SNP Discovery and Chromosome Anchoring Provide the First Physically-Anchored Hexaploid Oat

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Department of Agronomy Faculty Publications Department of Agronomy
3-22-2013
SNP Discovery and Chromosome Anchoring
Provide the First Physically-Anchored Hexaploid
Oat Map and Reveal Synteny with Model Species.
Rebekah E. Oliver
Nicholas A. Tinker
Gerard R. Lazo
Shiaoman Chao
Eric N. Jellen
See next page for additional authors
Follow this and additional works at:http://docs.lib.purdue.edu/agrypubs
Recommended Citation
Oliver, Rebekah E.; Tinker, Nicholas A.; Lazo, Gerard R.; Chao, Shiaoman; Jellen, Eric N.; Carson, Martin L.; Rines, Howard R.;
Obert, Donald E.; Lutz, Joseph D.; Shackelford, Irene; Korol, Abraham B.; Wight, Charlene P.; Gardner, Kyle M.; Hattori, Jiro; Beattie,
Aaron D.; Bjornstad, Asmund; Bonman, J. Michael; Jannink, Jean-Luc; Sorrells, Mark E.; Brown-Guedira, Gina L.; Mitchell Fetch,
Jennifer W.; Harrison, Stephen A.; Howarth, Catherine J.; Ibrahim, Amir; Kolb, Frederic L.; McMullen, Michael S.; Murphy, J. Paul;
Ohm, Herbert W.; Rossnagel, Brian G.; Yan, Weikai; Miclaus, Kelci J.; Hiller, Jordan; Maughan, Peter J.; Redman Hulse, Rachel R.;
Anderson, Joseph M.; Islamovic, Emir; and Jackson, Eric W., "SNP Discovery and Chromosome Anchoring Provide the First
Physically-Anchored Hexaploid Oat Map and Reveal Synteny with Model Species." (2013).Department of Agronomy Faculty
Publications.Paper 4.
http://dx.doi.org/10.1371/journal.pone.0058068
This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for
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Authors
Rebekah E. Oliver, Nicholas A. Tinker, Gerard R. Lazo, Shiaoman Chao, Eric N. Jellen, Martin L. Carson,
Howard R. Rines, Donald E. Obert, Joseph D. Lutz, Irene Shackelford, Abraham B. Korol, Charlene P. Wight,
Kyle M. Gardner, Jiro Hattori, Aaron D. Beattie, Asmund Bjornstad, J. Michael Bonman, Jean-Luc Jannink,
Mark E. Sorrells, Gina L. Brown-Guedira, Jennifer W. Mitchell Fetch, Stephen A. Harrison, Catherine J.
Howarth, Amir Ibrahim, Frederic L. Kolb, Michael S. McMullen, J. Paul Murphy, Herbert W. Ohm, Brian G.
Rossnagel, Weikai Yan, Kelci J. Miclaus, Jordan Hiller, Peter J. Maughan, Rachel R. Redman Hulse, Joseph M.
Anderson, Emir Islamovic, and Eric W. Jackson
This article is available at Purdue e-Pubs:http://docs.lib.purdue.edu/agrypubs/4
SNP Discovery and Chromosome Anchoring Provide the
First Physically-Anchored Hexaploid Oat Map and Reveal
Synteny with Model Species
RebekahE.Oliver1.,NicholasA.Tinker2*.,GerardR.Lazo3.,ShiaomanChao4,EricN.Jellen5,
MartinL.Carson6,HowardW.Rines7,DonaldE.Obert8,JosephD.Lutz1,IreneShackelford9,
AbrahamB.Korol10,CharleneP.Wight2,KyleM.Gardner2,JiroHattori2,AaronD.Beattie11,
A˚smundBjørnstad12,J.MichaelBonman9,Jean-LucJannink13,MarkE.Sorrells14,GinaL.Brown-
Guedira15,JenniferW.MitchellFetch16,StephenA.Harrison17,CatherineJ.Howarth18,AmirIbrahim19,
FredericL.Kolb20,MichaelS.McMullen21,J.PaulMurphy22,HerbertW.Ohm23,BrianG.Rossnagel11,
WeikaiYan2,KelciJ.Miclaus24,JordanHiller24,PeterJ.Maughan5,RachelR.RedmanHulse5,
JosephM.Anderson23,EmirIslamovic12,EricW.Jackson1.
1GeneralMillsCropBiosciences,Kannapolis,NorthCarolina,UnitedStatesofAmerica,2EasternCerealandOilseedResearchCentre,AgricultureandAgri-FoodCanada,Ottawa,
Ontario,Canada,3WesternRegionalResearchCenter,GenomicsandGeneDiscovery,UnitedStatesDepartmentofAgriculture-AgriculturalResearchService,Albany,California,
UnitedStatesofAmerica,4BiosciencesResearchLab,UnitedStatesDepartmentofAgriculture-AgriculturalResearchService,Fargo,NorthDakota,UnitedStatesofAmerica,
5DepartmentofPlantandWildlifeSciences,BrighamYoungUniversity,Provo,Utah,UnitedStatesofAmerica,6CerealDiseaseLaboratory,UnitedStatesDepartmentofAgriculture-
AgriculturalResearchService,SaintPaul,Minnesota,UnitedStatesofAmerica,7DepartmentofAgronomyandPlantGenetics,UniversityofMinnesota,SaintPaul,Minnesota,United
StatesofAmerica,8LimagrainCerealSeeds,Lafayette,Indiana,UnitedStatesofAmerica,9SmallGrainsandPotatoGermplasmResearchUnit,UnitedStatesDepartmentof
Agriculture-AgriculturalResearchService,Aberdeen,Idaho,UnitedStatesofAmerica,10DepartmentofEvolutionaryandEnvironmentalBiologyandInstituteofEvolution,
UniversityofHaifa,Haifa,Israel,11CropDevelopmentCentre,DepartmentofPlantSciences,UniversityofSaskatchewan,Saskatoon,Saskatchewan,Canada,12DepartmentofPlant
andEnvironmentalSciences,NorwegianUniversityofLifeSciences,A˚s,Norway,13RobertW.HolleyCenterforAgricultureandHealth,UnitedStatesDepartmentofAgriculture-
AgriculturalResearchService,Ithaca,NewYork,UnitedStatesofAmerica,14DepartmentofPlantBreedingandGenetics,CornellUniversity,Ithaca,NewYork,UnitedStatesof
America,15EasternRegionalSmallGrainsGenotypingLaboratory,NorthCarolinaStateUniversity,UnitedStatesDepartmentofAgriculture-AgriculturalResearchService,Raleigh,
NorthCarolina,UnitedStatesofAmerica,16CerealResearchCentre,AgricultureandAgri-FoodCanada,Winnipeg,Manitoba,Canada,17SchoolofPlant,EnvironmentalandSoil
Sciences,LouisianaStateUniversity,BatonRouge,Louisiana,UnitedStatesofAmerica,18InstituteofBiological,EnvironmentalandRuralSciences,AberystwythUniversity,
Aberystwyth,Ceredigion,UnitedKingdom,19DepartmentofSoilandCropSciences,TexasA&MUniversity,CollegeStation,Texas,UnitedStatesofAmerica,20DepartmentofCrop
Sciences,UniversityofIllinoisatUrbana-Champaign,Urbana,Illinois,UnitedStatesofAmerica,21DepartmentofPlantSciences,NorthDakotaStateUniversity,Fargo,NorthDakota,
UnitedStatesofAmerica,22DepartmentofCropScience,NorthCarolinaStateUniversity,Raleigh,NorthCarolina,UnitedStatesofAmerica,23DepartmentofAgronomy,Purdue
University,WestLafayette,Indiana,UnitedStatesofAmerica,24JMP,SASInstituteIncorporated,Cary,NorthCarolina,UnitedStatesofAmerica
Abstract
Aphysicallyanchoredconsensusmapisfoundationaltomoderngenomicsresearch;however,constructionofsuchamapinoat
(AvenasativaL.,2n=6x=42)hasbeenhinderedbythesizeandcomplexityofthegenome,thescarcityofrobustmolecularmarkers,
andthelackofaneuploidstocks.ResourcesdevelopedinthisstudyincludeamodifiedSNPdiscoverymethodforcomplexgenomes,a
diversesetofoatSNPmarkers,andanovelchromosome-deficientSNPanchoringstrategy.Theseresourceswereappliedtobuildthe
first complete, physically-anchored consensus map of hexaploid oat. Approximately 11,000 high-confidence in silico SNPs were
discoveredbasedonninemillioninter-varietalsequencereadsofgenomicandcDNAorigin.GoldenGategenotypingof3,072SNP
assays yielded 1,311 robust markers, of which 985 were mapped in 390 recombinant-inbred lines from six bi-parental mapping
populationsranginginsizefrom49to97progeny.Theconsensusmapincluded985SNPsand68previously-publishedmarkers,
resolving21linkagegroupswithatotalmapdistanceof1,838.8cM.Consensuslinkagegroupswereassignedto21chromosomes
usingSNPdeletionanalysisofchromosome-deficientmonosomichybridstocks.Alignmentswithsequencedgenomesofriceand
Brachypodiumprovideevidenceforextensiveconservationofgenomicregions,andrenewedencouragementfororthology-based
genomicdiscoveryinthisimportanthexaploidspecies.Theseresultsalsoprovideaframeworkforhigh-resolutiongeneticanalysisin
oat,andamodelformarkerdevelopmentandmapconstructioninotherspecieswithcomplexgenomesandlimitedresources.
Citation: OliverRE,TinkerNA, LazoGR, ChaoS,JellenEN,etal.(2013)SNPDiscoveryand ChromosomeAnchoring Provide theFirstPhysically-Anchored
HexaploidOatMapandRevealSyntenywithModelSpecies.PLoSONE8(3):e58068.doi:10.1371/journal.pone.0058068
Editor:MingliangXu,ChinaAgriculturalUniversity,China
ReceivedOctober17,2012;AcceptedJanuary30,2013;PublishedMarch22,2013pb?
Thisisanopen-accessarticle,freeofallcopyright,andmaybefreelyreproduced,distributed,transmitted,modified,builtupon,orotherwiseusedbyanyonefor
anylawfulpurpose.TheworkismadeavailableundertheCreativeCommonsCC0publicdomaindedication.
Funding:ThisworkwasfundedbygrantsfromtheUnitedStatesDepartmentofAgricultureNationalFoodResearchInitiative,theNorthAmericanMillers’
Association,thePrairieOatGrowersAssociation,GeneralMillsInc.,theUnitedStatesDepartmentofAgriculture-AgriculturalResearchServiceCRISproject301,
andbyAgricultureandAgri-FoodCanada.ScientistsREOandEWJcontributedtodatacollection,decisiontopublish,andpreparationofthemanuscriptpriorto
employmentatGeneralMillsInc.,andtheseauthorsdeclarethattheworkpresentedwasnotinanywayinfluencedbytheircurrentrelationshipwiththeir
employer.StaffscientistJDLfromGeneralMillsInc.engagedindiscussionsleadingtoexperimentaldesign,andparticipatedinmanuscriptpreparation,butthis
wasentirelylimitedtoscientificinput.AuthorDEOiscurrentlyemployedatLimagrainCerealSeeds,butcontributedallworkexceptformanuscriptrevisionprior
tothisemployment.StaffScientistsKJMandJHfromSASInstituteInc.contributedscientificinputanddataanalysis.Innootherwaydidthefundersplayarolein
studydesign, datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.Thefundingfrom General Millsandthecontributionsof
employeesfromGeneralMills,LimagrainandSASdoesnotaltertheauthors’adherencetoallthePLOSONEpoliciesonsharingdataandmaterials.
PLOSONE | www.plosone.org 1 March2013 | Volume 8 | Issue 3 | e58068
SNP-BasedChromosome-AnchoredConsensusMapinOat
CompetingInterests:ScientistsREOandEWJcontributedtodatacollection,decisiontopublish,andpreparationofthemanuscriptpriortoemploymentatGeneral
MillsInc.,andtheseauthorsdeclarethattheworkpresentedwasnotinanywayinfluencedbytheircurrentrelationshipwiththeiremployer.StaffscientistJDLfrom
GeneralMillsInc.engagedindiscussionsleadingtoexperimentaldesign,andparticipatedinmanuscriptpreparation,butthiswasentirelylimitedtoscientificinput.
AuthorDEOiscurrentlyemployedatLimagrainCerealSeeds,butcontributedallworkexceptformanuscriptrevisionpriortothisemployment.Thefundingfrom
GeneralMillsandthecontributionsofemployeesfromGeneralMillsandLimagraindoesnotaltertheauthors’adherencetoallthePLOSONEpoliciesonsharingdata
andmaterials.
*E-mail:[email protected]
.Theseauthorscontributedequallytothiswork.
Introduction model grass genomes. These results open a new window of
scientificopportunitytoexploreothercomplexgenomes,andwill
Cultivated hexaploid oat (Avena sativa L.; 2n=6x=42,
accelerate the genetic improvement of oat, an important
AACCDD) is a nutritionally important cereal crop [1] produced
functional food[1].
for both food and animal feed in many parts of the world. Plant
breedersinmanycountriesstrivetodevelopimprovedoatvarieties
Results
thatincorporatebetteragronomicandnutritionaltraitsaswellas
improved suitability for milling and processing. This work would In Silico SNP Discovery
be facilitated by a better framework of genetic and genomic More than 35 million un-filtered SNPs were predicted from
informationthatcanbeusedtoenhancegermplasmimprovement cDNA reads using the single-template approach (STA) (Fig. 1).
in this crop. Unfortunately, genomic knowledge and resources in Stringent filtering based on insufficient read depth (,5 reads),
oathavelaggedbehindthoseinsomeothercropspeciesdespitea heterogeneity within a variety, insertion/deletion polymorphism,
long history of research in this important crop. Previously- or an ambiguous reference base left 75,974 candidate SNPs.
published hexaploid oat maps contain more than the predicted Remaining SNPs were filtered by Illumina design scores (.0.8)
21 linkage groups, and alignment among maps has been
and redundancy, then sorted in descending order by predicted
fragmentary [2,3]. Difficulties have included a lack of sequence
minor-allele frequency (MAF), as estimated from the number of
data, the large size and complexity of the genome [4], and
varietiesthatdifferedfromthealleleinthereferencegenome.For
instability of aneuploid mapping stocks. Unlike other common
example,if6varietiesdifferedoutofapossible20,theMAFwas
polyploids, colinearity among oat subgenomes is disrupted by
predicted as 30%. The top 2,270 cDNA-based SNP predictions
numerous chromosomal rearrangements [5–9], thus diploid
were incorporated into pilotassays (Table1).
relatives have provided limited guidance for map construction.
The composite-template approach (CTA) utilized a multi-step
PriorgenotypinginoathasreliedheavilyonDNAmanipulation,
procedure (Fig. 1): assembling within varieties, condensing to
hybridization, and size-discrimination [10–12], with heteroge-
regions represented by three or more reads, then reassembling
neousresultsthatarepoorlyintegratedamongdifferent mapping
across varieties to predict 18,396 cDNA templates and 12,180
populations. This problem has been confounded by sequence
DArT templates with average lengths of 560 and 300bp,
redundancy among polyploid sub-genomes, causing duplicate
respectively. Condensation reduced the computational load for
marker loci [13,14].
thecompositeassemblyanddiminishedtheimpactofread-errors.
Recently, a pilot study has demonstrated the potential to
Afterre-assemblingcondensedreadsagainstcompositetemplates,
overcomesomeoftheseissuesthroughdiscoveryandmappingof
126,235cDNAand53,974DArTSNPswerepredicted.Filtering
highly-filtered SNP markers [15]. Transcriptome data linked to
was applied to eliminate SNPs that were heterogeneous within
physically anchored genetic maps can facilitate genomic research
varieties, had less than 10% MAF, or had fewer than 50 non-
and crop improvement, especially in plant species without
variable bases on either side. This resulted in a highly enriched
sequenced genomes [16,17]. Development of this resource in
candidate set of 1,056 cDNA SNPs and 519 DArT SNPs. Based
orphanedcropshasbecomepossiblethroughaffordablesequenc-
on MAF, the top 336 cDNA-based SNPs and 300 DArT SNPs
ing technologies and high-throughput genotyping platforms
non-redundantwiththeSTAwereselectedforvalidation(Table1).
[18,19]. These resources could enable development of the first
An additional 66 sequences selected by manual inspection of
physically-anchored consensus mapin hexaploid oat.
Sangersequences,and100SNPsderivedfromgenomicreduction
Anchoring of a transcriptome-based genetic map to chromo-
oftetraploidoat,hadcomparableIlluminadesignscoresandwere
somesprovidesvalidationoflinkagegroups,integrationofgenetic
included intheSNP assay.
and cytogenetic data, and a foundation for genome sequencing
and comparative genomics. However, this too has been a
SNP Assays
challenge in oat. Chromosome-deficient cytogenetic stocks have
beenusedtoassignmolecularmarkerstocorrespondingchromo- Alleles from 1,311 of the 3,072 SNP assays were clearly
somes [20,21], but such stocks are limited and require frequent discriminated among the mapping progeny and/or a set of 109
monitoring to detect univalent shifts and disomic reversion diverse oat varieties, indicating a 43.7% conversion rate. Of the
[22,23]. A partial oat monosomic series has been developed [23] converted assays, 991 (44% conversion) were based on the STA,
and used to assign 22 linkage groups to 16 chromosomes [24]. while 144 (43% conversion) and 121 (40% conversion) were
However, this technique did not directly interrogate the F plant derived from cDNA and DArT sequences using the CTA
1
and did not accountforcytogenetic variations, twofactors which (Table 1). Overall, 1,169 of the 1,311 successful SNP assays
limit resolution. (89.2%)identifiedpolymorphicallelesinapanelof109diverseoat
The objectives of this current work were to develop, in lines (Table 1, Dataset S1). Among these, approximately 20%
hexaploid oat: (i) robust SNP assays; (ii) a new chromosome (n=234)ofSNPsdetectedminoralleles(,10%).Allelesfrom985
anchoring strategy; (iii) the first physically-anchored consensus of the 1,311 assays (75.1%) segregated in at least one of the bi-
map; and (iv) a comprehensive orthology-based comparison to parental populations (Table1).
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SNP-BasedChromosome-AnchoredConsensusMapinOat
Figure1.InsilicoSNPdiscoveryapproaches. Bothmethodsstartedwithaset ofquality-trimmed454sequencereadsidentifiedbysource
germplasmfromeitherthecDNAlibrariesorfromDArT-basedgenomiccomplexityreductions.A.Inthesingletemplateapproach(STA),thereads
wereassembledbyMIRAsoftwaretogenerateaconsensussequenceforeachcontig.Fourreferencegenomeswereselectedbasedonmembership
infourdifferentquadrantsofaprinciplecomponentanalysisthathadbeenconductedpreviouslyusingDArTmarkers.Consensussequencesfromall
varietieswereassembledagainsteachofthefourreferencegenomes,andcandidateSNPswerecalledusingRocheGSMapper.SNPswerefiltered
basedonseveralcriteria,asdescribedinthemethods.RedundantSNPswereidentifiedusingBLASTN.B.Inthecompositetemplateapproach(CTA),
readswereassembledwithinvarietiesatahighstringencyusingDNAstarSeqmanSoftware.Thentheconsensusreadswerefilteredandtruncatedto
includeonlythosepartshavingperfectalignmentwithgreaterthantworeads.Theconsensusreadswerethenconcatenatedandsubjectedtoa
singlecompositeassemblyatlowerstringency.TheconsensusfromthisassemblywasusedasacompositereferencegenometocallSNPs.Although
theSNPcallingandfilteringprocesswassimilartoCTA,thispipelinewasautomatedusingin-housesoftwarecalled‘‘Ace-of-Base’’.
doi:10.1371/journal.pone.0058068.g001
Map Construction A consensus map was constructed from framework SNPs
Separate linkage maps were constructed within six RIL developed in this study and reference loci from previous maps
populations based on 320 to 647 loci (Table 2). After removing (Table4,Fig.3,Fig.S1).Thefinalconsensusmapcontained1,054
lociwithunstablepositions,frameworkmapscontained111to370 loci, 254 of which were not resolvable using single map solutions
markers. Individual map sizes ranged from 903.8 to 1,775.7cM, with limited RILs, and covered 1,838.8cM with an average
with 23 to 37 linkage groups, and marker densities ranged from marker distance of 1.7 cM. Marker density differed between
genomes,withtheCgenomehavingmoremarkers(39.7%)thanA
1.71 cMin SolFi/HiFi to 3.42cM in Ogle/TAM O-301 (Fig. 2,
(31.2%)orD(29.1%)(Table4).DistributionofcDNA-andDArT-
DatasetS2).Atleast100frameworklociweresharedbetweentwo
based SNPs among genomes was similar, while microsatellites
ormoremaps(Table3),withallbut18markersexhibitingsimilar
mapped predominately to the A genome (64.9%). Tetraploid-
order and distance. Markers with dissimilar map locations
derived CCDD SNPs mapped predominately to the C genome
appeared tobea result of geneduplicationor homology.
(85.8%)andresistancegenesmappedexclusivelytotheDgenome.
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Table1. Summaryof SNPsby markerdiscovery method.
Discovery No. Total No.SNPs No.calls
method* Prefix tested good Conversionrate(%) Mapped % Diversity %
cDNA–STA GMI_ES01-17 2270 991 44% 757 33% 878 39%
cDNA–CTA GMI_ES_CC 336 144 43% 98 29% 133 40%
DArT–CTA GMI_DS_CC 300 121 40% 87 29% 108 36%
DArT–Sanger GMI_DS_A,oPt 66 48 73% 36 55% 43 65%
GenomicTetraploidGMI_grs 100 7 7% 7 7% 7 7%
Totals 3072 1311 44% 985 32% 1169 38%
*SNPdiscoverymethodsarebasedonSNPcallsusinganassemblyagainstatemplatemadeofcontigsfromasinglevariety(STA),contigsassembledfrommultiple
varieties(CTA),orSangersequencesfromDArTclones(Sanger).
doi:10.1371/journal.pone.0058068.t001
TwelveSNPlocithatmappedtotheAgenomeinatleasttwo DArTdilutionanalysisrevealed200SNPalleleshavingreduced
populationsmappedtotheCgenome(five)orDgenome(seven)in hybridizationsignalswithinamonosomicstock;thesewereusedto
the remaining populations, resulting in two consensus map anchor linkage groups 3C and 10D (Fig. 2) and to confirm
positions for these markers. Notably, four loci mapped to both assignments on 9D,10D, 3C,13A,14D, and15A.
15Aand9D,withconservedorderingbetweengroups(Fig.S2).In
addition,onelocuson8Amappedto5Cwhileadifferentlocuson Comparative mapping
8A mapped to 14D. These results provide evidence of homoeol- A set of 367 SNPs showed highly significant matches
ogousrelationships between chromosomes. (score.100 or E,5E-20) with genomes of model grass species
riceandBrachypodium.Ofthese,30werehighlyrepetitiveandwere
Chromosome Assignment ignored. The remaining 337 loci were colored to highlight
Forty-five F deletion hybrids were interrogated with Gold- collinearity between chromosomes (Fig. 4, Dataset S3). Although
1
enGateSNPassaysandallelesfrom234lociwereusedtoanchor this representation isbiased by the forceduniform spacing of oat
15ofthe21linkagegroups tochromosomes.Stocksrepresenting markers, it demonstrates extensive regions of oat collinearity to
2C, 6C, 9D, 12D, and 16A produced the most robust data (Fig. rice and Brachypodium chromosomes. For example, extensive
S3), with a mean of 26 anchors per chromosome (Fig. 2). Stocks regions of oat 2C correspond with Brachypodium 2 and rice 1,
representing4C,8A,11A,13A,18D,and19Aproducedamean while oat 3C matches Brachypodium 3 and rice 2. These and most
of14.8anchorsperchromosome,andstocksfor14D,15A,20D, other matches are highly consistent with known orthologies
and 21D produced two, five, six and two SNP anchor loci. betweenriceandBrachypodium[25].Collinearitybetweengenomes
Heterogeneous assignment was observed for stocks representing was not perfect (Dataset S3), highlighting geneticrearrangements
11A vs. 5C and 1C vs. 8A. In these cases, the linkage group was thatareexpectedinevolutionarilydivergentspecieswithdifferent
assigned to the stock with the greater number of anchored SNP ploidy levels.
markers. C-band staining of F progeny revealed substantial
2
chromosomerearrangement,asexpectedinsegregatinganeuploid
hybrids.
Table2. Populationsize,marker statistics,map characteristics, and keytraits forRILpopulations used forconsensusmapping.
Polymorphic Marker Framework Mapsize Largestgap
Population RILs markers type* loci{ (cM) (cM) No.LG Keytraits{
Kanota/Ogle 52 320 DS,ES 111 1,387.7 36.2 25 O/Cr/Sr/H
Hurdal/Z-595-1 51 410 BA,DS,ES 370 903.8 35.1 27 DON
Ogle/TAMO-301 49 647 AB,AF,AM, 214 1,576.2 39.0 37 Tocol/Cr/H
BA,BM,DS,
ES,Pc,M,TLP
Otana/PI260616 90 487 BA,DS,ES 323 1,516.7 32.6 23 PCr
Provena/94197A1-9-2-2-2-5 97 402 BA,DS,ES 278 1,775.7 30.3 24 PCr
SolFi/HiFi 51 401 BA,DS,ES 135 1,344.9 32.9 22 BG
*AB,AF,AM,STSmarkersbasedonoatsequence;BA,genomicSNPsbasedontetraploidoat;BM,genomicmicrosatellitebasedonenrichedoatlibraries;DS,genomic
SNPbasedonDArT;ES,genicSNPbasedonEST;Pc,diseaseresistancephenotypicmarkerbasedoncrownrust;TLP,microsatellitebasedonthaumatin-like
pathogenesis-relatedprotein.
{MarkersidentifiedasframeworkmarkersonthefinalmapsusingMultiPoint.
{O,highoil;Cr,crownrustresistance;Sr,stemrustresistance;H,historicmappingpopulation;DON,Deoxynivalenol(toxinofFusariumheadblight);Tocol,high
tocopherol;PCr,partialcrownrustresistance;BG,highbeta-glucan.
doi:10.1371/journal.pone.0058068.t002
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Figure2.Markerdensityandpositionsforindividuallinkagemaps(Otana/PI260616,A;Provena/94197A1-9-2-2-2-5,B;Ogle/TAM
O-301,C;Hurdal/Z-959-1,D;SolFi/HiFi,E;andKanota/Ogle,F)inrelationshiptotheconsensus.Markerpositionsintheconsensusmap
are indicated by the scales on the left axes; positions of each corresponding marker in the component maps are indicated by a color gradient
describedinthekey.
doi:10.1371/journal.pone.0058068.g002
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SNP-BasedChromosome-AnchoredConsensusMapinOat
Table3. Pairwisecomparison ofcommonSNP markers across hexaploidoat populations.
RILpopulation SolFi/HiFi Provena/94197A1-9-2-2-2-5 Otana/PI260616Ogle/TAMO-301 Hurdal/Z-595-1
Kanota/Ogle 100 113 133 214 111
Hurdal/Z-595-1 163 141 162 178 --
Ogle/TAMO-301 203 172 201 -- --
Otana/PI260616 164 184 -- -- --
Provena/94197A1-9-2-2-2-5 148 -- -- -- --
doi:10.1371/journal.pone.0058068.t003
Discussion The first high throughput oat SNP assay
Discovery and application of SNPs in polyploid species is a
This work describes novel methods for SNP discovery in
relatively new challenge; therefore, we wished to compare the
polyploids, andanovelphysicalchromosomeanchoring strategy.
successofvariousapproaches.TheCTAwouldtheoreticallyallow
These methods were applied to develop a new high-throughput
prediction of non-redundant SNPs indexed to a single reference
SNP platform inoat, and thentoproduce thefirst oat consensus
genome.ThesuccessrateofCTAcalls(29%)waslowerthanthat
map assigned to physical chromosomes. These resources then
oftheSTA-basedSNPs(33%)butthisislikelybecausesomeofthe
enabled the first integrative summary of chromosome similarities
best CTA-based SNPs that were redundant with selected STA-
among oat, riceand Brachypodium.
based SNPs were eliminated. However, the consensus sequences
Table4.Consensusmapstatisticsofchromosomelength,markertype,andnumberofmarkersperchromosomeandgenome.
Genome Chromosome* Length(cM) Markers ESTSNP DArTSNP CCDDSNP SSR STS Resistancegenes Duplicateloci
A 8A 87.1 35 28 8 0 1 0 0 2
11A 41.9 24 20 2 0 2 1 0 0
13A 134.6 64 50 8 0 6 0 0 1
15A 85.7 31 26 3 0 2 0 0 4
16A 85.6 85 75 5 0 5 0 0 1
17A-7C 53.1 11 10 1 0 0 0 0 3
19A 115.5 78 58 13 0 7 1 0 1
Total 603.5 328 267 40 0 23 2 0 12
C 1C 74.8 89 80 8 0 1 0 0 0
2C 76.2 64 53 9 0 2 0 0 0
3C 93.5 55 45 7 2 1 1 0 0
4C 97.0 26 19 6 1 0 0 0 0
5C 126.0 94 85 6 1 2 0 0 3
6C 95.4 52 41 10 0 1 0 0 1
7C-17A 83.9 43 33 7 2 1 0 0 1
Total 646.8 417 356 47 6 8 1 0 5
D 9D 105.4 100 84 11 0 2 0 3 4
10Da 4.8 8 8 0 0 0 0 0 0
10Db 23.2 4 2 0 0 0 0 2 0
12D 133.1 63 50 13 0 0 0 0 1
14D 132.5 48 41 4 0 3 0 0 1
18D 47.2 44 40 1 0 0 0 0 1
20D 76.7 28 23 4 1 0 0 0 0
21D 65.6 11 8 3 0 0 0 0 0
Total 588.5 306 256 36 1 5 0 5 7
Total 1,838.8 1,053 879 123 7 36 3 5 12
*Chromosomeanchoringof8A,17A-7C,and7C-17A,arebasedonalignmentsfrompreviouswork;allotherchromosomesarebasedonmonosomichybriddeletion
analysis.
doi:10.1371/journal.pone.0058068.t004
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SNP-BasedChromosome-AnchoredConsensusMapinOat
Figure3.Chromosomeanchoringofconsensuslinkagemap.Greydotsindicatepositionsofnon-anchoredmarkersoneachlinkagegroup,
whilecoloreddotsindicatepositionsofphysicallyanchoredSNPs(bluedots),DArTs(reduparrows),andRFLPs(greendownarrows).
doi:10.1371/journal.pone.0058068.g003
from composite assemblies contained many ambiguities that conversion but a high rate of success in marking identical loci.
confoundedSNPidentification,whiletheSTAapproachprovided Higherconversionratesmayrequireastrategytoaccessdiagnostic
cleaner templates and was more amenable to automation. polymorphisms at theends of DArT sequences.
Therefore the STA was used for the majority of cDNA-based As demonstrated in previous SNP genotyping studies, homo-
SNPs,and werecommendthisstrategy forfuture work. eology and gene duplication affect hybridization and cluster
TheCTAwasusedforallDArT-basedSNPcalls.Thischoice resolution,complicatinggenotypecallsanddecreasingconversion
wasmadebecausecompositeassembliesofDArTsequenceswere frominsilicotophysicalassays[19,26,27].Conversionratesinthis
less complex, and because sequence redundancy among varieties study(43.7%)werelowerthaninlarge-scalestudiesinbarleyand
waslowerduetoselectivityofDArTcomplexityreduction.Itwas maize(approximately90%)[27–29],likelyreflectingthecomplex
also hoped that DArT sequences might provide a higher rate of genomic structure of oat. However, variations in SNP source,
polymorphism than cDNA-derived sequences because they populationstructure,andcropbiologymakeimpartialcomparison
contain less coding sequence. Results from the CTA suggested a of resultsdifficult.
slightly higher SNP rate in DArT sequences than in cDNA In this study, approximately 20% of converted SNPs detected
sequences but this was negated by a higher attrition rate after alleles with MAF,0.10. For comparison, selected barley SNPs
filtering.AlthoughfewerSNPswerecalledintheDArTsequences, also showed 20% with a MAF,0.08 [18], while 16% of maize
the conversion rate of called SNPs was similar to those based on markershadMAF,0.10acrossapanelof154diverseinbredlines
cDNA. [27].Thesefrequenciescanbehighlydependentonascertainment
SpecialattentionwasgiventoSNPsbasedonpublishedDArT bias. In another study, barley SNPs tested across 102 predomi-
marker clones that could provide a bridge to DArT-based maps. nately European accessions revealed that 50% of SNPs were
Althoughthesemarkershadthehighestsuccessrate,only66met monomorphicorhadMAF,0.10[29].Theoatlinesfromwhich
the stringent filtering criteria. More disappointing was that only the current SNPs were developed represent a diverse global
five pairs of DArT/SNP markers could be mapped in the same germplasm collection and were expected to be relatively free of
population; however,allcorrespondingpairs mappedtoidentical ascertainment bias. More than 70% of converted oat SNPs were
locations (Table S1) suggesting a low rate of DArT-to-SNP polymorphic in at least one of six mapping populations, while in
PLOSONE | www.plosone.org 7 March2013 | Volume 8 | Issue 3 | e58068
SNP-BasedChromosome-AnchoredConsensusMapinOat
Figure4.RegionsofsequencesimilaritybetweenSNPmarkerson21chromosomesfromanoatconsensusmap(8Ato21D)and
chromosomesfromthesequencedgenomesofBrachypodiumdistachyon(Bd)andOryzasativa(Os).Regionsofsubstantialcolinearity
havebeeninterpolatedusingchromosome-specificcolors.
doi:10.1371/journal.pone.0058068.g004
maize,69%ofsuccessfulSNPassayswerepolymorphicinatleast size [31,32]. Based on this study, which utilized an iterative
oneoftwomappingpopulations[27].Theseresults,comparedto mapping approach to remove problematic loci and multiple
previouswork,underlinethesuccessofinsilicoSNPidentification crossovers,weestimatethetotalgeneticlengthoftheoatgenome
methods used inthisstudy. tobecloser to2,000cM.
The two physically smallest chromosomes, 11A and 18D,
The first oat consensus map producedshortgeneticmapsasexpected[7].Thelackofmarkers
Until now, it has not been possible to resolve 21 oat linkage and short genetic distance on 17A–7C may be a result of the
groups or to merge individual maps into a single consensus. reciprocal translocation [33] which caused inconsistency among
Previous maps in hexaploid oat have been incomplete, or have populations and elimination of many markers. There was no
resolved to substantially more than 21 linkage groups, and the obvious explanation for lack of markers and short map distances
scarcityofhighthroughputmethodsprovidingreliablesingle-locus on 10Dand21D.
assays that segregate in multiple populations has provided only
fragmentary alignment among linkage groups from different Assignment to physical oat chromosomes
populations [11]. In the current work, SNP assays produced Integration of genetic and physical map data in other crop
stable grouping and ordering in all six maps. Most encouraging species has depended on the availability of cytogenetic stocks. In
wasthehighlevelofsharedmarkersandco-linearityamongmaps. hexaploid wheat, homoeologous chromosome buffering has
This result has been elusive in previous oat research due to the allowed development of telocentric chromosomes, sub-arm dele-
heterogeneity of marker assays, and because many assays have tionstocks,andmonosomics,allofwhichhavefacilitatedphysical
detectedvariantsatmultipleloci.Thesuccessofthecurrentwork mapping [20,21]. Although oat is also a hexaploid, chromosome
is likely because these SNP markers have been highly filtered for rearrangement and fragmented homoeologies have resulted in
consistency and reliability such that most of them directly weaker genomic buffering, which could explain the relative
interrogateaspecificbi-allelicvariantwithinasingledefinedlocus. difficulties of developing and maintaining oat aneuploid stocks.
Frameworkmapsforallsixpopulations wereusedtobuild the Inthisstudy,monosomichybriddeletionstocksrepresenting18of
consensus map consisting of 1,054 loci defining 21groups with a the 21 oat chromosomes were developed. Although a complete
totallengthof1,838 cM.Thisdistancewassimilartotheprevious monosomic series had been reported [23], monosomic progenies
Kanota/Ogle (1,890 cM) and Ogle/TAM O-301 (2,049 cM) were not recovered for 3C, a possible indication of minimal
maps,whichwereabout1,000cMshorterthananestimatedsize buffering and intolerance of monosomy for this chromosome.
(3,100 cM) [30]. However, previous maps likely had incorrect Monosomicswere alsounavailable for7C,whichwas nullisomic,
marker orders due to genotyping errors, inflating this estimated andfor10D,forwhichthemonosomicappearedtohaveshiftedto
PLOSONE | www.plosone.org 8 March2013 | Volume 8 | Issue 3 | e58068