Identification Of New Powdery Mildew Resistance Gene In Wheat Brock By AFLP - SCAR Method

Powdery mildew, caused by Erysiphe graminis DC.f.sp.tritici, is one of the most serious diseases of common wheat in China and in many other countries of the world. Most of the powdery mildew resistance genes have already been overcome by new virulent E. graminis strains owing to the single genetic resistant source and the repeatable use of it. Discovery and utilization of novel resistance genes, pyramiding of existing ones, and breeding for resistant cultivars have been proved effective ways to control the disease.In this study, a total of 225 primer combinations were screened to identify polymorphisms between the resistant parent Brock, susceptible parent Jing 411 and the NILs of Jing411 using AFLP technique.The main results are as following:1. Response type of plants to powdery mildewBrock, Line 015, Jing411 and 106 individuals which were obtained from hybrid F₂ population （BrockxJing411 ） , and their cross, backcross progenies （ Line Brock/015//Jing411⁷） were artificially inoculated with the race NO.15. Cultivated wheat Brock was immune to the race NO.15. Common wheat cultivar Jing411 and Line 015 were highly susceptible to that powdery mildew pathogen. Brock/015//Jing411⁷ was highly resistant to powdery mildew. F₁ （BrockxJing411） was resistant to powdery mildew. In the test of 106 F₂ plants, 79 individuals were resistant and 27 individuals were susceptible, which showed the expected Mendelian ratio of 3: 1.2. Identification of AFLP markers linked to the powdery mildew resistance gene in BrockA total of 225 AFLP primer combinations were screened to identify polymorphism between the resistant parent Brock, susceptible parent Jing411 and the resistant Line Brock/015//Jing411⁷, using AFLP technique. All the AFLP primer combinations could amplify clear bands. Among them, only 21 primer combinations could generate reproducible polymorphic amplification patterns between Brock, Jing411 and NILs.3. Linkage analysis of the specific fragments generated by 28 primer combinations to the powdery mildew resistance gene in BrockIn order to test whether the specific fragments generated by 21 primer combinations were linked to the powdery mildew resistance gene or not , 106 progenies , including 79 resistant and 27 susceptible individuals , which had been tested by powdery mildew infection, were tested by 21 primer combinations screened in last step . We found that only primer combinations P6/M3₁₆₃、P6/M3₁₆₆and P3/M6₃₉₅ could generate specific fragments tightly linked to powdery mildew resistance gene in Brock.4. Cloning , sequencing and sequence analysis of P6/M3₁₆₃、P6/M3₁₆₆ and P3/M6₃₉₅The DNA fragment was purified and cloned into pGEM-T Easy Vector . The sequencing result confirmed that AFLP marker P6/M3₁₆₃、P6/M3₁₆₆ and P3/M6₃₉₅ has 163、166 and 395 base pairs. The result of sequence analysis was that there was no homology like this sequence in GenBank, so it was a new molecular marker tightly linked to new powdery mildew resistance gene in Brock.The molecular marker plays an important part in crop breeding, because it is a stable DNA symbol of some special character or gene of crop plants. As a result, molecular markers can be used in plants screening, indicating the aggregation of different resistance genes in one plant, and setting foundation for the cloning of some genes. In this study, we got AFLP molecular markers P6/M3₁₆₃、P6/M3₁₆₆ and P3/M6₃₉₅ which were tightly linked to the new powdery mildew resistance gene in common wheat Brock. It meant that we had greatly approached the new powdery mildew resistance gene in Brock.5. SCAR marker transformationSCAR marker is usually transformed from RAPD or AFLP marker.To improve the stabilization of RAPD or AFLP marker’s application,we can reclaim the marked fragment from the gel, then clone and sequence the fragment,design a pair of specific primer based on the sequence.We can also just sequence the terminal of the segment,add about 14 bp to the primary 10bp primer,make it become a pair of specific primer which matches the primary marked segment terminal.We designed three pairs of primers according to the sequencing results of P6/M3₁₆₃、P6/M3₁₆₆ and P3/M6₃₉₅.The result of SCAR-PCR amplification showed that the specific fragments appeared among the resistent individuals while they didn’t appear among the susceptible individuals.That is exactly the some as the AFLP amplify result which indicated we have successfully transformed AFLP markers into SCAR markers.