Fining-mapping Of Rf3, A Restorer Of Fertility For S-CMS In Maize (Zea Mays L.) And Identifying Of Its Candidates

Utilization of heterosis has facilitated the improvement of yield and quality of crop.Cytoplasmic male sterility (CMS) has been used extensively in breeding for theproduction of a range of F₁ hybrid crops because of its advantages on decreasing the costof labor and improving seed quality. Moreover, the system of CMS/Rf is a perfect modelfor studying molecular mechanism of the interaction between nucleolus and cytoplasm.Maize is one of the pioneer crops whose heterosis is enhanced through this system. Theunstanable fertility of maize S-CMS limits its utility in practice. For using CMS in maizeproduction by the greatest extent, it is necessary to theoretically elucidate the mechanismsof steility and restoration of fertility. Map-based cloning is an effective method of genecloning and the genetic linkage map in the vicinity of target gene with high density is thebasis to map-based cloning. Several attemps have been made to tag Rf3 with molecularmarkers, however, the distances between them and Rf3 are not yet near enough to clonethis gene.In the present study, we screened AFLP and SSR markers polymorphic between nearisogenic lines (NILs). A BC₁F₁ population from a pair of NILs with different Rf3 locus wasconstructed for linkage analysis. Additionally, the differentially expressed profile indifferent tissues and times of S-MO17^Rf3Rf3/S-MO17^rf3rf3 was analyzed throughcDNA-AFLP technology. And some primary results are listed as follows:1. The BC₁F₁ generation yielded 178 semi-fertile (having 50% or more stainablepollen) and 165 sterile plants. This segregation ratio fits a monogenic Mendelianinheritance model of 1 fertile (Rf3rf3): 1 sterile (rf3rf3), it confirmed that fertilityrestoration was conditioned by one dominant restorer gene in these maize materials.2. One of 13 SSRs on chromosome 2.09 bin, UMC2184, was detected to showcodominant polymorphism between parents and bulks. Thus UMC2184 were employed toanalyze linkage relationship between itself and rf3 and perform anchor point to othermarkers. The total 528 AFLP primer combinations (PC) were screened for theirpolymorphism between parents and bulks. Only 4 polymorphic fragments from E3P1,E7P6, E10P7 and E12M7 were confirmed as candidate markers tightly linked to Rf3 andanalyzed subsequently using the BC₁F₁ population.3. Through designing primer and detecting in the parents and bulks, AFLP markersE3P1 and E12M7 are converted into codominant CAPS and dominant SCAR marker,respectively.4. A linkage map was constructed around the Rf3 locus, which was mapped on the distal region of chromosome 2 long arm with the help of SSR marker UMC2184. Thetotal size of this map is 11.1 cM and E7P6 and SCARE12M7 delimited a 2.7cM windowwith 0.9 cM and 1.8 cM from Rf3, respectively5. BLASTx analysis showed that the marker SCARE12M7 was high homologouswith resistance protein. Through comparing and assembling the mapped results of thisstudy and others, we assumed that marker SCARE12M7 was tightly linked orcosegregant with gene resistant to head smut. The potential of SCARE12M7 in assessingand selecting both Rf3 gene and gene resistant to head smut was discussed.6. Differential expression of genes in young leaves and pollen between S-Mo17^rf3rf3and S-Mo17^Rf3Rf3 was investigated via cDNA-AFLP. 2500 fragments distributing80-800bp were displayed by 64 primer combinationa. 33 TDFs which expressed only infertile pollen or expressed in fertile materials specifically were sequenced successfully.By BLAST analysis, these sequences were assigned into 8 categories based on theirbiology function: protein synthesis and fate (9%); regulation on the transcript level (15%);signal transduction (12%); energy metabolism (9%); cell developing and dividing (9%);transposable element (3%); unknown function protein (28%); novel ESTs (15%).7. Through comparing functions of 33 differentially expressed genes with thosederived from analysis of cDNA-microarray (Zhang, 2004), protein synthesis and faterelative genes account for 9% and 13%, respectively. Among them, 26S proteasomeregulatory particle expressed high in fertile pollen in two studies above mentioned.Through in silico cloning strategy, the candidate cDNA sequence of maize 26Sproteasome regulatory particle was obtained, it covered 1451bp in which coding sequencewas 1329bp and coded 443 amino acids. Clustering analysis using the software of ClustalW shows that the homologic rate between 26S proteasome regulatory subunit5 in maizeand its homology in rice, arabidopsis, human is 91%, 77% and 41%, respectively.8. Regulation on the transcript level relative genes occupy 15% in the sequenceddifferentially expressed genes. BLASTx analysis for sequence E1 indicates therepresented gene coding protein containing PPR model. Through expressed patternanalysis in different tissues and times by RT-real-time PCR, this gene expressed in thepollen on the highest level for S-Mo17^Rf3Rf3 but on the highest level in the leaves oftasseling time for S-Mo17^rf3rf3.Differentially expressed patter of this gene indicates the itsrelationship with between it and fertility restoring in maize S-CMS.