Fine Mapping Of The Locus On Chromosome 7 Conferring Resistance To Maize (Zea Mays L.) Rough Dwarf Disease

Maize(Zea mays L.) is an important food and fodder crop in the world, occupying a very crucial position in the national economy.Maize rough dwarf disease (MRDD) is a viral disease that is widely spread in China in recent years and has caused great losses in maize yield.Taking full advantage of existing resources of resistance to locate and clone the rough dwarf disease resistance genes and making an intensive study of disease-resistant mechanism are fundamental and effective ways to solve the problem.In our research,Wang Fei has located three resistance loci on chromosome 6,7, 8 respectively.The Mrdd1 locus was approximately mapped between the SSR markers umc1656 and bnlg2191.The Mrdd2 locus was approximately mapped between the SSR markers umc1401 and umc1666.The Mrdd3 locus was approximately mapped between the SSR markers bnlg1823 and umc1268.In the molecular marker genetic linkage map building using the(Ye478×90110)F₂,the distances of the three regions on chromosome 6,7,8 were 4.5cM,11.1cM,5.8cM, respectively.According to the mapping results of the of locus on chromosome 7 conferring resistance to MRDV,two recombinant inbred lines F038141 and F072141 that were from disease-resistant inbred line 90110 and susceptible inbred line Ye478 were chosen as parents to build their F2 population,which was the mapping population. The electrophoretic bands of F038141 and F072141 were consistent with Ye478 when checking with the linkage marker umc1656 on chromosome 6.The electrophoretic bands of F038141 and F072141 were consistent with 90110 when checking with the linkage markers bnlg1823 and umc1268 on chromosome 8.Differences existed at the locus on the chromosome 7,where the electrophoretic bands of F038141 and F072141 were consistent with Ye478 and 90110 respectively when checking with the linkage marker umc1401 on chromosome 7.According to the maize genome sequences provided by NCBI,47 pairs of SSR primers between umc1401 and umc1666 were designed in order to screen the SSR markers showed polymorphisms between the two parents 90110 and 478 as well as the F₂ population.After analyzing the mapping population with 13 markers selected with clear product band,the resistance locus on the chromosome 7 was mapped in a 1.4cM region between the SSR markers umc1401 and L6,apart from 0.7 cM and 0.7 cM,respectively.424 RAPD primers were used to detect the polymorphisms between 90110,478, resistant bulk and susceptible bulk of(Ye478×90110)F₂.In 405 markers with clear product bands,257(58%) showed polymorphisms between the two parents,in which the polymorphism of S217 between the parents was consistent with that between the resistant bulk and susceptible bulk.The specific RAPD-amplified products of S217 was about 340bp.Sequencing results showed that the length of the fragment was 329bp which was located on the BAC contig AC196110.4 of chromosome 4.The sequence of this fragment was matching with parts of the sequence of the forecast gene FG14 and its 3'end.The full-length of this forecast gene was 2151bp,which was made up of four exons and three introns.The encoded protein of this gene was belong to cysteine protease inhibitor family,and the full-length of this protein was 184aa.By using the maizesequence bioinformatics database and the conserved domain procedure of the NCBI,we found four more likely candidate genes in the interval of umc1401 and L6.The encoded protein of FG034 in AC203054.4 contained the conserved nucleotide binding site domain of the resistance gene.The encoded protein of GRMZM2G131926 in AC206581.2 contained two conserved domains of the resistance gene:serine / threonine protein kinase and leucine repeats.The encoded protein of GRMZM2G094867 in AC196440.3 belonged to the harpin-induced family protein.The encoded protein of GRMZM2G100012 in AC225799.2 belonged to the cytochrome P450 protein family.According to the known structural characteristics of the disease resistance genes,these four genes were worth being paid more attention to. In addition,we did research on the SSR-based triplex PCR reaction system and found one system that was fit for detecting three MRDV disease-resistance loci at a time,which can be used in the marker-assisted selection breeding in the future.We fine mapped the locus on chromosome 7 conferring resistance to maize rough dwarf disease,screened some candidate genes and did research on the SSR-based triplex PCR reaction system.These work laid a preliminary foundation for the cloning of the maize rough dwarf disease resistant gene and the molecular marker-assisted breeding.