was likely susceptible to sulfosulfuron (Fig. S5). These observations coincide well with other studies, where a point mutation at Ser653 resulting in Asn substitution provided resistance against imidazolinone but not against sulfosulfuron (17).

Transcription Data.

For relative quantification of HvAHAS transcripts in Bob and the mutant, four replicates of each sample were used in analyses, and the same experiment was repeated two times (Fig. S6 A and B). The results of relative quantification suggested that HvAHAS transcripts were less abundant compared with Actin, and interestingly, the mutant expresses ∼4.5 ± 0.3-fold more enzyme compared with the WT Bob (Fig. S6 A and B).

Enzyme Extraction, Characterization, and Quantification.

Presence of AHAS in the extract was confirmed by the red color obtained in the colorimetric assay in the presence and/or absence of imazamox solution and also by its molecular mass of ∼65 kDa on SDS/PAGE gel (Fig. 6 A and B). The relative quality of AHAS against known quantities of BSA in Bob and mutant was estimated from Bradford assay and RP-HPLC analyses (SI Materials and Methods). On the basis of Bradford assay, the mutant (2.05 μg/mL) accumulated relatively more enzyme than Bob (1.41 μg/mL), and this result was complemented by RP-HPLC quantifications (Fig. S7 A–C), where AHAS enzyme in mutant (1.85 μg/mL) quantified more than Bob (1.54 μg/mL).

Fig. 6.

(A) SDS/PAGE analysis of AHAS enzyme extracted from Bob and mutant. (B) Colorimetric assay of AHAS enzyme in the presence and absence of imazamox (beyond = concentration 3.2 μM). M, protein molecular weight marker; arrowhead, band of interest.

The barley mutant reported in the present study produces relatively more enzyme in comparison with WT, and the segregation ratios obtained from the reciprocal crosses between the mutant and Bob revealed single-gene inheritance for herbicide resistance. Taken together the two results suggest that the isoenzyme produced by the mutation may influence its feedback regulation or catalytic properties (2). Because the mutant plants produce slightly more enzyme compared with WT, it may compensate for a reduced functionality of the enzyme isoform and contribute to the full viability of the plant and its survival under application of a 10-fold field dose of the herbicide (40 oz/acre; 8,180 μM).

In conclusion, the single nucleotide mutation reported has been identified in the gene of the barley AHAS enzyme as providing resistance to IMIs, which are used extensively to keep spring and winter wheat fields in the PNW free of grass weeds. The herbicides are retained in the soil, preventing current barley cultivars from being planted as alternative crops in normal crop rotations. The induced mutant in the cultivar Bob has retained the phenotype of the parent Bob and shows that these mutations can restore barley as a suitable alternative crop. The specificity of amino acid substitutions in AHAS to individual herbicides shows the possibility to select resistance to individual herbicides by Targeting Induced Local Lesions in Genomes (TILLING) as well as transgenic technology.

Materials and Methods

Genomic DNA Extraction, PCR Amplification, and Sequence Analyses.

DNA was extracted from 1-mo-old seedlings of each of the six barley genotypes using the modified hexadecyl(trimethyl)arasimum bromide (CTAB) method (38). The PCR reactions were carried out in 20-μL reaction mixtures, each containing 50 ng template DNA, 0.25 μM primers, 200 μM dNTPs, 1.5 mM MgCl₂, 1× PCR buffer, and 0.5 U Ex Taq DNA polymerase (TAKARA) using the following PCR profile: initial denaturation at 95 °C for 5 min followed by 40 cycles at 95 °C for 30 s, 56–60 °C for 30 s (Table S2), 72 °C for 45 s, and a final extension at 72 °C for 10 min. The amplification products were resolved on 2% agarose gels. A 100-bp ladder was used as a size marker (New England BioLabs). Bands of expected sizes were excised from the gel, and DNA was eluted from the bands using the Geneclean kit following the manufacturer's instructions (MP Biomedicals). The eluted DNA was used as a template for the sequencing reaction using either forward or reverse primers in separate reactions. The sequencing reactions were carried out at the DNA Sequence Core, Washington State University, Pullman, WA. Sequence identity searches were performed at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov) using BLAST. Alignment of the deduced amino acid sequences was performed using the Vector NTI AdvanceTM 9.1 (Invitrogen).

RNA Isolation, cDNA Synthesis, and Real-Time PCR Assays.

RNA was extracted from 1-wk-old seedlings of Bob and mutant. The seedling leaves were harvested in liquid nitrogen, and about 0.5–1 g tissue were pulverized to fine powder using pestle and mortar. Total RNA was isolated using 8 mL TRIZOL reagent (Invitrogen Corp) per sample according to the manufacturer's recommendations. RNA was quantified by absorbance at 260 nm using a Bio-Rad SmartSpec 3000 (Bio-Rad Laboratories).

RNA (5μg) was combined with 2.5 mM oligo dT₂₀ (Invitrogen) primer in a total volume of 11 μL diethylpyrocarbonate (DEPC)-treated nuclease free water, heated at 65 °C for 5 min, and chilled on ice. The RNA mixture was supplemented with 8 μL mixture containing 1× First Strand buffer (Invitrogen), 10 mM dithiothreitol (DTT), 500 mM each dNTP, and 40 units RNaseOUT (Invitrogen) in a final volume of 25 μL, and the mixture was heated for 1 min at 55 °C in a thermocycler. After 1 min, cDNA synthesis was initiated by adding 200 units SuperScript III reverse transcriptase (Invitrogen) to the mixture and incubating it for 2 h at 55 °C. The cDNA was precipitated in 2.5 volumes ethanol in the presence of 0.3 M sodium acetate (pH 5.2) and resuspended in 20 μL nuclease-free water.

Real-time quantitative PCR (qPCR) analysis of HvALS transcripts was performed using the DNA Master SYBR Green 1 chemistry on The LightCycler 480 Real-Time PCR System (Roche Diagnostics). PCR primers for HvALS and Actin (used as internal controls) are listed in Table S2. Each PCR consisted of about 50 ng cDNA, 5 pmol each forward and reverse primers, 3 mM MgCl₂, and 10 μL SYBR Green I reagent (Roche) in a total reaction volume of 20 μL. The amplification profile for all cDNAs was 95 °C for 10 min followed by 35 cycles of 95 °C for 10 s, 57 °C for 5 s, and 72 °C for 10 s. Amplicon melting profiles were generated over a range of 65 °C to 98 °C, with a temperature change of 0.1 °C s⁻¹ and fluorescence monitoring every 0.3 °C s⁻¹. HvALS mRNA level was normalized to Actin using the DDC_T method (39). Transcript levels were expressed as a ratio of HvALS transcripts (normalized to Actin) in mutant and Bob.

Enzyme Extraction, Characterization, and Quantification.

AHAS enzyme was extracted from Bob and mutant using 5 g seedling leaves using the method of Singh et al. (40). Extracted enzyme was loaded on 10% SDS/PAGE followed by staining with Coomassie Brilliant Blue G-250 (41, 42). A prestained protein molecular weight marker (SM0441; Fermentas) was loaded in each gel for size estimation. Presence of AHAS enzyme was confirmed by Westerfeld reaction (40). Enzyme was quantified using Quick Start Bradford 1× dye reagent (500–0205; BioRad) following the manufacturer's instructions. BSA was used as standard (six standards from 0.2 to 2.2 μg/mL), and absorbance was measured at 595 nm on a BioRad SmartSpec Plus spectrophotometer. Values were plotted on a graph, with the dependent variable (in micrograms per milliliter) on the x axis and the independent variable (abs 595 nm) on the y axis; linear regression was performed, and the amounts of enzyme in Bob and mutant were calculated.

Acknowledgments

The authors would like to thank Nuan Wen for laboratory assistance and Vadim Jitkov and Max Wood for field and greenhouse assistance. This work has been performed with financial support from the Washington Grain Commission and National Institutes of Health Grant 1R01GM080749-01A1.

Footnotes

↵¹H.L. and S.R. contributed equally to this work.
²To whom correspondence may be addressed. E-mail: diter{at}wsu.edu or ullrich{at}wsu.edu.

Author contributions: S.R., K.S.G., D.v.W., and S.E.U. designed research; H.L., S.R., N.K., and I.B. performed research; J.P.Y. contributed new reagents/analytic tools; H.L., S.R., and N.K. analyzed data; and H.L., S.R., D.v.W., and S.E.U. wrote the paper.
The authors declare no conflict of interest.
Data deposition: The sequences reported in this paper have been deposited in the NCBI GenBank database (accession nos. HQ661102–HQ661107).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1105612108/-/DCSupplemental.

Freely available online through the PNAS open access option.

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↵
1. Powles SB,
2. Yu Q
(2010) Evolution in action: Plants resistant to herbicides. Annu Rev Plant Biol 61:317–347.
OpenUrl CrossRef PubMed
↵
1. Stewart NC Jr.
1. Preston C
(2009) in Weedy and Invasive Plant Genomics, ed Stewart NC Jr. (Blackwell, Oxford).
↵
1. Saghai-Maroof MA,
2. Soliman KM,
3. Jorgensen RA,
4. Allard RW
(1984) Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc Natl Acad Sci USA 81:8014–8018.
OpenUrl Abstract/FREE Full Text
↵
1. Livak KJ,
2. Schmittgen TD
(2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408.
OpenUrl CrossRef PubMed
↵
1. Singh BK,
2. Stidham MA,
3. Shaner DL
(1988) Assay of acetohydroxyacid synthase. Anal Biochem 171:173–179.
OpenUrl CrossRef PubMed
↵
1. Fling SP,
2. Gregerson DS
(1986) Peptide and protein molecular weight determination by electrophoresis using a high-molarity tris buffer system without urea. Anal Biochem 155:83–88.
OpenUrl CrossRef PubMed
↵
1. Schägger H,
2. von Jagow G
(1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379.
OpenUrl CrossRef PubMed

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Proceedings of the National Academy of Sciences: 108 (21)

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Biological Sciences
Sustainability Science

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Stewart NC Jr.
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[139] Preston C

[140] ↵
Saghai-Maroof MA,
Soliman KM,
Jorgensen RA,
Allard RW
(1984) Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc Natl Acad Sci USA 81:8014–8018.
OpenUrl Abstract/FREE Full Text

[141] Saghai-Maroof MA,

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[143] Jorgensen RA,

[144] Allard RW

[145] ↵
Livak KJ,
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(2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408.
OpenUrl CrossRef PubMed

[146] Livak KJ,

[147] Schmittgen TD

[148] ↵
Singh BK,
Stidham MA,
Shaner DL
(1988) Assay of acetohydroxyacid synthase. Anal Biochem 171:173–179.
OpenUrl CrossRef PubMed

[149] Singh BK,

[150] Stidham MA,

[151] Shaner DL

[152] ↵
Fling SP,
Gregerson DS
(1986) Peptide and protein molecular weight determination by electrophoresis using a high-molarity tris buffer system without urea. Anal Biochem 155:83–88.
OpenUrl CrossRef PubMed

[153] Fling SP,

[154] Gregerson DS

[155] ↵
Schägger H,
von Jagow G
(1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379.
OpenUrl CrossRef PubMed

[156] Schägger H,

[157] von Jagow G

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Single nucleotide mutation in the barley acetohydroxy acid synthase (AHAS) gene confers resistance to imidazolinone herbicides

Abstract

Results and Discussion

Screening Procedure and Selection of Resistant Lines.

Molecular Characterization of the Mutation.

Transcription Data.

Enzyme Extraction, Characterization, and Quantification.

Materials and Methods

Genomic DNA Extraction, PCR Amplification, and Sequence Analyses.

RNA Isolation, cDNA Synthesis, and Real-Time PCR Assays.

Enzyme Extraction, Characterization, and Quantification.

Acknowledgments

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References

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Single nucleotide mutation in the barley acetohydroxy acid synthase (AHAS) gene confers resistance to imidazolinone herbicides

Abstract

Results and Discussion

Screening Procedure and Selection of Resistant Lines.

Molecular Characterization of the Mutation.

Transcription Data.

Enzyme Extraction, Characterization, and Quantification.

Materials and Methods

Genomic DNA Extraction, PCR Amplification, and Sequence Analyses.

RNA Isolation, cDNA Synthesis, and Real-Time PCR Assays.

Enzyme Extraction, Characterization, and Quantification.

Acknowledgments

Footnotes

References

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