AssessingEcoTILLINGasaMethodologyforTargeted GenotypingandSNPDiscovery

(IRRIRef.No:DPPC200468)

Proposal
submittedtothe

GenerationChallengeProgram CommissionedResearch (SP1)

25November2004
Contact: Dr.MichaelT.Jackson DirectorforProgramPlanningandCoordination(DPPC) Telephone:+63(2)5805600ext.2747or2513;Direct:+63(2)5805621;Fax:+63(2)8127689or5805699 Emailaddress:dppcirri@cgiar.orgMailingaddress:DAPO7777,MetroManila,Philippines

Title: Assessing EcoTILLING as a Methodology for Targeted Genotyping and SNP Discovery TargetedSubprogramme(s): PrincipalInvestigator: CoPrincipalInvestigator: ParticipatingInstitutions: SP1 IRRI(KennethL.McNally) AgropolisCIRAD(ClaireBillot) IRRI(K.L.McNally,N.R.SackvilleHamilton,H.R.Lafitte) AgropolisCIRAD(C.Billot,I.Hippolyte,F.C.Baurens,JF Rami) Luca Comai, Dept. of Biological Sciences, University of Washington,Seattle,WA,USA JeffHarford,LiCor,Inc.,Lincoln,NE,USA Abdelhafid Bendahmane, URGV (Unit de Recherche en GnomiqueVgtale,INRAandCNRS),Evry,France November16,2004 1year $150,000

ExternalCollaborators:

SubmissionDate: Fundingperiod: Fundingrequest:

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TaskTitle AssessingEcotillingasamethodologyfortargetedgenotypingandSNPdiscovery Involvedinstitutions IRRI,Agropolis ProposedTaskLeader KenMcNally RationaleandObjective TILLING(TargetingInducedLocalLesionsINGenomes)isanewtechniquethatcan identify polymorphisms in a target gene by heteroduplex analysis. A variation of this technique(EcoTILLING)representsameanstodeterminetheextentofnaturalvariationin selectedgenesincrops.EcoTILLINGmaybeacosteffectiveapproachforhaplotypingand SNPdiscovery. Theobjectivesoftheprojectsarei)toassessEcotillingasareliableandcosteffective method to detect SNP in a large number of accessions, ii) to test for validity in triploid species,andiii)toestablishEcotillingtransfertechnologyplatformsatIRRIandAgropolis Cirad.Thesewillbe performedthroughthestudyof10orthologousgenesinthreerelated species,twodiploid(riceandsorghum)andonepresentingdifferentploidylevels(Musa). Foreseenactivities Choice of 10 candidate genes in agreement with current projects (projects accepted as grantedproject,genesinrelationtodroughttoleranceforrice,sorghumandMusa, andtosomaclonalvariationinMusa)andeasytocrossamplifyamongspecies; Assessment of EcoTILLINGdetected haplotype diversity in relation to previously SNP discoverythroughsequencinginamicrocoresampleforeachofthethreespecies. Searchforallelicdiversityinawiderangeofgermplasminriceandsorghumandassess EcoTillingasamethodtoprovideeasilynewalleles.

Indicativebudget 150k$(includingsequencing)

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 TABLEOFCONTENTS ProjectDescription IntroductionandRationale PreliminaryResults Objectives,Activities,andIntendedSpecificOutcomes ApproachandMethods GermplasmIdentification CandidateGeneTargetsandPrimerDesign SNPValidation EcoTILLINGTechnology SNPConfirmation TechnologyTransfer 1 1 2 2 3 3 4 5 5 6 6 6 6 6 8 9 9 11 13 13 14 14

Partners BudgetbyPartner(summary) BudgetNotesandJustification Workplan(TimelineandMilestones) APPENDICES DescriptionofPartners Table1.InitialDroughtCandidateGenes. Figure1.EcoTILLINGofTPPcdsonOryzasativa. Figure2.EcoTILLINGofDREB1cdsonwildOryza. Figure3.SequenceConfirmationofputativemismatches References

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PROJECTDESCRIPTION IntroductionandRationale Theidentificationofnovelallelesforvarietalimprovementisaprimaryobjectiveof theGenerationChallengeProgram(GCP).Thisemphasisprovidestherationaletodevelop platformsatIRRIandAgropolisCIRADfortheefficientdiscoveryofSNPsandhaplotypes in Oryza, Sorghum, and Musa by the technique of EcoTILLING, a broadly applicable and lowcosttechnology. Oryza, Sorghum, and Musa are three of the most important monocot crops having relativelysmallgenomesizes(450,800,and500600Mbp,respectively).WhileOryzaisthe worlds most important cereal in terms of cultivation, distribution, and contribution to human nutrition, progress remains to be made in developing highyielding varieties with significantdroughttolerance.Sorghum,ontheotherhand,isrelativelydroughttolerantand typically is grown in more arid areas. Both Oryza and Sorghum are predominantly inbred (lessthan1%outcrossingforOryza,020%dependingonvarietiesforSorghum).Musaisthe fourth most important crop of developing countries. Cultivated Musa may either have differentploidylevelsorbehybridsatdifferentploidylevelsbetweentheAgenomeofM. accuminataandtheBgenomeofM.babisianaspecies,withAA,BB,AAA,BBB,AB,AAB,and ABB combinations known. Musa is vegetatively propagated because of the combination of parthenocarpy with sterility. Several groups of cultivars are known that are likely derived from somatic mutations from each other and display high phenotypic variations that are particularlycrucialforthedevelopmentoftheflowerandthusthefruit.Therelativelysmall genomesizesofthesethreecropsandtheirclosetaxonomicdistancesmaybeadvantageous forcomparativegenomics,allowingcomparisonofdivergedorthologs. Inthisproject,IRRIsrecentexperienceinestablishingandoptimizingEcoTILLING methodology for rice, through ongoing collaboration with Drs. Luca Comai and Steven Henikoff, will be transferred to CIRAD for use on Sorghum and Musa. CIRAD has begun pilotstudiesonSorghumwiththeEuropeanCROPTILprojectthroughAgropolisURGV,led byDr.Bendahmane.Furtheroptimizationofthemethodologyforriceandeffectivetransfer ofthetechnologyto CIRAD will befacilitated through collaboration byboth Dr. Comai at theUniversityofWashingtonandDr.BendahmaneatURGV. ByestablishingEcoTILLINGwithoptimizedprotocolsforthreespeciesrepresenting a range of ploidy level and heterozygocity, IRRI and CIRAD will be able to serve as referencecentersandsitesforthefuturetransferofEcoTILLINGtootherGCPpartnersand NARES collaborators. In this one year project, capacity building will be achieved through trainingworkshopsheldatIRRIinearly2005andlaterintheyearatCIRAD. Important considerations beyond optimization of the technology are the choices in germplasm for discovery of SNP and in the candidate genes underlying traits of interest. The germplasm chosen for screening will consist i) of a microcore set for Oryza, Sorghum, and Musa used to validate SNP and ii) from 50% to 100% of the core germplasm sets as defined during GCPSP1 1st year commissioned projects (over 1500 for rice, over 700 for sorghumand200forMusa)totestforthecapacityofEcoTILLINGtodiscovernewSNPs.

PreliminaryEcoTILLINGResults Through collaboration with Drs. Comai and Henikoff, IRRI has established an EcoTILLINGplatformforrice.Thoughatanearlystageofdevelopment,resultshavebeen producedinricetargetinggenesinvolvedindiseaseresistance(Hanetal2004,abstract)and candidate genes for drought (Wang et al 2004, abstract). CIRAD has embarked on pilot studiesforsorghumwith CROPTIL,aEuropeanTILLING network,led by Dr.Abdelhafid Bendahmane of AgropolisURGV and based on the analysis of the variability at the Waxy geneinvolvedingrainstarchcontent. Oryza:AtIRRI,wehavefocusedonaseriesofgenesforprimarilydroughttolerance(Table 1).InitialresultshavebeenobtainedforcodingsequenceprimersfromDREB1andtrehalose 6phosphatase(TPP)usingcultivatedandwildgermplasm.Someoftheseresultsareshown in Figure 1 for TPPcds on cultivated germplasm and in Figure 2 for DREB1cds on wild germplasm (Appendix). We have been able to detect mismatches (putative SNPs) for both genesacrosscultivatedandwildgermplasm,usingceleryjuiceextract(CJE)asdescribedby Tilletal(2004). A subset of the identified strong and putative (less clear) mismatches for DREB1 upstreamhavebeensequencedacrossselectedsativaandwildgermplasm,andSNPswere identifiedinallofthese(Figure3,Appendix,multiplesequencealignmentfortheconsensus readsfor3regionsoftheamplicons). Wehavealsotestedtheabilitytodetectmismatchesinpoolsofgermplasm(Fig.1). Pooling is an approach that is routinely used in TILLING and may be advantageous for EcoTILLING as a firstpass screen among lines that are considered closely related. Our resultsindicatethatpoolsofupto8foldarepossible,andwithfurtheroptimization16fold pools may be possible. Eightfold pools would allow 768 accessions to be screened in a singlerunforpreliminaryanalysis. Sorghum:Thepilotstudywasrelatedtothemechanismsunderlyinggrainquality,focusing on starch and using the granule bound starch synthase (GBSS) gene (locus of the waxy mutation)asthecandidategene. Based on the maize sequence of the gene (6,000 bp), an alignment was constructed withapartialsorghumsequence.Primershavebeendefinedforthemostconservedregions, and 4 PCR fragments have been amplified. PCR and sequencing of 2,554 bp has been performed on 22 varieties chosen from different cultivar genetic groups. Some 21 SNPs groupedin7haplotypeshavebeenidentified,oneofwhichispresentin11varietiesoutof the 22 tested, and 4 rare haplotypes have been encountered in one variety each. Recombinantshavebeenidentifiedamongsomeofthehaplotypes. Arepresentativecollectionof210cultivarshasbeengenotypedbyEcoTILLING.The firstresultsareencouragingfortheyidentifythepresenceofmostmutations,althoughthese need to be confirmed and the technique optimized for the detection of consecutive SNPs. ClonedCel1(A.Bendahmaneslab)hasbeenusedforthisstudy. SpecificObjectives,Activities,andIntendedSpecificOutcomes The overall goal of the project is to discover SNPs/haplotypes by establishing a technical platformforEcoTILLING,aninexpensiveandquickapproachtoSNPgenotyping.Thiswill be achieved through the exploration of genetic variability of a set of 10 potentially

orthologous candidate genes across rice, sorghum, and Musa, and candidate genes for developmentinMusa. ThespecificobjectivesoftheAssessingEcotillingasamethodologyfortargetedgenotypingand SNPdiscoveryare: Objective1: Objective2: Objective3: CompareEcoTILLINGtechnologiesusedinOryzaandSorghum. Establishareliableprotocoltobeusedwithpolyploidspecies(Musa). GenerateSNP/haplotypedataforOryzasativa,Sorghumbicolor,andMusafor 10orthologouscandidategenesbyEcoTILLINGandverifybysequencingon amicrocorecollection.

Objective4: ValidateEcoTILLINGasamethodtoquicklyassessfornewhaplotypesona largecollection. Objective5: Build reference centers for EcoTILLING at IRRI and CIRAD for future technologytransfer,training,andshuttleresearch.

Theseobjectiveswillbemetbyaccomplishingthefollowingactivities: Activity1: Identify 10 potentially orthologous candidate genes for Oryza, Sorghum, and Musa,severalofwhicharedevelopmentallyregulatedgenesrelatedtoflower developmentinMusa(IRRIandCIRAD). Optimizeprotocolsforhaplotypediscoveryandaccomplishhighthroughput EcoTILLINGinOryza(IRRI,CIRAD,),Sorghum(CIRAD),andMusa(CIRAD). Verifyputativemismatchesacrossthe10locibyconfirmatorysequencing. IdentifyanddeploysoftwarefordataanalysisofEcoTILLINGprofiles(IRRI andCIRAD). Hold workshops on SNP discovery through the use of EcoTILLING dedicatedtotheNARESandotherpartners(withthehelpofGCPSP5).

Activity2: Activity3: Activity4: Activity5:

ApproachandMethods 1.GermplasmIdentification Choiceofthegeneticmaterialisakeyfactorforthesuccessoftheapproach. Foreachspecies,twogermplasmsetsneedtobechosenfromtheaccessionsgenotypedwith neutralSSRmarkersduringthefirstyearoftheGCPcommissionedresearch. The first one (microcore collection) will be chosen in order to maximize genetic diversityandwillserveasreferencelines.Thiswillbeusedforprevioussequencinginorder toi)validatetheorthologousstatusoftheselectedgenesandii)toassessforexpectedEco tilling profiles. This set will be based on the first set of 48 accessions used to test for SSR congruencybetweenlaboratories(GCP1styear). A second set will be chosen from 50100% of the varieties genotyped by 50 SSR markers during SP11st year commissioned research. This will be used for the high throughput tests. In the case of Oryza germplasm, 1536 diverse accessions including landraces, wild relatives of the AA genome type, and improved varieties will be chosen fromthe3000thatweregenotypedby50SSRmarkersduringSP1commissionedresearch. The landraces include representatives from the 6 variety groups indica, aus/boro, deepwatertypes3and4,aromatic,andjaponica(temperateandtropical).Thevarietiesused forimplementationwerechosenfromthissetof3,000.
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 The full set of around 700 Sorghum accessions, genotyped with 30 SSR markers duringthefirstyearoftheGCP,willbeusedforhighthroughputanalyses.Thissetincludes wildrelatives,landraces,andbreedinglines. ForMusa,afirstsetof96diploidaccessionsfromthefullsetof200Musagermplasm characterizedduringGCPyear1bySSRswillbeselectedforthetransferofthetechnology from Oryza/Sorghum to Musa. Once the technology has been optimized for use in Musa, a second set of 96 triploid accessions will be used to test and optimize the procedure for mismatchdetectionintriploidbackgrounds.Halfofthesewillalsohavebeencharacterized during GCP year 1 by SSRs and the other half will consist of groups of somatic plantain mutants(AAB,presentinganarrowgeneticorigin)thatdisplaylargephenotypicvariation (CARBAPcollection,Cameroon). 2.CandidateGeneTargetsandPrimerDesign Forthisproject,alistofgeneslinkedtodroughttoleranceinriceandsorghumwill be chosen based on consolidation of information, including gene function and annotation, expression, colocalization with drought QTLs, shifts in allelic frequency under selection, existing literature incorporating results obtained from other species and studies (e.g., Arabidopsis: Finkelstein et al 2002; maize: Ribaut et al 2004; rice: Lanceras et al 2004, Kathiresanetal2004),andresultsofthefirstyearGCPSP2commissionedresearch,Thisset ofgeneswillincludethegenesunderlyingHD1andHD3inricewithknownhomologuesin maize and sorghum. It will also include target genes being used in competitively funded GCPprojects,inordertoopentheprojectafterwardstocomparisonwithotherspecies. Through this process, a first set of target genes listed in Table 1 (Appendix) has already been chosen for implementation in rice. These genes include 2 loci for drought responsive elementbinding protein 1 (DREB1) that are located within QTL intervals for yieldcomponents underdroughtonCH1, 4trehalose 6phosphatase (TPP) loci, andthe 9 cisepoxycarotenoiddioxygenaselocusonCH12(NECD,vp14homologueinvolvedinABA biosynthesis).Outofthese,abilitytocrossamplifyinSorghumandMusawithoutdetecting paralogs will be a criteria for the choice of the final studied set across the three species. Several (up to three) candidate genes related to development in Musa will be chosen. We envision that these genes will have homologs for development in Oryza and Sorghum, e.g. candidategenesinvolvedinflowerdevelopmentsuchasAPATELA2homologs.Suchgenes may be candidate genes for reproductive stage drought tolerance in Oryza and Sorghum. However,theeaseatwhichEcoTILLINGcanbescoredwillnotbetakenintoconsideration, soasnottobiastheassessmentoftheusefulnessofthemethod. Aftertargetgeneshavebeenidentified,thefulllengthgenomicclonescolocalizing toaQTLwillberetrievedfromGenBank.Primerswillbedesignedfromtheannotatedgene structures for the coding and upstream regulatory regions. These primer pairs should overlapbyabout100200bptomaximizetheirutilityforSNPdetectionandsequencing.We willfollowaprocedureanalogoustoCODDLE(McCallumetal2000,Greeneetal2003), butnotreliantonthepresenceofcodonslikelytobechemicallymutagenizedbyEMSwitha concomitantchangeinproteinfunction. ForSorghumprimerdesign,comparativegenomicsbetweenrice,maize,sorghum(if available)andothergrasssequenceswillbeusedtopredicttheconservedregionsthatmay besuitableforamplificationofthehomologs.

 AsimilarapproachwillbeusedforMusa,usingavailableESTinformation,including 30,000 ESTs from Syngenta, and, in the absence of clear Musa orthologs, grassderived primerswillbetested.AspecificsetofgenesforuseonMusaisalsobeingdevelopedby Dr.Baurens(AgropolisCirad)onthebasisthattissuecultureinducedsomaclonalmutants mimic natural phenotypic variation among spontaneous clonal propagation derivatives. Four SSH (suppressive subtractive hybridization) libraries derived from somaclonal and normal in vitro cell suspension and plant flower cDNA have been constructed and differentially expressed genes are currently under investigation. Primers specific to a gene locusandgenometypewillbedesignedforgeneshavingafunctionrelatedtoMusaflower developmentthathaveanannotatedgenestructureforahomologinOryza. 3.SNPValidation Selectedgeneswillbesequencedinthefullmicrosetcollection.Thisstepwillenable thedetectionofSNPandpredicttheexpectedhaplotypes. 4.EcoTILLINGTechnology The critical approach and method to be used is based on TILLING (for Targeting Induced Local Lesions IN Genomes), a reverse genetics approach developed at the University of Washington and Fred Hutchinson Cancer Research Center by the groups of Drs.StevenHenikoffandLucaComai(Colbertetal2001,Tilletal2003,Greeneetal2003). This technique was originally designed to detect mutations in Arabidopsis thaliana treated withthechemicalmutagenEMS.Throughtheuseofdualfluorescentlabelingofsitespecific ampliconsfrompooledsamplesofmutantlines,anddigestionwithasinglestrandspecific endonuclease, CEL1, that cleaves mismatch positions in heteroduplex molecules, the locationofthemismatchposition(s)canbeidentified.Thisprocessallowsanallelicseriesof pointmutationsatanygeneofinterestinagenometobediscovered. AnextensionofTILLINGisEcoTILLING,whichallowsnaturalallelesatalocusto be characterized across many germplasm accessions, enabling both SNP discovery and haplotyping at these loci (Comai et al 2004). This can be done at a fraction of the cost of SNP/haplotypingmethodsrequiringlargescalesequencing.EcoTILLedhaplotypesacrossa range of germplasm can be binned and confirmatory sequencing done on the unique haplotypes.RecentreviewsbyHenikoffetal(2004)andStemple(2004)illustratethebroad adoptionofthesetechniquesindiversespecies,includingrice,maize,Lotus,poplar,Brassica, zebrafish, Drosophila, Caenorhabditis, and ratdemonstrating their broad applicability as a toolforSNPdiscoveryandallelemining. RecentworkbyTilletal.(2004)hasindicatedthatthemismatchcleavageactivityof CEL1 is general to singlestrand specific nucleases. Furthermore, they obtained higher specificactivitywithaneasilypreparedcrudeextractofceleryjuicethanwithpurifiedCEL1 or commercially available cloned CEL1 (Transgenomics Surveyor). Additionally, approaches to optimize cleavage conditions were shown that may help in optimizing the protocolforOryza,Sorghum,andMusa. Technology transfer to a triploid species, such as Musa, is of particular importance because it opens this technology to other polyploid species of agronomic interest and to DNApoolingstrategiesthatarefrequentlyusedintheTILLINGprocess,yetsofarsparsely in EcoTILLING (but see above on rice). In the case of polyploids, a first test should be performed to test for mutation between the genomes, and a second normal test within a
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genome(eitherAorB).Themainconcernsforthetechnologytransferliefirstindetecting the heteroduplex number in a possibly heterozygous polyploid, and second in detecting bands of different intensities. The first concern would also occur for heterozygous diploid individuals.Besides,ifmutationsarediscoveredbetweengenomes,difficultywilllieinthe quantification of the intensity of the bands, which will require software optimization for interpretingprofiles. 5.SNPConfirmation Once the broader sets of germplasm genotyped, accessions representative of independentandnewhaplotypes(patternsofmismatches)willbechosenandampliconsfor theseregionssequencedinordertovalidatethenewSNPs. 6.TechnologyTransfer Transfer of the technology between IRRI and CIRAD will occur through an initial visit of CIRAD scientists to IRRI. Workshops will be held at IRRI and CIRAD for training NARESandGenerationCPconsortiumpartnersonthetechnologyanduseofEcoTILLING withthehelpofGCPSP5. Partners The external collaborators from ARIs and private industry are Luca Comai (BiologicalSciences,UniversityofWashington),JeffHarford(LiCor,Inc.),andAbdelhafid Bendahmane(AgropolisURGV).Throughcollaboration withLiCor,IRRIandCIRADwill be established as LiCor reference sites for advancing EcoTILLING technology on a worldwide basis. IRRI is working closely with LiCor to adapt the Saga genotyping software for the analysis of TILLING/EcoTILLING runs. This modified software will be testedasanalternativetothemanualprocedurecurrentlyinuse(Colbertetal2001;Tilletal 2003;Comaietal2004). BUDGETINUS$

IRRI PersonnelCosts Supplies&Services Travel Training,MeetingandWorkshop TotalDirectCosts IndirectCosts TotalCosts InKindContribution TOTAL
BudgetNotesandJustification

CIRAD 22,000 28,060 7,000 6,500 63,560 11,440 75,000 75,000 150,000

Total 38,500 56,120 13,000 19,500 127,120 22,880 150,000 150,000 300,000

16,500 28,060 6,000 13,000 63,560 11,440 75,000 75,000 150,000

AtIRRI,fundsforsalarywillbeusedtohireNRStechnicalstaffforthelaboratory.Fundsfor supplies and services will be attributed for the materials needed for the EcoTILLING and sequencing. Travel funds will be used to facilitate the technology transfer from IRRI to

CIRAD and to participate in a workshop held at CIRAD. The training, meeting, and workshop funds will support a workshop dedicated to the NARES and other partners, includingfoodandlodgingfortheattendeesofworkshops. AtAgropolisCIRAD,fundsforsalarieswillcover0.7ofthetimeforatechnician.Supplies andservicesincludetheuseandmaintenanceoflabequipmentandbenchfeesforpersonnel working in the project, from which $5000 will be allocated to AgropolisURGV. Travel includes missions to IRRI to facilitate comparison of technology and technology transfer. Theallocation fortraining,meeting,and workshopincludespart of(1)theorganizationof training sessions (part of bench fees, and food and lodging for the attendees) and (2) the organizationofaworkshopdedicatedtoNARESandoutsiders(benchfeesforexperiments in the context of the workshops, and assistance for travel, food, and lodging for the attendees).ThesewillbeperformedwiththehelpofGCPSP5. Inkindcontributions IRRI

Highthroughput laboratories for molecular biology, including 3 LiCor genotypers for EcoTILLING Germplasmcollectionofmorethan107,000riceaccessions Bioinformatics infrastructure, including an 8node Opteron cluster and software for sequenceanalysisandprimerdesign DatabaseformolecularcharacterizationandEcoTILLINGprofiles 30%timecontributionforMcNallyand5%eachforHamiltonandLafitte

CIRAD

Wellequipped laboratories for molecular biology, including 6 LiCor genotypers for EcoTILLING Collectionsofrice,sorghum,andMusagermplasm
Musalibrariesforidentifyingcandidategenesrelatedtodevelopment

LaboratoryfacilitiesdevotedtoNARESpartners(tobeformedatCIRAD) Bioinformaticsinfrastructureforcomparativesequenceanalysis 20% time contribution for Billot, 10% each for Hippolyte, Baurens and Rami, and 70% timecontributionfortechnicalsupportstaff

Workplan
ACTIVITIES MilestonesperQuarter 1. Identify10candidategeneswithhomologsin Oryza,Sorghum, and Musaseveralofwhichare developmentallyregulated genesrelatedtoflowerdevelopment(IRRIandCIRAD). Identify 7 candidate stress tolerance genes for Oryza, Sorghum, Setsofstresstolerantcandidategenesfor Oryza, Sorghum, andMusa,anddesignprimers. andMusa,andverifiedprimers. Identify 3 regulatory genes related to flower development in Set of developmentally regulated floral genes from Musa MusawithhomologsinOryzaandSorghum,anddesignprimers. with homologs in Oryza and Sorghum, and verified primers. 2.SequencelociforreferencemicrocoresandmismatchesfromEcoTILLING(IRRIandCIRAD). Sequence 10 candidate gene loci in microcore collections from SNPs identified/confirmed in reference microcore Oryza,Sorghum,andMusa. collections. 3. Optimizeprotocolsforhaplotypediscoveryandaccomplishhighthroughput(HT)EcoTILLING in Oryza(IRRI,CIRAD), Sorghum(CIRAD),andMusa(CIRAD). FurtheroptimizeprotocolforOryza(IRRI). RobustprocedureinplaceforOryza. Compare technology used in Sorghum and Oryza and transfer OryzaprotocoladaptedforuseonSorghum technologyforOryzafromIRRItoCIRAD. OryzaEcoTILLINGimplementedatCIRAD. In Musa, establishprotocolfordiploidthenoptimizeontriploid OryzaandSorghumprotocolsadaptedforuseonMusa. germplasm(CIRAD). EstablishprotocolforpolyploidMusa(CIRAD). FirstEcoTILLINGprotocolfornondiploidspecies. HT EcoTILLING for Oryza (IRRI and CIRAD), for Sorghum SNPs/haplotypesdiscoveredfor10candidategenesatloci (CIRAD), and for Musa (CIRAD) for microcore and 50100% of inOryza,Sorghum,andMusa. GCPcorecollectionspereach. SequencenewhaplotypesforOryza,Sorghum,andMusa. SNPsidentified/confirmedinlargegermplasmcollection. 4.IdentifysoftwarefordataanalysisofEcoTILLINGprofiles(IRRIandCIRAD). Alternativemethodsfordataanalysisexplored. Proceduresuitableforsemiautomatedprofileanalysis. 5.HoldworkshopsonSNPdiscoveryusingEcoTILLINGdedicatedtotheNARESandotherpartners(IRRIandCIRAD). WorkshopsheldatIRRIandCIRAD(withthehelpofGCPSP5). NARESandotherpartnerstrainedinthetechnology. 1 2 3 4 X X

X X X X X X X X X X X X X

X X X X X X X

APPENDICES DescriptionofPartners IRRI The International Rice Research Institute (IRRI) is an autonomous, nonprofit agriculturalresearchandtrainingorganizationwithofficesin11nations.IRRIsmaingoalis tofindsustainablewaystoimprovethewellbeingofpresentandfuturegenerationsofpoor rice farmers and consumers while simultaneously protecting the environment. Most of IRRIsresearchisdoneincooperationwithnationalagriculturalresearchanddevelopment institutions, farming communities, and other organizations of the worlds riceproducing nations. IRRIs headquarters has laboratories and training facilities on a 252hectare experimental farm on the main campus of the University of the Philippines in Los Baos. These laboratory facilities include three LiCor sequencers for EcoTILLING, two MJR BaseStations for genotyping, and equipment for highthroughput DNA extraction, molecularmarkerdevelopment,andbioinformatics. Particularly relevant to this project is IRRIs large collection of germplasm, specializedgeneticstocks,andbreedinglinesthatrepresentthesourceofgeneticvariation forgenediscovery.TheInternationalRiceGenebankCollectionhousedatIRRIistheworlds largest collection of rice germplasm, with more than 107,000 accessions. IRRI is strongly committedtocollaborativeresearchinalleleminingandfunctionalgenomicsandconsiders it a driving force for future genetic improvement of rice. IRRI has a multidisciplinary researchteamthatintegrateslaboratoryandfieldresearchinbreeding,genetics,physiology, biochemistry, cereal chemistry, quality evaluation, and bioinformatics. IRRI leads the International Consortium for Rice Functional Genomics and the Asian Rice Biotechnology Network, with partners in national research programs and advanced research institutes throughoutthe world.Finally,IRRI has an extensivecollaborative varietaltestingnetwork throughout Asia to ensure adequate testing of promising germplasm in multiple environmentsanddeliveryoftheproductsforriceimprovementthroughouttheworld. CIRAD CIRAD, Centre de coopration internationale en recherche agronomique pour le dveloppement, member of Agropolis, has a long history of genetic diversity and genome analyses in tropical crops to assist breeding programs. Its facilities are located in Montpellier,inthesouthofFrance. The research unit consists of about 2,000 m2 of laboratory space, where all basic molecularbiologytechniquescanbeapplied,includingaroboticsstationforhandlinglarge numbers of materials. The unit hosts regional facilities (Genopole Montpellier Languedoc Roussillon)forcontainmentgreenhouses,robotics,andgenotyping.Genotyping(directedby ClaireBillot)isaccomplishedonsixLiCorsequencers.

The unit has produced pioneering results (SSR marker development, diversity studies, genetic maps, synteny conservation analysis, QTL mapping, BAC libraries, chromosome walks) on Musa (banana), sugarcane, cocoa, rice, sorghum, rubber tree, oil palm,coconutpalm,Eucalyptus,andcotton(publicandprivatefundingsources),aswellasa collection(Genoplanteproject)ofmorethan30,000insertionmutantsofrice,ofwhichmore than half have been characterized with flanking sequence tags. It is now coordinating internationalprojectsonESTproductionincitrusandcocoa, beinginchargeoflibraries and bioinformatics, and is involved in comparative Musa/rice genome analysis using selectedBACclones.

 AlargeroomisdedicatedtotrainingscientistsfromdevelopingcountriesforM.S.or Ph.D.research(fromover15countriesinthepastthreeyears),throughshortdurationgroup training sessions (e.g., for coconut specialists trained for SSR application within the COGENT network) and for onsite ad hoc courses on the use of molecular markers (involvingscientistsfromCtedIvoire,Mali,Madagascar,andVietnam,amongothers).
Thegrouphasinitiatedallelediversityanalysesforassociationstudiesinthecaseof Eucalyptus for candidate genes of the lignin pathway, and of sorghum and rice, so far focusing on candidate genes related to grain quality within a GenoplanteGabi (French/German)projectalsoinvolvingwheat,barley,andmaize. CIRADhasbeenengagedformanyyearsin Musaresearchprogramsandpublished the first Musa genetic map in 1993. To date, CIRAD has developed a broad range of capacities in Musa genetic diversity, cytogenetic and genome structure studies, as well as phenotyping and agronomy. Hence, the results from the firstyear GCP activities (comparativemapping,geneexpressionprofiling,andgenotypingin Musa)willcontribute totheMusacomponentofthisproject. ExternalCollaborators The external collaborators on this project are the University of Washington (Luca Comai), LiCor, Inc. (Jeff Harford), and AgropolisURGV (Abdelhafid Bendahmane). As a developerofEcoTILLING,LucaComaibringsinvaluableexpertiseinthetechnologytothe project. He will assist in the ongoing optimization and technology transfer to CIRAD. Abdelhafid Bendahmane has implemented EcoTILLING at AgropolisURGV and is contributingtopilotstudiesonEcoTILLINGinsorghumwithCIRAD.ThroughJeffHarford, LiCor,Inc.hasagreedtoprovidetechnicalsupportbyattendingandparticipatinginoneor more training courses held at IRRI and CIRAD, to provide support (either directly or through appropriate distribution channels) for NARES partners who are in a position to invest in LICOR 4300 instruments, and to use IRRI and CIRAD as reference sites for advancing EcoTILLING technology on a worldwide basis. Hence, all of these external collaboratorsbringexpertisetotheproposedprojectcrucialtoitssuccess.

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Table1.Initialdroughtcandidategenes.TargetsforEcoTILLINGimplementationinOryza(forprospectiveuseinSorghumandMusa).
Gene Bioprocess protein phosphatase Evidence Pimer_Name Primer_sequence ggTTggggCATATCTCCTCgTggT TCCTAggAgCTggTTCAAACTgCAA TIGR_pm2_chr 9631_chr03_osa1 9631_chr03_osa1 9638_chr10_osa1 9638_chr10_osa1 ACgTCAAAACCCAACCCCAACCAT gATTgAATCAgggCCATCgCTTTTC CCgTTgATTgCTgATAgCCTCCTTgA TgAAATATTCCTATTgACCCgCAgCA 9629_chr01_osa1 9629_chr01_osa1 9629_chr01_osa1 9629_chr01_osa1 9629_chr01_osa1 9629_chr01_osa1 9630_chr02_osa1 9630_chr02_osa1 9630_chr02_osa1 9630_chr02_osa1 9630_chr02_osa1 9630_chr02_osa1 9630_chr02_osa1 9630_chr02_osa1 9630_chr02_osa1 9630_chr02_osa1 9635_chr07_osa1 9635_chr07_osa1 9640_chr12_osa1 9640_chr12_osa1 9640_chr12_osa1 9640_chr12_osa1 9630_chr02_osa1 9630_chr02_osa1 9630_chr02_osa1 9630_chr02_osa1 9630_chr02_osa1 9630_chr02_osa1 Start 3640223 3639579 Stop 3640247 3639604 strand Amplicon + + + + + + + + + + + + + + + + 619 928 988 969 984 947 998 1001 1014 1001 815 1064 1013 1009 1005 838

Pp2a4

pp2a4L Up-regulated under pp2a4R drought on Syngenta array pp2a4L pp2a4R Transformation of homologue results in enhanced survival under stress; cM 137 locus is closest hit to 16.1 cM locus DREB1upL DREB1upR DREB1-16cdL DREB1-16cdR

13756718 13756742 13757621 13757646 3353301 3354264 3354092 3355035 3353325 3354289 3354118 3355061

DREB1

drought responsive element binding protein 1

DREB1-137cdL ACACCCAAACCCAACCTCCCAAAAC DREB1-137cdR CATCgCCggCATgATCTgTTCTAAT TPPL TPPR ggCACACTgTCgCCTATTgTggATg gTTTACgAgCCgTgCgACCAgTTTC CgAgCACACACACTCACTCCCTgTC gTTggAgCTTggTCCCTTgATgTCC ACCgCCTgggTggTgAgTgTTgTA gTggACggACAggCAgAACTTgTTg CCTCgTCCgTTCTTATCCACCTCCA TgTAgCCgCgCTTggACTggTAATC CAAggTCggTTTCTTCCTCCACTCg TCATCCCAAACCCAATACCCCAATg CgCACTACATTTCAgCCCACgAgAA TgAAAACTgCTgCTgACCTCCAgACA gCCCATTgCAAAgCCCAggTTgTAT CCCATCCTTCTTCTTgCCACACCTC TggCAAgAAgAAggATgggCTgAAC TCCACAggTggAAgCAgAAgCAgTC CCATggCAgTTgCCCTATTgTTCAC CTCAgCAggCTgCgACATCTTCACT ggCATgCTTgATTTgggACATgAgA TCTCCTCATACCTCTCggCCTgCTC TgAgCTTTCCCgTgAggAgAATgTg CAAgATTgCAAgCACggTCAggAgA

33545491 33545516 33546450 33546475 26336946 26336971 26335974 26335999 31222704 31222729 31223677 31223702 31223053 31223077 31224029 31224054 33128991 33129016 33129980 33130005 33129811 33129836 33130787 33130812 27905484 27905509 27906273 27906299 25844561 25844586 25845600 25845625 25845606 25845631 25846594 25846619 21952232 21952257 21953216 21953241 21952300 21952325 21953280 21953305 21953237 21953262 21954050 21954075

TPP

Trehalose 6phosphate phosphatase

Up-regulated under drought on IR64 panicle arrays; 2 loci on CH2 are best top hits to E.coli sequence that on transformation enhances survival

TPP5082uxL TPP5082uxR TPP5082aL TPP5082aR TPP5438uxL TPP5438uxR TPP5438aL TPP5438aR TPP4712aL TPP4712aR

VP14

viviparous14 (9-cis Last-step in ABA epoxycarotenoid synthesis; QTL at dioxygenase); same position ABA biosynthesis

VP14-1L VP14-1R VP14-2L VP14-2R CG18upL CG18upR

14-3-3

Membrane associated signal cascade

CG18-1L CG18-1R CG18-cd2L CG18-cd2R

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Gene

Bioprocess Transcriptional control of stress response

Functional Evidence

Pimer_Name MAPk-1L MAPk-1R MAPk-2L MAPk-2R Ext-L Ext-R TRAB1-L TRAB1-R CDPK-L CDPK-R

Primer_sequence CACCATCTCCTTCAgCCTCCgTTTC CACACCTCCACCCCAATCAAATTCC ATgggCATgAAgATgAgCCTCTTgg gggTgCCggCTATggTACgCTAgAT AggAgAAgATggCgATggCCAATAA gAgCTCgAgATgCCTgATgATgTCC gCAggggTCgATCTACTCACTgACg TCAACACAgCAAggACCCAATCTCg ATTTTgATTCCgATCCCTggCCTgT CAAggAACCAggTggCAgACTTTCA CCTCTCTTgCCACCggCCTTATCTT CAgggAAgAAATTggAAgggCAgTg CgCTCAgCgAgTgCgTgAgACTATT ggCgATTgTACACTgCACATgATgg ATggCATCggAgATgAgCAAgAACg TTCggAgCAACATgTgTACCTgCTTT CCTCCTTgCAgggCCTgTCTgT TggCAgTgAggCATTCTCATAAAA AAggAACAATgCTgCTTTTggTCA ACCATATgACgCCACggCCTTT CTTTggggTggCgACTTgCTTg gCCCggTTTCCCCTCAAAACCT TgACCCAggTTTTgAggggAAACC AAgCCgTAgggCACggTgAACA

TIGR_pm2_chr 9635_chr07_osa1 9635_chr07_osa1 9635_chr07_osa1 9635_chr07_osa1 9638_chr10_osa1 9638_chr10_osa1 9636_chr08_osa1 9636_chr08_osa1 9630_chr02_osa1 9630_chr02_osa1 9631_chr03_osa1 9631_chr03_osa1 9635_chr07_osa1 9635_chr07_osa1 9629_chr01_osa1 9629_chr01_osa1 9630_chr02_osa1 9630_chr02_osa1 9630_chr02_osa1 9630_chr02_osa1 9635_chr07_osa1 9635_chr07_osa1 9635_chr07_osa1 9635_chr07_osa1

Start

Stop

strand Amplicon + + 927 + + + 1000 947 + + + + + + + 1004 991 954 878 1170 881 982 975 1006

MAPK

23069803 23069828 23070753 23070778 23071388 23071413 23072369 23072394 17603398 17603423 17602446 17602471 23061210 23061235 23062167 23062192 27647309 27647334 27648284 27648309 34051918 34051943 34050946 34050971 25355903 25355928 25356882 25356907 36901422 36901447 36902387 36902413 26490266 26490288 26489288 26489312 26489168 26489192 26488268 26488290 11676840 11676862 11675648 11675670 11675652 11675676 11674749 11674771

Extensi n

Cell-wall protein for growth expansion ABA responsive TRAB1 TF Signal transduction, CDPK regulatory cascade Inositol FIERY1 polyphosphate 1phosphatase

Ca+2 dependent signal cascade

FIERY1-L FIERY1-R Suc-L Suc-R Bzip-L Bzip-R ADFCH02aL ADFCH02aR

SucSas sucrose synthase e BZIP Transcriptional control

ADF

Actin depolymerizing factor

up-regulated in proteomics of drought; QTL on CH7 is for leaf rolling

ADFCH02bL ADFCH02bR ADFCH07IaL ADFCH07IaR ADFCH07Ib4L ADFCH07Ib4R

Additional primers will be designed for genes linking to other projects, such as the SP3 funded work on phosphate deficiency and salt tolerance,theprojectinvolvingaluminumtoxicity,andnonGCPprojectsinvolvingsubmergencetolerance.

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Fig.1.EcoTILLINGofTPSonOryzasativa.Trehalose6phosphatase(TPS)primerstestedon panel of 46 Oryza sativa germplasm accessions contrasted to IR64. Circles indicate a clear SNP detected in the IRD700 (a) and IRD800 (b) channels for Kun Min Tsieh Hunan. This germplasm was pooled with IR64 and one or more others at 1:2, 1:4, 1:8, 1:12, and 1:16. Mismatchdetectedin1:16poolforIRD700(c)andin1:8poolforIRD800(d).

Fig. 2. EcoTILLING of DREB1 on wild Oryza. DREB1 amplicons of a similar size were produced from 47 wild Oryza species using primers designed from Nipponbare (a). MixturesoftheseproductswerecombinedwitheitherIR64(leftsetoflanes)orNipponbare (right set of lanes). The IRD700 channel is shown in panel b and the IRD800 in c. Circles indicate clear difference present in both channels. Arrows indicate a mismatch present in japonicabutabsentfrommostwildandindica.

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Fig.3.SequenceconfirmationofputativemismatchesfromEcoTILLINGofDREB1upstream onsativaandwildOryza.ThetopbarshowsthegenestructureforDREB1at16.1cMonCH1 (RGPmap).SNPsareshownforthreeregionsoftheupstreamarea.Basesintheconsensus readsidenticaltotheNipponbarereferencesequenceareshownasdots.Lowercasebases hadsupportbyonestrandwiththeotherstrandindeterminate.

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