Analysis Of Evolutionary Trajectory And Intrinsic Function Of CMS-WA Restorer Gene Rf4 In Rice(Oryza Sativa L.)

Cytoplasmic male sterility（CMS）and its fertility restoration（Rf）are vitally important in utilization of plant heterosis.At present,rice（Oryza sativa L.）wild-abortive type cytoplasmic male sterility（CMS-WA）and its fertility restoration system are widely used for hybrid rice production.One of the restorer Rf4 encoding pentatricopeptide repeat proteins 9（PPR9）has been identified.The WA352 transcript of CMS-WA is decreased by PPR9 leading to fertility restoration.In order to study the evolutionary trajectory and intrinsic function of Rf4,different Rf4 allele sequences were amplified wild rice and cultivated rice accesions,and analyzed to trace the evolutionary trajectory of Rf4 in Oryza.On the other hand,I verified the function of RF4 in RNA editing of mitochondrial genes by transcriptome analysis.The main results are summarized as follows:1.Three type of genomic sequence variations of Rf4 alleles,namely functional Rf4、nonfunctional-japonica-type rf4j and nonfunctional-indica-type rf4i,were identified.The rf4j showed high similarity in nucleotide to Rf4（62 SNPs）.However,rf4i contained two insertions and this allele may be derived from homologous recombination between Rf4/rf4j and PPR⁸³⁰.2.Investigation of the presence of Rf4,rf4j and rf4i in wild rice and cultivated rice accesions by PCR using specific primers demonstrated that Rf4 and rf4j might appear earlier than rf4i,and Rf4 widely existed in wild rice and cultivated rice.Furthermore,rf4i may evolves through homologous recombination in the wild rice of AA-genome.3.Different types and copy number variations of PPR genes in Rf4 locus were identified in different rice cultivars carrying functional restorer（Rf4）or non-functional restorer（rf4）.There are three functional types in restorer lines:Rf4-I is a single copy type of Rf4（Rf4-a^I and rf4-b）;Rf4-II is another single copy type of Rf4（Rf4-b and rf4-a）;Rf4-III is a two-copy type of Rf4（Rf4-a^M and Rf4-b）.4.Sequence alignment analyses of RF4 and rf4j amino acids in different cultivars indicated that RF4 has 14 amino acids substitutions compared with rf4j,distributing on PPR₁₃,PPR₁₄ and PPR₁₅ three PPR motifs.These PPR motifs may be critical for fertility restoration.5.RNA-seq analysis demonstrated that the RNA editing efficiency of six mitochondrial genes m RNA are different in sterile lines J23A（WA352,rf3rf3rf4rf4）and restorer lines Rf4-J23A.The results of Sanger sequencing and the next generation sequencing（NGS）further confirmed that Rf4 has effect on the editing of ccm Fn,which may not be related to fertility restoration.6.RF4 interacts with RNA editing factors MORF1,MORF8 and MORF9 in Y2H system,suggesting that RNA editing may happen in a‘editosome’complex composed of RF4 and RNA editing factors.In conclusion,this study preliminarily revealed the origin and evolutionary of Rf4 and deepened the understanding of RF4 PPR protein function in RNA editing.These findings lay foundation for further investigation on the co-evolution of sterility genes and restorer genes.