| Sclerotinia (stem rot) disease in rapeseed, caused by fungal pathogen Sclerotinia sclerotiorum, is most important disease in the wordwide and especially seriously taking place in the southeast coastal area and Yangtze River Area of China and has restricted the development of rapeseed industry for a long time.Till now, rapeseed cultivars with complete resistance to this disease haven’t been identified, nor have related species. Resistance to Sclerotinia sclerotiorum in rapeseed belongs to quantitative trait and easily affected by environment condition and other factors. There is no widely accepted method to identify resistance. These questions have made it difficult for breeders to select really resistance materials. The objectives of our study were to1) screen more precise identification methods,2) QTL mapping the resistance to Sclerotinia,3)find and analysis resistance related genes, which will be excepted to help understand the mechanisms of disease resistance, and can be used for selecting high-quality cultivars with strong resistance to Sclerotinia.In this study, we determined resistance of30rapeseed varieties from home and abroad to Sclerotinia sclerotiorum, then made the comparison between the different methods for identification. We chose a pair of the big resistant difference of the material as the resistant and susceptible parents to configurate hybrid combination and build F2group; We obtained one F2population derived from a cross between NingRS-1and APL01. We did QTL mapping of resistance to Sclerotinia by utilizing the resistance performance of F2:3population at seeding stage and molecular markers such as SSR and SRAP. The main results are followed below:1Comparison of different identification resistance methodsseedling mycelium agar to living leaves, seedling mycelium agar to leaves in vitro, seedling mycelium agar to petiole and dipping leaf into oxalic acid all showed effective for identifying resistant and susceptible plants,but there was not significant correlation between these methods.mycelium agar disk inoculation to axil and toothpick inoculation to stem during flowering period could well represent varieties’resistance and susceptibility. Especially, the correlation during the two methods was highly significant. According to the comparison results of the resistance identification methods,combined with the rule of disease occurrence, the two methods in adult stage can effectively reflect the differences between varieties and toothpick inoculation to stem during flowering period was relatively more reliable. It was suggested that the resistance identification be done during flowering period and seedling identification was used only as supplement as the resistance to S. sclerotiorum was different at the diferent growth stages.2QTL Mapping resistant genes conferring Sclerotinia sclerotiorum in B.napusBased on the F2population with154plants derived from the cross of NingRS-1X APL01, a genetic linkage map of Brassica napus L. was constructed, which was made up of19linkage groups including28SSR and158SRAP makers, covering2780.4cM in total with17.8cM between adjacent loci. Five QTL controlling resistance were detected by Composite Interval Mapping Method (CIM) in Windows QTL Cartographer V2.0with a LOD score more than2.5. Five QTLs were called SRL10、SRL11、SRL14、SRS1-1and SRS1-2. SRL10was located in the region of m16e26-m29e12on (LOD2.6), which could explain10.51%of the phenotypic variation. SRL11was located in the region of m30e1lb-m28e36a on (LOD3.3), which could explain14.52%of the phenotypic variation. SRL14was located in the region of m16e37-Ni2-C03on (LOD3.3), which could explain21.8%of the phenotypic variation. SRS1-1was located in the region of ml8e42-ml8e7a on (LOD3.7), which could explain16.79%of the phenotypic variation. SRS1-2was located in the region of ml6e7a-m19e32on (LOD3.2), which could explain14.58%of the phenotypic variation.3the expression analysis of resistance related genesAfter inoculation, The expression of the PDF1.2in resistant cultivar enhanced, but in susceptible variety the expression were not significantly changed and has been at a low level; The expression of PR-1in resistant and susceptible varieties overall were in the down-regulation of state;The expression of LOX、EnIN3in disease-resistant variety changed with the marker gene PDF1.2of ET/J A pathway and reached the maximum value in the48h, in addition the expression of LOX、PDF1.2in resistant variety was higher than that in susceptible variety; EDS1in resistant variety was induced to express, in the24h reached the maximum expression, but expression in the48h after inoculation was at down-regulated state, and expression of EDS1in susceptible ones after inoculation was in up-regulated state; After inoculation, PAL in resistant and susceptible cultivars were induced to express, then the expression in the disease-resistant variety increased rapidly at later stage, which is obviously higher than that in susceptible variety; The expression level of FeSOD in resistant and susceptible varieties was low and the difference between the two varieties was not significant, but expression of Cu/ZnSOD enhanced in resistant variety after inoculation. The expression level in48h was significantly higher than that in susceptible variety after inoculation; The expression of GLP and OXO remain consistent, and the two genes in resistant and susceptible cultivars were not significantly different in the expression; The expression difference of PGIP in the resistant cultivar Ning RS-1was extremely significant after inoculation, expression amount in the24h was170.45times than that before inoculation and in the susceptible cultivar APL01expression was not significant. The expression in the24h was only3.5times than that before inoculation, at the same time, the expression level of PGIP in Ning RS-1was much higher than that in APL01, which was1299.49times at the same period. |
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