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SupportingInformation

GeneticrecombinationvariationinwildRobertsonianmice:ontheroleofchromosomalfusionsandPrdm9allelicbackgroundLaiaCapilla1,2,NuriaMedarde2,AlexandraAlemanySchmidt3,MariaOliverBonet3,JacintVentura2,AuroraRuizHerrera1,41InstitutdeBiotecnologiaiBiomedicina(IBB),UniversitatAutnomadeBarcelona,CampusUAB,08193,Barcelona,Spain.2DepartamentdeBiologiaAnimal,BiologiaVegetaliEcologia,UniversitatAutnomadeBarcelona,CampusUAB,08193,Barcelona,Spain.3UnitatdInvestigaci,HospitalUniversitariSonEspases,CtraValldemossa79,07010,Palma,Spain,4DepartamentdeBiologiaCellular,FisiologiaiImmunologia,UniversitatAutnomadeBarcelona,CampusUAB,Barcelona,Spain.

1.MaterialandMethods________________________ Page22.SupplementaryTables________________________ Page53.SupplementaryFigures________________________ Page84.References__________________________________ Page18

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1.MaterialandMethodsa)Animalsandchromosomalcharacterization

Atotalof31wildmalemicewerelivetrappedincommensalhabitatsfrom10different

localitiesrepresentativesoftheBarcelonaRbsystem(TableS1andFig.S1).Threemalesfrom

thelaboratorystrainC57BL6werealsoincludedintheanalysisforcomparison.Mitotic

metaphasespreadswereobtainedfromfibroblastculturesandbonemarrowcells.Cell

cultureswerederivedfromfreshtissuessamplesandtheharvestingofcellsfollowedstandard

procedures.KaryotypesweredeterminedbyanalyzingGbandedchromosomalpreparations

[1].WhenGbandingidentificationwasnotsufficientforchromosomalidentification,weused

commercialmousechromosomespecificpaintingprobes(Cambio,Cambridge,UK)toconfirm

Gbandinghomologiesfollowing[2].TheEthicsCommitteeoftheUniversitatAutnomade

Barcelonaapprovedtheanimaltreatmentprotocolsadoptedherein.

b)Immunoflourescence

Immunofluorescence(IF)ofspermatocytespreadswasperformedfollowing[3].Differentsets

ofantibodieswereused:mousemonoclonalantibodyagainstMLH1(BDPharmigen)forthe

detectionofcrossovers(COs),rabbitpolyclonalserumagainstcentralelementproteinofthe

synaptonemalcomplex(SC)(SYCP3,Abcam),humancalcinosis,Raynaudsphenomenon,

oesophagealdysfunction,sclerodactylyandtelangiectasia(CREST)serumforthedetectionof

thecentromeres,mouseH2AX(Millipore),rabbitH3K9me3(Abcam)andmouseRPA(Abcam).

Correspondingsecondaryantibodieswere:antimouseconjugatedwithFITC,antimouse

conjugatedwithCy3,antirabbitCy3andantihumanCy5(allpurchasedfromJackson

Immunoresearch).

c)Fluorescenceinsituhybridization

WiththeaimofidentifyingthechromosomesimplicatedinRbfusions,fluorescenceinsitu

hybridization(FISH)wasperformedonpreviouslyimmunostainedpreparationsusingBacterial

ArtificialChromosome(BAC)probesforspecificmousechromosomes(chromosomes4,9,11,

12,13and14).BACcloneswhereselectedfromtheUCSCgenomebrowser

(http://genome.ucsc.edu)andpurchasedfromCHORI(http://bacpac.chori.org)(TableS3).DNA

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waspurifiedfrombacterialpelletsusingaMidiprepextractionkit(Quiagen).FISHwas

performedonbothmetaphasechromosomesandspermatocytespreadsaspreviously

described[2,3],withmodifications.Briefly,1goftheplasmidDNAwaslabeledwithdUTP

digoxigeninusinganicktranslationkit(Abbot)andethanolprecipitatedwithcompetitorDNA

(Cot1MouseDNA,Invitrogen,1mg/ml),salmonspermDNA(Invitrogen,10mg/ml)and1/10

volumeof3mol/Lsodiumacetateovernightat20C.Theslidesweredehydratedinan

ethanolscalecontaining70%formamideat74Cbefore,andafterthedenaturation.Theprobe

wasprecipitatedandwashedtwicewith70%ethanolandthendilutedin15ulofhybridization

buffer(50%deionizedformamide,10%dextransulfate,2xSSCand0.5mol/Lphosphate).

Chromosomesweredenaturedat74atwhichpointtheprobehybridizationmixwasplaced

directlyontheslides,whichwerecoverslippedandincubatedO/Nat37Cinahumid

chamber.Washeswereperformedwith2Xsalinesodiumcitrate(SSC)and50%formamide

solutionbeforeapplyingtheantidigoxigeninconjugatedwithFITC.

d)Imageprocessinganddataanalysis

PreparationswerevisualizedusingaZeissAxioskopepifluorescencemicroscopeequippedwith

theappropriatefiltersandachargedcoupleddevicecamera(ProgResCS10plus,Jenoptik).

ImageswerecapturedandproducedbytheProgRessoftware(2.7.7).Imagesforeach

specimenweretakenblindly(i.e.,specimenswerenumericallyassignedirrespectivelyoftheir

chromosomalcomposition)inordertoavoidagainstunintentionalbiasesinMLH1scores.

TheMicromeasure3.3software[4]wasusedfortheanalysisofrecombinationmaps

(basedonthedistancesbetweenadjacentMLH1andRPAfoci).Foreachchromosome

analyzed,thepositionofeachfocus(MLH1andRPA)wasrecordedasarelativeposition

(percentageofthesynaptonemalcomplextotallength)fromthecentromere,identifiedbythe

CRESTsignalineachpreparation.ThepositionsofMLH1/RPAfociwerecalculatedforeach

chromosomeusingthecentromereasreferencepoint,(i.e.,fromthecentromeretothe

telomereintheqarm).Thusforcomparisonamongchromosomes,thepositionsofMLH1foci

wereexpressedastherelativepositionofeachCOtothelengthofthechromosome(the

lengthofeachSCwasdividedinto10%intervals).Thesedatawereusedtoconstruct

cumulativefrequencydistributionplotsandwecomparedthedifferentdistributionsusinga

KolmogorovSmirnovtest(KStest).Moreover,weanalyzedthefrequenciesofMLH1focialong

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SCsbydividingtheminthreeregionsdependingontherelativedistancefromthecentromere:

(i)proximal(fromthecentromereto30%oftheSC)(ii)interstitial(between30%70%ofSC)

and(iii)distal(from70%totelomericregion).FortheanalysisofH3K9me3chromosomal

distribution,theMicromeasuresoftwarewasusedtocalculatethesignalarea(measuredin

micrometers)overlappingtheSCforeachchromosomalarmanalyzed.

ThestatisticalanalysiswascarriedoutbymeansofPAWSStatistics18andJMP7

softwarepackage.Giventhatthedistributionofcrossoversperspecimendidnotfollowa

normaldistribution(ShapiroWilktest,pvalue0.001),weappliednonparametricanalysis,

suchasMannWhitneyUtestorKruskalWallistestformeancomparisonandSpearmantest

forcorrelations.

e)Prdm9genotyping

GenomicDNAwasextractedandpurifiedfromcellculturesderivedfrommiceoftheRb

polymorphismzone(n=27)followingthestandardphenolchloroformprotocol.Wesequenced

thePrmd9exon12,fromZnrepeat+3towardsthecterminaldomain.Thisexoncontainsthe

ZnfingerdomainarraythatrecognisesandmethylatesthespecificDNAsequences[5,6].

PRDM9zincfingerarrayswereamplifiedbyPCRusingExTaq(Takara),with4%DMSO,

primersfl1500U20and2848L23[5]asfollows:95C(3min),30cyclesof95C(30s);56C(30

s);72C(90s).PCRproductswererunina2%agarosegelinordertodistinguishdifferent

haplotypes(Fig.S5).Whenmicehadallelesofthesamelength(lengthhomozygotes,n=20),

PCRamplifiedfragmentswerepurifiedwiththeGeneJETPCRpurificationkit(Fermentas).For

micewithallelesoftwodifferentlengths(lengthheterozygotes,n=7),bandswerecutfromthe

agarosegelandpurifiedusingtheGeneJETGelExtractionKit(Fermentas).Inbothcases,

purifiedgeneproductsweresubjectedtobidirectionalSangersequencingwithprimers

fl1822U24and2848L23[6]andreadsweresubsequentlyanalyzedusingtheCLCbioMain

Workbenchprogram,version6.8(Aarhus,DK).Forthoseanimalsfoundheterozygotesfor

allelesofthesamelength,twodifferentsoftwareswereusedinordertodistinguishallelic

variantsanddefinethehaplotypes:Phase

(http://stephenslab.uchicago.edu/software.html#phase)andChampuruv1.0

(http://www.mnhn.fr/jfflot/champuru/).Inanycase,allnewallelicvariantswereconfirmedby

resequencingandreanalyzed.

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SupplementaryTables

TableS1:Listofspecimens,localities,diploidnumber(2n),chromosomalcharacteristics,meannumberofMLH1foci/cell(standarddeviation,SD),numberofcellsanalyzedintherecombinationstudy(N)andPrdm9genotypingofmicefromthechromosomalpolymorphismareaofBarcelonaincludedinthestudy.St=micewithstandardkaryotype(2n=40).Rb=micewithRobertsonianfusions.n.a.=DNAnotavailableforgenotyping.

Specimen Location 2n FusionsMeanMLH1foci/cellSD

N Prdm9alleles

St1 Arbeca 40 21.652.43 55 n.a.

St2 Vacarisses 40 21.162.23 89 n.a.

St3 CastellfollitdelBoix 40 20.901.74 20 10A/12B

St4 CastellfollitdelBoix 40 20.801.43 21 12B/12B

St5 CastellfollitdelBoix 40 20.681.35 22 10A/10A

St6 CastellfollitdelBoix 40 20.681.43 25 10A/12B

St7 CastellfollitdelBoix 40 20.570.87 21 10A/12C

St8 CastellfollitdelBoix 40 20.571.45 14 10A/12C

St9 CastellfollitdelBoix 40 20.271.40 26 12B/12B

St10 CastellfollitdelBoix 40 20.161.17 25 10A/10A

St11SantaPerptuade

Mogoda40 20.581.02 31 10B/11B

Rb1 SantSadurnd'Anoia 39 Rb(12.13) 20.261.68 27 n.a.

Rb2 SantSadurnd'Anoia 37 Rb(4.14)+2(9.11) 19.712.22 40 10A/10A

Rb3 SantSadurnd'Anoia 38 Rb(4.14)+(9.11) 19.711.65 46 10A/10A

Rb4 SantSadurnd'Anoia 37 Rb(4.14)+(9.11)+(12.13) 20.571.90 46 n.a.

Rb5 SantSadurnd'Anoia 37 Rb(4.14)+(9.11)+(12.13) 20.261.86 47 10A/10A

Rb6 ElPapiol 38 Rb(4.14)+(12.13) 19.391.19 37 10A/10A

Rb7 ElPapiol 38 Rb(4.14)+(12.13) 19.641.27 11 10A/10A

Rb8 AmetlladeSegarra 39 Rb(4.14) 18.930.68 16 10A/10A

Rb9 Castelldefels 29 Rb2(4.14)+2(9.11)+2(12.13)+2(3.8)+2(5.15)+(6.10) 19.951.67 44 10A/10A

Rb10 Castelldefels 32 Rb2(4.14)+(9.11)+2(12.13)+2(5.15)+(6.10) 19.921.59 40 10A/10A

Rb11 Castelldefels 30 Rb2(4.14)+(9.11)+2(12.13)+2(3.8)+2(5.15)+(6.10) 19.851.61 27 10A/10A

Rb12 Castelldefels 30 Rb2(4.14)+2(9.11)+2(12.13)+(3.8)+2(5.15)+(6.10) 19.591.53 37 10A/10A

Rb13 Castelldefels 28 Rb2(4.14)+2(9.11)+2(12.13)+2(3.8)+2(5.15)+2(6.10) 20.231.81 18 10A/10A

Rb14 Castelldefels 30 Rb2(4.14)+2(9.11)+2(12.13)+(3.8)+2(5.15)+(6.10) 19.000.94 19 10A/10A

Rb15 Castelldefels 29 Rb2(4.14)+2(9.11)+2(12.13)+2(3.8)+2(5.15)+(6.10) 18.951.25 22 10A/10A

Rb16 Castelldefels 30 Rb2(4.14)+2(9.11)+2(12.13)+(3.8)+2(5.15)+(6.10) 18.73