| Wheat(Triticum aestivum L.) is the second most important crops in the world, serving as staple food for 30% of the world’s population, and China is the largest country in the world’s wheat production. However, wheat production has been threatened by variety of adversity conditions. Wheat stripe rust, as one of the most important diseases on wheat, widely spread in the world, can cause 10–70 % crop loss commonly. China as the largest stripe rust epidemic area, there are about 4 million hectares of wheat were affected by stripe rust. In history, four times of national epidemics were broken out, every time had caused more than 1million tons of production losses. Growing resistant varieties is considered to be one of the most economic and effective approaches to control stripe rust. From 2001 to 2013, the main prevalent stripe rust pathotypes were CYR32 and CYR33. Therefore, the stripe rust resistance gene Yr26 which confer effective resistance to these two races was widely used in wheat breeding. While the pathogens could escape the identification of resistance genes through virulence variation, which result in new epidemic. From the first time detected since 2008, the frequency of V26(a new virulence race of Yr26 resistance gene) was rising constantly. Up to2012, its frequency achieved 16.10%, only behind CYR32 and CYR33 in Longnan, The sum frequency of these three races up to 70%. Cultivars Chuanmai 42, Neimai 8, Neimai 9,Neimai 10, Neimai 11, Mianmai37, and so on which carry Yr26 gene become to susceptible cultivars of new race, which result in a high potential of nationwide epidemic of stripe rust.To solve these problems, on the one hand we want to clone Yr26 gene by map-based cloning,and through RNA-seq of near isogenic line of Yr26 gene inoculated with CYR32 to explore the molecular interaction mechanism of Yr26 gene and the stripe rust race CYR32. On the other hand, to make clear which genes still confer effective resistance to these three races, and though reasonable layout of resistance genes and predictability resistance breeding to reach the purpose of control disease by the using of host resistance. Therefore, at first this study built BAC library of 92R137(The donor of Yr26 gene), which lay a foundation for Yr26 gene map-based cloning. Then by RNA-seq of Yr26 nearly isogenic line inoculated with CYR32 to uncover resistance signaling pathways mediated by Yr26 gene, and get the sequence information which can be used in the development of Yr26 linked markers for fine mapping and function analysis of Yr26 gene. Finally, to respond to the present situation of newvirulence race emerge, 92 varieties with known resistance genes were inoculated with CYR32,CYR33 and V26 respectively, combined with field investigation, got the information of which resistance genes can be used in the production practice. These researches have achieved the following results:1) For map-based cloning of genes conferring important traits in the hexaploid wheat line 92R137, a bacterial artificial chromosome(BAC) library, including two sub-libraries,was constructed using the genomic DNA of 92R137 digested with restriction enzymes HindIII and Bam HI. The BAC library composed of total 765,696 clones, of which 390,144 were from the HindIII digestion and 375,552 from the BamHI digestion. Through pulsed-field gel electrophoresis(PFGE) analysis of 453 clones randomly selected from the HindIII sub-library and 573 clones from the BamHI sub-library, the average insert sizes were estimated as 129 and 112.6 kb, respectively. Thus, the HindIII sub-library was estimated to have a 3.01-fold coverage and the BamHI sub-library a 2.53-fold coverage based on the estimated hexaploid wheat genome size of 16,700 Mb. The 765,696 clones were arrayed in1,994 384-well plates. All clones were also arranged into plate pools and further arranged into5-dimensional(5D) pools. The probability of identifying a clone corresponding to any wheat DNA sequence(such as gene Yr26 for stripe rust resistance) from the library was estimated to be more than 99.6%. Through polymerase chain reaction screening the 5-D pools with Xwe173, a marker tightly linked to Yr26, six BAC clones were successfully obtained. These results demonstrate that the BAC library is a valuable genomic resource for positional cloning of Yr26 and other genes of interests.2) To explore the molecular interaction mechanism of Yr26 gene and the stripe rust race CYR32, this study carried on the RNA-seq of near isogenic line of Yr26 gene inoculated with CYR32 in six time points. After quality control, about 2.9 billion reads and 365 G bp of the clean data were obtained. All of the clean data was mapped to the Chinese spring reference genome, there are 68985 expression genes identified among all of the 112496 genes in the reference genome. Based on the contrast of the same time point post inoculation between NIL-R and NIL-S, a total of 14449 significantly differentially expressed genes(DEG) were identified. Among these DEG, most of the genes which part in the photosynthesis signaling pathway were down-regulated expression, the genes which belong to the hormone metabolism had different expression patterns, while most of the genes which participated in the defense response were up-regulated expression. This work provides novel and comprehensive perspectives and establishes bases for questions that should be explored in next step.3) To evaluate which resistance genes are still effective to the prevalent pathotypes, 92 cultivars with known resistance genes were inoculated with prevalent stripe rust pathotypesCYR32, CYR33 and V26(Pingnan17-5) separately in both the seedling stage and the field test in Yangling and Tianshui. The result showed that the single gene Yr1,Yr2, Yr6-Yr8, Yr17,Yr18, Yr21, Yr27-Yr29, Yr31, Yr36, Yr39, Yr41, Yr43, Yr44, YrA, YrExp2, YrSP and the Yr-gene combinations in the cultivar Joss Combier, Heines Ⅶ, Heines Peko, Strubes Dikkopf,Capelle Desprez, Stephens, Fielder, Heinese Kolben, Clement, Paha were susceptible in all of the tests. Among all of the tested, three genes Yr5, Yr15 and Yr61 confer effective resistance in all the test, had all-stage resistance(ASR). Three other genes Yr32、YrTr1 and YrTye which were resistant during all of the adult stage but susceptible in at least one test in the seedling stage were effective adult-plant resistance(APR) genes. Nine cultivars in carrying multigene,i.e., Mega, Ibis, Hyak, Maris Huntsman, Hobbit, Carstens V, Express, Lee and Compair also showed effective APR in this study. These three classes genes can be used in the practice. |
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