| Cucumber, Cucumis sativus L.,is an economically important fruit vegetable crop globally ranking 4th in quantity of world vegetable production. Powdery mildew is one of the main diseases in cucumber production, frequent occurrence of powdery mildew causes serious wilt or death to cucumber plants, seriously effect the yield and fruit quality of cucumber. But the progress of cucumber powdery mildew resistance breeding is slow, which is because the genetic mechanisms especially the molecular mechanisms of powdery mildew resistance in cucumber have been not well understood. Prior to this experiment, cucumber line D8 (susceptible to powdery mildew, dwarf) were crossed with Jin5-508 (highly resistant to powdery mildew, sprawl) and then backcrossed twelve times from 2004 to 2009. A 0.7 cM single segment substitution line (SSSL0.7) was successfully developed using phenotypic and marker assisted selection. In this study, to further narrow down the substituted segment and obtain the gene governing PMR, a secondary F2 segregating population from a cross between SSSL0.7 and the recurrent parent D8 was developed. Candidate genes were predicted and analyzed by qRT-PCR and subcellular localization. This study laid a foundation for the innovation of cucumber powdery mildew resistance germplasm and cucumber powdery mildew resistance breeding of new varieties. The main results are as follows:1. The SSSL0.7 was crossed with D8 to develop a secondary F2 segregating population, which contained 3,600 individuals. Frequency distribution of disease index (DI) of powder mildew on the 3,600 secondary F2 segregating individuals was calculated; two flanking SSR markers including SSR16472 and SSR16881 were used to identify recombinants,7 recombinants (SD1, SD2, SD3, SD4, SD5, SD6 and SD7) were screened out,SD6, SD1 performance is high sensitive, the rest is high resistance.2. Based on the whole genome resequence results of Jin5-508 and D8,20 pairs SNP-dCAPs were screened out between SSR16472 and SSR16881. Of which, three polymorphic markers, SNP02, SNP09 and SNP20 were screen out between Jin5-508 and D8.3. All the 7 recombinants (SD1 to SD7) were genotyped by the three SNP-dCAPs markers. Based on the genotype of marker loci, the target gene locus for PMR was mapped between SNP09 and SNP20 with physical distances of 41.1Kb. Cucumber genome annotation system analysis of this 41.1Kb DNA fragment predicted 8 candidate genes:Csa1M064720.1, Csa1M064730.1,CsalM064740.1, Csa1M064750.1, Csa1M064760.1, Csa1M064770.1, CsalM064780.1 and CsalM064790.1.4. We investigated expression dynamics of all eight genes in D8, SSSL0.7 and Jin5-508 after artificial inoculation with spore suspension by qRT-PCR. The relative expressions of Csa1M064780.1 and Csa1M064790.1 were low and stable in D8 before and after inoculation; both were up-regulated in SSSL0.7 and Jin5-508 12h after inoculation.On the other hand, no consistent trends in expression levels were found among the three cucumber lines for the remaining six genes.The results of qRT-PCR showed that Csa1M064780.1 and Csa1M064790.1 might be the key candidate genes for PMR in cucumber.5. To provide further evidence for the potential roles of Csa1M064780.1 and Csa1M064790.1 in transcriptional regulation, the localization of Csa1M064780.1 and Csa1M064790.1 protein were investigated in onion epidermal cells transiently expressing gene fusions to the green fluorescent protein (GFP) by particle bombardment. As shown, Csa1M064780.1 was observed in plasma membrane and nucleus, but CsalM064790.1 was observed exclusively in the plasma membrane. |
PDF Full Text Request