Efficient Method For Screening Stable Reference Genes And Cloning Of Restorer Gene Rf3 For CMS-WA In Rice

Cytoplasmic male sterility(CMS)and its fertility restorer are the genetic basis for the heterosis utilization.At present,rice(Oryza sativa)wild-abortive cytoplasmic male sterility(CMS-WA)and its fertility restoration system are the most widely used in hybrid rice production.The sterility of CMS-WA is caused by WA352,and restored by Rf3 and Rf4.In this study,I fine-mapped the restorer gene Rf3 with a large population,and verified Rf3 by complementation or knockout of the candidate genes in the mapping region.q RT-PCR(Quantitative reverse-transcription PCR)is a well-established method for quantification of gene expression.However,few stable,reliable reference genes are available for accurate quantification of gene expression in reproductive stages in rice.In this study,I established an efficient strategy for reference gene screening.Two reliable reference genes were identified by this strategy and were applied to quantify the expression analysis of Rf3 candidate genes.The main results of this study are summarized as follows:1.In this study,I established an effective strategy for rapid selection of robust reference genes using public database IC4R(Information Commons for Rice)and Rice XRro(Rice Expression Profile Database).2.This study revealed that genes in different tissues/organs,different developmental stages or different treatment conditions,harbor a Cq value ranging less than 2.5 are potentially stable internal reference genes.3.Two new reference genes UFC1(Homolog of UFM1-Conjugating Enzyme 1)and Fha B(Homolog of Adhesin Fha B),were identified as stable reference genes for quantitative verification of gene expression in reproductive stage.4.Fine mapping indicated that Rf3 is delimited to a 11 kb-region by two molecular markers M5565640 and M5576246 on chromosome 1.This region includes the first two exons of the MADS3 gene,and three lnc RNAs(lnc RNA-1,lnc RNA-2,lnc RNA-3)that are opposite to the orientation of MADS3 transcript.In Addition,the restorer line harboried a 1kb-restorer specific insertion(RSI)in this region.5.During the microspore mother cell(MMC)stage in which the WA352 protein specifically accumulates,the expression levels of MADS3 and lnc RNA-2 in the Rf3 single gene restorer line ZSR1(WA352/Rf3Rf3/rf4rf4)were higher than those in the sterile line ZS97A(WA352/rf3rf3/rf4rf4)and the maintainer line ZS97B(rf3rf3/rf4rf4).However,the expression level of lnc RNA-1 was higher in ZS97 A than ZS97 B and ZSR1.6.Subcellular localization experiments indicated that GFP-tagged MADS3 protein is localized in the nucleus and mitochondrion of rice.7.Complementation of the lnc RNA-Rf3 genomic fragment restored the fertility of CMS-WA line J23 A,and also reduced the accumulation of WA352 protein in the transformant.8.Knockout of MADS3,lnc RNA-2,lnc RNA-3 and RSI in ZSR1 did not affect the fertility in related lines,thus excluding the possibility that MADS3,lnc RNA-2,lnc RNA-3and RSI fragment are Rf3.Thus,long noncoding RNA,lnc RNA-1 is the candidate of Rf3.In conclusion,this study established a new strategy for efficient screening of robust reference genes.Moreover,the CMS-WA restorer gene Rf3 was likely a noncoding RNA lnc RNA-1,which is different from current known restorters that encoding PPR proteins.