Studies On The Genetic Mechanism Of The Restorer Gene Rf4 Of C-type Cytoplasmic Male Sterility And MiRNAs Regulating Length Of Intemodes Under Ear In Maize

With the rapidly increasing demands for food and feed,maize has been one of the most important crops in the world.It plays an important role in ensuring the national food security.The production of hybrid seeds is the key links in promoting the high and stable yield in maize.The application of cytoplasmic male sterility is an effective way in heterosis breeding to simplify the seed production procedures and reduce the production cost.Illustrating the restorer mechanism of maize cytoplasmic male sterility can provide the theoretical basis and technical support for the application of sterile seed production technology.Lodging has become an important factor in affecting maize yield.In crop breeding programs,plant height and ear height are two of the most important agronomic traits and closely related to lodging.Internode length is one of the decisive factors affecting ear height.It is crucial to elucidate the genetic mechanism of the internode elongation and development.On the basis of previous study,the genetic mechanism of the restorer gene Rf4 of C-type cytoplasmic male sterility was studied.The miRNAs and their targets involved in the intemode elongation and development were also identified and analyzed by high-throughput sequencing.The results are as follows:1.The Rf4 gene was mapped by genome-wide association study.Nine significantly associated SNP loci were detected in the short arm of chromosome 8.All of the SNPs located in the gene encoding the protein,transcript_factor_SBP_box.Combined with the result of the early stage of the fine mapping,the gene was identified to be the candidate gene of Rf42.Based on the sequence of B73,3 transcripts were found about the candidate.The transcript Rf4-T1 was verified by PCR and it contained lexon.However,the transcript Rf4-T2 could not be amplified.The transcript Rf4-T01 was amplified and it contained 11 exons.The transcript Rf4-T1 had also been proved to be not restorer gene by transgenic.It could not restore male fertility.3.The full-length cDNA of Rf4-T01 were obtained by RACE.The results of sequencing indicated the sequences obtained from the tetrad of restorer line and sterile line were shorter than the sequence of Rf4-T01.There was one amplified fragment(r-RACE 2043bp)in the tetrad of sterile line,however,there were two amplified fragments(R-RACE-1 1942bp and R-RACE-2 1794bp)in the tetrad of restorer line.The ORF(1545bp)of r-RACE was the same as the ORF(1545bp)of R-RACE-2.The ORF(1545bp)of r-RACE and the ORF(1545bp)of R-RACE-2 were longer than the ORF(996bp)of R-RACE-1.The three ORFs were found to contain only one ANK domain by the prediction of domain structure.However,the transcript of Rf4-T01 contained two domain structures.Two domain structures were SBP domain and ANK domain.The results provided valuable information for the functional verifivation of Rf4.4.qRT-PCR indicated that R-RACE-2 was constitutive expression in different stages of different materials.But the expression level was also different.The expression level in the spikelet and floret of sterile line were the highest by comparing with it in the other stages of three lines.The expression level in the tetrad of restorer line was higher than them by comparing with the corresponding stage of the other two lines,respectively.The expression level in the monokaryotic stage of restorer line was higher than it the other two lines.5.The polyclonal antibodies were designed by analysis of the two domain structure and the the 5'end of Rf-T01.Five materials with each containing three stages were used for western bolt.Two materials with C cytoplasm and nuclear background of 87-1 carried different genotypes of Rf genes:CMS-C(rf4rf4rf5rj5)and CMS-C(Rf4Rf4rf5rf5).The other one material with N cytoplasm and nuclear background of 87-1 carried genotypes of Rf genes:N(rf4rf4rf5rf5).The other two materials with C cytoplasm and nuclear background of 6233 and F1 carried different genotypes of Rf genes:CMS-C(rf4rf4Rf5Rf5)and CMS-C(Rf4rf4Rf5rf5),respectively.The three stages were the earlier stage of tetrad,tetrad and monokaryotic stage,respectively.There was only one hybridization band(40KD)for the antibody of the 5' end of Rf-T01.According to the analysis of the hybridization band,the transcript of Rf-T01 might not encode protein in each materials.The results indicated that there were two hybridization bands(about 70KD and 90KD)for the antibody of SBP domain structure in the monokaryotic stage of five materials.There were not significant changes about the 90KD hybridization band in different materials.The hybridization band about 70KD was detected in the other two stages of five materials with no significant changes.The transcripts encoded the protein about 70KD and 90KD were not the restorer gene.There were three hybridization bands(50KD,65KD and 75KD)for the antibody of ANK domain structure in the three stages of five materials.There were not significant changes about the hybridization bands with 50KD and 75KD in the three stages of different materials.However,there was significant change about the hybridization bands with 65KD in the three stages of different materials.Besides,combined with the results of RACE,the transcripts of r-RACE ORF and R-RACE-2 ORF were predicted to contain ANK domain structure and encode the proteins about 60KD.The proteins were similar with the the hybridization band about 65KD.Thus,the R-RACE-2 ORF was speculated to be the restorer gene.Combined with the results of RACE and weatern blot,the ORF of R-RACE-2 was the restorer gene.The protein encoded by this gene had a mask effect on the rf4rf4..6.Six miRNA and two degradome libraries were constructed using the 7th,8th and 9th internodes of two inbred lines,'Xun928' and 'Xun9058',which had significantly different internode lengths.A total of 45 and 54 miRNAs showed significant changes for each pairwise comparison among the 7th,8th and 9th internodes of 'Xun9058' and 'Xun928',respectively.The expression of 31 miRNAs showed significant changes were common to the corresponding comparison groups' of the 7th,8th and 9th internodes of'Xun9058'and 'Xun928'.Based on the sequencing data,20 miRNAs related to hormone signaling among the candidates,belonging to five conserved miRNA families,were selected for expression profiling using quantitative reverse-transcription polymerase chain reaction(qRT-PCR).The five miRNA families,zma-miR160,zma-miR167,zma-miR164,zma-miR169 and zma-miR393,targeted the genes encoding auxin response factor,N-acetylcysteine domain containing protein,nuclear transcription factor Y and auxin signaling F-BOX 2 through degradome sequencing.Combined with the previous research results,the miRNAs might regulate their targets to respond to hormone signaling,thereby regulating the internode elongation and development under maize ear.