Studies On Biochemistry And Molecular Biology Of Genetic Diversity Of Grape Cultivar And Resistant Appraisal Of Downy Mildew

The grape is the second largest fruit tree in the world. Its variety reaches as many as 8000. There are more than 1000 in China. In recent years, the plantation of grapes develops rapidly from the North to the South. In 2003, the total area of its plantation adds up to 421,000 hm2 and the output reaches 517,600t. The south accounts for 80,900 hm2 with an output of 97,090t, Covering 13 provinces. Due to the megathermal and rainy climate in South China, along with severe damage of plant diseases and insect pests, the downy mildew resulted in a 50% loss or so in Sichuan province. Besides, on account of the mixed types of the grape, it is difficult to select those disease-resisting varieties.In view of above-mentioned factors and the major problems of grape plantation in the south at present, by making a systematic study of grape varieties from the standards of morphology, bio-chemistry and molecular biology as well as grapes' diverse properties, POD isozyme bands, PAL, PPO, CAT, SOD activities, molecule signs of RAPD type and RAMP type and quality characteristics of superior seedless grapes under the different growing conditions, this research aims to find a quick method to identify grape varieties and their resistance to downy mildew and provide theoretical bases for reasonable application and protection of grape resources as well as grapes' high-quality disease resistance and breeding. The major results are as follows:l.The result by plumb plate polyacrylamide gel electrophoresis shows that 22 varieties electrophoresis 6 POD isozyme bands and 17 kinds of bands type , each of which consists of 3-5 bands; that POD isozyme bands can not identify grape varieties. Band with 0.6089 Rf is the common characteristic of 22 varieties; Band with 0.3333 Rf is of grapes with susceptibility or mid-suseptibility to downy mildew ; band with 0.2661, 0.2978 and 0.4455 Rf is of grapes with resistance or mid resistance to downy mildew. SPSS analyses and UPGMA cluster analyses indicate that the experimented varieties can be classified into 5 groups: group 1 and 2 are of 12 V. vinifera such as centennial seedless, Wuhe 8611 etc., showing susceptibility or mid-susceptibility to downy mildew; group 3,4 and 5 are of 10 V. vinifera 1abrusca , such as Fujiminori, Fanganghei, kyoho etc., showing resistance or mid-resistance to downy mildew.The result of identifying grape's resistance and susceptibility to downy mildew by means of POD isozyme characteristic bands is more or less in accordance with the fieldresult, so it can be used as an effective method for early identifying grape's resistance to downy mildew.2. One high resistant variety, Fujiminori, and eight middle resistant variety, that is Fanganghei ShingnoSmile FlameSeedless Hemi Youxuanpiaonai Takatsuma Jinxiu Fenghou were selected from 22 grape varieties tested through the inoculation test in pots and natural infestation experiments in field. There were very significant correlation between disease indexes of downy mildew and several enzyme activities including PPO and PAL as well as CAT specific activity in these 22 varieties tested before and after inoculation. But SOD enzyme activity was not quite correlated with disease index of grape downy mildew. PPO and PAL enzyme activities and CAT specific activity of grape were related to its resistance of downy mildew very significantly. The results suggested that these three enzyme activities could be used as one of the standard for resistance estimation of grape to downy mildew.3. Through the rectifying research into the RAPD reaction system of grape varieties and the genetic diversity test on 18 grape varieties by using RAPD marks, in 25 1 reaction systems, the best one bears the following qualities: template DNA is 20ng,dNTP is 150 mol/L, Taq DNA polymerase is 2.0 U , primer is 0.2 ~ 0.25 u mol/L, mg2+ is 1.5- 2.0 u mol/L, 94°C pre-denaturation is 3min, 1 min anneal temperature is 36°C, 72°C extended time is 2.0 min, 50 circulations, and 72°C heat preservation is 10 min. RAPD marks indicate that in 80 random primers, 29 primer amplified products are polymorphic, gaining 114 DNA amplified hands, 92 of which are polymorphic. Each primer amplifies 2-10 polymorphic bands, averaging 3.9 bands. When the GS is 0.8090, three groups are gathered: in group 1, only Fujiminori is resistant to downy mildew; in group 2, 8 varieties including Fenghou are mid-resistance to downy mildew, Jinxiu and ShingnoSmile having close genetic relations; in group 3 , among 9 varieties, except 8612 and Ruby seedless showing mid-susceptibility to downy mildew, RAPD mark can separate all varieties and has the same analysis result as that of pots inoculation, thus can be the effective way to identify grape varieties and their susceptibility to downy mildew.4. The RAMP molecule mark analysis of 18 grape cultivars indicates that among 80 primer combinations, 33 produce 152 DNA amplified bands, 124 of which are polymorphic. 1~8 polymorphic bands can be amplified from each primer combination, with an average of 4.6 bands. Four groups can be formed: group 1 is Fujiminor only, showing resistance to downy mildw; group 2 includes 9 cultivars such as Fenghou, showing mid-resistance to downy, mildew, JingXiu and ShingnoSmile having the close genetic relation; group 3 are crimson seedless and wuhe 8612, showing mid-susceptibility to downy mildew ; group 4 includes 6 cultivars such as superior seedless, showing susceptibility to downy mildew. Cluster analysis demonstrates that RAMP markers can separate 18 cultivars entirety the results of RAMP and RAPD analyses are identical.Therefore, this method can effectively identify grape varieties and their susceptibility and resistance to downy mildew.5. Meanwhile by means of systematic investigation into the botany and cultivation characteristics and fruit habits of 18 varieties of grapes under the humid and hot growing conditions in the south as well as analyses, identification and appraisal of the inward and outward qualities of the berry, it turned out that 17 varieties show strong growth, 12 better adaptability and 7 weak adaptability, mainly due to severe occurrence of downy mildew. Fruit habits indicate that florescence is in early May; detachable time is from early July to late September; bearing branch rate is 44.60%~ 89%; bearing coefficient is 0.62-1.89; all fruit grains are medium-sized or giant with surface colors of purple-red, green-yellow and black blue; SSC is 14.5%~19.3%; total sugar is 13.2%~18.5% and total acid is 0.36%~0.60%. Most of varieties have such good qualities as moderate bearing branch rate, high fruit-bearing coefficient, oval or round shapes, large grains, bright colors, high sugar and high total acid.To sum up, POD isozyme characteristic bands can be supplementary appraisal of identify grape's resistance and susceptibility to downy mildew, but not the effective method to identify grape varieties. Meanwhile, PPO, PAL and CAT activities can be supplementary standards of appraising grape's resistance to downy mildew, while SOD activity can not be appraisal standard. This result is the same with the results in field .Beside, RAPD and RAMP molecule markers can be effective ways to appraise grape varieties and their resistance to downy mildew, Due to its more exact mark results, RAMP element-markers can better reveal the genetic relations of grape varieties than RAPD and thus applicable to genetic diversity research.