Characterization Of The Maize CMS-C Fertility Restoration Mechanism And Its Restorer Lines

C-type cytoplasmic male sterility（CMS-C）in maize can be widely employed in maize hybrid seed production in the future because of its complete pollen abortion and stable male sterility.However,the complex fertility restoration mechanism and the lack of strong restorer line limit the promotion of CMS-C in agricultural application.As a dominant restorer gene for CMS-C,Rf4 is able to restore most of CMS-C sterile lines and has a potential value in hybrid seed production.Until now,Rf4 has not yet been cloned and its functional mechanism remains unclear.To reveal the functional mechanism of Rf4 and identify Rf4 restorer lines,two research aspects were conducted in this research.First,the fertility restoration mechanism of CMS-C was analyzed by exploring the molecular function of the Rf4 candidate gene ZmbHLH16 and combining with the analysis of the genome and transcriptome data of a near-isogenic line of Rf4（NIL＿Rf4）.Secondly,to discover some strong Rf4 restorer lines,the genotypes on the Rf4 locus of 18 maize inbreds were examined by restoring-maintaining relationship testing,genetic analysis of F₂,molecular marker mapping and allelic testing.The main findings were listed as follows:1.ZmbHLH16 is a candidate gene of Rf4.A total of 34 nucleotide variants of genomic sequence of ZmbHLH16 were detected among four CMS-C lines（rf4rf4）and five restorer lines（Rf4Rf4）,and four amino acid mutations could be caused.Further analysis revealed that the S1596 mutation（TTT/TAC）tended to be important:this locus was TTT in the above five restorer lines,which encodes phenylalanine,and in the four CMS-C sterile lines,it was TAC,encoding tyrosine.Moreover,the following allelic test showed that when cross with A619 or Guang10-2,all the allelic testing populations of 28 inbred lines with TTT at S1596 locus were complete male fertile.In comparison,for other 10 inbred lines with TAC at S1596 locus,nine allelic populations appeared male fertility segregation,except the allelic test population of SAM1001.All the plants in the SAM1001 corresponding allelic population were male fertile.In conclusion,these results suggested that the nucleotide variation at S1596 locus of ZmbHLH16 is closely related to Rf4 restoring function.Finally,we developed a CAPS marker based on the S1596 variation.When S1596 was TAC,the PCR product could be digested with the restriction enzyme Tat I to obtain two fragments,but when S1596 was TTT,the PCR product could not be digested by Tat I.In total,the CAPS marker might be helpful for the Rf4 restorer line selection.2.The coding sequence（CDS）of ZmbHLH16 was cloned and a conserved bHLH domain was found in it.Transcriptional activation activity analysis in yeast showed that the full-length protein of ZmbHLH16 did not possess transcriptional activation activity.Interestingly,through a series of truncated protein analysis,we found that the 1～80th amino acids of the N-terminus of ZmbHLH16 had transcriptional activation activity,and the 161-365th amino acids of ZmbHLH16 could inhibit transcriptional activation activity.Yeast one-hybrid analysis demonstrated that the ZmbHLH16 protein could not be bind to the G-box（CACGTG）element.Genome-wide coexpression analysis showed that 395genes were coexpressed with ZmbHLH16.Among these coexpressed genes,72 and 54genes might be located in mitochondrial and chloroplast,respectively.q PCR analysis illustrated that ZmbHLH16 and the other three male fertility-related transcription factors including ZmbHLH51,ZmbHLH122,and Zm MYB74 were all mainly expressed at the mononuclear and binuclear stages in anthers.Transient expression in rice protoplasts showed that three bHLH proteins,ZmbHLH16,ZmbHLH51,and ZmbHLH122 were mainly located in the nucleus.Yeast two-hybrid assay proved that ZmbHLH16 could interact with three transcription factors,ZmbHLH51,ZmbHLH122 and Zm MYB74,respectively,and ZmbHLH51 could interact with Zm MYB74.In addition,yeast two-hybrid analysis indicated that the bHLH domain of ZmbHLH16 could interact with ZmbHLH51,and some regions without the bHLH domain including the 81～160th,241～365th amino acid could also form a dimer with ZmbHLH51.3.Tassel fertility of the backcross populations（NIL＿Rf4）were investigated for three generations（BC₄F₁,BC₅F₁,and BC₆F₁）and the male fertility segregation ratio of fertile and sterile plants in all the backcross populations fit to 1:1.At the same time,we found that the male fertility of all the plants in the BC₅F₁ population co-segregated with the Rf4 locus genotypes.The above results suggested that male fertility of the near-isogenic line NIL＿Rf4 is mainly controlled by Rf4.Whole-genome resequencing revealed that genomic difference between NIL＿Rf4 and NIL＿rf4 mainly clustered in the interval of 0～1 Mb of chromosome 8,which is also the mapping region of Rf4.The above result indicated that the near-isogenic line for Rf4 was successfully constructed.In addition,those unmapped clean reads were assembled and 2,890 novel genes were identified.Among all the novel genes,645 genes could be targeted to mitochondria and one gene belonged to the PPR protein family.These novels genes might be useful for uncovering any Rf4 candidate.4.Comparative transcriptome analysis of NIL＿Rf4 and NIL＿rf4 showed that they shared 7,125 DEGs in developing spikelets.These DEGs were then subjected to GO analysis.Within the biological process,these DEGs were mainly related to the metabolic process,cellular process,and single-organism process.In addition,the molecular function of those DEGs was associated with binding and catalytic activity.Moreover,100 DEGs were found to be involved in the four pollen formation-related GO terms:pollen tube development（GO0048868）,pollen tube growth（GO0009860）,pollen development（GO0009555）,gametophyte development（GO0048229）.KEGG pathway enrichment analysis showed that those DEGs were mainly involved in carbon metabolism and energy metabolism.In cell energy metabolism processes,58 DEGs were found to participate in glycolysis,pentose phosphate pathway,and pyruvate metabolism.Transcriptome data showed that 14 DEGs were involved in the mitochondrial tricarboxylic acid（TCA）cycle.Transcriptome sequencing and q PCR indicated that all the TCA-related DEGs were up-regulated in male fertile NIL＿Rf4 and down-regulated in male sterile NIL＿rf4.Among the above 14 TCA-related DEGs,six genes were associated with the oxoglutarate dehydrogenase（OGDH）enzyme complex,and three genes were involved in the composition of the isocitrate dehydrogenase（IDH）complex.The results of enzyme-linked immunosorbent assay（Elisa）showed that the abundance of OGDH and IDH in male fertile NIL＿Rf4 spikelets were significantly higher than those of male sterile NIL＿rf4.The above results implied that the mitochondrial TCA cycle was closely related to the fertility restoration of maize CMS-C,and OGDH and IDH enzymes might be the crucial factors.5.Five TCA-related DEGs were cloned,including GRMZM2G064695（Succinate-Co A ligase subunit）,GRMZM2G079538（OGDH subunit）,GRMZM2G120857（IDH subunit）,GRMZM2G176397（Aconitate hydratase 1）,and GRMZM2G466833（Malate dehydrogenase 3）.First,they were transiently expressed in tobacco leaves for subcellular localization analysis.It was noted that their protein products were mainly localized to mitochondria,but the translational products of GRMZM2G079538 and GRMZM2G176397 genes were also distributed in the nucleus.Further,we explored the interaction among the above five genes by using bimolecular fluorescence complementation（BIFC）technology.It was turned out that GRMZM2G176397 and GRMZM2G466833 could form homodimers respectively,and the homodimers were mainly distributed in mitochondria,but not any heterodimers were noticed among these above five genes.Finally,several overexpressed Arabidopsis lines for the above five genes were respectively constructed.We found that the GRMZM2G120857 overexpressed plants showed reduced fertility compared to the wild type（Col0）,but the other overexpressed plants showed no obvious phenotype alteration.6.Restoring and maintaining relationship testing showed that 10 of 18 inbred lines could fully rescue the CMS-C sterile line CHZS.Subsequently,all the fully restored F₁were self-crossed to form several F₂populations.Genetic analysis of the above F₂population showed that DAN598,PHT77,78551S and LH212Ht only contain one dominant Rf gene,and PHN82 contains two pairs of major Rf genes.Through map-based cloning,we found that the dominant Rf gene in DAN598,PHT77,78551S and LH212Ht were all located at the terminal of the short arm of chromosome 8.Results of the allelic testing showed that one Rf gene in 12 inbred lines including DAN598,PHT77,78551S,LH212Ht,PHN82 and so on,were allelic with one restorer gene in A619.In summary,it could be concluded that,for DAN598,PHT77,78551S and LH212Ht,the fertility restoration of the CMS-C line CHZS was mainly controlled by Rf4.