Study On Resistance Identification And SSR Molecular Marker Screening Of Downy Mildew In Different Grape Germplasm Resources

The resistance to downy mildew(Plasmopara viticola)among 120 grape germplasm resources was investigated.The leaf discs inoculation test was used to identify the resistance to downy mildew in 120 grape germplasm resources.In addition,real-time fluorescence quantitative PCR(qRT-PCR)was performed to evaluate the relative expression level of key genes in six wild grape strains.At the same time,SSR molecular markers were screened for resistance to downy mildew in grapevines,which provided basis for molecular assisted breeding of grapes.The main results are as follows:(1)The disease index ranges of 120 text varieties(strains)were 0~78.70,which showed differences in resistance of downy mildew among different grape germplasm resources.What’s more,Vitis rotundifolia,including Pride,Jumbo,Fry,Summit and Triumph,was immune to downy mildew,of which the disease index range was 0.Interestingly,Chinese wild strains Yunnan-yuanmou 2,Yunnan-2 and Muzhaling-3 were highly resistant to downy mildew and the disease indexes were 1.81,4.40 and 1.62,respectively.Besides,Thirty-eight of them were resistant to downy mildew and the disease index ranges were 5.32~25.00,such as Moldova,Palmata,Yingyu-linxian and DVIT2437 cinerea Barrett.Three of all the grape germplasm resources,such as Italian Riesling,Medoc Noir and Gamay Teinturier,were highly susceptible to downy mildew and the disease indexes were 78.70,55.56 and 50.93,respectively.Furthermore,the remaining varieties(strains)were susceptible to downy mildew and the disease index ranges were 25.93~49.54.The results indicated that a number of wild grape strains showed highly resistant to downy mildew,such as Yunnan-yuanmou 2,Yunnan-2 and Muzhaling-3,which could be used as original materials for disease resistance breeding.(2)The agarose gel electrophoresis results were compared with the identification of leaf discs inoculation.Thirty pairs of SSR molecular markers were used to verify the resistance of 120 grape germplasm resources to downy mildew.What’s more,phenotypic variation manifests in individuals with the same genotype that can develop different phenotypes due to external environmental conditions.Eight pairs of SSR markers explained 53.33%~80.83% of the phenotypic variation,such as RGA10,RGA9,RGA8,UDV-097,RGA4 and RGA11,of which the marker RGA9 had the highest phenotypic variation of resistance to downy mildew(80.83%),and it is expected to become a standard marker for detecting disease resistance of grape downy mildew,which will be conducive to provide a basis for rapid identification and molecular assisted breeding of resistance to downy mildew in grape germplasm resources.In contrast,22 pairs of SSR markers were less than 50% of the phenotypic variation for resistance to downy mildew,which could not be used to identify resistance of grape downy mildew,such as VMC8g9,VMC1g3.2,UDV-370,and the marker Rgvvin180 had the lowest phenotypic variation rate for downy mildew resistance,which was 24.17%.(3)The results of clustering revealed that the gene expression patterns of disease resistance in wild strains were various,and the peak of their expression level was concentrated on inoculation of Plasmopara viticola for 12~24 h.After inoculating Plasmopara viticola for 12 h,the expression level of TGA1 reached 50.45 in Pride.And expression level of WRKY27 was 400.94 in the disease-resistant strain Yingyu-linxian.In addition,expression level of STS1 was 35.60 in Qiu-songxian,a susceptible strain.After inoculating Plasmopara viticola for 18 h,the expression level of NPR1 was 209.87 in Yunnan-yuanmou 2 and the expression levels of PAD4 and ICS2 were 32.30 and 104.94 in Pride.Moreover,expression level of EDS1 was 22.78 in Qiu-songxian for 18 h.After inoculating Plasmopara viticola for 24 h,the expression level of PAL was the highest in Pride and expression level of TLP was 382.73.In contrast,the change of expression level of WRKY33 was more slowly in all strains.Above all,the results showed that TLP,NPR1,PAL and PR1 had rapidly response and high expression level in different disease-resistant strains,which may play a significant role in resistance to downy mildew.