Abstract: Cellulose synthase cataly.c subunit genes (CesAs) are involved in the biosynthesis of cellulose, an insoluble fiber that is the main component of the plant cell wall. In all land plants, cellulose microfibrils are synthesized from a roseAe complex known as the cellulose synthesis complex, embedded in the plasma membrane. Each complex consists of six globules that all contain mul.ple cellulose synthase cataly.c subunits (CesAs). CesA6 is a member of the cellulose synthase cataly.c subunit family and research shows it is specialized for cellulose synthesis in the primary cell wall. In this study, EST sequence data from several oat germplasms were u.lized to amplify and clone the CesA6 cataly.c subunit gene from mul.ple Avena species, including the diploid species A. strigosa (AA), A. canariensis (DD), and A. ventricosa (CC). Comparison of the A. sa2va sequences to the sequences of the diploids allowed for the determina.on of genomic origin. The CesA6 gene in Oat is ~ 5kb with 12 introns and a coding sequence of 3.2 kb. Materials and Methods: Germplasm was acquired from Na.onal Small Grains Collec.on at the USDAARS in Aberdeen, ID, and from the Laren Robinson Memorial Genbank at Brigham Young University Provo, UT. Assembly of CesA6 Expressed Sequence Tags (ESTs) from a database created by the Collabora.ve Oat Research Enterprise (CORE) was done using the barley CesA6 coding sequence as a reference. Primers were designed from this assembly and used to amplify the CesA6 gene. Clones of two different lengths were created for each line. The first clone (f3r7) began at the 5’ end and proceeded into the tenth exon, while the second (f7r2) shorter clone contains sequence through the next three exons un.l the end. Sequencing was performed at the BYU DNA Sequencing Center using Applied Biosystems 3730xl DNA Analyzer and BigDye v3.1 chemistry. Sequences were assembled and aligned using Geneious Pro assembly so]ware by BiomaAers LTD. available at www.geneious.com . Results and Discussion: Gene Ontology: The CesA6 gene in Oat is approximately 5 kb with 12 introns and a coding sequence of approximately 3.2 kb. The CesA6 gene of Oat is similar to the CesA6 gene of Barley. The size and loca.on of the exons are very similar. The size of the introns are more variable, as would be expected. An alignment showed a 89.4% homology between Oat and Barley. Allele varia:on: Sequencing of A. sa2va revealed three dis.nct alleles of the CesA6 gene (Fig2). Different alleles are presumed to be from each of the three genomes A, C and D. Consistently, two of the alleles were closely related while the third allele was more divergent from the other two. There is a 98.6% iden.ty between the three Hifi homeologs. As expected the intron regions had the greatest sequence diversity including frequent small dele.ons and inser.ons. Differences in the coding regions between alleles consisted almost exclusively of single nucleo.de polymorphisms. Separa.on of A. sa2va homeologs is being done by comparison of sequence data from diploid strains such as A. canariensis, which is a D genome diploid, allowing for genome allele assignment (Fig 3). A 92.6% iden.ty was found between Hifi and A. canariensis. Sequencing is currently being conducted on mapping parents Provena, Mn84, HiFi, Solfi, GS7 and Assiniboia which feeds into the physicallyanchored hexaploid oat linkage map 2 . With homeolog specific SNPs it is now possible to conduct allele specific expression analysis with RNAseq reads. References: 1. Ding SY, Himmel ME (2006) The maize primary cell wall microfibril: a new model derived from direct visualiza.on. J. Agric. Food Chem. 54:597606. 2. Oliver RE, Tinker NA, Lazo GR, Chao S, Jellen EN, et al. (2013) SNP Discovery and Chromosome Anchoring Provide the First PhysicallyAnchored Hexaploid Oat Map and Reveal Synteny with Model Species. PLoS ONE 8: e58068. doi: 10.1371/journal.pone. 0058068 3. Mutwil M, Debolt S, Persson S (2008). Cellulose synthesis: a complex complex. Curr. Opin. Plant Biol. 11: 252257. Characteriza.on of CesA6 in Oat Cassandra Anne Dohse, Melissa C. Fogarty, Eric N. Jellen Brigham Young University, Department of Plant and Wildlife Sciences, Provo, Utah Figure 1: Cellulose synthases, such as CesA6, form hexameric roseAes known as cellulose synthesis complexes. 1C shows a roseAe array in the plasma membrane 1 . Figure 3: Alignment of Hifi (high fiber A. sa2va cul.var) homeologs and A. canariensis sequence. Mul.ple SNPs can be seen. Base pair 345 is of par.cular interest as it infers the first Hifi homeolog is of the D genome like A. canariensis. Figure 2: Alignment of Hifi (high fiber A. sa2va cul.var) homeologs represen.ng various polymorphisms. Unique polymorphisms can be seen at bp 709, 713, 759 and 796.
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.
