| Powdery mildew,caused by Blumeria graminis f.sp.tritici(syn.Erysiphe graminis f.sp.tritici),is one of the most important diseases of wheat(Triticum aestivum L.)worldwide.The most feasible means of controlling the disease and reducing yield loss is the breeding of resistant cultivars.So far,85 genes/alleles for resistance to powdery mildew at 61 loci have been identified and mapped on 19 different chromosomes,but only a few,including Pm3 b,Pm8,Pm21,Pm60,Pm2 a and Pm24,have been cloned due to the size and complexity of the wheat genome.Thinopyrum species,which are immune or highly resistant to powdery mildew,stripe rust and leaf rust of wheat,have been considered as excellent gene donors in wheat genetic improvement.Two common wheat lines CH1357 and CH006 recently developed in our laboratory are immune or highly resistant to major diseases including powdery mildew,and were derived from the hybrids between wheat and wheat-Thinopyrum amphiploid.This study was attempted to reveal the inheritance mode of the resistance to powdery mildew in these lines,to determine the chromosomal locations of and positionally clone their resistance gene(s),and to evaluate the validity and accuracy of the molecular markers closely linked to resistance genes used in marker-assisted breeding practice,which should be useful in facilitating the study of molecular mechanisms underlying disease resistance and greatly enhancing the efficiency of wheat resistance breeding.The main results are as the following:1.The preliminary mapping of Pm CH1357.CH1357 confers the excellent resistance to powdery mildew and is immunity or highly resistant to 27 Bgt isolates at the seedling stage.Genetic analysis of the F1,BC1,and F2:3 populations from two crosses(Taichang 29/CH1357 and Mianyang 11/CH1357)revealed that resistance was controlled by a single dominant allele,temporarily named Pm CH1357.The gene responsible for powdery mildew resistance was mapped by the linkage analysis of two segregated F2:3 populations.This gene was located on chromosome arm 5DS and flanked by SSR markers Xcfd81 and Xbwm8 at 2.0/1.5 and 11.3/8.9 c M,respectively.Pm CH1357 differs from other Pm genes reported on chromosome 5DS in the pedigree and resistance spectrum,and should be a new source of resistance.2.Evaluation for selection efficiency of the linked markers.Eight markers(Xcfd81?Xbwm20?Xbwm21?Xbwm25?Xmp510?Xbwm8?Xbwm9 and Xgwm190)associated with Pm CH1357 were ued for analysing the correlations between the PCR amplification products and the response to powdery mildew in the population from the cross between susceptible cultivar ‘Jinmai66' and breeding line CH1357.The result showed that the selection efficiency of four markers Xcfd81,Xbwm20,Xbwm21 and Xbwm25 exceeded 93%,which could be used in the select of resistance gene Pm CH1357 in breeding program.It was also found that using the flanked multiple marker combination could significantly improve the accuracy of marker-assisted selection for resistance gene,while the selection efficiency will be reduced when using the multiple marker combination on one side of the gene.Therefore,the combination of markers Xcfd81 and Xbwm8 had higher selection efficiency,and could be used in screening of the resistance gene Pm CH1357 thereby achieving of more plants carrying target genotype.3.Map-based cloning of Pm CH1357.Based on the preliminary mapping of Pm CH1357,the F2 plants and F2:3 families that were heterozygous at the resistance locus were chosen to generate a fine-mapping population of 2252 F3:4 plants.Using this population,Pm CH1357 was delimited in a physical interval of 526 kb containing Traes CS5D01G044600 only.The Traes CS5D01G044600 sequence of the susceptibility allele in susceptible parent Taichang 29(TC29)was identical to that in Chinese Spring(CS),whereas the sequence of the resistance allele in resistant parent CH1357 was identical to Pm2 a previously cloned from the germplasm Ulka/*8Cc.The susceptibility allele in TC29 contained a 7 bp deletion in exon 1,resulting in loss of 856 of the 1277 amino acids in the predicted nucleotide-binding domain leucine-rich repeat containing Pm2 a protein.Pm CH1357/Pm2 a sequence was also isolated from the Chinese wheat landraces and cultivars that were previously reported to possess the resistance gene Pm2 b,Pm2c,Pm LX66,or Pm ND399.The Pm CH1357/Pm2 a resistance allele was present in 10 of 495 accessions in core germplasm and contemporary cultivars from China and the USA.A newly developed diagnostic marker for the 7 bp In Del in the resistance gene could be used for marker-assisted selection in wheat breeding programs.4.Molecular mapping of Pm CH006.The wheat-Th.intermedium introgression line CH006 exhibited all-stage resistance to powdery mildew.To identify the resistance gene,genetic analysis was conducted using F1,BC1 and BC4F2:3 family populations derived from the cross of CH006 and the susceptible cultivar TC29.Segregation ratios from inoculation with Bgt isolate E09 indicated that the resistance was conferred by a single dominant gene,temporarily designated Pm CH006.In order to initially determine the chromosomal location of Pm CH006,A 90 K SNP array of 81,587 SNP markers was used for testing the parents and the contrasting DNA bulks,and found that 22 SNPs were putatively associated with the resistance phenotype,9(41%)of which were located on chromosome arm 2AL,suggesting that the resistance gene was located on 2AL.Subsequentely,thirteen new polymorphic markers were developed in 762-768 Mb,the corresponding genomic region of CS 2AL and used for testing the secondary population of BC4F2 consisting of 415 plants.Pm CH006 was placed at a nearby location of 762.318 Mb,and closely linked to proximal co-dominant SSR marker Xsnp10 at 0.7 c M.And four new polymorphic markers were then developed in the 761-762 Mb of corresponding physical region of CS 2AL and used to genotype the secondary population.The final linkage analysis delimited this gene to a 1.2 c M interval flanked by the developed markers SSR01/SSR04(distal)and 2AL-17(proximal)at 0.3 and 0.9 c M,respectively,covering about 1.0 Mb on 2AL in the CS reference sequence.Several other powdery mildew resistance genes/alleles,including Pm4a-e,pm50 and Pm65,have been mapped on 2AL,but it is unknown whether Pm CH006 was allelic with or linked to any of these genes.Further studies are needed to determine the relationship of Pm CH006 with the reported genes on 2AL,and its source. |
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