Identification Of Resistance To Downy Mildew And Its Related Gene Cloning In Radish (Raphanus Sativus L.)

Radish (Raphanus sativus L.), belonging to the Brassicaceae family, was an annual or biennial herb, and it was originated from China. Downy mildew (DM) is a worldwide disease of Brassicaceae vegetable induced by the oomycete Peronospora parasitica. Recently, radish downy mildew was increasingly serious at cotyledon, seedling and propagation stages, especially in temperature suitable and humid environment. Therefore, the radish yields and qualities declined seriously.So it is imperative for radish breeding of resistance to downy mildew. The research of Brassicaceae plants resistance to downy mildew were only focus on brassica vegetables,such as Chinese cabbage, brassica oleracea L.,Cauliflower,and Broccoli.The research of resistance to downy mildew in radish had just started,and the study of physiological,inheritance,gene cloning were not reported recently.In the study, a systematic analysis of radish resistance to downy mildew was conducted in four areas in order to provide theoretical and technical supporting for radish resistance breeding.Downy mildew IP classes of radish cotyledon, seedling and adult-plant established through screening germplasms with field naturally infection and artificial inoculation identification methods. In field 500 radish inbred lines were identified. The result suggested that the radish with flat-foliage, red or green succulent root were more susceptible to downy mildew.20 radish inbred lines were identified in lab by artificial inoculation, and the result suggested that highly resistance materials performed good characteristics through all stages, while materials susceptible to downy mildew were especially serious at cotyledon stage, which consistent with the identification result in field. Correlation analysis was established between cotyledon and adult stages, and between seedling and adult stages. The correlation coefficients (R2) were 0.9778 and 0.8519 respectively. Thus, we could reflect the radish resistance characteristics through identifying the radish plants at cotyledon stage. The PPO, POD, CAT and SOD activities were significantly positive correlated with the downy mildew resistance in resistance lines,and reached maximum at different post-inoculation stages, respectively. The variance analysis shows that there is extremely significant difference between maximum and the activities at other stages. Nevertheless, in susceptible line, the enzyme activities were relatively stable after inoculation and the differences of each stage were not significant. It was suggested that these protective enzyme activities could be used as the index for resistance evaluation of downy mildew in radish.The objective of the study was to determine the mode of inheritance of resistance to downy mildew (Peronospora parasitica) in radish at seedling stage. Six populations of two cross combinations (NAU-XBY06×NAU-SH08,NAU-DHP07×NAU-BJQ09) were researched.The F1 was completely resistant, and the F2 segregated a clear 3 resistant to 1 susceptible. the BC populations using the resistant parent as the recurrent parent contained all resistant plants, and the BC to the susceptible parent segregated 1 resistant to 1 susceptible. These results indicate that resistance is controlled by a single dominant gene. All F1, F2 and BC plants (including reciprocals) were no difference, indicating no maternal effect for this trait.The genomic DNA sequence of NAU-XBY06 was amplified with four specific primer pairs, and we get four fragments of 2372bp,924bp,636bp and 607bp, and named Rs-dml, Rs-dm2,Rs-dm3,Rs-dm4 respectively.Nucleotide and deduced amino acid sequences analysis indicted that Rs-dml is highly homologous with RPP8 of Arabidopsis thaliana (61%). It contained conservative domains of NBS-LRR resistance genes and belonged to the NBS-LRR resistance gene. BLAST analysis in NCBI database suggested that Rs-dm2, Rs-dm3, Rs-dm4 is highly homologous with RPP1, RPP5,RPP27 of Arabidopsis thaliana respectively,and they also contained conservative domains of NBS-LRR or Receptor-like Protein(RLP). The resistant related genes of resistant to radish downy mildew we cloned in the study provided technical and theoretical basic for further research of resistance to downy mildew in molecular mechanisms.