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BASE Biotechnol. Agron. Soc. Environ.201418(1),3-10

Bulksegregantanalysisandmarker-traitassociationrevealcommonAFLPmarkersforresistancetoseptorialeafblotchinTunisianolddurumwheatMaherMedini(1),SahbiFerjaoui(2),BochraBahri(3),WissemMhri(3),SarrahHattab(3),SoniaHamza(3)(1)NationalBankofGenes(BNG).BoulevardduLeaderYasserArafat.Z.I.LaCharguia.TN-1080Tunis(Tunisia).(2)RegionalFiledCropsResearchCenter(CRRGC).RoutedeTunis.TN-9000Beja(Tunisia).(3)NationalInstituteofAgronomyofTunisia(INAT).AvenueCharlesNicolle,43.TN-1082ElMahrajène,Tunis(Tunisia).E-mail:[email protected]

ReceivedonApril29,2013;acceptedonFebruary3,2014.

Septoriatriticiblotch(STB)causedbythefungusMycosphaerella graminicolaiscurrentlythemajorfoliardiseaseofdurumwheatinTunisia.MostoftheTunisianelitecultivarsshowlittleornoresistancetothispathogen.Recently,someTunisianlandracesincluding‘Agili’havebeenidentifiedashighlyresistanttothevirulentSTBpathotype,‘Tun06’.Inordertoidentifyamplifiedfragmentlengthpolymorphism(AFLP)markersassociatedwithresistancetoSTB,weusedbulksegregantanalysis(BSA)intwoF3biparentalpopulationsderivedfromcrossesbetweentheresistantdurumwheataccession‘Agili’andthesusceptiblecultivars‘Karim’and‘Khiar’andAssociationMapping(AM)inacollectionof110olddurumwheatlandraces.Outof the24EcoRI/MseIprimercombinationstested,4werepolymorphicbetweenresistantandsusceptibleDNApools.Screenedontheparentsand9to12progeniesfromeachbulk,thesepolymorphicAFLPprimercombinationsrevealedEcoACT/MseCTC-241,EcoACA/MseCTT-128,andEcoAGG/MseCTC-104asspecificallelestoresistantindividualssuggestingtheirlinkage toagene for resistance toSTB.Binary logistic regressionanalysison thediversitypanel showed that twoAFLPmarkersoutofthethreeidentifiedbyBSAweresignificantlyassociatedwiththeresistance.EcoACT/MseCTC-241wasthesignificantlyassociatedalleleatP<7.10-8andwasidentifiedatageneticdistanceof1.2cMtotheresistancegene.Thisresultindicatestheefficiencyofusinglandracepopulationstodetectpotentialmarkersforqualitativeresistance.Keywords. Mycosphaerella graminicola, amplified fragment length polymorphism, genetic markers, disease resistance,Tunisia.

Les méthodes d’analyse de ségrégation en mélange et d’association marqueur-trait révèlent des marqueurs AFLP communs de résistance à la septoriose chez un ancien blé dur de Tunisie. La septoriose, causée par le champignonMycosphaerella graminicola, est actuellement lamaladie foliaire du blé dur la plus répandue enTunisie. La plupart descultivarsélitestunisiensmontrentpeuoupasderésistanceàcepathogène.Récemment,certainesvariétéstunisiennesdont‘Agili’ontétéidentifiéescommehautementrésistantesaupathotypevirulent,‘Tun06’.Afind’identifierlesmarqueursAFLPderésistanceàlaseptoriose,nousavonsutilisél’analysedeségrégationenmélangedansdeuxpopulationsbiparentalesF3issuesdecroisementsentrel’accessionrésistantedeblédur‘Agili’etlescultivarssensibles‘Karim’et‘Khiar’,etlacartographied’associationenutilisantunecollectionde110accessionsanciennesdeblédur.Surles24combinaisonsd’amorcesEcoRI/MseItestées,4ontétépolymorphesentrelespoolsd’ADNrésistantsetsensibles.L’analysedupolymorphismeAFLPutilisantcescombinaisonsd’amorcessurlesparentsetles9à12descendancesdechaquemélangearévéléquelesallèles,EcoACT/MseCTC-241, EcoACA/MseCTT-128 et EcoAGG/MseCTC-104 sont spécifiques aux individus résistants suggérant leurliaison avec le gène de résistance à la septoriose.Une régression logistique binaire sur la population locale de blé dur amontréquedeuxdesmarqueursAFLPidentifiésparBSAontétésignificativementassociésàlarésistance.L’allèleEcoACT/MseCTC-241aétésignificativementassociée(P<7.10-8)etsituéeàunedistancegénétiquede1.2cMdugènederésistance.Cerésultatindiquel’efficacitédel’utilisationdespopulationslocalespourdétecterlesmarqueurspotentielsderésistancedenaturequalitative.Mots-clés.Mycosphaerella graminicola,polymorphismedelongueurdesfragmentsamplifiés,marqueurgénétique,résistanceauxmaladies,Tunisie.

4 Biotechnol. Agron. Soc. Environ. 201418(1),3-10 MediniM.,FerjaouiS.,BahriB.etal.

1. INTRODUCTION

Septoriatriticiblotch(STB)causedbytheascomyceteMycosphaerella graminicola(anamorphZymoseptoria tritici)istheprimaryfungaldiseasethataffectsdurumwheat throughout the area around theMediterraneanSea(Boukefetal.,2012).Chemicalcontroliscurrentlyone of the main approaches used to manage STB(Jorgensenetal.,2008).However,fungicideresistanceand its associated environmental impact is now awidespread problem, thereby necessitating geneticwheat improvementand thedevelopmentof resistantvarieties of wheat (Jorgensen, 2008; Cools et al.,2012).SelectionforSTBresistancecanbefacilitatedby tagging effective genes with molecular markersthatcansubsequentlybeusedinbreedingprogramstotracetheusefulalleles(Varshneyetal.,2007).Thesemarkers have traditionally been identified throughsegregation analyses using bi-parental populations.Bulked segregant analysis (BSA), which consists ofcomparing two pooled DNA samples of individualsfrom a segregated population originating from asinglecross,isconsideredtobeaquickermethodforidentifying potential markers linked to monogenicqualitative trait (Michelmore et al., 1991). Recently,a new approach has been suggested that involvesgenotype-phenotype correlations in multiparentalpopulations with broader genetic diversity (Flint-Garciaetal.,2005;Zhuetal.,2008;Halletal.,2010).This approach, known as associationmapping (AM)orlinkagedisequilibrium(LD)mapping,successfullyaddresses the challengeofdetectionandexploitationofDNApolymorphismsinplantsystems(Ingvarssonet al., 2011). The use of multiparental populationspresentsclearadvantagesoverbiparentalpopulations,andthetwoareoftenappliedinconjunctiontofurthervalidate the associations and to reduce the spuriousones(Tommasinietal.,2007).

AnimportantprerequisiteforAMistheavailabilityofaccessionswithsuitablelevelsofgeneticvariationfor target traits (Maccaferri et al., 2006). Severalmarker-trait association analyses have shown thegreaterefficiencyofwildrelative/landracepopulationscompared to modern cultivar panels, especially formapping disease resistance genes (Jing et al., 2007;Mazzucato et al., 2008). In fact, genetic diversity ofmodernwheatgermplasmisshowntobesignificantlylowerthanthatoflandraceaccessions(Rousseletal.,2004; Hao et al., 2006). Therefore, wheat researchtargeting disease resistance improvement considerswildrelative/landracepopulationstobehighlyvaluableresourcesforintroducingnewresistancegenesandforbettermarker-traitdetectionsandmapping resolution(Sodkiewicz et al., 2004). Severalmarkers linked toSTB resistance genes have been identified in breadwheatusingBSA,includingrestrictionfragmentlength

polymorphism (RFLP), simple repetitive sequence(SSR) and amplified fragment length polymorphism(AFLP) (Adhikari et al., 2003; McCartney et al.,2003;Adhikarietal.,2004a;Adhikarietal.,2004b).Todate,18majorstb genesandQTLsfor resistanceto STB in bread wheat have been mapped (Tabib-Ghaffary, 2011). In durumwheat, resistance to STBhasneverbeen investigatedat thegenetic leveluntilthe recent identification of a single dominant geneconferringtotalresistanceintheadultstagein‘Agili’,an old Tunisian durum wheat accession (Ferjaouietal.,2011).Inaddition,Gharbietal.(2011)identifiedaccessions tolerant to STB in Tunisian elite durumwheatgermplasm.

TheaimofthepresentstudywastoidentifypotentialAFLPmarkersforresistancetoSTBdiseaseindurumwheatintheadultstageusingtwoapproaches:– BSA in segregating F3 progenies generated from crosses between a resistant accession ‘Agili’ and twosusceptiblecultivars‘Khiar’and‘Karim’;– AMinadiverseold localdurumwheatcollection harboring phenotypic variation for traits including resistancetoSTB.

Thereliabilityofmarker-traitassociationanalysiswouldbetheidentificationofthesamemarkerslinkedto the gene of interest. Investigation of landracepopulations to detect major genes for resistance isdiscussed.

2. MATERIALS AND METHODS

2.1. Plant material

F2-derivedF3 families,originating from twocrossesbetween a resistant durum wheat parent ‘Agili’ andtwosusceptibledurumwheatmodernvarieties‘Khiar’and‘Karim’,wereusedfortheBSAapproach.Thesebiparentalpopulationsconsistedof8to12homozygousresistant and 9 to 13 homozygous susceptibleprogenies.IntheAManalysis,thediversitypanelusedconsistedof9completelyresistantand101susceptibleaccessions of old durum wheat landraces. TheresistancetoSTBwasevaluatedintheadultstageonboth populations. Inoculation and disease evaluationmethodsaredescribedbelow.

2.2. Inoculum preparation and plant inoculation

‘Tun6’,themostvirulentpathotypeofSTBinTunisia,wasusedinthisstudy(Medinietal.,2008).TestedontheCanadiandifferentialhosts,thispathotypehasbeencharacterizedbyitsvirulenceonthreedifferentialdurumwheatlinesoffourtested.Inoculumwaspreparedbyinoculating250mlofliquidyeast-glucosemedia(10g

IdentificationofAFLPmarkersforresistancetoleafblotchindurumwheat 5

ofyeastextractand30gofglucosein1lofdistilledwater)in500mlErlenmeyerflaskswithfreshZ. triticicoloniesinsolidyeastglucosemediacontainingagar(20g.l-1). The Erlenmeyer flasks were incubated for7 to 10days with shaking (100rpm). The resultinginoculum suspensions were filtered and adjusted to107spores permlwith distilledwater. Ten drops ofTween20 (polyoxyethylene-sorbitanmonolaurate)were added per liter of spore suspension to reducesurface tension. Plants were inoculated twice in thefieldatstemelongation(Zadoksscale37)withamotorsprayer.

2.3. Disease assessment

Symptoms of STB were assessed 28days after thesecondinoculation(dpi).Susceptibilityandresistancewere measured using a qualitative scale, i.e., plantswerescoredassusceptible if leaveswerecoveredbynecrotic lesions bearing pycnidia, or as resistant ifleavesofthewholeplanthadnopycnidia.F3familieswereclassifiedashomozygousresistantwhenallplantswithin the familywere resistant, heterozygouswhenthefamilysegregatesforresistance,andhomozygous-susceptible when all plants within the family weresusceptible(Ferjaouietal.,2011).

2.4. DNA extraction

ForbothBSAandAMpopulations,totalgenomicDNAwas isolated from leaf tissues of adult plants usinga modified CTAB (hexadecyltrimethyl ammoniumbromine) method (Saghai-Maroof et al., 1994).DNAextractedwasvisualizedon1%agarosegelforqualityassessment,quantifiedonaspectrophotometerat a wavelength of 260nm and adjusted to a finalconcentrationof50ng.μl-1.

2.5. Bulk segregant analysis

Bulked segregant analysis (BSA) was performedas described by Michelmore et al. (1991). Nine to12homozygous-resistant and 9 to 12homozygous-susceptible individuals of each F3 progeny wererandomlyselectedtoconstructresistantandsusceptiblebulks(Table 1),respectively.Eachbulkwasatafinalconcentration of 50ng.μl-1. Band patterns of bulkswerecomparedwiththosefromtheparentstoidentifypotentialmarkerslinkedtotheresistance.

2.6. AFLP procedure

TheAFLPprocedurewasconductedaccording to theprotocol developed by Vos et al. (1995). GenomicDNAwasdigestedwithEcoRIandMseIat37°Cfor4h. DigestedDNA fragmentswere ligated to EcoRIandMseI adapterswithT4DNA ligase at 16°C for12h.After ligation, the reactionmixturewasusedastemplateDNAforpre-amplification.Pre-amplificationwas performed using primers with one selectivenucleotide, followed by selective amplification usingprimerswiththreeselectivenucleotides.Thereactionswere conducted in a 25µl reaction containing2.5ml10×PCRbuffer, 0.2mMdNTPs, 1UTaqpolymerase,and0.5µMEcoRIfluorescent-labeledselectiveprimer(AppliedBiosystems)and1µMMseIselectiveprimer.Selectiveamplificationwasinitiatedbyadenaturationstep at 94°C for 2min, followed by 10cycles ofdenaturation at 94°C for 20sec, annealing at 65°Cfor30secandextensionat72°Cfor2min,andthenfollowedby20cyclesofdenaturationat94°Cfor30sec,annealingat56°Cfor30secandextensionat72°Cfor2min,andafinalextensionat72°Cfor10min.PCRproductswereanalyzedusingcapillaryelectrophoresisABIPRISM3130(AppliedBiosystems).

Table 1. DistributionofAFLPmarkersinthehomozygous-resistantandhomozygous-susceptibleF3progeniesofthecrosses‘Agili’x‘Khiar’and‘Agili’x‘Karim’—Distribution des marqueurs AFLP chez les descendants F3 homozygotes résistants et homozygotes sensibles issus des croisements ‘Agili’ x ‘Khiar’ et ‘Agili’ x ‘Karim’.Individuals AFLP marker Resistant Susceptible

Total number Number displaying the marker

Total number Number displayingthe marker

‘Agili’x‘Khiar’ 1.Eco-ACT/Mse-CTC-241 12 12 13 02.Eco-ACA/Mse-CTT-128 12 12 13 03.Eco-AGG/Mse-CTC-104 12 12 13 04.Eco-ACA/Mse-CTG-250 12 6 13 5

‘Agili’x‘Karim’ 1.Eco-ACT/Mse-CTC-241 8 7 9 02.Eco-ACA/Mse-CTT-128 8 8 9 03.Eco-AGG/Mse-CTC-104 8 8 9 04.Eco-ACA/Mse-CTG-250 8 5 9 4


2.7. Linkage, marker trait association, and linkage disequilibrium

ThepotentialmarkersidentifiedbyBSAwerescoredineachhomozygousresistantandsusceptibleindividualof each F3 progeny. Mapping was performedconsidering the population as RILs because thehomozygous progenies were fixed for the resistancelocus. Linkage analysis was performed with theQTL IciMapping version 3.1 (www.isbreeding.net).TheKosambimapping functionwasused to convertrecombinationdataintocentimorgans(cM).

The association between molecular markers andresistance to STBwas determined by the procedureofbinary logistic regressionbasedonbinarydiseasescoreandtheAFLPmarkersusingtheSPSSsoftwarepackage (version 9.1). The AFLP allele (1; 0) wastreatedasan indicatorvariableand thephenotypeasdependent variable. We fitted a multiple regressionmodel of the trait response on the set of theAFLPalleletreatedastheindicator.Tocontrolformultipletesting,thecorrectionofBonferroni(http://mathworld.wolfram.com/BonferroniCorrection.html)wasappliedfor the markers that were associated at P<0.05.MarkerswithprobabilitiesofassociationslessthantheBonferronithresholdof0.00035wereretained.

Linkagedisequilibriumwasestimatedbetweenpairsof AFLP markers that were significantly associatedwiththeresistanceusingr2coefficient(Pritchardetal.,2001), with the GDA software, version 1.1 (Lewiset al., 2001). Significant associationwas determinedusing a chi-square test. Pairs of markers that haveP<0.001weredeclaredsignificantlyassociated.

2.8. Segregation analysis of the resistance

InordertoassesstheindependenceofSTBresistancegenes,crossesbetweentheresistantaccession‘Agili’and seven other resistant landraces identified werecarriedout.Inoculationsusing‘Tun06’pathotypewereperformed according to the methodology describedabove. Accordance of the observed segregations to1(resistant):0(susceptible) expectation was testedusingachi-squaretesttodetectpossibledistortion.A1:0 ratio is expectedwhen identical resistancegenesare involved;a15:1ratio isexpectedfor twoclearlydistinctdominantresistancegenesintheparent.

3. RESULTS

3.1. Bulk Segregant Analysis (BSA)

Twenty-four primer combinations were assessed onhomozygous-resistant and homozygous-susceptiblepoolsoftheF3familiesderivedfromthecrosses‘Agili’

(resistant)x‘Khiar’(susceptible)and‘Agili’x ‘Karim’(susceptible).Thetotalnumberofbandsvariedfrom27 (for EcoAGC/MseCAG primer combination) to155(forEcoACA/MseCTCprimercombination).Thecomparison between resistant and susceptible bulksofeachcrossrevealedthatfourprimercombinationsproduced a distinct peak (band) in resistant bulksandinresistantparentsandwasabsentinsusceptiblebulksandsusceptibleparents,indicatingtheirlinkagewithresistancetrait.Thesefourprimercombinationswere further analyzed for segregation in individualsof resistant and susceptiblebulks.Only threeprimercombinationsproducedapeak(markerband)specifictotheresistantbulk(namely,EcoACT/MseCTC-241,EcoACA/MseCTT-128,andEcoAGG/MseCTC-104),co-segregatedwiththeresistancein‘Agili’x ‘Khiar’progenies,whereasthemarkerEcoACA/MseCTG-250didnot.ThealleleEcoACT/MseCTC-241wasabsentinoneresistantprogenyof‘Agili’x‘Karim’,allowingus to estimate recombination distance between theresistance locus and the AFLP marker. Linkageanalysis of the F3 progenies showed that EcoACT/MseCTC-241mappedat1.2cMfromthestbgene.

3.2. Marker-trait association and linkage disequilibrium

The three successful primer combinations thatidentified three potential markers for resistance toleafblotchbyBSAwereassessedona collectionof9fullyresistantand101susceptibleaccessionstotheM. graminicolaisolate‘Tun06’.Multivariateanalysisshowed that 15markers from a total of 110wereassociated at P<0.05 with the resistance (Table 2).The marker EcoCT/MseCTC-241 was associatedat a high significant probability (P<7.10-8). ThismarkerwasretainedafterBonferronicorrection.TheEcoACA/MseCTT-128 marker was associated withresistance but at a lower significance level than themarkerEcoACT/MseCTC-241.Infact,theEcoACA/MseCTT-128 allele was present in three susceptibleaccessions, whereas the EcoACT/MseCTC-241allele was present in only one susceptible accession(Table 3). The presence of the resistance markerallele (band) in the susceptible progenies could beoriginated from another part of the genome andco-migrated with identical mobility to the resistantmarkerallele.Inaddition,theEcoACA/MseCTT-128allelewasfoundinsignificantlinkagedisequilibrium(LD) with EcoACT/MseCTC-241 as demonstratedabovebylinkageanalysis(Table 2).However,markertraitassociationanalysisdidnotshowanassociationbetweenresistanceandEcoAGG/MseCTC-104alleleas revealed by linkage analysis. In fact, the markerEcoAGG/MseCTC-104 was found in 42susceptibleaccessionstotheisolate‘Tun06’.


4. DISCUSSION

For the past 20years, investigation for diseaseresistancehasbeenanimportantobjectiveinmolecularbreeding of wheat. In this study, two approacheswereusedtoidentifyAFLPmarkerslinkedtoaSTBresistancegene:– BSA in biparental F3 populations derived from crosses between a resistant accession ‘Agili’ and twosusceptiblecultivars,‘Khiar’and‘Karim’;– associationmappingindurumwheatlandraceswith ahighlydiversegeneticbackground.

Both strategies identified two reliable AFLPmarkerslinkedtothestbgenethatprovidesaqualitative

resistance against the most frequent pathotype ofM. graminicolareportedinTunisia.

Molecular markers linked to disease resistancetraitshavegreatimportancebutaredifficulttorevealespecially when the disease is difficult to score duetothemulti-componentofpathogenfitnessandwhenthetraithasasmalleffectonphenotypicvariationandis influenced by environmental factors (Sukhwinder-Singh et al., 2012). In addition, available geneticresources are difficult to obtain and are not alwaysefficient in detecting loci underlying qualitative/quantitative traits. This study provides evidence thattheexistingassociationpanelanddiseaseassessmentprocedure can be used to detect AFLP markers forSTBresistanceoncethetraitisqualitative.Despitethe

Table 2. Significant(P<0.05)markertraitassociationsfoundin110durumwheataccessionsforresistancetoMycosphaerellagraminicolapathotype‘Tun06’aftermultivariateanalysisandsignificantLDbetweenassociatedmarkers—Associations marqueur-trait significatives (P < 0,05) pour la résistance au pathotype ‘Tun06’ deMycosphaerella graminicola chez 110 accessions de blé dur après analyse multivariée et déséquilibre de liaison significatif entre les marqueurs associés.Marker P value R2 (%) Loci in LD(P<0.001)EcoACT/MseCTC-60 0.012 6.30EcoACT/MseCTC-64 0.015 5.82EcoACT/MseCTC-83 0.029 4.74EcoACT/MseCTC-132 7.646E-4 11.32EcoACT/MseCTC-138 0.041 4.13EcoACT/MseCTC-182 0.029 4.74EcoACT/MseCTC-241a 6.805E-7* 24.66 EcoACA/MseCTT-128ECoACA/MseCTT-56 0.032 4.57ECoACA/MseCTT-58 0.045 4.00ECoACA/MseCTT-65 0.012 6.27ECoACA/MseCTT-109 0.028 4.76ECoACA/MseCTT-124 7.646E-4 11.32ECoACA/MseCTT-128a 0.007 7.05 EcoACT/MseCTC-241EcoAGG/MseCTC-105 0.020 5.38EcoAGG/MseCTC-204 0.029 4.74a:markeridentifiedbylinkageanalysis—marqueuridentifiéparanalysedeliaison;*:markerretainedafterBonferronicorrection—marqueur retenu après correction de Bonferroni.

Table 3.Occurrence ofAFLPmarkers in resistant and susceptible accessions toMycosphaerella graminicola pathotype‘Tun06’—Présence de marqueurs AFLP chez les accessions résistantes et sensibles au pathotype de Mycosphaerellagraminicola ‘Tun06’.Marker Resistant (total N : 9) Susceptible (total N : 101) Resistant accessions carrying the markerACT/MseCTC-241 3 1 ‘Agili’;‘JenahKhotifa’;‘Derbassi’ACA/MseCTT-128 2 3 ‘Agili’;‘JenahKhotifa’AGG/MseCTC-104 6 42 ‘Agili’;‘JenahKhotifa’;‘Derbassi’;‘Aziz’;

‘Mahmoudi’;‘Sbei’


small population size, low frequency of the resistantphenotypes in the tested population (less than 10%),andnottakingintoconsiderationpopulationstructure,theassociationstudywasconclusiveandinagreementwiththeBSAresults.Thisisattributedtothequalitativeapproachoftheresistanceanditssimpleinterpretationusing a binary disease scoring (1: susceptibility;0: resistance) that enable the use of binary logisticregression to analyze the association.Here, commonissues related tomarker-trait association analysis areavoided, e.g., false positive associations caused bypopulation subdivision (Pritchard et al., 2001) andlimited power to detect rare loci within populations(Flint-Garcia et al., 2005; Breseghello et al., 2006;Soto-Cerdaetal.,2012).

The AFLP markers identified by associationanalysis were at aminimal distance of 1.2cM fromstb gene.This genetic distance is not significant dueto small number of segregating individuals andmayincreaseby enhancing thepopulation size.However,wecanassumethataminimalintervalof2.4cM(LDextent) is required between markers for associationmappingusingthisdurumwheatlandracepopulation.This low LD may probably inhibit marker-aidedselectionandthusrequirealargenumberofmarkerstodetectanassociatedregion.ThelevelofLDdependson population structure, cross/self pollination ratios,effective population size, and selection pressure andmay vary across genome regions (Maccaferri et al.,2005).TheLDextentobservedhere issimilar to theLDpatterndescribedinlandraceaccessionscomparedto wheat-inbred lines (Caldwell et al., 2006). TheextentofLDlessthan1cMwasdescribedinlandracepopulations because of high recombination eventsin the ancestry of the accessions (Comadran et al.,2009).However,theaverageLDwithin189cultivarsand advanced lines of durum wheat accession fromtheMediterranean areawas decayed over a distance

of10cMduetogeneselectionduringplantbreedingprograms(Maccaferrietal.,2006),

Inthisstudy,sevenresistantaccessionslackedtheidentifiedAFLP markers for the resistance to STB.Twohypotheticalexplanationscanbesuggested:– the linkage could have been broken between themarker and the stb genewithin these resistant accessionsduetorecombinationeventsafterthestb geneintrogression;– these resistant accessions may carry different resistanceallelesfromtheonesidentifiedin‘Agili’.

The allelic test for resistance to the ‘Tun06’pathotypeusingF2progeniesderived from thecrossbetween‘Agili’,andtheseresistantaccessionsrevealedsusceptible individuals. The segregation pattern ofSTB resistance was approaching a 15:1 ratio in thecross,confirmingthepresenceof twoclearlydistinctdominant resistance genes in the parent (Table 4).The‘Agili’x‘JenahKhotifa’crosswastheexception,where no susceptible progeny was shown indicatingthe presence of identical resistance genes in theparent.‘Agili’and‘JenahKhotifa’landraceaccessions,which share the EcoACT/MseCTC-241, EcoACA/MseCTT-128 and EcoAGG/MseCTC-104 alleles,probably represent common ancestry. The resistancegenes identified likelyderivefromwildrelativesandwere introgressed through natural outcrossing eventsinto the durumwheat landraces.Evidence of naturalgene flow between wheat and wild relatives havebeendetected(Arrigoetal.,2011),aswellasseveraldisease resistance genes to rusts, powdery mildewand fusarium head blight in domesticated wheatgenomecultivarsderivedfromwildrelativesincludingAegilopsandTriticumspeciesandotherdiploidwheatgrasses(Monneveuxetal.,2000;Jauharetal.,2009).These findings emphasize the need for further studyofresistancemechanismsinordertofullyexploitthe

Table 4.SegregationpatternofSTBresistanceto‘Tun06’pathotypeusingF2progeniesderivedfromthecrossbetween‘Agili’andresistantlandraces—Ségrégation à la septoriose au pathotype ‘Tun06’ utilisant les descendants F2 dérivés du croisement entre ‘Agili’ et les accessions locales résistantes.Crossa Total plants Observed Calculated Expected ratio χ2*‘Agili’/‘Agili41’ 185 172:13 173.43:11.56 15:1 0.011(0.179)‘Agili’/‘J.Kottifa’ 175 175:0 175:0 1:0 0(0)‘Agili’/‘Agili38’ 75 70:5 70.31:4.68 15:1 0.0013(0.021)‘Agili’/‘Azizi’ 160 149:11 150:10 15:1 0.0066(0.1)‘Agili’/‘Agili37’ 140 128:12 131.25:8.75 15:1 0.080(1.207)‘Agili’/‘Mahmoudi’ 110 102:8 103.12:6.87 15:1 0.12(185)

*:χ2criticalvaluesatP=0.05and0.1with1degreeoffreedomare3.84and2.71,respectively—les valeurs critiques de χ2 à P = 0,05 et 0,1 à 1 degré de liberté sont respectivement de 3,84 et 2,71;a:‘Agili’/‘Derbassi’and‘Agili’/‘Sbei’crossfailedandarenotshowninthistable—les croisements ‘Agili’/‘Derbassi’ et ‘Agili’/‘Sbei’ ont échoué et ne sont pas représentés dans ce tableau.


landrace reservoir for STB resistance in cultivateddurumwheat.

5. CONCLUSION

Finally, this study demonstrates relevant preliminaryresults in identifying STB resistance genes andleads us to increase the research interest in pursuingadvanced studies. The next step will be to movetowards more precise localization of the identifiedstb resistance gene by using a large population ofrecombinantinbredlinesinordertosuccessfullyapplymarker-assisted selection in breeding program fordurumwheat. In additiondurabilityof the resistanceandtheevolutionarypotentialofthepathogen,majorcomponents for a successful virulence management,should be considered. Potentially durable resistancegenes are valuable tools for breeding, especially ifcombinedwithotherstbgenesand/orresistanceQTLsaspartofapyramidingstrategy.

Acknowledgements

This studywas supportedbyTunisiangovernment fundedproject untitled “Breeding for resistance to biotic stressin durum wheat”.We thank the National Gene Bank forprovidingfacilitiesforconductingAFLPgenotyping.

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