| Powdery mildew(Pm), caused by Blumeria graminis f. sp. tritici(Bgt), and stripe rust(Yr), caused by Puccinia striiformis f. sp. tritici(Pst), are the devastating diseases in common wheat(Triticum aestivum L.) worldwide. The breeding resistant varieties are the most feasible means of controlling the disease and reducing yield losses. To date, 57 Pm resistance genes and 59 Yr resistance genes have been identified. However, many resistance alleles become ineffective because of frequent changes in the pathogen population, especially when a single resistance gene is deployed over a wide area. Therefore, it is necessary to search for new sources of effective a nd durable resistance for resistance breeding.Thinopyrum intermedium and Th. ponticum are valuable sources for wheat improvement. They have excellent resistances for many wheat diseases. To transfer of disease-resistant genes from the two Thinopyrum species into common wheat, the productions of wheat-Thinopyrum partial amphiploids and wheat-Thinopyrum derivatives were cultivated in our laboratory. In this thesis, we focus our studies mainly on(i) evaluating the powdery mildew and stripe rust resistance of wheat-Thinopyrum derivatives;(ii) identifying the wheat-Thinopyrum derivatives by molecular and cytological analysis;(iii) determining the inheritance mode, chromosome location of the resistance genes in the three wheat-Thinopyrum derivatives. The main results of this study are summarized as follows:1. Cytological analysis of new multi-resistance Thinopyrum intermedium-derived partial amphiploids. Genomic in situ hybridization(GISH), using total genomic DNA from Pseudoroegneiria spicata as probe and C hinese Spring(CS) DNA as blocker were used. We found that TAI8331 contained 1 St chromosome, 2 J chromosomes, 3 JS chromosomes and 2 St/J chromosomes; TAI8332 contained 3 St chromosomes, 1 J chromosome, 2 JS chromosomes and 1 St/J chromosomes originated from Th. intermedium genomes.2. Resistance evaluations of 147 wheat-Thinopyrum derivatives to powdery mildew and stripe rust. Among them, 80 derived lines were immune to races E09, 66 lines were immune to races E21, 44 lines were immune to races CYR31 and 76 lines were immuneto races CYR32 in seeding plants. About 83 and 70 derived lines were immune to races E09 and CYR32 in adult plants, respectively. Other 20 derived wheat lines were immune to powdery mildew and stripe rust in whole growing period. 4 stable lines CH13-21, CH5383, CH5382 and CH7086 with excellent agronomic traits werescreened for further investigation.3. Molecular and cytological analyses of wheat-Thinopyrum derivatives CH13-21, CH5383, CH5382 and CH7086.(1)GISH analysis was performed on somatic chromosomes of CH13-21 using Th.intermedium genomic DNAas the probe and the CS as the blocker. A pair of Th. Intermedium chromosomes was detected in CH13-21. Multicolor-GISH(mc-GISH), multicolor- fluorescence in situ hybridization(mc-FISH) and PLUG markers analysis revealed that the CH13-21 contained the Th. intermedium segments involving B- genome chromosome. Therefore, the CH13-21 is a new T6BS?6Ai#1L compensating Robertsonian translocation line with high resistance to powdery mildew and stripe rust.(2)GISH of root-tip chromosome of CH5383 was probed by genomic DNA of Th. intermediumand blocked with CS genomic DNA. No translocated chromosome was visible in C H5383, suggesting that the size of Th. intermedium was possibly too small to be detected by GISH if the translocation presented. Strong hybridization signal in the distal region of wheat chromosome 3BL was observed in FISH using p Hv G38 containing the simple repeats(GAA)7 as a probe. 135 PLUG primer pairs were used to detect in CS, CH5383 and wheat-related species. The results showed that TNAC1383 displayed unique bands of CH5383 and Th. intermedium. CH5383 was a small wheat-alien introgression line and the alien Th. intermedium DNA segments were located in wheat bin 3BL-0.81-1.00 region.(3)GISH analysis using Th. intermedium genomic DNA as probe was conducted to determine the Th.intermedium fragment in CH5382. No signal was observed in mitotic metaphase of CH5382. This is similar to that of CH5383. Markers TNAC 1102 and TNAC1567, from wheat homologous group-2 short arm and group-5 long arms, respectively, generated additional bands in resistance donor TAI7044 and CH5382 compared to CS. Therefore, CH5382 may containsmall fragments introgressionof Th. Intermedium chromatin.(4)GISH using Th. ponticum genomic DNA as probe was performed on the line CH7086, no GISH signals were observed in CH7086 and Giemsa-C banding indicated that CH7086 contained typical wheat chromosomes without visible bands presenting Thinopyrum chromatin. FISH patterns used p Hv G38 containing the simple repeats(GAA)7 repeat as a probe. The chromosome 2BL arm in CH7086 displayed abundant hybridization bands with strong signals, in particular in the 2BL0.80-1.00 regions compared to CS. Therefore, CH7086 was also likely a cryptic wheat-Th. ponticum translocation line.4. Molecular mapping of the new stripe rust resistance gene Yr C H5383. To investigate the inheritance of stripe rust resistance, CH5383 was crossed to susceptible cultivars to yield segregating populations. The F1, F2 and F2:3 populations were tested for segregation of stripe rust resistance. Genetic analysis revealed that the resistance was controlled by a single dominant gene inoculating Pst race CYR32 in adult stage. SSR markers screening results showed that five polymorphic markers were linked to the resistance gene. Based on marker loci and the origination, Yr C H5383 might be a new gene towheat stripe rust on 3BL chromosome.5. Genetic analysis and molecular mapping of powdery mildew resistant genes Pm CH5382. The F1, F2 and F2:3 populations from across between CH5382 and susceptible lines were tested for segregation of powdery mildew resistance. Genetic analysis revealed that resistance of Th. Intermedium introgressions line CH5382 was controlled by only one dominant gene. Six markers were found linking to resistance gene Pm CH5382. Through the detection by nullisomic-tetrasomic and ditelosomic lines, the resistance gene Pm CH5382 was located on 1BL chromosome.6. Chromosomal location and comparative genomics analysis of new powdery mildew resistancegene Pm51. To study the location of resistance gene in CH7086, F2:3 population of C H5241/CH7086 was used for mapping the resistance gene. Five SSR markers were associated with resistance gene Pm CH86. Based on the locations of the markers by nullisomic-tetrasomic and deletion lines of CS, Pm CH86 was located in deletion bin 2BL-0.89-1.00. Conserved orthologous markers analysis indicated that the genomic region flanking the resistance gene has a high level of collinearity to that of rice chromosome 4 and Brachypodium chromosome 5. Due to origination, position, resistancespecificities and tests of allelism, it appeared that Pm CH86 was different from other known powdery mildew resistance genes on chromosome 2BL and represented a new powdery mildew resistance locus, and was therefore designated as Pm51. The closely linkedmarker BQ246670(1.5 c M distal) may be useful in exploiting this putative wheat-Thinopyrum translocation line for rapid transfer of Pm51 to wheat breeding programs.In summary, a new T6BS?6Ai#1L compensating Robertsonian translocation line and three cryptic wheat- Thinopyrum introgression lines with multi- resistance have been developed. These new wheat-Thinopyrum derivatives were helpful for further utilizing the wheat-related species to the wheat disease breeding. The new genes from Thinopyrum were analyzed by genetic analysis and molecular mapping. Among them, a new powdery mildew resistance gene carried by C H7086 was formally designated Pm51. Pm51 had independent intellectual property rights in C hina. Some molecular markers linked with resistance gene were developed by comparative genomics analysis. It will be beneficial for resistance breeding and fine genetic mapping of this target gene. |
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