Single nucleotide mutation in the barley acetohydroxyacid synthase (AHAS) gene confers resistance toimidazolinone herbicidesHyejin Leea,1, Sachin Rustgia,1, Neeraj Kumara, Ian Burkea, Joseph P. Yenisha, Kulvinder S. Gilla, Diter von Wettsteina,b,2,and Steven E. Ullricha,2

aDepartment of Crop and Soil Sciences and bSchool of Molecular Biosciences, Washington State University, Pullman, WA 99164

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

Induced mutagenesis can be an effective way to increase variabil-ity in self-pollinated crops for a wide variety of agronomicallyimportant traits. Crop resistance to a given herbicide can be ofpractical value to control weeds with efficient chemical use. Insome crops (for example, wheat, maize, and canola), resistance toimidazolinone herbicides (IMIs) has been introduced throughmutation breeding and is extensively used commercially. How-ever, this production system imposes plant-back restrictions onrotational crops because of herbicide residuals in the soil. In thecase of barley, a preferred rotational crop after wheat, a period of9–18 mo is required. Thus, introduction of barley varieties showingresistance to IMIs will provide greater flexibility as a rotationalcrop. The objective of the research reported was to identify resis-tance in barley for IMIs through induced mutagenesis. To achievethis objective, a sodium azide-treated M2/M3 population of barleycultivar Bob was screened for resistance against acetohydroxy acidsynthase (AHAS)-inhibiting herbicides. The phenotypic screeningallowed identification of a mutant line showing resistance againstIMIs. Molecular analysis identified a single-point mutation leading toa serine 653 to asparagine amino acid substitution in the herbicide-binding site of the barley AHAS gene. The transcription pattern ofthe AHAS gene in the mutant (Ser653Asn) and WT has been ana-lyzed, and greater than fourfold difference in transcript abun-dance was observed. Phenotypic characteristics of the mutantline are promising and provide the base for the release of IMI-resistant barley cultivar(s).

imidazolinone resistance | crop rotation | improved transcription

Barley (Hordeum vulgare L.) is an important annual crop in thePacific Northwest (PNW) for 2- or 3-y rotations with winter

wheat (Triticum aestivum), pea (Pisum sativum), lentil (Lensculinaris), or fallow (1). One of the major reasons for theworldwide decline in barley acreage is its sensitivity to commonlyused herbicides. Many of the widely used herbicides imposingbarley plant-back restrictions belong to the group B herbicidesthat were first commercialized in 1982 (2). The advantages ofthese herbicides are low application rates, broad spectrums ofweed control, soil residual activity, high margins of crop safety,and low mammalian toxicity (3). This herbicide group consistsof five different chemical families: imidazolinones (4), sulfony-lureas (5), triazolopyrimidines (6), pyrimidyloxybenzoates (7),and sulfonlyaminocarbonyl-triazolinones (8). These herbicidestarget acetolactate synthase (ALS; EC 2.2.1.6), also known asacetohydroxyacid synthase (AHAS), which is an octameric en-zyme with four catalytic and four regulatory subunits (9). AHAScatalyses two parallel reactions in the synthesis of branched-chain amino acids. The first reaction is condensation of twopyruvate molecules to yield acetolactate, leading to the pro-duction of valine and leucine, and the second reaction is thecondensation of pyruvate and α-ketobutyrate to yield acetohy-droxybutyrate, leading to the production of isoleucine (10). TheAHAS-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 acidscauses a decrease in protein synthesis, which in turn, slows downcell division rate (5, 14). This process eventually kills the plantafter showing symptoms in meristematic tissues, where bio-synthesis of amino acids primarily takes place (15). Resistantplants, in most cases, depend on reduced sensitivity to these her-bicides by an isoform of AHAS, which does not severely affect itscatalytic activity. Most AHAS isoenzymes resistant to the herbi-cides carry substitutions for the amino acid residues Ala122,Pro197, Ala205, Asp376, Trp574, or Ser653 (amino acid num-bering refers to the sequence in Arabidopsis thaliana). The aminoacid residues Ala122, Pro197, and Ala205 are located at theN-terminal end of the enzyme, whereas Asp376, Trp574, andSer653 are located at the C-terminal end (16, 17). Amino acidsubstitutions at Ala122 and Ser653 confer high levels of resistanceto imidazolinone herbicides (IMIs), whereas substitutions atPro197 endow high levels of resistance against sulfonylureas andprovide low-level resistance against IMIs and triazolopyrimidineherbicides (18–22). Substitutions at Trp574 endow high levels ofresistance to imidazolinones, sulfonylureas, and triazolopyrimidines(19, 23–26), whereas substitutions at Ala205 provide resistanceagainst 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 varietiescultivated in the PNW. Thus, introduction of barley varietieswith resistance to IMI will provide greater flexibility as a rota-tional crop. The objective of the research reported was toidentify resistance in barley for IMIs through induced muta-genesis of barley cultivar Bob followed by phenotypic, genetic,and molecular characterization of the identified mutation(s).Development of an IMI-resistant barley cultivar through inducedmutagenesis in Bob will have many inherent advantages; for in-stance, the line is adapted to the PNW, it has excellent feedquality, and it will be nontransgenic in origin. In addition, IMI-resistant barley has less risk of introducing herbicide resistanceto the most common monocotyledonous weeds in the barleyfields. In contrast, IMI-resistant wheat has a potential risk oftransferring herbicide resistance to jointed goat grass (Aegilopscylindrica), which shares its D genome with common wheat (28,29). Also, ryegrass (Lolium perenne), both as forage crop and

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., andN.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.

Freely available online through the PNAS open access option.

Data deposition: The sequences reported in this paper have been deposited in the NCBIGenBank database (accession nos. HQ661102–HQ661107).1H.L. and S.R. contributed equally to this work.2To whom correspondence may be addressed. E-mail: diter@wsu.edu or ullrich@wsu.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1105612108/-/DCSupplemental.

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weed, has recently been shown to develop IMI resistance be-cause of gene flows from IMI-resistant crops through inter- and/or intraspecies hybridizations (30).

Results and DiscussionScreening Procedure and Selection of Resistant Lines. The two-rowspring feed barley cultivar Bob (31) was mutagenized using so-dium azide (NaN3) using a modified procedure developed byKueh and Bright (32). Approximately 500 g seeds were pre-soaked for 16 h at 0 °C followed by 8 h soaking at 20 °C withaeration in a water bath. The seeds were treated with 1 mMNaN3 (pH 3) for 2 h, rinsed three times, and dried. The muta-genized seeds were planted in isolation at the Spillman Agron-omy Farm, Pullman, WA, in the spring of 2007. The plants fromthese seeds, the M1 generation, were selfed, and M2 seeds werebulk harvested at maturity. The M2 seeds were screened forimidazolinone resistance using a modified procedure developedby Newhouse et al. (33). About 250 seeds were placed ona sterile 10 × 1.5-cm Petri dish and soaked in 25 mL imazethapyrsolution [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-hdark (16 °C) cycle. Each flat contained ∼1,000 seeds or fourtreated Petri dishes, and each flat was watered as needed.Emerged seedlings were visually evaluated after 4 wk. PutativeM2 mutants were transferred to pots and grown under the sameglasshouse conditions to maturity. The M3 seeds from putativeM2 mutants were harvested individually, and 5–10 M3 seeds fromeach plant were planted to increase seeds for further validation.The M4 seeds were used for verification using imazamox. The M4seeds from each line were individually placed into the cells ofa 25 × 50-cm, 72-celled flat. Two identical setups with barleycultivar Bob (WT) were used as controls: one with imazamoxtreatment and the other without herbicide treatment. Imazamoxwas applied over foliage when most plants were at the two-leafstage in an herbicide chamber. The spray solution included 818μM (34.7 g ai·ha−1) imazamox with nonionic surfactant (NIS) at0.25% of the solution volume. A moving nozzle cabinet sprayerwith 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, oneeach 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 M4plants derived from this line survived with little symptoms afterherbicide application (Fig. 1). This homozygous line showed nodifference in aboveground biomass compared with nontreatedcontrols of the susceptible WT Bob (Fig. 2A). The imazamox re-sistance did not seem to interfere with mutant fitness in general,although a difference in plant height was observed (Fig. 2B). Thismight be explained by a slightly delayed germination of themutants as a possible result to additional mutations induced by themutagen treatment. The mutant barley line was reciprocallybackcrossed to its progenitor Bob. Segregation ratios of F2 prog-enies confirmed the semidominant nature of the mutation andmonogenic inheritance of the IMI resistance (Table S1).

Molecular Characterization of the Mutation. The molecular natureof the mutant conferring IMI resistance in barley was de-termined by PCR amplification and subsequent sequencing of an∼850-bp fragment from the genomic DNA of the mutant and fivebarley cultivars: Bob, Morex, Steptoe, Champion, and Baron-esse. The fragment was amplified using the wheat AHAS-specificprimers CM-F and CM-R (Table S2) (34). DNA sequencesobtained from five susceptible barley cultivars (including WTBob) and the mutant line were aligned and searched for anypotential mutation in the gene sequence. The sequence analysis

revealed a single transition from G to A at around nucleotide1742 when based on the wheat AHAS gene sequence AY210406(Fig. S1). The point mutation leads to an amino acid substitutionfrom serine 653 (AGC) to asparagine (AAC) at the herbicide-binding site of the acetohydroxyacid synthase protein. The mu-tation (Ser653Asn) seems to be identical to the mutation con-ferring resistance against IMIs in Arabidopsis, tobacco (Nicotianatobaccum), maize (Zea mays), rice (Oryza sativa), oilseed rape(Brassica napus), and wheat (35). To eliminate the possibility ofmutations other then G to A transition in HvAHAS, sequences of1,845 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 pointmutation(s) between Bob and mutant (Fig. S3). However, twotransitions leading to synonymous changes between AF059600and the sequences obtained from Bob and mutant were observed(Fig. S3). The changes seem to be varietal differences, and theycould be used to develop SNP markers. The phylogenetic anal-ysis of the amino acid sequences around the amino acid sub-stitution 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 thecatalytic subunit of the AHAS enzyme. These subunits aggregate

Fig. 1. Phenotypic confirmation of resistance in the selected M4 mutantline. Negative control is the Bob without imazamox treatment (rows 1 and2), positive control is Bob with imazamox treatment (rows 3 and 4), Morexwith imazamox treatment is in rows 5 and 6, and mutant plants with ima-zamox treatment are in rows 7 and 8 (70 g ai·ha−1).

A B

Fig. 2. Phenotypic observations on the mutant line with positive and neg-ative controls. (A) Above-ground biomass was represented as percentage ofnontreated control plants. The values are averages and obtained from plantsdried 21 d after imazamox treatment. (B) Plant height was represented aspercentage of nontreated control plants. The values are averages andobtained from plants 21 d after imazamox treatment. Bobtrt, Bob withimazamox treatment; Bobnontrt, Bob without imazamox treatment; mutant,mutant with imazamox treatment. Double asterisks indicate significanceat ≤0.01; ns indicates no significant at ≤0.05.

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to form a tetramer and complex with another tetramer of fourregulatory subunits to build the AHAS apoenzyme (9). Theamino acid location of the point mutation detected in the currentstudy was also modeled on the crystal structure of the catalyticsubunit of the Arabidopsis AHAS enzyme by matching the partialamino acid sequence of the barley mutant AHAS with the Ara-bidopsismutant AHAS sequence (http://www.rcsb.org/pdb/home/home.do) (Fig. 4) (13). A one-step allele-specific assay for thebarley AHAS mutation (Fig. 5) allows identification of the mu-tant in production trials and breeding of resistant cultivars.The C-terminal ends of the catalytic subunits form the sub-

strate access channel, and substitutions at 6 (A122, F206, Q207,K256, W574, and S653) of 12 aa residues in the catalytic sub-unit are known to provide resistance against imidazolinone her-bicides (9). Four different substitutions were reported at Ser-653,replacing Ser with Asn, Thr, Phe, or Ile (9, 36), but to date, nonegative impact of substitutions at Ser653 on plant performancehas been reported, giving these point mutations a great com-mercial value for the development of IMI-resistant crops (37).The crystal structure of Arabidopsis AHAS with the IMI herbi-cide imazaquin indicates that a replacement of Ser653 with thelarger side chain carrying amino acids Asn or Thr likely obstructsbinding of the quinoline ring of imazaquin to the enzyme andthereby, leads to the herbicide resistance in the mutant plants(Fig. S4) (13). Single amino acid substitutions at one of six aaresidues (A122, F206, Q207, K256, W574, and S653) are knownto be sufficient to convert AHAS from a sensitive to resistantform (17). In addition, a limited initial test of the mutant linewith sulfosulfuron [used rate = 534.4 μM (35.2 g ai·ha−1) with

0.25% NIS] indicated that the line was likely susceptible to sul-fosulfuron (Fig. S5). These observations coincide well with otherstudies, where a point mutation at Ser653 resulting in Asn sub-stitution provided resistance against imidazolinone but notagainst sulfosulfuron (17).

Transcription Data. For relative quantification of HvAHAS tran-scripts in Bob and the mutant, four replicates of each sample wereused in analyses, and the same experiment was repeated two times(Fig. S6 A and B). The results of relative quantification suggestedthatHvAHAS transcripts were less abundant comparedwithActin,and interestingly, the mutant expresses ∼4.5 ± 0.3-fold more en-zyme compared with the WT Bob (Fig. S6 A and B).

Enzyme Extraction, Characterization, and Quantification. Presence ofAHAS in the extract was confirmed by the red color obtained inthe colorimetric assay in the presence and/or absence of imaza-mox solution and also by its molecular mass of ∼65 kDa on SDS/PAGE gel (Fig. 6 A and B). The relative quality of AHAS againstknown quantities of BSA in Bob and mutant was estimated fromBradford assay and RP-HPLC analyses (SI Materials and Meth-ods). On the basis of Bradford assay, the mutant (2.05 μg/mL)accumulated relatively more enzyme than Bob (1.41 μg/mL), andthis result was complemented by RP-HPLC quantifications (Fig.S7 A–C), where AHAS enzyme in mutant (1.85 μg/mL) quanti-fied more than Bob (1.54 μg/mL).The barley mutant reported in the present study produces

relatively more enzyme in comparison with WT, and the segre-gation ratios obtained from the reciprocal crosses between the

AAO53548-T. aestivumAAO53550-T. aestivumACR55069-Ae. tauschiiAAO53549-T. aestivumAAO53551-T. aestivumAAC14572-H. vulgareAAM03119-B. tectorumACR55067-A. fatuaAAG30931-L. multiflorumAAX14282-O. sativa Japonica ACD74789-O. sativa IndicaNP_001151761-Z. maysABM92357-C. difformis1YBH_Chain A-A. thaliana

E H V L P M I P S GG A F K DM I 598E H V L P M I P NGG A F K DM I 598E H V L P M I P S GG A F K DM I 572E H V L P M I P S GG A F K DM I 598E H V L P M I P NGG A F K DM I 598E H V L P M I P S GG A F K DM I 541E H V L P M I P S GG A F K D I I 583E H V L P M I P S GG A F K D I I 572E H V L P M I P S GG A F K D I I 640E H V L P M I P S GG A F K DM I 644E H V L P M I P S E G A F K DM I 644E H V L P M I P S GG A F K DM I 547E H V L P M I P S GG A F K DM I 569E H V L P M I P S GG T F N D V I 590* * * * * * * * . * : * : * : *

*

*

Fig. 3. Comparative sequence alignment of AHAS showing phylogenetic relationships among members of grass lineage and Arabidopsis; the rectangularbox indicates the position of the amino acid substitution providing resistance against imidazolinone herbicides. *Sequences obtained from seed mutagenizedwheat 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. The2D topology diagram was modified from Duggleby et al. (9). [Reprinted from Plant Physiology and Biochemistry, 46(3), RG, Duggleby, JA McCourt, LWGuddat, Structure and mechanism of inhibition of plant acetohydroxyacid synthase, 309–324, Copyright (2008), with permission from Elsevier.]

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mutant and Bob revealed single-gene inheritance for herbicideresistance. Taken together the two results suggest that the iso-enzyme produced by the mutation may influence its feedbackregulation or catalytic properties (2). Because the mutant plantsproduce slightly more enzyme compared with WT, it may com-pensate for a reduced functionality of the enzyme isoform andcontribute to the full viability of the plant and its survival underapplication 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 asproviding resistance to IMIs, which are used extensively to keepspring and winter wheat fields in the PNW free of grass weeds.The herbicides are retained in the soil, preventing current barleycultivars from being planted as alternative crops in normal croprotations. The induced mutant in the cultivar Bob has retainedthe phenotype of the parent Bob and shows that these mutationscan restore barley as a suitable alternative crop. The specificity ofamino acid substitutions in AHAS to individual herbicides showsthe possibility to select resistance to individual herbicides byTargeting Induced Local Lesions in Genomes (TILLING) aswell as transgenic technology.

Materials and MethodsGenomic DNA Extraction, PCR Amplification, and Sequence Analyses. DNA wasextracted from 1-mo-old seedlings of each of the six barley genotypes usingthe modified hexadecyl(trimethyl)arasimum bromide (CTAB) method (38).The PCR reactions were carried out in 20-μL reaction mixtures, each con-taining 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 fol-lowing PCR profile: initial denaturation at 95 °C for 5 min followed by 40cycles at 95 °C for 30 s, 56–60 °C for 30 s (Table S2), 72 °C for 45 s, and a finalextension at 72 °C for 10 min. The amplification products were resolved on2% agarose gels. A 100-bp ladder was used as a size marker (New EnglandBioLabs). Bands of expected sizes were excised from the gel, and DNA waseluted from the bands using the Geneclean kit following the manufacturer’sinstructions (MP Biomedicals). The eluted DNA was used as a template forthe sequencing reaction using either forward or reverse primers in separatereactions. The sequencing reactions were carried out at the DNA SequenceCore, Washington State University, Pullman, WA. Sequence identity searcheswere performed at the National Center for Biotechnology Information (NCBI;http://www.ncbi.nlm.nih.gov) using BLAST. Alignment of the deduced aminoacid sequences was performed using the Vector NTI AdvanceTM 9.1 (Invi-trogen).

RNA Isolation, cDNA Synthesis, and Real-Time PCR Assays. RNA was extractedfrom 1-wk-old seedlings of Bob and mutant. The seedling leaves were har-

vested in liquid nitrogen, and about 0.5–1 g tissue were pulverized to finepowder using pestle and mortar. Total RNA was isolated using 8 mL TRIZOLreagent (Invitrogen Corp) per sample according to the manufacturer’s rec-ommendations. 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 ina total volume of 11 μL diethylpyrocarbonate (DEPC)-treated nuclease freewater, heated at 65 °C for 5 min, and chilled on ice. The RNA mixture wassupplemented with 8 μL mixture containing 1× First Strand buffer (Invi-trogen), 10 mM dithiothreitol (DTT), 500 mM each dNTP, and 40 unitsRNaseOUT (Invitrogen) in a final volume of 25 μL, and the mixture washeated for 1 min at 55 °C in a thermocycler. After 1 min, cDNA synthesis wasinitiated by adding 200 units SuperScript III reverse transcriptase (Invitrogen)to the mixture and incubating it for 2 h at 55 °C. The cDNA was precipitatedin 2.5 volumes ethanol in the presence of 0.3 M sodium acetate (pH 5.2) andresuspended in 20 μL nuclease-free water.

Real-time quantitative PCR (qPCR) analysis of HvALS transcripts was per-formed using the DNA Master SYBR Green 1 chemistry on The LightCycler480 Real-Time PCR System (Roche Diagnostics). PCR primers for HvALS andActin (used as internal controls) are listed in Table S2. Each PCR consisted ofabout 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 35cycles of 95 °C for 10 s, 57 °C for 5 s, and 72 °C for 10 s. Amplicon meltingprofiles were generated over a range of 65 °C to 98 °C, with a temperaturechange of 0.1 °C s−1 and fluorescence monitoring every 0.3 °C s−1. HvALSmRNA level was normalized to Actin using the DDCT method (39). Transcriptlevels were expressed as a ratio of HvALS transcripts (normalized to Actin)in mutant and Bob.

Enzyme Extraction, Characterization, and Quantification. AHAS enzyme wasextracted from Bob andmutant using 5 g seedling leaves using themethod ofSingh et al. (40). Extracted enzyme was loaded on 10% SDS/PAGE followedby staining with Coomassie Brilliant Blue G-250 (41, 42). A prestained proteinmolecular weight marker (SM0441; Fermentas) was loaded in each gel forsize estimation. Presence of AHAS enzyme was confirmed by Westerfeldreaction (40). Enzyme was quantified using Quick Start Bradford 1× dyereagent (500–0205; BioRad) following the manufacturer’s instructions. BSAwas used as standard (six standards from 0.2 to 2.2 μg/mL), and absorbancewas measured at 595 nm on a BioRad SmartSpec Plus spectrophotometer.Values were plotted on a graph, with the dependent variable (in microgramsper milliliter) on the x axis and the independent variable (abs 595 nm) on they axis; linear regression was performed, and the amounts of enzyme in Boband mutant were calculated.

ACKNOWLEDGMENTS. The authors would like to thank Nuan Wen forlaboratory assistance and Vadim Jitkov and Max Wood for field andgreenhouse assistance. This work has been performed with financial supportfrom the Washington Grain Commission and National Institutes of HealthGrant 1R01GM080749-01A1.

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Mutant

120kDA

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5µl 7µl 9µl

Bob

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Fig. 6. (A) SDS/PAGE analysis of AHAS enzyme extracted from Bob andmutant. (B) Colorimetric assay of AHAS enzyme in the presence and absenceof imazamox (beyond = concentration 3.2 μM). M, protein molecular weightmarker; arrowhead, band of interest.

M Bob

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Fig. 5. (A) One-step allele-specific assay for AHAS mutation using a combi-nation 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_Fand ALS_O_R) used as loading controls. Primers used are described in TableS1, and their putative locations are depicted in Fig. 3. M, size markers (100bp ladder; New England Biolab); arrowhead, expected product size.

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