Genome-wide Cloning And Mapping Disease Resistance Gene Candidates In Maize

Up to date, more than 50 resistance genes(R gene) have been cloned from 12 different plant species. In this study, the degenerate primer designed on the NBS motif conserved among most R genes was used in the modified AFLP and 3'RACE methods to amplify resistance gene analogs (RGAs). In addition, data mining method was used to recover R-gene-like ESTs from the maize EST databases available with BLAST method. The identified RGAs from the above three methods were mapped on maize genome to construct the R-gene linkage map for accelerating cloning of resistance genes.A modified AFLP method was developed by combining the AFLP method with the NBS degenerate primer to amplify RGAs from maize gemonic DNA. A total of 64 primers combinations between 64 AFLP selective primers and the NBS degenerate primer were used to amplify maize genome DNA, resulting in a lot of PCR amplicons. The PCR products were purified, and then cloned into pGEM-T vector. After sequencing and analysis, 285 non-redundant sequences were obtained. Of which 23 sequences were proved to be RGAs through tblastx in GenBank databases, and this accounts for up to 8% sequenced PCR products..A modified 3'RACE method was used to amplify RGAs from cDNA templates. Three main PCR bands ranging in sizes of 1.5kb, 0.75kb and 0.5kb were appeared using the primer pair of 3'RACE primer upm and NBS degenerate primer. The amplified bands of 1.5—2.0kb in size were excised from the gel, and then ligated into pGEM-T vector. After transformed and sequenced, 246 unique sequences were obtained. In all, there are 12 RGAs recovered from these sequences through tblastx in GenBank databases. About 5% of these sequences are RGAs.A data mining method was used to identify R-gene-like ESTs from maize EST databases. Hitherto, more than 550,000 maize EST entries were deposited in the GenBank and MaizeGDB databases. Our results showed that the EST databases covered most of the expressed regions in maize genome. The full-size putative protein sequences of the 48 known R genes were used to search for the homologous ESTs form the GenBank and MaizeGDB database through tBLASTn, and the recovered R-gene-like ESTs were compared with GenBank database by tBLASTx to confirm their resistant function. Finally, 110 R-gene-like unique genes and 75 singleton ESTs were obtained. Among the 185 ESTs/Unigenes, 66 contained putative NBS-LRR domains, 59 contained putative LRR domains, and 60 contained putative PK or PK/PK domains.In order to map the RGAs on maize genome, STS, CAPS and SNP markers tagged to RGAs were developed based on RGA sequence divergences between two parent lines 87-1 and Zong3. A RIL population derived from the tenth self-pollinated generation of the cross 87-1×Zong3 were used as mapping population. In all, 53 markers tagged to RGAs were developed and were used to investigate the RIL population. By using Mapmaker3.0 soft, the marker data integrated into the high-density SSR linkage map to construct a RGA linkage map. Finally, 48 RGAs have been mapped to 49 loci on maize genome (note: the RGA mla2 was mapped to two different loci in maize genome). But the five RGAs cannot be mapped on any loci by using their marker data.On analysis of the RGAs linkage map, the RGAs were not evenly distribution on the maize genome. The number of RGAs located on chromosome 1, 4 & 10 are far more than those on chromosome 5 & 7. These results are in accordance with the distribution of major resistance genes or resistant QTL on maize genome reported by other investigators.