Genotyping by Sequencing in Almond

12 Pages·2017·2.26 MB·English

INVESTIGATION
Genotyping by Sequencing in Almond: SNP
Discovery, Linkage Mapping, and Marker Design
Shashi N.Goonetilleke,*TimothyJ.March,*Michelle G.Wirthensohn,* PereArús,†
AmandaR.Walker,‡andDiane E.Mather*,1
*SchoolofAgriculture,FoodandWine,WaiteResearchInstitute,TheUniversityofAdelaide,GlenOsmond,5064,
Australia,†InstitutdeRecercaiTecnologiaAgroalimentàries,CentredeRecercaenAgrigenòmica,ConsejoSuperiorde
InvestigacionesCientíficas–InstitutdeRecercaiTecnologiaAgroalimentàries–UniversitatAutònomadeBarcelona–
UniversityofBarcelona,CampusUniversitatAutònomadeBarcelona,08913,Spain,and‡AgricultureandFood,Waite
Campus,CommonwealthScientificandIndustrialResearchOrganisation,GlenOsmond,5064,Australia
ORCIDIDs:0000-0001-7539-011X(S.N.G.);0000-0002-2066-9495(T.J.M.);0000-0003-1174-6580(M.G.W.);0000-0003-0939-8038(P.A.);
0000-0002-7596-8484(A.R.W.);0000-0001-7506-2589(D.E.M.)
ABSTRACT Incropplantgenetics,linkagemapsprovidethebasisforthemappingoflocithataffectimportant KEYWORDS
traitsandfortheselectionofmarkerstobeappliedincropimprovement.Inoutcrossingspeciessuchasalmond Prunusdulcis
(PrunusdulcisMill.D.A.Webb),applicationofadoublepseudotestcrossmappingapproachtotheF progeny singlenucleotide
1
of a biparental cross leads to the construction of a linkage map for each parent. Here, we report on the polymorphisms
applicationofgenotypingbysequencingtodiscoverandmapsinglenucleotidepolymorphismsinthealmond allele-specific
cultivars“Nonpareil”and“Lauranne.”Allele-specificmarkerassaysweredevelopedfor309tagpairs.Applica- molecular
tion of these assays to 231 Nonpareil · Lauranne F progeny provided robust linkage maps for each parent. markers
1
Analysisofphenotypicdataforshellhardnessdemonstratedtheutilityofthesemapsforquantitativetraitlocus composite
mapping.Comparisonofthesemapstothepeachgenomeassemblyconfirmedhighsyntenyandcollinearity linkagemap
between the peach and almond genomes. The marker assays were applied to progeny from several other shellhardness
Nonpareil crosses, providing the basis for a composite linkage map of Nonpareil. Applications of the assays
to a panel of almond clones and a panel of rootstocks used for almond production demonstrated the broad
applicabilityofthemarkersandprovidesubsetsofmarkersthatcouldbeusedtodiscriminateamongaccessions.
Thesequence-basedlinkagemapsandsinglenucleotidepolymorphismassayspresentedherecouldbeuseful
resourcesforthegeneticanalysisandgeneticimprovementofalmond.
Almond(PrunusdulcisMill.D.A.Webb)isanimportantnutcropwith progeny, with only limited use of molecular information (Scorza
anannualglobalproductionof1.2milliontons(UnitedStatesDepart- 2001; Sánchez-Pérez et al. 2007; Gradziel 2009; Koepke et al. 2013).
mentofAgriculture2015).Almondbreedingreliesmostlyonpheno- Development and implementation of modern molecular tools could
typic assessment of parents, crossing between selected parents, support genetic mapping and the precision of the almond breeding
vegetative propagation of progeny, and phenotypic selection among process.
Almond is an outcrossing species with a gametophytic self-
Copyright©2018Goonetillekeetal. incompatibility system. Genetic mapping in almond has therefore
doi:https://doi.org/10.1534/g3.117.300376 usedthepseudotestcrossstrategy(Arúsetal.1994;Virueletal.1995;
ManuscriptreceivedJuly20,2017;acceptedforpublicationNovember4,2017; Tavassolianetal.2010;FontiForcadaetal.2012,2015;Fernándezi
publishedEarlyOnlineNovember15,2017. Martíetal.2013),whichprovidesalinkagemapforeachparent.The
Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommons first almond linkage maps to include all eight linkage groups were
Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted use, distribution, and reproduction in any medium, constructedbasedonapplicationofisozymeandrestrictionfragment
providedtheoriginalworkisproperlycited. lengthpolymorphism(RFLP)markerstoprogenyfromacrossbetween
Supplementalmaterialisavailableonlineatwww.g3journal.org/lookup/suppl/ the almond cultivars “Ferragnès” and “Tuono” (Viruel et al. 1995).
doi:10.1534/g3.117.300376/-/DC1.
Subsequently,areferencelinkagemapforalmondwasestablishedwith
1Correspondingauthor:SchoolofAgriculture,FoodandWine,WaiteResearchInstitute,
theapplicationofisozymeandRFLPmarkerstoprogenyfromacross
TheUniversityofAdelaide,PMB1,GlenOsmond,SouthAustralia5064,Australia.
E-mail:[email protected] between the almond cultivar “Texas” and the peach (P. persica
Volume8 | January 2018 | 161
L. Batsch) cultivar “Earlygold” (Joobeur et al. 1998). Simple sequence theDNAsamplesusedwereresidualsamplesfromtheearliermapping
repeat(SSR)markerswerelateraddedtothismap(Aranzanaetal.2003; work.Thesesamples,whichhadbeenextractedfromyoungleavesusing
Dirlewangeretal.2004).Othermarkertypesthathavebeenmappedin theLamboyandAlphaDNAextractionmethod(Lamboy1998),were
almondincluderandomamplifiedpolymorphicDNA(RAPD)markers, checkedforDNAqualitybyelectrophoresison1%agarosegels,quan-
inter-SSR (ISSR) markers, sequence characterized amplified region tifiedusingPicoGreenintercalatingdye(Invitrogen,Carlsbad,CA),and
markers,andsinglenucleotidepolymorphism(SNP)markers(Joobeur normalized to a working concentration of 20 ng/ml. For the other
etal.2000;Wuetal.2009,2010;Tavassolianetal.2010;Donosoetal. 231N·Lprogeny,andforalloftheotheralmondclonesmentioned
2016). Among these, SNPs are particularly promising as they are the above,DNAwasextractedfromyoungleavesusinganOktopureDNA
mostabundantsequencedifferencesinplantsandtheyareusuallybial- extractionprotocolthathadbeenoptimizedforalmond(LGCLimited,
lelicandcodominant.Theyhavebeenusedinmanyplantspecies,in- Teddington).
cludingalmond(Wuetal.2009,2010;Donosoetal.2016;Sorkhehetal. In addition, some use was made of rootstock materials that are
2017). availableinAustralia:“Adafuel,”“Atlas,”“Bright’sHybrid1,”“Corner-
Withnext-generationsequencing(NGS),itispossibletodiscoverand stone,”“Felinem,”“Garnem,”“GF557,”“Hansen536,”“Krymsk86,”
directlyassaylargenumbersofsequencepolymorphismswithoutprior “Monegro,” “Nemaguard,” “Nickels,” “Penta,” “Tetra,” and “Viking”
knowledgeaboutthepolymorphismsortheirgenomicpositions.Given (Table S2 in File S1). For these materials, DNA was extracted from
the size and complexity of plant genomes, NGS-based polymorphism youngleavesusingthemethodofThomasandScott(1993)followedby
discovery and genotyping benefit from the preparation of reduced sodiumchloride/ethanolprecipitation.
representation libraries (Miller et al. 2007; Baird et al. 2008; Elshire
et al. 2011; Peterson et al. 2012; Poland et al. 2012). Among various Libraryconstruction andsequencing
availablelibrarypreparationprotocols,themethodproposedforgeno- Toselectarestrictionenzymethatmightbesuitablefordigestionofthe
typingbysequencing(GBS)byElshireetal.(2011)issimpleandmakesit almond genome, in silico restriction of the peach whole genome se-
possibletodiscoverthousandsofSNPs.Thismethodhasbeenappliedin quenceassemblyv1.0(www.rosaceae.org)wasconductedusingBiopy-
manyplantspecies,includingpeach(Bielenbergetal.2015),sweetcherry thon (Cock et al. 2009) for each of three methylation-sensitive
(P.aviumL.)(Guajardoetal.2015),Japaneseplum(P.salicinaLindl.) restrictionenzymes:ApeKI,PstI,andHpaII.TheenzymeApeKI,which
(Salazaretal.2017),andapricot(P.armeniacaL.)(Gürcanetal.2016). hasbeenusedinGBSforotherplants(Elshireetal.2011;Luetal.2013;
WhileGBScanbeacost-effectiveapproachfortheinitialdiscovery Bielenbergetal.2015;Guajardoetal.2015;Kujuretal.2015;Gürcan
andmappingoflargenumbersofSNPs,thequalityofthelinkagemaps etal.2016;Salazaretal.2017),wasselected.Ofthethreeenzymes,it
producedissomewhatlimitedbygenotypingerrorsandmissingdata. waspredictedtoyieldthehighestnumberoffragmentswithinthesize
Onewaytoaddressthislimitationistodevelopallele-specificassaysfor range that isconsidered suitable forGBS (between150 and 500 bp)
SNPsdiscoveredbyGBSandtoapplythesetothemappingpopulationto (TableS3inFileS1).Further,ithadbeenreportedtogenerateuniform
obtainmoreaccurateandcompletedata.Suchassaysmayalsobeuseful librarieswiththedegreeofcomplexityreductionthatisrequiredfor
forapplicationtomaterialsbeyondthosethatwereincludedintheGBS sequencing(Heetal.2014).
library.AssaysforindividualSNPsareparticularlyusefulwhenonlyone Barcode,primer,andadaptersequencesforApeKI(TableS4inFile
orafewmarkersaretobetested,asinmarker-assistedselectionforone S1)wereobtainedfromhttp://www.maizegenetics.net/genotyping-by-
orafewloci.Amongthemanytechnologiesthatcanbeusedtoassay sequencing-gbs.Barcodes,adapters,andprimersweresynthesizedby
SNPs, allele-specific Kompetitive Allele Specific PCR (KASP) assays Sigma Aldrich (Castle Hill, Australia). The complementary top and
(LGCGenomics,Teddington,UnitedKingdom)arenowwidelyusedin bottom strands ofeachbarcodeand adapterwerediluted to10mM
plantgeneticsandbreeding(e.g.,Babikeretal.2016;Rasheedetal.2016; with10·adapterbufferandannealedusingthefollowingPCRcondi-
Tanetal.2017). tions:95(cid:2)for1min,followedbyrampingdownto30(cid:2)by1(cid:2)percycle.
In this research, the GBS protocol was adapted for almond and Theresultingdouble-strandedbarcodeandadaptersweredilutedsep-
appliedtoF progenyfromacrossbetweenthealmondcultivars“Non- aratelyin1·TEto0.6ng/ml,quantifiedusingPicoGreenintercalating
1
pareil”and“Lauranne”todiscoverSNPsandconstructlinkagemaps. dye,andnormalizedto0.1mMwith1·TE.Eachbarcodesolutionwas
KASPassayswerethendevelopedforasubsetoftheSNPsandwere mixedwiththeadaptersolutionina1:1ratioinonewellofa96-wellplate.
appliedtotheoriginalmappingpopulationandtoadditionalmaterials. ToselectanappropriateratiobetweenadapterandDNAconcentra-
Quantitativetraitloci(QTL)forshellhardnessweremappedforNon- tions,atitrationexperimentwascarriedout.Forthis,apooledDNAsample
pareilandforLauranne. waspreparedbymixingequalamountsofDNAfrom10N·LF progeny.
1
Eight200-ngsamplesofDNAweredrawnfromthispooledsample.After
additionof3.2UofApeKIin2ml10·NEBbuffer(NewEnglandBiolabs,
MATERIALS ANDMETHODS
Ipswich,MA)andwatertobringthefinalvolumeto20ml,thesesamples
Plant materialsandDNAsamples wereincubatedfor2hrat75(cid:2).Oneofeightquantitiesofadapter(2,5,8,10,
The almond clones used in this research were Nonpareil, Lauranne, 12, 15, 18, or 20 ml of a 0.1 M adapter solution), 10 ml of a solution
“Chellaston,”“Constantí,”“Ferraduel,”“Glorieta,”“Johnston,”“Mandaline,” containing200UofT4DNAligase(NewEnglandBiolabs),and5mlof
“Marta,” R1065, “Somerton,” “Tarraco,” “Vairo,” “White,” and 12-350 10·ligationbufferwereadded.Sampleswereincubatedat22(cid:2)for2hrand
(Supplemental Material, Table S1 in File S1). In addition, 320 F thenat65(cid:2)for20min.LigationproductswerepurifiedusingaPureLink
1
progeny were used from crosses involving Nonpareil: 320 from PCRPurificationKit(Invitrogen)asperthemanufacturer’sinstructions.
Nonpareil·Lauranne(N·L),349fromNonpareil·Constantí(N· Eachpurifiedligationproductwasresuspendedinafinalvolumeof50ml.
C),207fromNonpareil·Tarraco(N·T)and198Nonpareil·Vairo For the final library, 10 ml of each purified ligation product was used
(N·V).Nonpareilisofparticularinterestbecauseitisamajorcultivarin ina25-mlPCRreactionwith2mlofthe10-mMpaired-endprimers
bothCaliforniaandAustralia. 59-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACA
Ofthe320N·Lprogeny,89hadbeenpreviouslyusedforgenetic CGACGCTCTTCCGATCT-39 and59-CAAGCAGAAGACGGCATAC
mappingbyTavassolianetal.(2010).FortheseclonesandforLauranne, GAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-39
162 | S.N.Goonetillekeetal.
whetherthetagswerehomozygousinNonpareilandheterozygousin
Lauranne,orviceversa.Eachofthesedatasetswasfurtherfilteredto
retainonlytheSNPsmissingnomorethan20datapointspermarker
andwithsegregation ratiosnotdeviatingsignificantlyfrom3:1(a =
0.05).SeparateparentallinkagemapswereconstructedforNonpareil
and Lauranne using a double pseudotestcross strategy implemented
using the backcross(BC) format inASMap R packageversion 1.0-1
(TaylorandButler2017)withthefollowingmapconstructionstrategy:
1. An initial framework linkage map was constructed using data from
progenyforwhichtherewerenomissingdatafortheselectedSNPs.
DataforafewSSR,RAPD,andISSRmarkersthatTavassolianetal.
(2010)hadreportedtobehomozygousinoneparentandheterozygous
intheotherparentwereincludedinadditiontodatafortheselected
SNPs.Linkagemappingwascarriedoutusingtheminimumspanning
treemapalgorithm(MSTmap)(Wuetal.2008)asimplementedin
ASMaptoassignmarkerstolinkagegroupsandtoorderthemwithin
linkage groups. A P-value of 0.0001 was used to declare whether
markers belong to the same linkage group. The Kosambi mapping
function(Kosambi1944)wasusedtocalculategeneticdistancesincM.
Figure1 Numberofuniquesequencetagsvs.numberofsequence 2. Foreachlinkagegroup,ASMapwasusedtogenerateaheatmap
reads.Relationshipbetweenthenumberofsequencereadsobtained (rf/LODplots)toevaluatepairwiseassociationsbetweenmarkers.
andthenumberofunique64-bpsequencetagsobtainedfromGBSof For cases in which markers appeared to have had their alleles
89N·LF1progeny. assignedtotheincorrectparents,genotypedesignationswerereas-
signed using the“switchAlleles”function oftheR/qtl Rpackage
alongwith12.5mlofTaq2XMasterMix(NewEnglandBiolabs).ThePCR version1.41-6(Bromanetal.2003).Mapswerethenreestimated
conditionsusedwereasfollows:30secat95(cid:2),15cyclesof30secat95(cid:2), usingthemstmap.crossfunction.
20secat65(cid:2),30secat68(cid:2),followedbyafinalextensionat72(cid:2)for5min. 3. To further improve the quality of the Nonpareil and Lauranne
Eachamplifiedlibrarywaspurifiedasdescribedaboveandelutedinafinal linkage maps, markers were checked for segregation distortion
volumeof30ml.Eachlibrary(2ml)wasrunon2%agaroseat90Vfor and numbers of double crossover events involving adjacent
markerintervals.Markerswereremovediftheirsegregationratio
30mintoevaluatethelibraryandtheadapterdimerpeaks.Anadapter
concentration of 4.5 ng in a volume of 15 ml was selected because it deviated significantly from 1:1 (a = 0.05) and/or if they were
associatedwithhighnumbersofapparentdoublecrossoverevents.
providedasatisfactorylibrarywithnoadapterdimerpeak.
Librarypreparationwascarriedoutusing200ng(10mlof20ng/ml)of Mapswerethenreestimatedusingthemstmap.crossfunction.
DNAfromeachoftheinitial89N·Lprogenyandeachofthreealiquotsof 4. TheorientationofeachlinkagegroupoftheNonpareilandLauranne
maps was established by comparing the maps constructed
DNAfromNonpareilandLauranne.Thesameprocedurewascarriedout
usingtheSNPdatawiththepublishedmapsofTavassolianetal.
forawatersampleasanegativecontrol.Initialreactionswerecarriedoutin
(2010).ThiswasdoneusingtheAlignCrossfunctionofASMap.
a96-wellplateusingaseparatewellforeachsample.Afteradapterligation,
sampleswerepooledforpurification,PCRamplification,evaluation,and FromtheresultingframeworkmapsforNonpareilandLauranne,a
sequencing.Thepooledlibrarywassequencedusingsingle-endsequencing setofGBSmarkersfromtheeightlinkagegroupswasselectedforthe
(100-bpreads)ononeflow-celllaneofanIlluminaHiSeq2000instrument designofallele-specificassays,withtheobjectiveofobtainingmarkers
attheAustralianGenomeResearchFacility(Melbourne,Australia). spacedat(cid:1)10-cMintervalsthroughoutthegenome.
SNPdiscovery Primerdesign
TheGBSsequencedatawereanalyzedusingtheUniversalNetworkEnabled PrimersetsweredesignedforSNPsthathadbeendiscoveredandmapped
AnalysisKit(UNEAK)pipelineinTASSEL3.0software(Bradburyetal. basedontheGBSdata.SomeofthesewereheterozygousinNonpareiland
2007;Luetal.2013).ThispipelinepermitsSNPcallingbasedsolelyon homozygousinLauranne,andotherswereheterozygousinLauranneand
GBStagsequencedata,withoutrequiringareferencegenomesequence. homozygousinNonpareil.Forsomeofthese,itwaspossibletodesign
TheoutputwasfilteredtoselectSNPswithatleast80%coverageacross primersbasedsolelyontheGBSdata.Forothers,theSNPsweretoocloseto
samples,aminimumreaddepthof5,andaminimumrelativeheterozy- oneendoftheGBStags.Forthese,thetagswerealignedtoNonpareil
gosity value (ratio of heterozygotes to homozygotes) of 0.01. The level genomic contig sequences using the BLAST tool in Geneious software
ofalmondgenomecoverageobtainedwasestimatedusingtheLander– version9.1.3(Kearseetal.2012)toobtainsequencesof(cid:1)100bpwiththe
Watermanequation(LanderandWaterman1988). SNPslocatedneartheirmidpoints.EachSNP-bearingsequencewasused
todesignasetofthreeprimers(twoallele-specificprimersandonecom-
Construction oflinkagemaps monprimer)usingKrakensoftware(LGCLimited).Theprimersetswere
FortheconstructionofinitialframeworklinkagemapsforNonpareil namedusingtheprefixWriPdK,withWrireferringtotheWaiteResearch
andLauranne,tagpairswithminorallelefrequencies(MAF)between0.2 Institute,PdreferringtoP.dulcis,andKreferringtoKASPtechnology.
and0.3inthemappingpopulationwereselected.Thiswasbasedonthe
expectationofaMAFof0.25forthemostinformativeSNPs(thosethat ApplicationofKASPassays
areheterozygousinoneparentandhomozygousintheotherparent). Atotalof309primersets(146designedforSNPsthatwereheterozygousin
Theresultingdatasetwasseparatedintotwoparentaldatasetsbasedon Nonpareiland162designedforSNPsthatwereheterozygousinLauranne)
Volume8 January2018 | SNPDiscoveryandMappinginAlmond | 163
Figure2 Anchoringofalmondsequence
tags to peach genome. Numbers of
unique tags anchored to each 500-kbp
regionofeachoftheeightmainscaffolds
(Pp1–Pp8) of the peach whole genome
sequence assembly v2.01a1. For each
scaffold,thetotalnumberofuniquetags
isgiveninparenthesesandtheestimated
position of the centromere is indicated
withanarrow.
wereassayedonapanelconsistingofDNAsamplesofNonpareil,Lauranne, achievedusingapseudotestcrossmappingstrategywithdatacodedinthe
andsevenN·LF progeny,withawatersampleincludedasanegative BCformat,consideringonlythosemarkersforwhichNonpareilwashet-
1
control.Samplesof10ngofDNA(5mlof2ng/ml)weredriedat55(cid:2)for erozygous.Inpopulationsforwhichtheotherparentwashomozygousat
1hr.Aliquotsofaprimermixture(0.028ml,containing12mMofthe a marker, the codes “ab” and “aa” were assigned to heterozygous and
allele-specificprimersand30mMofthecommonprimer)andof1·KASP homozygousprogeny,respectively.Inpopulationsforwhichtheotherpar-
MasterMix(1.972ml;LGCLimited)wereaddedtoeachreactionsample. entwasalsoheterozygous,allprogenywerecodedashavingmissingdata.
PCRamplificationwasconductedusingthestandardKASPPCRprotocol Recombinationfractionsbetweenadjacentmarkerswereestimated
inaHydrocycler-16PCRsystem(LGCLimited).Fluorescencewasdetected usingtheR/qtlpackageversion1.41-6(Bromanetal.2003).Recom-
inaPherastarPlusplatereader(BMGLABTECH,Ortenberg,Germany). binationfractionswereconvertedtomapdistancesusingtheKosambi
DatawereanalyzedusingKrakensoftware(LGCLimited).Primersetsthat mappingfunction(Kosambi1944).Theresultingcompositemapwas
detectedpolymorphisminthevalidationpanelwereselectedandassayed comparedwithNonpareilmapsthathadbeenconstructedusingdata
on311N·Lprogeny:80ofthe89progenythathadbeenusedtoprepare fromindividualpopulations.Markersforwhichthereweresubstantial
theGBSlibrary,plus231others.Thesameprimersetswerealsoassayed inconsistenciesamongmapswereremovedandthemappinganalysis
onthepanelofalmondclones.Genotypiccallswerecomparedamong wasrepeated.Markersthathadbeenmappedinonlyonepopulation
clonesusingFlapjacksoftwareversion1.16(Milneetal.2010).Selected wereassignedpositionsinthecompositemapbasedontheirpositions
markers(thosethatwereheterozygousinoneparentandhomozygousin relativetoflankingmarkers.Thefinalcompositemapwasdrawnusing
theother)wereassayedontheN·C,N·T,and/orN·Vprogeny. MapChartversion2.3software(Voorrips2002).
LinkagemappingusingKASPmarkers Comparativemappingbetweenalmond andpeach
LinkagemapswereconstructedforeachparentusingKASPmarkerdata EachuniqueGBSsequencereadobtainedforNonpareilandLaurannethat
from80N·Lprogeny,231N·Lprogeny,349N·Cprogeny,207 wasatleast64-bplongandhadsequencecoverage$10wasalignedagainst
N·Tprogeny,and198N·Vprogeny,usingtheproceduresdescribed thepeach(P.persica)wholegenomesequenceassemblyv2.0.a1(www.
fortheconstructionof theinitialframeworklinkagemap.Mapswere rosaceae.org)usingtheBLAST+toolversion2.2.27(http://www.ncbi.nlm.
drawnusingMapChartversion2.3software(Voorrips2002). nih.gov/blast).Eachsequencereadwasconsideredtohavebeenanchored
Datafrom985progenyfromfourcrosses(N·C,N·L,N·T,and tothepeachgenomeifitmappedtoauniquesitewith.90%sequence
N·V)wereusedtoconstructacompositemapforNonpareil.Thiswas similarityandanE-value,1e215.Forsequencesthatmetthesecriteria
164 | S.N.Goonetillekeetal.
Figure 3 Examples of KASP assay re-
sults.IntensitiesofFAMandHEXfluores-
cence detected when two KASP assays
(WriPdK7andWriPdK69)wereappliedto
Nonpareil,Lauranne,andN·Lprogeny.
TheWriPdK7primersweredesignedfor
aSNPthatisheterozygous(G:C)inNon-
pareilandhomozygous(C:C)inLauranne.
TheWriPdK69primersweredesignedfor
aSNPthatishomozygous(C:C)inNon-
pareilandheterozygous(T:C)inLauranne.
(A) WriPdK7 applied to Nonpareil (in
duplicate), Lauranne (in duplicate), and
seven N · L F progeny. (B) WriPdK7
1
applied to 231 N · L F progeny. (C)
1
WriPdK69appliedtoNonpareil(indupli-
cate),Lauranne(induplicate),andseven
N·LF progeny.(D)WriPdK69applied
1
to231N·LF progeny.
1
andforwhichmarkerassayshadbeendeveloped,theCirclizeRpackage Dataavailability
version0.4.1(Guetal.2014)wasusedtocomparethegeneticpositionsin Information on the parentage and origin of the almond clones and
almondwithphysicalpositionsintheeightmainscaffoldsofthepeach rootstocksusedinthisresearchisinTablesS1andS2inFileS1.Sequence
genomeassembly. datafor89N·LprogenyhavebeendepositedintheNationalCenter
forBiotechnologyInformationShortReadArchive:studySRR5722967.
QTL mappingforshellhardness Informationonfragmentsizedistributionsfrominsilicodigestionof
Shellhardnesswasevaluatedin2015for180N·Lprogeny.Foreach the peach genome sequence is in Table S3 in File S1. Barcode and
tree,arandomsampleof10nutswasweighedtoobtainin-shellweight. primer sequences used for GBS are in Table S4 in File S1. Contig
Thenutswerethencrackedopenusinganutcracker,andkernelswere sequencesforNonpareilhavebeendepositedintheEuropeanNucle-
weighed.Theshell-hardnesspercentagewascalculatedassuggestedby otideArchiveunderaccessionnumberPRJEB23106.Primersequences
Rugini (1986): (kernelweight/in-shell weight)· 100%. Accordingto forKASPassaysareinTableS5inFileS1.LinkagemapsforNonpareil
thismeasure,almondnutsmaybeclassifiedaspapershell($55%),soft are in Table S6 in File S1. Linkage maps for Lauranne, Constantí,
shell(45–54%),semihardshell(35–45%),hardshell(25–34%),orstone Tarraco,andVairoareinTableS7inFileS1.Resultsobtainedfrom
shell(#24%).QTLforthistraitweremappedusingtheR/qtlpackage theapplicationofKASPmarkerstoalmondclonesandrootstocksare
version1.41-6(Bromanetal.2003),withthefunctionScanoneusedto inTablesS8andS11inFileS1,respectively.ThebestBLASThitsinthe
testforputativeQTLat1-cMintervalsthroughoutthegenome.Signif- peachgenomeforSNP-bearingtagsfromNonpareilandLauranneare
icance wasdeclared bycomparing LODvaluestoa threshold deter- inTablesS9andS10inFileS1,respectively.Thegenotypicandphe-
minedusing10,000permutationsandagenome-widesignificancelevel notypicdatausedforQTLanalysisareinTablesS12andS13inFileS1.
of0.05.
RESULTS
Polymorphismdetectionin rootstocks
UsingtheKASPassayproceduresdescribedabove,253SNPs(128hetero- Sequencedata
zygousinNonpareiland125heterozygousinLauranne)wereassayedon TheGBSlibrarygenerated21.6Gb of sequencedata,withatotalof
duplicatesamplesofDNAsamplesextractedfromtherootstockaccessions. 186millionsequencereads(ameanof2.1millionpersample).Linear
Volume8 January2018 | SNPDiscoveryandMappinginAlmond | 165
Figure4 Nonpareillinkagemaps.LinkagemapsconstructedforNonpareilusinggenotypicdatafromKASPassaysappliedto(A)231N·LF
1
progenyand(B)985F progenyfromfourcrosses(N·L,N·C,N·T,andN·V).
1
regressionanalysisindicatedastrongpositiverelationshipbetweenthe 295markers(279GBS,5SSR,8ISSR,and3RAPD),andis1371cM
numberofsequencereadsandthenumberoftagsforeachindividual long(TableS7inFileS1).
(R2=0.92,P,0.0001,Figure1). Ofthe149KASPprimersetsdesignedbasedonsequencetagsthat
Acrossallsamples,atotalof453,648uniquetagswasobtained.Of exhibited heterozygosityin Nonpareil (e.g., Figure 3A), 138 detected
these tags, 308,971 (68%)wereanchored tothe peachgenome, with polymorphismamongtheprogeny(e.g.,Figure3B).Ofthe162primer
between30,594and59,923mappingtoeachoftheeightmainscaffolds setsdesignedbasedonsequencetagsthatexhibitedheterozygosityin
(Pp1–Pp8)(Figure2)and6088mappingtootherscaffolds.Tagswere Lauranne (e.g., Figure 3C), 155 detected polymorphism among the
mappedthroughouttheentirelengthofeachmainscaffold,butwith progeny (e.g., Figure3D). Noneof the genotypic ratios observed for
somevariationinthemarkerdensity.Therewereafewregions(e.g.,on thesepolymorphismsdeviatedsignificantlyfromtheexpected1:1ratio.
Pp5andPp7)withveryhighdensity. RelativetotheinitialframeworkmapsthatwerederivedfromGBS
Fromtheuniquetags,11,936SNP-containingtagpairswereiden- data, the linkage maps constructed based on KASP marker data for
tified.Withtheapplicationofaseriesoffilters,.300tagpairsthatwere 231 N · L progeny had very similar marker orders, but were much
consideredsuitableformappingwere selectedforeachofNonpareil shorter(TablesS6andS7inFileS1).TheKASPmapforNonpareilhad
andLauranne. 138markersandatotallengthof609cM(Figure4A).TheKASPmap
forLaurannehad155markersandatotallengthof659cm(Figure5).
Linkagemaps forNonpareil andLauranne
AninitialframeworkmapconstructedforNonpareilbasedoncomplete Polymorphismsamongalmond clones
datafor 52progenyhad327 markers (310GBS, 9 SSR,5 ISSR, and Ofthe261KASPassaystested,239exhibitedpolymorphismamong14
3RAPD)oneightlinkagegroupswithatotallengthof1152cM(TableS6 almondclonesotherthanNonpareilandLauranne(TableS9inFileS1).
inFileS1).TheinitialframeworkmapconstructedforLaurannewas Ofthesemarkers,111hadbeendesignedbasedonheterozygosityin
basedoncompletedatafrom55progeny.Ithaseightlinkagegroups, Nonpareil,and128basedonheterozygosityinLauranne.Amongthe
166 | S.N.Goonetillekeetal.
Figure 5 Lauranne linkage map. A linkage map constructed for Lauranne using genotypic data from KASP assays applied to 231 N · L F
1
progeny.
14otheralmondclonesonwhichthesemarkerswereassayed,allexcept totallengthsof439,569,and553cM,respectively(TableS6inFile
MartaandSomertoncouldbedistinguishedfromallothersbyjustone S1),andwithmarkerordersverysimilartothoseobtainedwiththe
marker. A total of 11 KASP assays were selected (Figure 6) that, in N ·L population. Linkage maps developedfor Constantí, Tarraco,
combination,couldbeusefulfordistinguishingamongalloftheclones andVairohad65,39,and52markers,respectively,withtotallengths
thatwereexaminedhere. of382,295,and148cM(TableS7inFileS1).
Linkagemaps basedonN3C,N3T,andN3V QTLforshellhardness
Of the 138 KASP markers that were developed based on Nonpareil ForNonpareil,QTLforshellhardnessweredetectedintworegions,both
heterozygosity and mapped using N · L progeny, 92, 85, and onlinkagegroup5(LG5).ForLauranne,QTLforshellhardnesswere
103markersdetected polymorphismin N·C,N ·T, andN ·V, detectedinfourregions:oneonLG2,twoonLG5,andoneonLG8(Table
respectively. Of the 155 KASP markers that were derived based on 1). In all of these regions, Nonpareil-like genotypes were associated
Lauranne heterozygosityandmappedusingN·Lprogeny,68,40, with softer shells (higher mean shell hardness). Of the 180 progeny
and56markersdetectedpolymorphisminN·C,N·T,andN·V, thatwereevaluatedforshellhardness,just17hadtheNonpareil-like
respectively.Inaddition,severalmarkersthathadnotexhibitedpoly- genotypeinallQTLregions.LikeNonpareil,theseprogenyexhibited
morphismamongN·Lprogenywerefoundtobepolymorphicin the “paper-shell” trait (very high shell-hardness percentage). The
other populations. Linkage maps for Nonpareil that were based on genotypes at six markers (WriPdK251 and WPdK50 on LG2;
N·C,N·T,andN·Vhad90,82,and94markers,respectively,with WriPdK129,WriPdK18,andWriPdK264onLG5;andWriPdK282
Figure6 Genotypesof15almondclonesfor11KASPmarkersselectedbasedontheirabilitytodiscriminateamongtheseclones.Foreach
marker,theleastcommongenotypeisshowninwhitetextonadarkbackground.
Volume8 January2018 | SNPDiscoveryandMappinginAlmond | 167
n Table 1 QTL detected for shell-hardness percentage in
NonpareilandLaurannebasedonevaluationofnutsharvestedin
2015from180N3LF progeny
1
LinkageMap LinkageGroup Position(cM) LOD R2a
NonpareilKASPmap LG5 0 2.5 9
45 2.5 9
LauranneKASPmap LG2 26 3.4 9
LG5 0 4.2 11
43 3.2 9
LG8 87 3.4 9
a
PercentageofphenotypicvarianceexplainedbytheQTL.
onLG8)weresufficienttoseparateprogenywiththepaper-shelltrait
fromthosewithhardershells(Figure7).
CompositelinkagemapforNonpareil
Thecompositelinkagemapconstructedbasedondatafromtheprogeny
offourNonpareilcrosseshad129KASPmarkerswithatotallengthof
741cM(Figure4BandTableS6inFileS1).Somemarkersthathad
collocatedinNonpareilgeneticmapsconstructedforindividualNon- Figure7 Marker-baseddiscriminationofprogenywiththepaper-shell
pareilpopulations(N·L,N·C,N·T,and/orN·V)wereseparated trait.Shell-hardnesspercentagesandmeansfortwosetsofN·LF
1
inthecompositemap. progeny,oneselectedtohavetheNonpareilgenotypiccombination
across six markers (WriPdK251 and WPdK50 on LG2; WriPdK129,
Comparisonofalmond geneticmapswiththe WriPdK18,andWriPdK264onLG5;WriPdK282onLG8)areassociated
with shell hardness and the other consisting of progeny with other
peachgenome
genotypiccombinationsatthosemarkers.
ComparisonofmarkerpositionsonNonpareilandLauranneparental
mapswithpositionsonpeachgenomescaffoldsconfirmedtheexpected
highsyntenyandcollinearitybetweenthealmondandpeachgenomes discoverandassaySNPswithoutanypriorsequenceinformation.The
(Figure8andTablesS9andS10inFileS1).Almostallmarkersan- restriction enzyme used here, ApeKI, is a type-II endonuclease that
chored to the expected peach scaffolds. For the Nonpareil map, the recognizes a degenerate 5-bp sequence (GCWGC, where W is A or
exceptionsarea fewmarkers thatgenetically mapped on LG1,LG4, T).Itisusefulforthereductionofsequencecomplexity,becauseithas
LG6,andLG8butanchoredtopeachscaffoldsPp5,Pp1,Pp1,andPp4, relativelyfewrecognitionsitesinthemajorclassesofplantretrotrans-
respectively.FortheLaurannemap,thereweremarkersthatgenetically posonsandwillnotcutifthe39baseoftherecognitionsequenceonthe
mappedonLG2,LG3,andLG6butanchoredtopeachscaffoldsPp6, bottomstrandis59methylcytosine(Söllneretal.2006).Itcreatesa59
Pp6,andPp4,respectively.Therearealsoafewdiscrepanciesinmarker overhangof3bp,providingsitesforattachmentofadapterstowhich
orderbetweenthealmondgeneticmapsandthepeachscaffolds(e.g.,at primers can anneal toprovide a uniform libraryfor sequencing(He
eachendofNonpareilLG4andpeachscaffoldPp4).Afewareasofthe etal.2014).
peachgenomearenotwellrepresentedononeorbothalmondlinkage Themeannumberofsequencereadspersamplethatwasobtained
maps.Forexample,onlytwomarkersfromtheNonpareilLG7map here(2.1million)issimilartowhathasbeenreportedforotherPrunus
andnomarkersfromtheLauranneLG7mapanchoredbetween0and species:1.8millionforsweetcherry(Guajardoetal.2015),2.4million
7MbponpeachPp7scaffold.Therearealsosomeregionsinwhich for peach (Bielenberg et al. 2015), 2.3 million for Japanese plum
markersthatarecloselylinkedinalmond(e.g.,at27cMonNonpareil (Salazaretal.2017),and3.5millionforapricot(Gürcanetal.2016).
LG2andat20cMonLauranneLG8)anchoredtophysicallydistant Ofthesequencesgenerated,68%wereanchoredtouniquepositionsin
positionsonpeachscaffolds. thepeachgenomesequenceassembly.Thisishigherthanwasreported
forapricot(43%,Salazaretal.2017),whichisnotascloselyrelatedto
Polymorphisms amongrootstocks peach.Withinatotalof224Mbofthepeachgenometowhichalmond
Ofthe220KASPassaystestedonrootstockmaterials,169werepoly- tags were anchored, the densityof almond–peach SNPs was (cid:1)1 per
morphic and 66 were monomorphic (Table S11 in File S1). Of the 22kb.Similarresultshavebeenreportedforsweetcherry(Guajardo
169 polymorphic assays, 93 had been designed based on Nonpareil etal.2015).Somevariationwasobservedinthenumbersofsequence
heterozygosity, and 76 based on Lauranne heterozygosity. In most tagsandSNPsmappedtoeachscaffoldandintheanchorpositionsof
cases,justonemarkerwassufficienttodistinguishaparticularroot- tagswithinscaffolds.Possiblereasonsforthisvariationcouldinclude:
stockfromallothers.PentaandTetra,bothofwhicharederivedfrom (1) variation in the distribution of ApeKI restriction sites across the
European plum, were very similar but there were four markers that almondgenome,(2)variationinDNAmethylationacrossthealmond
distinguishedbetweenthem.Atotalof10KASPassayswereselected genome,and(3)structuraldifferencesbetweenthealmondandpeach
(Figure 9) that, in combination, could be useful for distinguishing genomes. The unusually high numbers of sequence tags obtained in
amongalloftherootstockmaterialsthatwereexaminedhere. someregions(e.g.,oneat6.5MbponPp5andoneat0.5MbponPp7)
mayindicatethattheseregionsaremorepolymorphicormorerepet-
DISCUSSION itiveinalmondthaninpeach.Consistentwiththis,oneoftheseregions
Inthisresearch,implementationofaGBSprotocolenableddiscovery (at6.5MbponPp5)correspondswitharegionofLG5inwhichmany
ofthousandsofSNP-bearingGBStags,providinganeasymethodto polymorphismsweregeneticallymappedforNonpareil(albeitnotfor
168 | S.N.Goonetillekeetal.
what was obtained for apricot (18,322; Gürcan et al. 2016) or plum
(42,909;Salazaretal.2017).Oneapproachtoincreasethenumberof
SNPsdiscoveredwouldbetouseless-stringentfiltersinthesequence
analysis. The TASSEL GBS 3.0 SNP-calling pipeline, which was de-
velopedmainlyforhighlyhomozygousmaterials,isconsideredtobe
sensitive to low sequence depth in highly heterozygous materials
(Hymaetal.2015).Therefore,astringentreaddepthcutoffvalueof
fivewasappliedpermarkercall.Withavalueofthree,alargernumber
ofGBStags(.600)couldhavebeenselectedformapping,butmaps
constructedonthisbasis(datanotshown)hadverylonglinkagegroups
(.300cM);someoftheadditionalSNPsmayhavebeenspurious.
AnotherapproachtoincreasethenumberofSNPsdiscoveredcould
betoincreasesequencedepth.Inthisanalysis,therewasastrongpositive
relationship(R2=0.92)observedbetweentotalreadnumberandthe
totalnumberofuniquetags,indicatingthatadditionaluniquetagsand
SNPsmighthavebeendiscoveredbyincreasingsequencingdepth.
AthirdapproachtoincreasethenumberofSNPsdiscoveredcouldbe
touseanenzymeorcombinationofenzymesthatwouldprovidealarger
numberofdigestedfragmentsandincreasingthedepthofsequencing.
To investigate this, we extended the in silico analysis of the peach
genome sequence to include consideration of two-enzyme combina-
tions.Theresults(TableS3inFileS1)indicatedthatthecombinationof
ApeKIandHpaIImightprovideasubstantiallyhighernumberoffrag-
mentsofsuitablelengththanApeKIalone.
WhileGBScangeneratelargenumbersofpolymorphicmarkers,it
cansufferfromincorrectassignmentofparentalphase,underestimation
ofheterozygotes,andhighproportionsofmissingdata(Luetal.2013).
Here,technicalreplicatesoftheparentswereincludedinthegenomic
library and very stringent filters were applied to select subsets of
markers and progeny for initial mapping. Duringmap construction,
diagnostictestswereconductedtodetectandcorrectphasingerrors.
TheseapproachescontributedtoaveryhighsuccessrateinKASPassay
design.Onlyoneincorrectlyphasedmarkerwasdetected.Thatmarker
(GBS tag pair TP37439), which had originally been assigned to the
Nonpareilmap,wasreassignedtotheLaurannemapbasedonresults
obtainedwiththeWriPdK92primerset.Threemarkers(GBStagpairs
TP15642,TP16449,andTP18643)thatwereoriginallyassignedtothe
Laurannemapweredeterminedtobeheterozygousinbothparentsand
were not used for map construction. Three other markers (GBS tag
pairsTP11609,TP12109,andTP25403)thathadoriginallybeenscored
asheterozygousinoneparentweredeterminedtobehomozygousin
bothparentsandwerenotusedformapconstruction.WiththeKASP
Figure8 Comparisonsofalmondgeneticmapswiththepeachgenome
markers, it was possible to obtain complete and accurate data for a
sequence.(A)Nonpareillinkagegroups1–8comparedwithpeachscaf-
largernumberofprogenythanhadbeenusedfortheinitialGBSmap.
foldsPp1–Pp8.(B)Laurannelinkagegroups1–8comparedwithpeach
scaffoldsPp1–Pp8.Onthealmondlinkagegroups,geneticdistancesare Thereforeitisnotsurprisingthattherearesomedifferencesinmarker
orderbetweentheinitialandKASPmaps;theKASPmapsshouldbe
given in cM. On the peach scaffolds, physical distances are given in
Mbp.Linksbetweenlinkagegroupsandscaffoldsindicatethepositions consideredasmorereliable.
atwhichmarkersgeneticallymappedinalmondanchortothegenomic ThenumbersofGBStagpairsusedtoconstructtheinitialNonpareil
sequenceofpeach. andLaurannelinkagemaps(310and282,respectively)weresimilarto
numbersthathavebeenusedforJapaneseplum(232foroneparentand
Lauranne).However,anotherone(at0.5MbponPp7)isonachro- 324fortheother;Salazaretal.2017)andforsweetcherry(443forone
mosome arm for which only two polymorphisms were mapped in parentand474fortheother;Guajardoetal.2015).Theinitialgenetic
Nonpareil and none were mapped for Lauranne. Lack of polymor- mapsconstructedusingGBSdataareabouttwiceaslongastheNon-
phisminthisregionmaynotbelimitedtothesematerials,noreven pareilandLaurannemapspublishedbyTavassolianetal.(2010),but
toalmond,assimilarobservationshavebeenreportedforthisregion thefinalmapsconstructedusingdatafromKASPassaysaresimilarin
for the sweet cherry cultivars Riverdale and Rainer (Guajardo et al. lengthtothepreviouslypublishedmaps.This“shrinkage”wasdueto
2015).Theconsistentlackofpolymorphismincertaingenomicregions correctionofgenotypesthathadbeenerroneouslycalledintheGBS
couldreflectfixationoffavorableallelesduetoselection. analysis.Inmostcases,correctionswerefromhomozygoustohetero-
Whilethetotalnumberofhigh-qualitySNPsobtainedforalmond zygous,indicatingthatalthoughtwoalleleswerepresent,onlyoneof
(11,936) was sufficient for genetic mapping and higher than was them had been sequenced in sufficient depth. Of a total of 12,720
obtainedforsweetcherry(8476;Guajardoetal.2015)itislowerthan heterozygous calls in the final KASP data set, 1526 (12%) had been
Volume8 January2018 | SNPDiscoveryandMappinginAlmond | 169
Figure 9 Genotypes of 15 rootstocks for 10 KASP markers selected based on their ability to discriminate among these rootstocks. For each
marker,theleastcommongenotypeisshowninwhitetextonadarkbackground.
miscalledashomozygousintheGBSanalysis.Thistypeoferrorwas marker genotypes were associated with softer shells. Depending on
evenlydistributedamongmarkers.These observations aresimilarto whetherthepaper-shellcharacteristicofNonpareilisconsideredfavor-
whathasbeenreportedforGBSanalysisinswitchgrass(Luetal.2013). ableorunfavorable(toosoft),selectioncouldbeimposedfororagainst
The success rate in converting SNP-bearing tag pairs to useful Nonpareil-likegenotypesatsixmarkers(twoonLG2,threeonLG5,and
fluorescence-basedmarkerassayswasveryhigh.Ofthe309SNP-bearing oneonLG8).IntheN·Lpopulation,thiswouldhaveeitherfixedor
GBS sequences that wereselected for assay design, 293(95%) were eliminatedthepaper-shelltrait.
successfullyconvertedtoKASPassaysandmapped(138forNonpareil Giventhatthismapwasconstructedusingsequence-basedmarkers,
and 155 for Lauranne). However, not all of these could have been itwaspossibletoanchorittothepeachgenomesequenceassembly.It
designedsolelybasedontheGBSdata.Inmanytags,theSNPposition willbepossibletoanchorittoalmondgenomesequenceassembliesas
was too close to one end of the tag for primer design. These tag they become available and to connect it with other sequence-based
sequenceshadtobealignedagainstpreexistingcontigsequencesto linkagemapsastheyaredeveloped.Thus,thiscompositemapcould
obtainflankingsequences. provideaplatformforunificationofgeneticandgenomicresourcesfor
Untilnow,linkagemappinginalmondhasreliedondatafromthe thealmondresearchcommunity.
progeny of individual biparental crosses. Here, progeny from four Thisisthefirstreportofgenome-wideanchoringofalmondgenetic
Nonpareilcrosseswereused,providingfourlinkagemapsforNonpareil. maps to the peach whole genome sequence assembly. The results
There should be no differences in the true biological positions of confirmedtheexpectedhighsimilaritybetweenthealmondandpeach
the SNPs on these four maps, as each map is based on estimates of genomes, with only a few of the mapped markers anchoring to un-
recombinationfrequenciesforthesameparent,Nonpareil.Whilethere expectedpositions.Inmostpartsofthegenome,markerpositionson
was very good agreement among the maps, there were also some Nonpareiland Lauranne genetic maps were linearly relatedwith the
differences, presumably due to sampling error. Given that the SNPs physicalpositionstowhichtheywereanchoredinthepeachgenome.
mappedinallfourpopulationshadbeenpreselectedbecausetheywere Thereweresomeregionsofthepeachgenomeforwhichfewalmond
informativeforN·L,themapderivedfromthatpopulationhasmost polymorphismswerediscovered.Basedthedatageneratedhere,itisnot
markers and the best genome coverage. Among the four individual possible to distinguish whether these are simply regions in which
Nonpareilmaps,theonefromN·Lshouldbeconsideredasthemost NonpareilandLaurannearebothhighlyhomozygous,orwhetherthese
reliable. Nevertheless, the quality of any genetic map is limited by regionsarestructurallydifferentbetweenpeachandalmond.
samplesize.Here,theavailabilityofadditionalNonpareilpopulations Thepolymorphismsdetectedhereamongalmondclonesandroot-
providedanopportunitytoimprovethemapaccuracyandresolution. stocksprovideinformationaboutthetransferabilityofSNPsdiscovered
The Nonpareil composite linkage map constructed here is the first inonepopulationtoothermaterials.Ofthemarkersdesignedbasedon
almondlinkagemapconstructedbasedongenotypicdatafrommulti- heterozygosity in Nonpareil or Lauranne, 70% were useful for poly-
plecrosscombinations.Theuseoffourcrosseswithacommonparent morphism detection among almond rootstocks. Although SNPs are
(Nonpareil)providedanopportunitytoexploitalargertotalpopula- generallyusedforpolymorphismdetectionwithinspecies,manyofthe
tionsizetoresolvetheorderofsomecloselylinkedmarkers.Giventhat SNPmarkersdevelopedherebasedonalmondpolymorphismdetected
NonpareilisthepredominantalmondcultivarinboththeUnitedStates polymorphismsinmaterialthatoriginatedfrompeach,apricot,plum,
and Australia, this linkage map should be a particularly useful new cherry,andcomplexbackgrounds.Themarkersdevelopedherecouldbe
resource for almond research, including QTL mapping and marker- broadlyusefulfordetectinggeneticdifferencesamongaccessionsofP.
assistedbreeding. dulcis,relatedspecies,andinterspecificcrosses.Applicationsofthese
TodemonstratetheutilityofthelinkagemapsforQTLmappingand markerscouldincludeassessmentof cultivar verification,geneticdi-
toprovideexamplesofmarkersthatcouldbeusedforselection,QTL versity assessment, genetic mapping, and marker-assisted selection.
resultsarepresentedhereforshellhardness.Forthattrait,QTLwere From among the markers that were assayed here, 11 were selected
detectedonthreelinkagegroups:LG2,LG5,andLG8.QTLforthistrait based on their ability to differentiate among 15 almond clones and
hadpreviouslybeenmappedonLG2(Sánchez-Pérezetal.2007)and 10 were selected based on their ability to differentiate among
LG8(Arúsetal.1998),butnotonLG5.AtalloftheseQTL,Nonpareil-like 15rootstocks.
170 | S.N.Goonetillekeetal.