Gene Mapping for Powdery Mildew Resistance and Related Study of Programmed Cell Death Caused by Powdery Mildew in Wheat
|School||Chinese Academy of Agricultural Sciences|
|Course||Biochemistry and Molecular Biology|
|Keywords||wheat powdery mildew wild relatives of wheat molecular marker programmed cell death (PCD) hypersusceptible response (HR)|
Powdery mildew caused by Blumeria gramini f. sp. tritici, is one of the most devastating diseases in common wheat （ Triticicum aestivum L.em Thell.）. In the long run, the most economic and environmental methods to control this disease is developing resistant cultivars. In view of high genetic variability of Blumeria graminis f.sp. tritici, the narrow genetic basis and limited effective resistance sources of common wheat cultivars. The objectives of the present studies were: 1) to identify and tag new powdery mildew resistance genes;2) to map the known gene Pmlc which has good resistance;3) to detect the programmed cell death （PCD） and cloning the PCD related genes of wheat after induced by powdery mildew in an attempt to get more information about resistance mechanism. The results obtained are as followed:A dominant major gene conferring resistance to powdery mildew, originating from accession Y201 of Ae. Tauschii, was identified in a F2 population derived from a cross between Ae. Tauschii acc. Y201 and Y2272. Six microsatellite markers Xgwm174, Cfd26, Cfd57, Cfd102, Xgwm639 and Xgwm583 were identified to link to the gene with the genetic distance of 5.2, 7.7, 9.6, 12.5, 20.2, 22.1 cM respectively. According to the location of the linked markers, the gene was located on the region of Ae. Tauschii chromosme 5DL. Based on the chromosomal location and the resisitant pattern of the gene, it was suggested that the gene should be a novel powdery mildew resistance gene. It was temporarily designated as PmY201.A dominant gene conferring resistance to powdery mildew, originating from accession Y150 of Ae.longissima, was identified in a population of Lanzhou9533/Y150F6//Lanzhou953F2. Four Ae.longissima-specific microsatellite markers Xwmc419, Barc60, Xgwm44, Cfd30 were identified to linke to the gene with the genetic distance of 4.2, 3.3, 3.3, 3.3 cM respectively. According to the location of the markers on wheat chromosome （Xwmc419, Barc60 on 4B, Xgwm44, Cfd30 on 4A） and the homology relationships of Ae.longissima and common wheat, the gene was probably located on the region of Ae.longissima chromosme 4S1. Using the four markers to amplified the wheat Chinese spring nulli-tetrasomic lines, it was suggested that the line Lanzhou9533/Y150F6 used in this study is a addition line. According to the chromosome location, the origin and the resisitant pattern, the gene should be a new powdery mildew resistance gene. Temporarily, it was named PmY150-2.Using the near-isogenetic lines （NIL） of wheat: Pm1c/Bainong32177 to screen the molecular markers linked to the known powdery mildew resistance gene Pm1c. The markers which showed polymorphism in iso-genetic lines were used to screen two F2 populations. The genetic map of Pmlc and the six linked molecular markers was constructed. One of these markers was microsatellite marker Barc121, the genetic distance was 1.2 cM;One AFLP marker P37M77 linked to Pm1c with the genetic distance of 0.4 cM;Three TRAP markers were W28T13, W24T10, W11T10 at 2.2, 6.1, 8.6 cM;The last one was an EST which located on 7A linked with Pmlc with the distance of 5.3 cM.Blwneria gramini f. sp. tritici （BgtE09）was used to inoculate the susceptible parent Bainong3217, two resistant NILs: Pm2/ Bainong 32177F5, Pm21l Bainong 32177F5 which possesses Pm2 and Pm21 genes respectively. The wheat leaves were detected by DAPI dyeing and TUNEL hybridization at 0, 3, 7dai （dai after inoculation）. There were evidently features of PCD in compatible （Bainong3217） and HR （Pm2/ Bainong 32177F5） interaction at 7dai. This showed that HR caused in wheat-powdery mildew interaction was a form of PCD. This also indicated that there was no necessary relationship between resistance and HR in the interaction system. But whether the mechanism of PCD occured was same in compatible and HR interaction still not clear.Cloned the cDNA sequence of three PCD related genes from wheat by homologue cloning method: TaLSDl, TaPDCD5, TaDAD. Expression analyses were further confirmed via RT-PCR after powdery mildew inoculation. The results were obtained:TaLSDl The full length cDNA sequence is 901bp. It was predicted that there was a complete opening reading frame （ORF） （441bp） including 147 amino acids , the presumed TaLSDl protein contains three conserved zinc fingers with the majority of the consensus CxxCxxLLMYxxGAxSVxCxxC, and has 95%, 57% and 85% identity with rice OsLSDl protein, arabidopsis LSD1 protein and LOL1 protein, respectively. RT-PCR result showed that TaLSDl is a negative PCD related gene in wheat.TaDADl The full length cDNA sequence is 586bp. It was predicted that there was a complete opening reading frame （ORF） （345bp） including 115 amino acids, the presumed TaDADl protein contains three transmembrane domains. The identities compared with the homology proteins of rice, arabidopsis, mouse and human’s are 93%, 86%, 51%, 51% respectively. The results showed that DAD1 gene is very conserved in evolution, implying that DAD1 gene is very important in development. RT-PCR result showed that TaDADl is a negative PCD related gene in wheat.TaPDCDS The full length cDNA sequence is 558bp. It was predicted that there was a complete opening reading frame （ORF） （396bp） including 132 amino acids. The identities compared with the homology proteins of rice, arabidopsis, mouse and human’s are 92%, 73%, 41% and 40% respectively. The RT-PCR result showed TaPDCDS is a positive PCD related gene in wheat.The new genes and the molecular markers linked to the powdery mildew resistance genes were discovered in this study enriched the resistance gene’s library and found a basement for fine mapping, positional cloning and markers- assisted selection （MAS） these genes. The studies on wheat PCD induced by powdery mildew and the related genes cloning were very important for the next study on resistance mechanism and plant PCD.