Molecular Mapping And Allelism Test Of Leaf Rust Resistance Gene In Chinese Wheat Lines

Leaf rust, caused by Puccinia triticina, is an important wheat disease in many countries worldwide. It is adapted to a wide range of environments, occurs wherever wheat is grown, and can cause significant yield losses. As the global climate warms, temperature conditions in the future will be more suitable for the occurrence and epidemic of wheat leaf rust. Planting resistant wheat cultivars is an efficient, economic and environmentally safe means to reduce the damage caused by leaf rust. To use the leaf rust resistance genes for controlling leaf rust, it is very impont to continually identify and tag new wheat leaf rust resistance genes.Many analytical method for rust resistance genes were used in researching wheat leaf rust, the appropriate methods can be selected according to the different test conditions and research purposes. It has proven that the EST and SSR markers are very effective in the assisted breeding for the wheat leaf rust resistance genes. The Chinese wheat line Zhou 8425B developed in 1984 is still resistant to leaf rust, stripe rust and powdery mildew under field conditions in China. Previously, we identified the dominant resistance gene LrZH84 in Zhou 8425B, and it was linked to SSR markers gwm582 and barc8 with genetic distances of 3.9 and 5.2 cM, respectively. The objectives of this study were to fine map LrZH84 in a large F₂ from Zhou 8425B/Chinese Spring using EST and STS markers as a prelude to its cloning and possible use in more precise marker assisted selection, and to test the allelism of LrZH84 with Lr44, LrG98 and LrXi. The two Chinese wheat (Triticum aestivum L.) lines Tian 95HF₂ and SW8588 showed high resistance to most of Chinese current Puccinia triticina pathotypes at the seedling stage. It is very important to identify leaf rust resistance genes in these lines for breeding wheat cultivars with durable resistance. In this study, some researches were conducted on Tian 95HF₂ and SW8588. The main results showed as follows:1. Zhou 8425B, Chinese Spring, and their F₂ populations of 2086 were tested for wheat leaf rust resistance with Chinese Puccinia triticina races THTT in greenhouse. The results showed that the resistance of Zhou 8425B was controlled by one dominant gene to Puccinia triticina race THTT. A total of 70 pairs of EST primers were used to test the parents and their resistant and susceptible bulks. Results indicated that the resistance gene LrZH84 was closely linked to the three known EST loci on the 1B with genetic distance ranging from 0.7 cM to 1.7 cM. The two closest linked EST loci flanking the resistance gene were BE497107-1B and BF474863-1B, with genetic distances of 0.7 cM and 0.7 cM, respectively. 2. Based on the sequences of PCR fragments amplified with EST marker BF474863, an STS marker, Hbsf-1, was designed. It generated a polymorphic band with an expected size of 1,006 bp in the resistant parent and resistant bulk. DNA alignments showed that the sequences of PCR products generated by Hbsf-1 were consistent with those produced from the corresponding EST marker. When Hbsf-1 was used to genotype the F₂ population, the results were identical with those obtained with the corresponding EST marker BF474863.3. STS marker Hbsf-1 was tested on 40 Thatcher NILs and genetic stocks with different resistance genes, and a 1,006 kb PCR fragment was produced only in line RL6078 (Lr26). Because that Lr26 and LrZH84 are both present in the Zhou 8425B and their loci are very closely linked, the STS marker also can be used to detect Lr26 in these lines. The STS marker was tested on 38 cultivars and lines derived from Zhou 8425B. The STS marker was detected in 31 resistant lines, but was not present in 6 susceptible lines. Resistant line Pu 96105 did not have the specific band for LrZH84, indicating that the line may carry a different gene for resistance to THTT or it could be a recombinant. These results indicated that the STS marker Hbsf-1 is effective in detecting LrZH84 in lines derived from Zhou 8425B.4. To test the allelism of Lr44 and LrZH84, pathotype PHTS was used to inoculate 606 F₂ plants from Zhou 8425B/RL6147 (Lr44) and the two parents. The results showed that RL6147 (Lr44) and Zhou 8425B was resistant (IT 1 or 2), and the F₂ population segregated with 605 resistant plants (IT 0-2) and 1 susceptible plant (IT 4), indicative of close linkage between Lr44 and LrZH84, and the genetic distance between Lr44 and LrZH84 was estimated to be 8.19±2.4 cM.5. All 837 F₂ plants from Xinong1163-4/Zhou 8425B and 838 F₂ plants from Guizhou 98-18/Zhou 8425B were highly resistant to THTT (IT 0;-1), the maximum recombination between each of two genes and LrZH84 was estimated as 0.0036 at P=0.05, indicating that LrXi, LrG98 and LrZH84 were likely to be allelic or closely linked.6. In seedling tests with pathotype THTT, 145 F₂ plants from Zhoumai 11/Chinese Spring segregated in 137 with IT 0; to 2 (resistant) and 8 with IT 3 to 4 (susceptible) (χ215:1 = 0.133, 1df, P>0.5), indicating two dominant resistance genes in Zhoumai 11. Two markers (gwm582,ω-secalin/Glu-B3) closely linked with LrZH84 were therefore used to test the 8 susceptible plants. All susceptible plants contained Chinese Spring alleles, indicating that one resistance gene in Zhoumai 11 was likely to be LrZH84. In another seedling test, Zhou 8425B and 285 F₂ plants from Zhoumai 11/Chinese Spring were inoculated with pathotype FHTT. The F₂ population segregated monogenically. Two markers (gwm582,ω-secalin/Glu-B3) closely linked with LrZH84 were used to test all the F₂ plants, and the data showed that the gene was located at a different locus with LrZH84. It was therefore concluded that LrZH84 was susceptible to pathotype FHTT and that Zhoumai 11 carried a different unknown resistance gene.7. A total of 232 F₂ plants from Xinong 1163-4/Thatcher were inoculated with pathotype FHTT. The F₂ population segregated monogenically. Markers gwm582 andω-secalin/Glu-B3 were used to test all F₂ plants of Xinong 1163-4/Thatcher, and the data showed that the gene was linked to the markers and likely to be LrXi. Based on these data it was evident that LrXi was different from LrZH84.8. The wheat (Triticum aestivum L.) line Tian 95HF₂ showed in low infection types to 16 Chinese current pathotypes of Puccinia triticina at seedling stage and the reaction patterns of Tian 95HF₂ were different from the known Lr resistance genes used in the test.Based on the result from molecular mapping Tian 95HF₂ carried the 1BL·1RS translocation and contain the leaf resistant gene Lr26. F₂ population from the cross of Tian 95HF₂ and Zhengzhou 5389 was inoculated with pathotypes FHTT and PHTS in greenhouse. In seedling tests with pathotype FHTT, only one resistance gene was detected and in another seedling test with pathotype PHTS two resistance genes were indetifid in Tian 95HF₂. The result from molecular mapping indicated that the other two genes in Tian 95HF₂ were Lr1 and LrZH84.9. SW8588 and 30 differential lines with known leaf rust resistance genes were inoculated with 15 pathotypes of Puccinia triticina for postulating leaf rust resistance genes in the line. Based on the result from the gene postulation SW8588 did not contain the known leaf rust resistance gene Lr1.When inoculating with pathotype FHTT, only one resistance gene was detected in the resistance lines. Using genetic analysis and molecular markers it could be found that SW8588 carried a single dominant resistance gene, temporarily designated LrSW85, co-segregating with STS marker WR003, which may be an allele of Lr1 or was closely linked to Lr1.