Selection and Validation of Reference Genes for Quantitative Real-Time PCR in Buckwheat (Fagopyrum esculentum) Based on Transcriptome Sequence Data 英文参考文献.docVIP

SelectionandValidationofReferenceGenesfor QuantitativeReal-TimePCRinBuckwheat(Fagopyrum esculentum)BasedonTranscriptomeSequenceData NataliaV.Demidenko1,MariaD.Logacheva2,3,4,AlekseyA.Penin1,3,4 * 1Department of Genetics, Biological Faculty, M.V. Lomonosov Moscow State University, Moscow, Russia, 2Department of Evolutionary Biochemistry, A.N. Belozersky Institute ofPhysico-Chemical Biology, M.V.Lomonosov Moscow StateUniversity,Moscow, Russia, 3EvolutionaryGenomics Laboratory,FacultyofBioengineeringand Bioinformatics,M.V.LomonosovMoscowStateUniversity,Moscow,Russia,4A.A.KharkevichInstituteforInformationTransmissionProblems,RussianAcademyofScience, Moscow,Russia Abstract QuantitativereversetranscriptionPCR(qRT-PCR)isoneofthemostpreciseandwidelyusedmethodsofgeneexpression analysis. A necessary prerequisite of exact and reliable data is the accurate choice of reference genes. We studied the expressionstabilityofpotentialreferencegenesincommonbuckwheat(Fagopyrumesculentum)inordertofindtheoptimal reference for gene expression analysis in this economically important crop. Recently sequenced buckwheat floral transcriptome was used as source of sequence information. Expression stability of eight candidate reference genes was assessedindifferentplantstructures(leavesandinflorescencesattwostagesofdevelopmentandfruits).Thesegenesare the orthologs of Arabidopsis genes identified as stable in a genome-wide survey gene of expression stability and a traditionallyusedhousekeepinggeneGAPDH.Threesoftwareapplications–geNorm,NormFinderandBestKeeper-were used to estimate expressionstability andprovided congruent results. The orthologs of AT4G33380 (expressed protein of unknownfunction,Expressed1),AT2G28390(SANDfamilyprotein,SAND)andAT5G46630(clathrinadaptercomplexsubunit familyprotein,CACS)arerevealedasthemoststable.WerecommendusingthecombinationofExpressed1,SANDandCACS forthenormalizationofgeneexpressiondatainstudiesonbuckwheatusingqRT-PCR.Thesegenesarelistedamongfive themoststablyexpres