| Cucumber downy mildew, which is caused by Pseudoperonospora cubensis Rostow, is one of main diseases in cucumber product. It was firstly discovered in 1868, and had a serious development recently. Methods of control downy mildew included utilizing medicine and closing a greenhouse at high temperature, but were ultimately to produce resistant varieties. So cloning and marking the resistance gene of the downy mildew in cucumber are fundaments for disease resistance breeding of cucumber.The study consisted of three parts: (1) the resistance gene analogs of cucumber were isolated by performing PCR, in which degenerate primers designed according to conservative domain of resistance genes were used; (2) the separate population with resistant and susceptible traits for downy mildew were constructed and used for screening markers on downy mildew resistance gene among the cloned RGAs; (3) the novel RGAs isolated from cucumber were employed for investigation of cucumber germplasm resource and their potential of cluster analysis was tested.Firstly, the genome DNA as template from varieties JINCHUN 4 and 649 were extracted by using SDS method, and twelve degenerate primers were designed based on four substructure motifs, P-loop, kinase2, kinase3a and HD, within NBS conserved domain referring to some reports, and amplifying products, which spanned approximately 270bp, 340bp and 540bp, were obtained by performing PCR and purification technique, which were cloned into pMD18-T vector and transformed E. coli JM109. After blue and white plaque preliminary screening, each seventy-two recombinant clones were randomly picked and their plasmids were prepared and were classed by restriction endonuclease digesting and PAGE method. Sixty clones were sequenced and their homologous sequences were searched on GenBank with Blast. Finally, fifteen clones were distinguished as RGA of cucumber and named CsRGA1~15, which were submitted to GenBank and provided accession numbers as AY555482~AY555495 and AY545993. Among the CsRGAs, the CsRGA5 contains all of four substructure motifs and spans 520bp, and CsRGA2 involves three ones of them and spans 336bp, and other CsRGAs only include two ones of them and span 168~288bp extent. Ten CsRGAs have open reading frames (ORFs) and other five ones don't have because of stop codons in them. Performing PCR supported the truth of all CsRGAs exist in the genome of tested cucumbers. The ten CsRGAs having ORFs showed high similarities with the announced RGAs of Cucumis melo, which displayed a close relationship as both species are in the same genus.For screening markers of downy mildew resistance gene from the CsRGAs, four separate populations with resistant and susceptible traits for downy mildew were constructed. Two separate populations were F2 intercross and two other populations were F2 backcross. Many polymorphic bands were obtained on agarose gel electrophoresis pattern using special primers designed based on CsRGA1~15 sequences and performing PCR in above populations. Meantime, the PCR of SCBC519 and SCBC526, both of which were the markers for dm, were performed. The genome DNA of the individuals were extracted by SDS-mini-extracting method, and the level of disease resistance was evaluated by inoculating into the cotyledons of the individuals, and the linkage analysis was carried out by MAPMARKER/EXP version 3.0 program. The results showed that the separate ratios of both disease resistant and susceptible traits in the populations, which were F2 backcross one of L18×129 with L18 as backcross parent, F2 intercross one of 631×649 (the individual amounted to eighty-six), and F2 intercross one of L18×129 (the individual amounted to seventy-seven), matched up to Mendel's law; the linkage marker, CsRGA3, to the dm was picked out from fifteen CsRGAs and was not very close to the dm; the pm locus that being powdery mildew resistance gene existed between CsRGA3 and dm, which was detected and showed the linked relationship of both pm and dm.For the sake of knowing the research potential of analyzi... |
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