Genetic Mapping Of The Powdery Mildew Resistance Gene And Cloning The Relative Gene In Wheat

Powdery mildew, caused by Blumeria graminis (DC.) E.O. Speer f. sp. tritici (Bgt), is one of the most important diseases in common wheat (Triticum aestivum L.) worldwide. Growing resistant cultivars is the most economical and environmentally safe approach to eliminate the use of fungicides and to reduce production losses due to this disease. Molecular markers closely linked to the resistance gene not only facilitated identification and mapping of resistance genes, but also could be used for markers-assisted selection (MAS) of resistant cultivars/lines in breeding program. MAS greatly enhance the aggregation efficiency of genes and accelerate the breeding program. Cloning of plant resistance gene is greatly helpful for crop resistance breeding and the insight of resistance mechanism. Resistance gene analog (RGA) cloning provided the economic and quick approach for wheat resistance gene isolation.Genetic analysis of powdery mildew resistance was finished in this study at first. Molecular marker techniques such as SSR (simple sequence repeat), STS (sequence tagged site),SRAP (sequence-related amplified polymorphism) and RGA were used to develop the molecular markers closely linked to Pm4b gene and construct the high-density genetic map of PmZB90 gene. Some homologous fragments of resistance gene were cloned by RGA technique. RT-PCR analysis and TAIL-PCR sequence extension were further done in the research. All of these will provide the foundation for get the full-length gene and investigate the resistance mechanism in wheat.The main results are as follows:1. Adult resistance identification was done with different wheat Pro-genes carrying cultivars/lines. Six of twenty-nine known Pm-genes carrying cultivars are immune to Bgt. Their contained genes are Pm4b, Pm13, Pm20, Pm21, Pm23 and Pm24. In addition, Pm4a, Pm6, Pm17, Pro22, Pm5a＋6 and Pro33 have high or medium resistance. Other Pm-genes have lost resistance to powdery mildew. Those Pm-genes have good resistacne should be fully utilized in the wheat powdery mildew resistance breeding program.2. Bulk segregant analysis (BSA) was employed to identify SRAP, STS and SSR markers linked to the Pm4b gene, which confers good resistance to powdery mildew in wheat. Out of 240 primer combinations tested, primer combinations Me8/Em7 and Me12/Em7 yielded 220-bp and 205-bp band associated with Pm4b, respectively. STS-241 also linked to Pm4b with a genetic distance of 4.9 cM. Out of eight SSR markers located on wheat chromosome 2AL, Xgwm382 was found to be polymorphic and distally linked to Pm4b with a genetic distance of 11.8 cM. Further analysis was carried out using the four markers to investigate marker validation for marker-assisted selection (MAS).The results showed that Xgwm382 was special for Pm4 gene. Marker STS-241 and Me8/Em7-220 which can distinguish Pm4b from Pm4a should be used for MAS and gene pyramiding in wheat breeding. It will facilitate rapidly and valuably utilizing the Pm4 gene for powdery mildew resistance.3. Chinese native wheat variet ZB90 was an effective broad-spectrum powdery mildew resistance variety. Genetic analysis showed that the resistance was controlled by a single dominant gene at the seedling and adult stage. The resistance gene is temporarily designated as PmZB90. Among the 53 pairs of SSR primers tested, three polymorphic microsatellite markers Xgwm382, Xgwm311 and Xgwm312 on the long arm of chromosome 2AL were mapped in 144 F₂ segregating populations for the powdery mildew resistance gene in ZB90. PmZB90 was located on chromosome 2AL according to the genetic distance. STS marker STS-470 and RGA marker RGA-1102,SRAP marker Me5/Em5-650 and Me8/Em16-600 were also combined for map construction. The orders between PmZB90 and the seven markers were: Me5/Em5-650-RGA-1102-PmZB90-Xgwm382-Xgwm311-Me8/Em16-600-STS-470-Xgwm312. Genetic distances between seven markers and the resistance gene were: 3.7cM,9.2cM,2.9cM,4.5cM,2.3cM,20.8cM,49.6cM。The orders of these markers loci agreed well with the established map of chromosome 2AL.The relationship between PmZB90 and other powdery mildew resistance genes located on chromosome 2AL were distinguished and discussed. According to the gene origin, chromosome location and relation discussion, PmZB90 is a new Pm gene different from Pm4 and maybe allelic to PmDR147. Identification and genetic mapping of the new powdery mildew gene PmZB90 offered the new resistance resource for wheat powdery mildew resistance breeding program.4. In the study, different Pm-genes carrying cultivars/lines were amplified by arbitrary degenerate primers based on the conserved domain of plant diseases resistance genes. Some RGAs were cloned from wheat VPM and ZB90. Sequences analysis and function forecast showed six of RGAs belong to the resistance homologous fragments. RGA1231 and RGA1102 cloned from VPM and ZB90 respectively were analyzed in detail.RT-PCR technique was used to study the expression of Rc955 gene at different time intervals after inoculated with Bgt. The expression amount of Rc955 was influenced by Bgt induced, but the change was slight. It showed the expression of Rc955 maybe constitutive expression.Three nested primer pairs were designed according the 5' and 3' end sequence of RGA1102. RGA1102 were successfully extended to 5'end with 799bp and 3'end with 281bp by TAIL-PCR technique. Sequence analysis showed the 2182-bp extend fragment contain the full-length coding frame. It will be helpful for gene cloning and function research of PmZB90.