| Agropyron cristatum(L.) Gaertn.(2n = 28, PPPP), a wild relative of common wheat, possesses a large number of useful agronomical traits for wheat genetic improvement, such as resistance to powdery mildew, barley yellow dwarf virus, leaf rust, stripe rust and stem rust, tolerance to drought and low temperature, as well as more fertile tiller numbers per plant, spikelets and florets than wheat. The wheat-A. cristatum alien translocation lines could be useful for molecular cytogenetic study and used as an important bridge material to successfully transfer desirable gene to wheat for genetic improvement. In this study, the wheat-A. cristatum alien 2P disomic addition line was utilized to produce wheat-A. cristatum 2P alien translocation lines and A. cristatum 2P deletions induced by 60Co-γ irradiation. The wheat-A. cristatum alien translocation lines were characterized by genomic in situ hybridization(GISH), fluorescent in situ hybridization(FISH), EST-STS molecular markers analysis and agronomic traits evaluation for better utilization and more accurate mapping of the novel excellent gene on A. cristatum chromosome 2P. The main results are as follows:1. Production and GISH identification A. cristatum chromosome 2P structural aberrations. The wheat-A. cristatum alien 2P disomic addition line was used to produce wheat-A. cristatum 2P structural aberrations induced by 60Co-γ irradiation for the first time. Thirty-nine wheat-A. cristatum 2P alien translocation lines and six A. cristatum 2P deletions were detected by GISH. There were18, 20, 8 and 15 translocated chromosomes involving large alien segmental translocations, small alien segmental translocations, intercalary translocations and whole arm translocations, respectively. Of the total 69 breakage-fusion events, 54 ones involved interstitial regions and 15 ones involved centric regions. All different types of wheat-A. cristatum alien translocation lines would be valuable for not only identifying and cloning A. cristatum 2P-related genes and understanding the genetics and breeding effects of the translocation between A. cristatum chromosome 2P and wheat chromosomes, but also providing new germplasm resources for wheat genetic improvement.2. FISH analysis of the wheat-A. cristatum alien 2P translocation lines. The twice hybridization of two color FISH-GISH showed that the A. cristatum chromosome 2P was translocated to wheat ABD genome. Thirty-six types of translocated chromosomes were identified in the twenty-eight wheat-A. cristatum alien chromosomal translocation lines. A. cristatum chromosome 2P fragment was translocated to the wheat chromosomes 1A, 2A, 3A, 4A, 5A, 6A, 7A, 1B, 3B, 5B, 7B, 1D, 3D, 4D and 6D, respectively.3. Construction a molecular marker map of A. cristatum chromosome 2P. Seventeen 60Co-γ ray irradiation-induced wheat-A. cristatum alien 2P translocation lines and three A. cristatum 2P deletions with different fragment sizes and breakpoints derived from wheat-A. cristatum 2P disomic addition lines II-9-3 were analyzed using 83 chromosome 2P-specific EST-STS markers. After combining marker results with chromosome aberrations in each line, a fine physical map of A. cristatum chromosome 2P was developed which included thirty-four markers in nine bins of the short arm and forty-nine markers in eight bins of the long arm. The fine physical map of chromosome 2P developed in this research would lay a solid foundation for fast and accurate screening large numbers of samples. It played an important role in both theory and practice for transferring desirable genes designedly and developing novel translocation lines directly.4. Physical mapping a novel powdery mildew resistance gene on A. cristatum chromosome 2P. A mixture of the current prevalent Bgt isolates mainly composed of E09 and E20 were used to inoculate seven BC1F2 populations at the adult stage, it was found that the progenies of whole arm translocation line 2P-205 containing whole 2P chromosome long arm and two small segment translocation lines 2P-36 and 2P-173 corresponding to the FL 0.60-1.00 and 0.47-1.00 region of 2PL were highly resistant to powdery mildew in translocation individuals, while the others were powdery mildew susceptible in non-translocation individuals in BC1F2 populations; the progenies of whole arm translocation line 2P-43 containing whole 2P chromosome short arm and three small segment translocation lines 2P-35, 2P-122 and 2P-167 corresponding to the FL 0.00-0.60, 0.86-1.00 and 0.37-0.66 region of 2PL were all highly susceptible to powdery mildew whether in translocation individuals or in non-translocation individuals. Based on the resistance evaluation, cytogenetic and molecular marker analysis of the translocation lines, the novel gene conferring powdery mildew resistance was physically mapped to the FL 0.66-0.86 region of 2PL and twenty 2P-specific markers could be detected in this region, indicating that they could be used for marker-assisted selection in wheat breeding. The developed translocation lines 2P-36, 2P-173 and 2P-205 with powdery mildew resistance and co-dominant molecular markers would be valuable for the utilization in wheat powdery mildew resistant breeding program as well as further gene cloning.5. Physical mapping a novel leaf rust resistance gene from A. cristatum. Resistance evaluations of two BC1F2 populations with a mixture of the Puccinia triticina Eriks. isolates were performed at the adult stage in Xinxiang, Henan. It was found that the progenies of whole arm translocation line 2P-205 containing whole 2P chromosome long arm were immune to leaf rust in translocation individuals, while the others were highly susceptible to leaf rust in non-translocation individuals in BC1F2 populations; the progenies of whole arm translocation line 2P-43 containing whole 2P chromosome short arm were all powdery mildew susceptible whether in translocation individuals or in non-translocation individuals. Based on the resistance evaluation, cytogenetic and molecular marker analysis of the translocation lines, the novel gene conferring leaf rust resistance was physically mapped to the long arm of A. cristatum chromosome 2P. The developed translocation line 2P-205 with leaf rust resistance and other translocation lines with different fragment sizes and breakpoints of A. cristatum chromosome 2P long arm in this study would lay a foundation for the utilization in wheat breeding for leaf rust resistance as well as further cloning of the novel gene conferring leaf rust resistance.6. Physical mapping of important agronomical genes from A. cristatum. Phenotypic identifications of two BC1F2 populations were carried out. Variance analysis showed that the flag leaf length and width of the progenies of whole arm translocation line 2P-205 containing whole 2P chromosome long arm in translocation individuals were shorter and narrower than that of non-translocation individuals and all progenies of whole arm translocation line 2P-43 containing whole 2P chromosome short arm; but the highest spikelet density, which indicated that A. cristatum 2P chromosome long arm played an important role in regulating flag leaf length, width and spikelet density. The newly developed wheat-A. cristatum 2P alien translation lines could not only lay the foundation for the localization of A. cristatum excellent genes, but also provide bridge material for high-yield wheat breeding. |
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