Fine-mapping Of A Restorer Of Fertility Gene For S-CMS In Maize And Genome-wide Association Study (GWAS) Of Drought Tolerance In Maize (ZEA MAYS L.)

In higher plant, cytoplasmic male sterility (CMS) is a common phenomenon. The CMS/Rf system is not only widely used in hybrid breeding, but also a perfect model system for studying the molecular mechanism of the interaction between nucleus and cytoplasm. Maize is one of the earliest crops whose heterosis is improved through this system. S-CMS is the largest one of the three CMS groups in maize but utilization in breeding is limited by its unstable sterility. For using CMS in maize production by the greatest extent, it is necessary to clone the Rf3gene and theoretically elucidate the mechanisms of sterility and restoration of sterility.In the present study, we screened SSR and STS marker polymorphic between the near isogenic lines (NILs). Two sets of BC1F1populations were constructed from NILs with differences in Rf3locus for linkage analysis. The two populations were designated SSN for (S-Mol7rf3rf3xS-Mol7Rf3Rf3)xN-Mol7rf3rf3and SNN for (S-Mol7Rf3Rf3xN-Mol7rf3rf3)xN-Mol7rf3rf3. Additionally, we analyzed the sequences of maize2.09bin from B73genome (AGPv2). At the same time, a BAC library containing Rf3gene was also constructed. And some primary results are listed below:1. By examining the structure and development process of maize florets, we found it was more accurate to check pollens from the upper flowers inside the floret for determining whether the plant was sterile or not, which ensures the correction of fertility identification.2. The SSN population generated2781semi-fertile and2720sterile plants while the SNN generation yielded445semi-fertile and472sterile plants. The segregation retios of the two populations fit a monogenic Mendelian inheritance model of1fertile (Rf3rf3):1sterile (rf3rf3), it confirmed that the fertility restoration was conditioned by one dominant restorer gene in these maize materials.3. Total638pairs of primers were designed. Of those,198pairs were SSR markers,440were STS markers and only10SSR and4STS marker were polymorphic. The rates of the polymorphism for SSR and STS marker were10%and1%, respectively, relatively lower than usual, probably because markers were assayed between NILs in which most genomic region assayed were as the same as the recurrent, the other parent.4. Sequencing results of STS fragments confirmed the low polymorphism between the target region of the NILs. Only a small amount of SNP sites were discovered. Several could be converted to CAPS and dCAPS markers successfully.5. A linkage map was constructed around R/3locus, which was mapped on the distal region of chromosome2long arm. The total size of the map is19.9cM and all marker are mapped to one side of Rf3gene. The nearest marker to R/3gene is2043S-3, which is1.6cM away from the gene.6. A bacterial artificial chromosome (BAC) library was constructed from inbred line S-Mol7Rf3Rf3, and high-density filter set was prepared also. The library contains175,104clones with130kb average insertion fragment, which covers9.1times of the maize genome. The availability of this BAC library provides a useful tool for the cloning of Rf3gene.7.12PPR genes were discovered after rescanning the region of maize2.09bin. Of those PPR genes,10are belong to P subfamily and6PPRs of P subfamily located the mapping area of R/3locus. RFLP analysis revealed two PPR genes were polymorphic between tested lines containing R/3gene and lines containing rf3gene.8. the sequences of two PPR genes and the nearest marke were used as hybridization probes to screen the BAC library and48positive clones were obtained. Contig building and further analysis are under the way.Another area interested in our research is the discovery of maize genes related to response or tolerance to drought. Drought is one of the most important factors affecting the yield stability of maize (Zea mays L.) in China and many other areas in the world. In order to solve the drought and water shortage problem, breeding hybrids with drought tolerance is an important method besides water saving engineering. All350hybrids were phenotyped for9traits under water-stressed (WS) and well-watered (WW) conditions in multiple environments. Genes significantly related to drought were found through association analysis. These findings lay the foundation for further study on the mechanism of maize drought tolerance.1. Broad sense heritability of nine traits across the tested environments was calculated. Heritability of all but GY and KNO were high, which indicated the genotypic values of these traits were more consistent across environments. GY and KNO were more affected by the environments. These are in accordance with results from CIMMYT.2. Illumina MaizeSNP50array was used for genotyping the panel of350maize inbred lines. Statistical results showed that the SNP type of A/G was the most abundant in the population which was74.5%of total. All the other SNP types only held25.5%. This bias reflects the inherent connections among genomic structure, gene function, natural selection and artificial selection.3. In this study,275associated SNPs (P≤2.25x10-5|1/N) were identified, located i n221genes for126trait x environment x treatment combinations. under the Bonferroni correction of1/N.50genes were related to more than one trait. GO annotations showed that these genes were related to biosynthase, signaling, metabolism, etc. The results indicated that it is a very complex process of maize responsive to drought and drought tolerance of maize is a result by coordinating multiple pathways.4.38genes were co-localized to28Meta-QTL intervals which were known for drought tolerance of maize,9of them were found under WS,22under WW and7under both WW and WS.38genes were distributed over10chromosomes.7genes within5Meta-QTLs were on chromosome2while there was only one gene on chromosome3.