Targeted and efficient transfer of value-added genesinto a wheat variety

N. Kumar & Harpinder S. Randhawa &

Ryan W. Higginbotham & Xianming Chen &

Timothy D. Murray & Kulvinder S. Gill

Received: 24 October 2016 /Accepted: 7 March 2017# Springer Science+Business Media Dordrecht 2017

Abstract With an objective to optimize an approach totransfer value-added genes to a wheat variety whilemaintaining and improving agronomic performance,two alleles (Als1 and Als2) with mutations in theacetolactate synthase (ALS) gene located on the longarm of wheat chromosomes 6B and 6D providing toler-ance to imazamox herbicide were transferred to Eltan, apopular soft white common winter wheat cultivar in thePacific Northwest (PNW), USA. Four-step marker-assisted background selection and marker assisted for-ward breeding approaches were used to develop a wheatvariety carrying two genes (Als1 and Als2) forimazamox tolerance along with improvements in manyother agronomic traits. Screening of 1600 BC1 plants for

imazamox tolerance identified 378 plants that were fur-ther screened with markers to identify seven plants thatwere used to make a population of 1400 BC2 plants, andthe selection cycle was repeated. Progeny of 17 selectedBC2F1 plants was evaluated for various agronomic andquality parameters to select 12 plants that were in-creased for field testing. Field evaluation of these linesconducted over 58 location-years along with evaluationin the greenhouse/growth chamber led to the selection ofa line BWA8143^ carrying the two genes for imazamoxtolerance that yielded >3% higher than Eltan did. With96.8% similarity to the recurrent parent, WA8143 alsoshowed a better disease resistance package and grainquality needed in a successful Pacific Northwest wheatvariety and was subsequently released for cultivationunder the name of BCuriosity CL+.^

Keywords Triticum aestivumL. .Marker-assistedbackground selection . Two-gene imazamox tolerance .

Fast breeding . Forward breeding

AbbreviationsMABS Marker-assisted background selectionSRC Solvent retention capacitySSR Simple sequence repeatSWW Soft white winterWECVTP WSU extension cereal variety

testing programWSU Washington State UniversityWWQL Western Wheat Quality LaboratorySKCS Single kernel characterization systemRPG Recurrent parent genome

Mol Breeding (2017) 37:68 DOI 10.1007/s11032-017-0649-1

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11032-017-0649-1) contains supplementarymaterial, which is available to authorized users.

N. Kumar :K. S. Gill (*)Department of Crop and Soil Sciences, Washington StateUniversity, Pullman, WA 99164-6420, USAe-mail: ksgill@wsu.edu

H. S. RandhawaAgriculture & Agri-Food Canada, Lethbridge Research Centre,Lethbridge, AB, Canada

R. W. HigginbothamWSU Extension, ANR Program Unit, Washington StateUniversity, Pullman, WA 99164-6420, USA

X. Chen : T. D. MurrayUSDA-ARS, Wheat Health, Genetics and Quality Research Unitand Department Plant Pathology, Washington State University,Pullman, WA 99164-6430, USA

Introduction

Variety development is a time-consuming and expen-sive process especially in winter wheat (Triticumaestivum L.) crops that takes about a year to completea lifecycle. First introduced by Harlan and Pope (1922),backcross breeding is an effective method to transferdesirable genes into popular cultivars. Conventionalbackcrossing is an effective method for developinggermplasm carrying value-added genes, but it rarelyresults in successful cultivars even after six cycles ofbackcrossing. Marker-assisted background selection(MABS) is an effective method to develop release-ready cultivars in just two backcrosses (Randhawaet al. 2009). Most successful crop varieties are eventu-ally rendered unfit for cultivation because of one or twoundesirable traits, often caused by sudden populationshifts and/or mutations in ever-evolving, dynamic pestpopulations leading to disease susceptibility or changesin consumer preference. Rapid and precise introgressionof value-added genes is essential to alleviate these con-straints for continued success of a cultivar to protect andincrease yield potential and to benefit from newly avail-able value-added genes in a timely manner (Randhawaet al. 2009). There are only a few successful examples oftransferring gene(s) of interest using MABS. The germ-plasm lines were developed in wheat by transferringcomplex quality traits including grain protein contentand pre-harvest sprouting tolerance to a cultivar (Kumaret al. 2010, 2011). A major QTL controlling submer-gence tolerance was transferred using marker-assistedbackcrossing (Neeraja et al. 2007). In another study,QTL for submergence tolerance was also transferredthat eventually resulted in the release of a new ricevariety (Iftekharuddaula et al. 2015). Genes controllingbacterial blight and brown planthopper resistance weretransferred using marker-assisted backcrossing to im-prove existing rice cultivars (Sundaram et al. 2008;Basavaraj et al. 2010; Hu et al. 2016). In pepper (Cap-sicum annum) variety, a mutation in the (putative ami-notransferase) gene controlling capsinoid content wastransferred using marker-assisted backcrossing (Jeonget al. 2015). Originally designed using BPlabsim^ sim-ulations, a four-step MABS method was optimized andshown to be effective in transferring a stripe rust(P. striiformis f. sp. tritici) resistance gene while recov-ering 97% of the recurrent parent background in just twobackcrosses (Randhawa et al. 2009). Application of theprocedure to transfer two or more genes is not known,

however. It is also not known, if the resultant lines willhave the agronomic performance required for releasinga new cultivar. In the present study, we show that theoptimized method can also be used to simultaneouslytransfer multiple value-added genes to result in success-ful cultivars.

Annual grassy weeds are a major problem in manyparts of the world especially in the high and mediumrainfall regions, and they are responsible for significantyield and economic losses. Imidazolinone herbicidessuch as imazamox belong to a major group of herbicidesthat can provide a wide spectrum of weed control.Initially registered in 1998 BClearfield® Technology^allows the use of imidazolinone herbicide for effectiveweed control in wheat and other crops (Shaner et al.1996; BASF 2004). The recurrent parent for the currentstudy was Eltan, a very successful and popular softwhite winter (SWW) wheat cultivar (Peterson et al.1991), which still occupies >50,000 acres in the PNWafter more than 25 years of cultivation. For many years,it has been grown on more than 200,000 acres in thestate of Washington. Eltan is specifically adapted to themedium-low rainfall areas of Washington State and waschosen for this project in order to give the two-geneherbicide tolerance option to the farmers. It has excellentyield potential with very high levels of resistance tosnow mold (caused by Microdochium nivale,Sclerotinia boreali Pythium spp., and/or Typhula spp.)and Cephalosporium stripe (caused by Cephalosporiumgramineum) diseases though moderately susceptible tothe local races of Puccinia striiformis f. sp. tritici, thewheat stripe rust pathogen. The main objective of thepresent study was to optimize the MABS approach totransfer two different value-added genes into a popularcultivar and to incorporate forward breeding steps todevelop wheat cultivars with improvements over therecurrent parent in a short period of time. Transfer ofthe two genes for herbicide tolerance into cultivar Eltanwas used as an example.

Materials and methods

Plant material

The Australian hard red spring wheat line BCL0618^was used as a donor to transfer two genes for imazamoxtolerance. The line carries mutations in the large subunitof two of the three wheat ALS or AHASL (acetohydroxy

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acid synthase large subunit) genes, providing toleranceto the imidazolinone group of herbicides (imazamox).These two mutations are located on the long arm ofwheat chromosomes 6D (AhasL-D1) and 6B (AhasL-B1) and will be referred to as Als1 and Als2, respective-ly. The recurrent parent was the SWW wheat cultivarBEltan.^

AHAS-allele-specific marker analysis

Allele-specific markers were used to select plants carry-ing one or more of the Als mutant alleles using a gel-based test. For this procedure, two rounds of PCRreactions were performed using allele-specific primers.In the first round, a nested PCR was performed withcommon primers CM-F (5′-CCGCCGCAATATGCTATCCAG-3′) and CM-R (5′-GTAGGACAAGAAACTTGCATG-3′) to amplify the two AHAS genes fromchromosomes 6D and 6B. The PCR product from thefirst reaction was used as templates for the second PCRreaction using three primers: the nested forward andreverse allele-specific primers (for Als1 gene 1D-F,1D-R primers and for Als2 gene 1B-F, 1B-R) and anallele-specific primer (wild-type forward (WT-F) or mu-tant type forward (MU-F)) with the mutated/wild-typebased on its 3′ primer end for amplification of theresistant/susceptible locus, respectively (Perez-Joneset al. 2006). The larger PCR fragment between thegene-specific primers would always amplify, and thisconstant PCR band can be used as a positive control forthe PCR reaction (control band). The smaller PCRfragment (amplified by the allele-specific primer andreverse gene-specific primer) is an allele-specific PCRproduct (diagnostic band).

The first PCR reaction was performed in 25 μl reac-tions containing 1× PCR buffer, 0.2 μM of each primer,0.2 mMof each of the four deoxynucleotides, 0.5 unit ofTaq DNA polymerase (Roche Applied Science, IN,USA), and ∼100 ng of the template DNA. The secondPCR reaction was performed in 25 μl containing 1×PCR buffer, 0.16 μM of the forward primer, 0.2 μMof the reverse primer, 0.2 mM of each of the fourdeoxynucleotides, and 0.125 unit of BHotStar^ Taq po-lymerase (HotStar Taq-Polymerase Qiagen, Valencia,CA, USA). The first PCR amplification was performedin 96-well plates on a Bio-Rad iCycler (Bio-Rad labo-ratories Hercules, CA). The PCR cycling programconsisted of a denaturation step of 3 min at 94 °C,25 cycles (30 s at 94 °C, 1 min at 56 °C, 45 s at

72 °C, and final extension for 7 min at 72 °C. Thesecond-step PCR program consisted of one denaturationstep of 15min at 95 °C, followed by 8 touchdown cycleswith annealing temperature 1 °C decreased for eachcycle (1 min at 94 °C, 1 min at 68 °C, 1 min at 72 °C),then 26 cycles (1 min at 94 °C, 1 min at 60 °C, 1 min at72 °C) and final extension for 10 min at 72 °C. The PCRproduct was loaded on a 2% agarose gel prepared andrun in 1× Tris base, boric acid and EDTA (TBE) bufferand products visualized on a UV trans-illuminator.

SSR marker analysis

The four-step marker-assisted background selection ap-proach was followed as described earlier (Randhawaet al. 2009). The information of gene recombinationand distribution was used as described by Eraymanet al. (2004). Genomic DNA isolation and simple se-quence repeat (SSR) marker analysis was carried out asdescribed earlier (Randhawa et al. 2009). A total of∼715 wheat (SSRs) markers (Xbarc, Xwmc, Xcfd, Xcfaand Xgwm) covering the wheat genome were screenedfor polymorphic survey between Eltan and CL0618.Primer sequences of the SSR markers with prefixesBXbarc,^ BXwmc,^ Xcfd, and BXcfa^ were retrievedfrom the GrainGenes (http://wheat.pw.usda.gov/GG2/index.shtml), while sequences of BXgwm^ primerswere obtained from Röder et al. (1998). DNA fragmentanalyses were performed using either Li-COR DNAsequencer or an ABI 3730 Gene Analyzer (AppliedBiosystems, Foster City, CA). Marker analysis wascarried out using tailed-PCR, where the forward primerhad a 19-bp M13 sequence (5′-CACGACGTTGTAAAACGAC-3′) at the 5′ end and an M13 fluorescentlylabeled primer added to the reaction (Schuelke 2000).The 12 μl PCR reaction mix contained 10 ng DNA; 1.2μl of 10× PCR buffer; 0.48 μl of 25 mMMgCl2 (RocheApplied Science, USA); 0.24 μl of 250 μM each ofdCTP, dGTP, dTTP, and dATP (GenScript USA Inc.Piscataway, NJ); 0.06 μl of 10 μM M13-tailed forwardprimer; 0.3 μl of 10 μM reverse primer; 0.24 μl of 10μM M13 primer fluorescently labeled with 6-FAM (6-carboxifluoresceine), Hex (hexachloro-fluoresceine),NED [N-(1-naphthyl)-ethyletediamine], and PET (Ap-plied Biosystems, Foster City, CA); and 0.6 U TaqDNAPolymerase (New England Biolabs, Ipswich, MA). PETis an unpublished proprietary dye of AppliedBiosystems (Foster City, CA, USA) that provide redcolor. PCR amplification was performed in 96- or 384-

Mol Breeding (2017) 37:68 Page 3 of 11 68

well plates on a Bio-Rad iCycler (Bio-Rad laboratoriesHercules, CA) with an initial cycle of 94 °C for 3 min,42 cycles of 1 min denaturing at 94 °C, 1 min annealingtemperature 60 °C, 1 min extension at 72 °C, and thefinal extension at 72 °C for 10 min. The PCR productslabeled with different fluorophores were mixed anddetected on an ABI 3730 Gene Analyzer (AppliedBiosystems, Foster City, CA). Amplified PCR productswere size-separated by capillary electrophoresis on Ap-plied Biosystems fragment analyzers (3130xl GeneticAnalyzer or 3730xl DNA Analyzer). Raw data filesfrom fragment analyzers were imported intoGeneMarker v1.5 (SoftGenetics) to analyze ampliconsizes.

Phenotypic screening for herbicide tolerance

Screening for herbicide tolerance was performed both inthe greenhouse/growth chamber as well as in the field.For the greenhouse/growth chamber screening, plantswere grown in 96-celled, 25 × 50-cm trays containingSunshine mix, a commercial potting media (Pottingmedia, LC1 Mix. Sungro Horticulture DistributionInc., Bellevue, WA). Natural light of the greenhousewas supplemented with sodium halide lamps to providea 16-h day length with temperatures of 24 °C day and18 °C night (±3 °C). Plants were sprayed with theherbicide at 3 to 5 leaf stage using an air-pressurizedindoor spray chamber equipped with TeeJet 8002E flat-fan nozzles (TeeJet Spraying Systems, Wheaton, IL,USA) calibrated to deliver a volume of 186 L/ha at193 kPa. Wheat plants were treated with imazamoxherbicide at the rate ranging from 52.5 to 140 gai/hadepending upon the objective. The spray mix alsocontained 2.0% (v/v) methylated seed oil (MSO) and5.0% (v/v) urea ammonium nitrate (UAN). The treatedplants were placed back in the greenhouse. Untreated orcontrol plants were maintained in the same greenhouse,which were used for comparison to score percent phys-ical injury. Physical injury was recorded at 14, 21, 28,and 35 days after treatment (DAT). Physical injury wasrecorded on a 0–100% scale as described by Carter et al.(2007).

Field evaluation for herbicide tolerance was per-formed using uniform field plots of varying sizes withthree replications. Spray rates of 52.5 and 105 gai/ha ofimazamox were used along with controls. MSO wasused as a surfactant 2.0% (v/v) in the mix. Physicalinjury was recorded on a 0–100% scale, as mentioned

above. Eltan was used as a susceptible check, whereascommercial two-gene Clearfield variety BAP503CL2(PVP 200800322)^ was used as a positive control forherbicide tolerance.

Multi-location field evaluation

The agronomic performance of the selected lines wasevaluated under trials conducted by Washington StateUniversity, Extension Cereal Variety Testing Program(WECVTP; http://smallgrains.wsu.edu/variety). TheWECVTP annually evaluates more than 75 winterwheat lines, including popularly grown varieties alongwith experimental lines from both public and privatebreeding programs. Field evaluations were conducted at22 locations throughout the state of Washington usingan alpha-lattice design with three replicates. Plot sizevaried, depending on trial location. A mechanical smallplot combine (Nurserymaster Classic, Wintersteiger Co., Salt Lake City, UT) was used to harvest matured wheatplots. Grain volume weight was measured using aSeedburo filling hopper and stand (Seedburo EquipmentCo., Chicago, IL, USxA).

Evaluation of grain quality

Grain quality parameters including grain hardness andgrain protein content were analyzed using the Perten4100 instrument and by single kernel characterizationsystem (SKCS) (Perten NA, Springfield, IL). Prelimi-nary evaluation of grain quality was performed usingsingle plant seed harvested from greenhouse for micro-solvent retention capacity (micro-SRC) test with twosets of solvents (sodium carbonate and sucrose) as de-scribed by Bettge et al. (2002). The milling and bakingquality analyses were performed by the USDA-ARS,WesternWheat Quality Laboratory in Pullman,WA. Forquality analyses, ∼600 g grain samples were used bymixing 200 g wheat grains from each of the threereplications for each location.

Evaluation of disease resistance

Greenhouse evaluation for stripe rust resistance wascarried out in a growth chamber using freshly amplifiedurediniospores of stripe rust prevalent races in the PNW,i.e., PST-37, PST-45, PST-100 and PST-114, (Chen2005). Plants were inoculated at a two-leaf stage usingspores mixed with talcum powder as described by Chen

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and Line (1992). A fine mist of water was sprayed onthe plants and maintained high relative humidity(∼100%) by placing inoculated plants in a dew chamber.After 16 h of incubation at 10 °C, plants were moved toa growth chamber with a temperature regime of 8 and16 °C cycled every 6 h starting with 8 °C at midnight.Disease scoring was performed 21 days after inoculationusing the standard scale and method described by Chenand Line (1992). During the season crop of 2011–2014,stripe rust evaluationwas also carried out at various fieldlocations of the Washington State including theSpillman Agronomy Farm, Plant Pathology Farm,Mount Vernon, Walla Walla, and Lind Dryland Re-search stations. Infection type (IT) was recorded basedon the 0–9 scales, whereas percent severity was record-ed on the 0–100% scales as described by Chen and Line(1992, 1995). Generally, IT 0–3 was considered resis-tant, 4–6 intermediate or moderately resistant, and 7–9 israted as susceptible. WA8143 was also tested along withtheir recurrent parent Eltan for snow mold disease atspecific locations where snow persists for more than100 days. The data was also recorded on snow mold in2 years (2011 and 2013) under WECVTP trials.WA8143 was also evaluated along with Eltan forCephalosporium stripe under field nursery with artificialinoculation for crop season 2011 and 2013.

Statistical analysis

Statistical analysis of the yield data was performed usingProc GLM ANOVA in Agrobase and significant differ-ences were identified by least significant difference(LSD) test at 5% significance level using AgrobaseGeneration II (Agromix Software Inc., Winnipeg

Manitoba, Canada). Statistical analysis of quality datawas computed using SAS V9.3 (SAS Institute, Raleigh,NC, USA). Paired t test was performed to analyzequality parameters, and significant differences wereidentified by LSD test at the 5% significance level.

Results

The backcrossing steps

Approximately 715 wheat (SSRs) primers coveringwhole-wheat genome were screened for polymorphismbetween the two parents (Table S1). Approximately52.7% (377/715) of the markers were polymorphic. Ofthe 89 markers for the two-carrier chromosomes, 47were polymorphic including 21 for chromosome 6Band 26 for 6D. Among non-carrier chromosomes, thenumber of polymorphic markers per chromosomeranged from seven for chromosome 6A to 27 for 2Dand 5D each (Table S1). Selected polymorphic markerswere used for MABS, as mentioned below.

The overall MABS approach and the number ofplants used in each backcross generation is given inFigs. 1 and S1. Of the ∼1600 BC1F1 plants that weresprayed with imazamox at 105 gai/ha rate, 378 survivedand the remaining plants (1222) died. Out of the 378survived plants, 96 were excluded for further tests basedon the plant health and the remaining 282 were analyzedusing 11 and 12 SSRmarkers of the carrier chromosome6B and 6D, respectively. This analysis identified 44plants carrying the highest proportion of RPG, whichwere further analyzed with 42 markers covering theremaining 19 non-carrier chromosomes (Table 1). Com-bined analysis of the markers from carrier and non-carrier chromosomes resulted in the selection of 10

Fig. 1 PCR amplification of lines derived by MABS approach. aBanding pattern of allele-specific primers of Als1 gene. b Bandingpattern of allele-specific primers of Als2 gene along with donorand recipient parents. C CL0618, donor of two genes (Als1 andAls2); E recipient parent Eltan without ALS gene, 1–4 two-genelines derived in recipient background by MABS, 5–10 one genederivatives (Als1 or Als2)

Table 1 Recurrent parent genome (RPG) recovery of MABS

Loci analyzed BC1F1 BC2F1 BC2F2:3

No. of loci analyzed 65 149 377

Homozygous loci for RP 44 132 363

Homozygous loci for DP 0.0 0.0 10

Heterozygous loci 21 17 4

Percentage of RPG 83.8 94.3 96.8

Percentage of DPG 16.2 5.7 3.2

DPG donor parent genome, RP recurrent parent, DP donor parent

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plants carrying both (Als1 and Als2) genes along withthe maximum proportion of RPG. These 10 selectedplants were used to make 1400 BC2 plants.

Only 364 of the 1400 BC2 plants survived afterimazamox spray with 140 gai/ha rate. The remaining1036 plants died. Out of the 364 plants, 97 were furtherrejected on the basis of plant health and the remaining267 were analyzed using DNAmarkers for the carrier aswell as non-carrier chromosomes. Marker analysis ofthe 267 plants with the carrier chromosomemarkers (6Band 6D specific) identified 41 plants showing 26 of the31 markers to be recurrent parent type. Further markeranalysis of these 41 plants with 117 polymorphic SSRmarkers covering the non-carrier chromosomes identi-fied 17 plants with the highest proportion of the RPG.The seeds of the 17 selected plants formed 17 BC2F2lines.

About 100 seeds of each of the 17 selected plants(∼1700) were first screened for herbicide tolerance, asmentioned above. About 509 plants survived after theherbicide spray of which only 332 tolerant and healthy-looking plants were selected and used for DNA markerbased on background analysis with 60 SSR markers.These plants were also evaluated for SKCS and micro-SRC in order to identify plants with a desirable grainquality. Combined analyses of the quality parametersand DNA marker data identified 12 plants that werehomozygous for both of the mutations (Als1 and Als2)and had the desired agronomic characteristics and grainquality. These plants were further genotyped with 377SSRs in order to perform DNA marker-based forwardselection as well as to prioritize plants for cultivar re-lease. Progeny of these plants were grown in the fieldand greenhouse to evaluate agronomic performance anddisease reaction. Growth chamber screenings showedthese 12 plants to be resistant to the predominant racesof stripe rust founding in the PNW region of the USA.The micro-SRC evaluation of the 144 plants helped toreject several inferior plants and selected above 12plants including a line BWA8143^ that also showedfavorable SRC values. We selected the plants towardslower sucrose-SRC as well as carbonate-SRC but ahigher break flour yield and predicted flour yield(Fig. 2). The progeny of these plants was evaluatedunder multi-location field evaluation under differentprograms, as well as in the greenhouse. Combinedanalysis of various types of data resulted in the selectionof WA8143 that was eventually released as a varietyunder the name of Curiosity CL+. Detailed

characterization of WA8143 for various agronomiccharacteristics is given below.

DNA marker analysis

DNA marker analysis of the selected 12 plants withthe 377 polymorphic markers covering the entirewheat genome identified WA8143 to be carryingthe highest proportion of the carrier chromosome(Fig. 3). Out of the 377 markers, 363 were homo-zygous for the Eltan-type alleles, 10 were homozy-gous for donor (CL0618)-type alleles, and the re-maining four were heterozygous; therefore, WA8143was found to have a very high proportion of RPG(96.8%). We also found very high RPG recovery forcarrier chromosomes (6D and 6B); out of the 47 ofthe total polymorphic markers, 46 were homozygousfor the Eltan-type alleles and one marker was homo-zygous for donor (CL0618)-type allele and showsvery high recovery of RPG (97.9%). Analysis ofWA8143 with the allele-specific markers for theherbicide tolerance trait confirmed it to be homozy-gous for both of the mutant alleles.

Herbicide tolerance

In addition to the marker confirmation mentionedabove, herbicide tolerance of WA8143 was evaluated

Fig. 2 Binary plot distribution of two quality parameters (sucrose% and break flour yield %) of micro-solvent retention capacity(micro-SRC) test estimated from backcross population forherbicide tolerance along with the recipient parent Eltan

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under greenhouse as well as in field conditions(Table S2 and Figs. 1 and S2). Under greenhouse con-ditions, WA8143 had a herbicide tolerance responsesimilar to the two-gene Clearfield® control varietyBAP503CL2^ and was significantly better than thesingle-gene Clearfield® control variety BORCF-102^(PVP 200500337) (Table S2 and Fig. S2a, b, c). Thehighest physical injury on WA8143 at the rate 140 gai/ha was 15%, similar to that observed on the two-genecontrol line AP503CL2. The negative control Eltanshowed 100% damage of 35 DAT and the single-genecontrol BORCF102^ showed 50% damage.

Under field evaluation, response of WA8143 to her-bicide spray was similar to that observed in the green-house evaluation (Table S2). WA8143 showed 5% in-jury at the rate 52.5 gai/ha of imazamox spray on 14DAT that was similar to that observed for AP503CL2and was significantly lower than the single-gene controlBORCF-102^ (20%) and the negative control line Eltan(70%). For all lines, a greater physical injury was re-corded at 105 gai/ha rate of imazamox spray. The

physical injury of WA8143 and AP503CL2 was up to15% compared to 40% on ORCF-102 and 100% onEltan (Table S2). These results clearly indicated thatWA8143 has a very high level of herbicide toleranceas compared to negative control (Eltan) in greenhouse aswell as in field conditions.

Agronomic performance

The agronomic performance of WA8143 was comparedwith Eltan as well as with other popular varieties grownin PNW, under the WECVTP multi-location trials con-ducted during the 2011–2014 crop years (http//variety.wsu.edu/SWW/2011/2014) (Table 2). WA8143 showedsimilar agronomic characteristics as its recurrent parentEltan. Both lines are semi-dwarf, with awns and com-mon head type and white straw with white glumes. Theaverage plant height of WA8143 was 36 in compared to35 in for Eltan. Days to heading for both varieties wereabout 155 days. The average grain yield across 58location-years ofWA8143was 6447 kg/ha, significantlygreater than Eltan (6256 kg/ha) (Table 2). Of the 58location-years, WA8143 yielded significantly higher at22 location-years, whereas Eltan yielded significantlyhigher at four location-years and the rest of the location-years have no yield differences. WA8143 did particular-ly well in the low rainfall and colder climatic zones ofthe PNW. Currently available data across 4 years (2011–2014), WA8143 is the top-yielding two-gene Clear-field® variety available in the PNW region. Comparingwith other high-yielding varieties with or without Clear-field® traits especially in low-rainfall (<12 in annualprecipitation) areas of the Washington State, WA8143achieved first rank after 3 years of independent evalua-tion by WECVTP. Other useful traits of WA8143 in-cluding winter hardiness and seedling emergence weresimilar to that of Eltan.

Grain and end-use quality

The grain and end-use quality assessment of WA8143and Eltan was also conducted by the USDA-ARSWest-ern Wheat Quality Laboratory (WWQL) Pullman, WA(Table 3). The data averaged over 18 locations sug-gested that WA8143 has similar grain quality attributesto their recurrent parent Eltan such as test weight, grainprotein content, single kernel hardness, milling score,flour protein, flour sedimentation coefficient,mixograph absorption, and cookie diameter (Table 3).

Fig. 3 Graphical representations showing parental derivation,where each bar represents the wheat chromosome with A, B,and D genome; green bars represent homozygous alleles of recip-ient genotype; red bars indicate the homozygous marker allele fordonor, and heterozygous loci were marked with half red and halfgreen bar. Black star represents the approximate position ofherbicide-tolerant genes (Als1 and Als2) on carrier chromosome6D and 6B, and yellow lines on the bars show the approximateposition of flanking markers, whereas black lines on the bars showthe relative position of markers used for background selection

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The single kernel weight of WA8143 (39.5) was signif-icantly higher than Eltan (36.4), whereas the total flouryield was significantly lower (Table 3). Flour ash con-tent of WA8143 (0.34) was significantly lower thanEltan (0.36), which is one of the desirable attributes offlour quality for soft white wheat. The cookie diameterof WA8143 (9.20 cm) was similar to Eltan (9.25 cm).The sponge cake volume of WA8143 was significantlygreater than the check variety BStephens^ (CI 17596;Kronstad et al. 1978).

Disease resistance

WA8143 was evaluated along with its recurrent parentEltan for three major diseases (resistance for stripe rust,snow mold and Cephalosporium stripe) under differentdisease nurseries over multiple years. Stripe rust

screening under natural infection of the pathogen inthe field and with selected races under controlled con-ditions (crop season 2011–2014) showed that WA8143has better resistance than Eltan does (Tables S3–S4). Inthe seedling tests under greenhouse conditions (diurnaltemperature cycle gradually changing from 4 °C at 2:00a.m. to 20 °C at 2:00 p.m. with 16 h light/8 h darkcycles), WA 8143 was resistant to races PST-114,PSTv-4, PSTv-40, and PSTv-51, whereas intermediateresistant to PST-37, PST-100, PST-45, and PST-14 butsusceptible to PST-127. In adult-plant tests at high tem-peratures (diurnal temperature cycle gradually changingfrom 10 °C at 2:00 a.m. to 30 °C at 2:00 p.m. with 16 hlight/8 h dark cycles),WA 8143was resistant to (PST-40and PST-114), moderately resistant to PST-14, PST-100,PST-37, and PST-127, slightly better than Eltan. There-fore, the data indicated that WA 8143 has moderate

Table 2 Yield performance (kilograms/ha) of BWA8143^ with the recipient variety Eltan evaluated at multiple locations under WECVTPtrials in during crop growing season of 2011–2014 at different locations

Trial year Trial locations Precipitation (inches) BWA8143^ BEltan^ Significance

2011–2012 Colton >20″ 8777 8710 ns

Fairfield 5762 5360 +

Farmington 8174 7705 +

Moses Lake 9614 9045 +

Pullman 10,050 9849 ns

Mean 8442 8140 +

2011–2012 Dayton 16–20″ 8040 8040 ns

Mayview 8207 8073 ns

Reardan 7671 6298 +

St. John 7973 8040 ns

Walla Walla 5795 5192 +

Mean 7504 7135 +

2011–2014 Almira 12–16″ 7638 7370 +

Anatone 5849 5795 ns

Creston 6432 6432 ns

Dusty 6003 6325 −Lamont 5682 5628 ns

Mean 6321 6305 ns

2011–2014 Connell <12″ 2898 2613 +

Harrington 4774 4556 ns

Lind 2713.5 2814 ns

Ritzville 4271 4405 ns

Horse Heaven 2968 2834 +

Mean 3525 3444 ns

Overall mean 6447 6256 +

+ yield performance ofWA8143 significantly higher than Eltan,− yield difference ofWA8143was lower than Eltan, ns no significant differences

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high-temperature adult-plant (HTAP) resistance likeEltan but it has additional all-stage resistance to someof the tested races (PST-40 and PST-114) than theirrecurrent parent Eltan. Snow mold is also a devastatingdisease in winter wheat production in the colder regionsof the USA, especially where snow falls on unfrozen orlightly frozen soil and persists for more than 100 days.Two critical years (2011 and 2013) of testing for snowmold resistance under WECVTP showed WA8143 tohave very high level of resistance, similar to that presentin Eltan (Table S5). Similarly, Cephalosporium stripe isa fungal leaf disease of winter wheat. Disease screeningwas conducted in crop season 2011 and 2013 under fieldconditions with artificial inoculation and the resultswere evaluated as described by Wetzel and Murray(2013).WA8143 showed similar level of Cephalosporiumstripe resistance that of Eltan (Table S5). The comparisonswere made relative to well-known susceptible varietyStephens and resistant check variety Eltan.

Discussion

In an earlier study (Randhawa et al. 2009), we havereported a four-step marker-assisted background

selection (MABS) approach. By transferring a striperust resistance gene (Yr15) into a cultivar with onlytwo backcrosses (BC2s), we showed that the approachworks well to transfer a single gene into a cultivar whilerecovering agronomic performance of the recurrent par-ent. In this report, we show that the approach can also beused to transfer two genes located on different chromo-somes and high proportion of RPG can be achieved inonly two backcrosses. We are also reporting that theforward breeding methods used during the study helpedto make improvements over the recurrent parent. Wecombined our previously reported MABS method withthe new forward breeding approach to transfer twomutations in acetolactate synthase (Als) gene locatedon long arm of 6B and 6D chromosomes of wheatproviding imidazolinone herbicide tolerance to a popu-lar PNW cultivar Eltan. We recovered 96.8% RPGsimilar to the recurrent parent (Eltan) along with all thedesirable characteristics. Evaluated in multi-locationfield trials over 58 location-years (2011–2014),WA8143 yielded >3% higher than the recurrent parentEltan. All of the other agronomic traits of recovered lineWA8143 were either equal to or better than Eltan.Therefore, WA8143 was released for commercial culti-vation under the name of Curiosity CL+. There are onlyfew examples of releasing cultivars via MABS in rice(Neeraja et al. 2007; Iftekharuddaula et al. 2015). Intro-gression experiment was conducted in rice by transfer-ring a major QTL for submergence gene (sub-1), andRPG was recovered 96% in three backcross cycles(Neeraja et al. 2007). Similarly, another study was con-ducted in rice and recovered 96.1% RPG after twobackcrosses (Iftekharuddaula et al. 2015).

We consider some of the key reasons for the successof the present study. (i) Initial screening for the targettrait was done by herbicide spray which allowed rejec-tion of more than 70% of the backcross populations,reducing the genotyping cost and effort; (ii) used infor-mation about the physical location of the markers forbackground selection facilitating precise estimation ofthe RPG; (iii) micro-solvent retention capacity (Micro-SRC) test provided a reliable measure of grain qualityparameters, which is critical for the PNW wheat varie-ties; (iv) forward breeding using markers as well asphenotypic selection in the greenhouse was successfulin achieving significant improvement over the recurrentparent both for high grain yield as well as for diseaseresistance package; and (v) reliable markers were avail-able to differentiate homozygous from heterozygous

Table 3 Comparison between WA8143 and Eltan for grain andend-use quality parameters

Traita WA8143 Eltan LSD

Test weight 62.30 62.20 0.49

Grain protein content (%) 9.80 9.80 0.70

Single kernel hardness 31.00 30.00 1.96

Single kernel weight 39.50 36.40 0.97

Flour yield (%) 67.30 68.50 0.48

Milling score (%) 84.70 85.00 0.85

Flour ash (%) 0.34 0.36 0.01

Flour shallow volume 20.30 20.20 1.40

Flour protein (%) 8.30 8.40 0.62

Flour SDS (%) 14.90 14.10 2.10

Mixograph absorption 55.00 54.30 1.20

Cookie diameter (cm) 9.20 9.25 0.11

Sponge cake volume 1286.00 1249.00 13.0

SD standard deviationa Corresponds to the value averaged over 18 locations of theWashington State except Sponge cake volume, that is, an averageof 8 locations

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plants for the target traits. However, the value of thebreeding approach is obvious from the fact that thedonor used in this study was Australian hard red springwheat, whereas Eltan is a soft white winter. Such diversecrosses are rarely made to result in a cultivar. In additionto winter vs. spring difference, grain quality of the PNWwheat varieties is particularly important because most ofthe wheat from the region is exported needs to meetstrict quality requirements. Along with MABS ap-proach, micro-SRC test adapted to evaluate grain qual-ity at a single plant level can be credited to the fact thatthe end-use quality of Curiosity CL+ was at least asgood as the recurrent parent in spite of the cross with thehard red spring donor line.

We previously showed that the four-step MABSmethod could be successfully employed to transfer asingle gene into a cultivar without any yield reduction(Randhawa et al. 2009). Although transferring multiplegenes is feasible withMABS, combining forward breed-ing helped us in recovery of all the desirable traits alongwith target traits as two genes for herbicide tolerance,therefore employing several forward breeding steps thatcan be credited to the yield advantage seen in CuriosityCL+. For yield improvement, controlled condition se-lection of the plants and selecting for markers linked toknown chromosomal regions involved in controllingyield probably contributed to the yield recovery andperhaps slight advantage although we do not know theirrelative contribution. We also managed to slightly im-prove stripe rust resistance in Curiosity CL+, and itprobably was due to the extensive controlled conditionscreening of the BC generations at every step of the way.

In the MABS approach, the most difficult part is toremove donor type chromatin from the carrier chromo-somes, thus our major emphasis was to obtainrecombinants between the target gene and the flankingmarkers. For foreground selection, allele-specificmarkers for two-genes (Als1 and Als2) were used toselect donor-type alleles and background selection wascarried out using whole-genome SSR markers carefullyselected for the procedure to recover maximum RPG.Although the type of markers as such does not deter-mine the effectiveness of MABS, any new high-throughput marker platforms can be used for whole-genome background selection, such as genotyping bysequencing (GBS). However, GBS can be an alternativeto confirm the recovery of RPG of the final productrather than using it in every backcross generations thatincreases the cost of the procedure (Jeong et al. 2015;

Kim et al. 2015). As proposed by Randhawa et al.(2009), the uniformly selected two markers per armare sufficient to recover a high proportion of RPG. Inthe present study, we attempted a minimum of threemarkers per arm for non-carrier chromosomes and 10markers per arm for carrier chromosomes (6D and 6B)because we were dealing with two genes located ondifferent chromosomes. We further propose the use ofa large number of carrier chromosomemarkers to ensureselection against plants carrying unwanted recombina-tion events for the carrier chromosomes. We used 47markers from the two carrier chromosomes, thus as aresult, we were able to recover plants carrying very highproportion of the recurrent parent-type carrier chromo-somes; therefore, the carrier chromosome recovery(97.8%) was even higher than the non-carrier chromo-somes (96.3%).

Considering that the two value-added genes werepresent on different chromosomes (6D and 6B), theo-retical calculations suggested a requirement of a muchhigher number of plants for each backcross cycle. Fol-lowing the calculations of Randhawa et al. (2009), theprobability of a cross-over even on one side of the targetgene and the flanking marker in the two genes was oneout of 1000 at 1 centimorgan (cM) distance and slightlyhigher at 5 cM. Considering the number of plants need-ed to recover chromosomes other than the carrier chro-mosomes, about 20,000 plants were estimated to beneeded for each BC generation. Surprisingly, we man-aged to recover the desired RPG proportion with about1/9 of the plants for each generation and this was mainlybecause of the recovery of significantly higher recom-bination rate between the target gene and the flankingmarkers. This practical example of a combination ofMABS and forward breeding approaches clearly dem-onstrated the superiori ty over conventionalbackcrossing because the recovery of high proportionof RPG with superior agronomic performance alongwith value-added genes would be impossible using con-ventional backcrossing method.

Acknowledgements This work was supported by the USDANational Institute of Food and Agriculture, Hatch (project numberWNP00449), Washington Grain Commission, and US Agency forInternational Development Feed the Future Innovation Lab-Climate Resilient Wheat (Grant number AID-OAA-A-13-00008). The authors greatly appreciate the following personnelwho assisted this research in various ways, such as for qualityassessment, field testing, and disease screening: Dr. A. H. Carter,Dr. C. F. Morris, D. A. Engle (USDA-ARS), Dr. B. Baik (WSU),Dr. S. O. Guy, Dr. V. Jitkov, T. Harris, Dr. H. C. Wetzel, and J. S.

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Kuehner. We also thank Dr. D. See and the USDA-ARS RegionalSmall Grains Genotyping Laboratory, Pullman, WA, for providingfacility for marker genotyping.

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