Characterization of CesA6 in Oat copy

1. Abstract: Cellulosesynthasecataly.csubunitgenes(CesAs)areinvolvedinthebiosynthesisofcellulose,aninsolubleberthatisthemaincomponentoftheplantcellwall.Inallland plants,cellulosemicrobrilsaresynthesizedfromaroseAecomplexknownasthecellulosesynthesiscomplex,embeddedintheplasmamembrane.Eachcomplexconsistsof sixglobulesthatallcontainmul.plecellulosesynthasecataly.csubunits(CesAs).CesA6isamemberofthecellulosesynthasecataly.csubunitfamilyandresearchshowsitis specializedforcellulosesynthesisintheprimarycellwall.Inthisstudy,ESTsequencedatafromseveraloatgermplasmswereu.lizedtoamplifyandclonetheCesA6cataly.c subunitgenefrommul.pleAvenaspecies,includingthediploidspeciesA.strigosa(AA),A.canariensis(DD),andA.ventricosa(CC).ComparisonoftheA.sa2vasequencesto thesequencesofthediploidsallowedforthedetermina.onofgenomicorigin.TheCesA6geneinOatis~5kbwith12intronsandacodingsequenceof3.2kb. MaterialsandMethods: Germplasm was acquired from Na.onal Small Grains Collec.on at the USDA-ARS in Aberdeen, ID, and from the Laren RobinsonMemorialGenbankatBrighamYoungUniversityProvo,UT.AssemblyofCesA6ExpressedSequenceTags(ESTs) from a database created by the Collabora.ve Oat Research Enterprise (CORE) was done using the barley CesA6 coding sequenceasareference.PrimersweredesignedfromthisassemblyandusedtoamplifytheCesA6gene.Clonesoftwo dierentlengthswerecreatedforeachline.Therstclone(f3-r7)beganatthe5endandproceededintothetenthexon, while the second (f7-r2) shorter clone contains sequence through the next three exons un.l the end. Sequencing was performedattheBYUDNASequencingCenterusingAppliedBiosystems3730xlDNAAnalyzerandBigDyev3.1chemistry. Sequences were assembled and aligned using Geneious Pro assembly so]ware by BiomaAers LTD. available at www.geneious.com. ResultsandDiscussion: GeneOntology: The CesA6 gene in Oat is approximately 5 kb with 12 introns and a coding sequence of approximately3.2kb. TheCesA6geneofOatissimilartotheCesA6geneofBarley.Thesizeandloca.onofthe exonsareverysimilar.Thesizeoftheintronsaremorevariable,aswouldbeexpected.An alignmentshoweda89.4%homologybetweenOatandBarley. Allelevaria:on: SequencingofA.sa2varevealedthreedis.nctallelesoftheCesA6gene(Fig2).Dierent allelesarepresumedtobefromeachofthethreegenomesA,CandD.Consistently,twoof thealleleswerecloselyrelatedwhilethethirdallelewasmoredivergentfromtheother two.Thereisa98.6%iden.tybetweenthethreeHihomeologs.Asexpectedtheintron regions had the greatest sequence diversity including frequent small dele.ons and inser.ons.Dierencesinthecodingregionsbetweenallelesconsistedalmostexclusivelyof singlenucleo.depolymorphisms. Separa.on of A. sa2va homeologs is being done by comparison of sequence data from diploidstrainssuchasA.canariensis,whichisa Dgenomediploid,allowingforgenome allele assignment (Fig 3). A 92.6% iden.ty was found between Hi and A. canariensis. SequencingiscurrentlybeingconductedonmappingparentsProvena,Mn-84,Hi-Fi,Sol-, GS-7andAssiniboiawhichfeedsintothephysically-anchoredhexaploidoatlinkagemap2. WithhomeologspecicSNPsitisnowpossibletoconductallelespecicexpressionanalysis withRNAseqreads. References: 1. DingSY,HimmelME(2006)Themaizeprimarycellwall microbril:anewmodelderivedfromdirectvisualiza.on.J. Agric.FoodChem.54:597-606. 2. OliverRE,TinkerNA,LazoGR,ChaoS,JellenEN,etal.(2013)SNP DiscoveryandChromosomeAnchoringProvidetheFirst Physically-AnchoredHexaploidOatMapandRevealSyntenywith ModelSpecies.PLoSONE8:e58068.doi:10.1371/journal.pone. 0058068 3. MutwilM,DeboltS,PerssonS(2008).Cellulosesynthesis:a complexcomplex.Curr.Opin.PlantBiol.11:252-257. Characteriza.onofCesA6inOat CassandraAnneDohse,MelissaC.Fogarty,EricN.Jellen BrighamYoungUniversity,DepartmentofPlantandWildlifeSciences,Provo,Utah Figure1: Cellulose synthases, such as CesA6, form hexameric roseAes known as cellulose synthesis complexes. 1-C shows a roseAe arrayintheplasmamembrane1. Figure3: Alignment of Hi- (high ber A. sa2va cul.var) homeologs and A. canariensis sequence. Mul.ple SNPs can be seen. Base pair 345 is of par.cularinterestasitinferstherstHi-homeologisoftheDgenome likeA.canariensis. Figure2: AlignmentofHi-(highberA.sa2vacul.var)homeologsrepresen.ngvariouspolymorphisms.Uniquepolymorphismscanbe seenatbp709,713,759and796.