| Disease is one of the key factors which limits high and steady yield of wheat. Mining and cloning disease resistance genes and breeding resistant cultivars are the most economical, safe and effective approaches to control wheat diseases. Resistance gene analogues (RGAs) have a close relationship with disease resistance genes. RGAs not only can be used as molecular markers which play important roles in genetic map constructing, disease resistant breeding, germplasm resource genetic relationship analyzing, but also can be used as probes in genomic library screening and disease resistance genes cloning. The target of this study was to mine RGAs from D genome of wheat and to carry on research on the development and application of the RGAs markers. The main progresses were as follows:1. Most of the cloned disease resistance genes in plant contained five highly conserved domains.419 wheat RGA sequences were obtained from wheat EST databases in NCBI and TIGR using keywords such as NBS, NBS-LRR, STK, PK, CC, etc. By aligning these RGAs with wheat D genomic sequences, we got 201 best scaffolds. A total of 763 SSR primer pairs were designed based on these scaffolds which contained RGAs.2. A total of 763 RGA-SSR primer pairs were screened by using 20 pairs of parents. Among them,626 pairs could be amplified clearly and repeatability, which accounted for 82.04% of the total. The number of polymorphic primer pairs was 203, and the polymorphic primer frequency reached 32.43%. The average polymorphic frequency of these polymorphic primers was 8.2% in the 20 pairs of parents, with the highest value as 16.5% and the lowest value as 3.2%. The polymorphism of synthetic varieties was much higher than that of landraces and modern varieties.3. The polymorphic RGA-SSR distributed unevenly in five aspects, including different parents, the number of allele, major allele frequency, gene diversity index, and the polymorphism information content (PIC). The average PIC was 0.3442, with a maximum value as 0.699 and a mimimum value as 0.0866.The polymorphism of complex SSRs was higher than that of complete SSRs, and the longer the SSR sequnce was, the higher polymorphism the SSR marker obtained. (TC/AG)n and (AAC/TTG) n type RGA-SSR tends to present higher polymorphism than the other types.4. Three mapping populations (MOpop, NYpop, and ZYpop) were used and 101 pairs of polymorphic RGA-SSR primers (107 polymorphic loci) demonstrated repeated polymorphism among the mapping parents. Fifty four, thirty seven, and sixteen polymorphic loci were amplified from MOpop, NYpop, and ZYpop Respectively, At last, forty nine loci were located in Mopop linkage map, thirty three in Nypop linkage map and eleven in Zypop linkage map, sum to of 88 primers and 93 loci.5. Of the 93 mapped loci, nineteen, sixteen, and fifty eight were assigned to the A, B and D genome, respectively, with the most in genome D. The distribution of RGA-SSR loci among seven linkage groups was uneven with the most on group 1, then on group 2 and the least on group 6. Most of the mapped RGA-SSR makers were distributed in the chromosomes telomere or subtelomeric region, which appeared clustering phenomenon. These regions may be resistance gene-rich regions.6. In this study, the 93 mapped loci were adjacent to a total of seventeen resistance genes, which including three wheat powdery mildew resistance genes, Pm2, Pm3 and Pm34; five stripe rust resistance genes, Yr5, Yr15, Yr18, Yr24 and Yr45; seven leaf rust resistance genes, Lr10, Lr21, Lr22, Lr34, Lr39, Lr40 and Lr41; two stem rust resistance genes, Sr9 and Sr33. The genetic maps based on RGA-SSR markers can be used for both disease resistance gene cloning and marker asistant selection in disease resistance breeding. |
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