Inheritance, Fine Mapping And Expression Of Resistance Genes To Soybean Mosaic Virus In Soybean

Soybean mosaic disease, caused by soybean mosaic virus （SMV）, is one of the most broadly distributed viral diseases worldwide in soybean [Glycine max （L.） Merr.]. It causes yield loss and seed quality deficiency seriously. Utilization of resistant varieties is the most economical and environmentally safe approach to controlling this disease. The success and failure of breeding resistant varieties depend on resistant resources, the realization of inheritance mechanisms of resistance and molecular mapping of resistance genes to SMV strains.In China, SMV has been classified into21strains based on the reactions of SMV isolates on a set of soybean differentials. The objectives of this study were to identify resistant resources, to study the inheritance of resistance to the newly identified strains, to fine mapping the resistance genes, and to study feasibility of pyramiding of resistance genes for SMV using tightly linked molecular markers. Meanwhile, the mRNA expression profiles were analyzed by quantitative real-time polymerase chain reaction （QRT-PCR） in order to insight the resistance mechanism, to determine the soybean functional resistance genes. These will provide basis to cloning of resistance genes and improvement of the resistance of soybean through transgenic technique. The main results were as follows:1. Identification of resistant resourcesIn order to provide resistant sources for breeding resistant varieties to SMV strains, the resistance to SMV of334cultivars from the soybean regional test in2004-2007was evaluated after inoculation with two SMV prevalent strains, i. e. SC3and SC7. The results showed that148（44.31%） and71（21.26%） cultivars were resistance to strains SC3and SC7respectively,55（16.47%） cultivars were resistance to both SC3and SC7. These resistance cultivars can not only be used directly in soybean production, but also be used as resistance resources in breeding programs. The study also showed that the cultivars from Northwest China and Huang-Huai-Hai valleys were more resistant to SMV than those from the other regions.2. Analysis of inheritance and allelism of resistance genesCrosses of resistant （R） and susceptible （S） parents were made and selfed to construct F1, F2and F2:3populations. They were used to determine the inheritance for resistance to SMV strain SC8. All F1showed resistant, F2generation segregated in3resistant:1susceptible ratio, and1R:2Seg.:1S in F2:3families. The results indicated that Kefeng No.1, PI96983, Qihuang No.1, Dabaima, Jinda74, Fendou56, Zhongzuo229, and NY58carried single resistant gene for SC8strain. Reciprocal crosses were made with Qihuang22and Nannong1138-2to investigate the inheritance of resistance to SC8strain. No cytoplasmic effect was found in the two kinds crosses. It indicated that Qihuang22carried a dominant resistant gene. The test of the allelism of the resistance genes showed that the single dominant gene in Jinda74and Fendou56were alleles or very closely linked; the resistance genes between Qihuang No.1and Kefeng No.1, between Kefeng No.1and Fendou56were not at same locus.Ten soybean cultivars with resistance to SMV strain SC4, was crossed respectively with a susceptible cultivar （Nannong1138-2or8101） to determine the inheritance of their resistance reaction to SC4strain. Each R parents were also crossed with each other to test the allelism of the resistance genes. The results showed that a dominant gene controlled the resistance to SC4strain in each of Ji LD42, Kefeng No.1,Xudou No.1, Jinda74, P196983, Qihuang22, Yuejin No.4, and Fendou56, respectively. Reciprocal crosses were made with Dabaima and Nannong1138-2to investigate the inheritance of resistance to SC4. The result indicated that Dabaima carried single dominant resistant gene, no cytoplasmic effect was found in the two kind’s crosses. The test of the allelism of the resistance genes showed the resistance genes between Dabaima and Fendou56, Kefeng No.1, Qihuang No.1, Zaoshul8; between Ji LD42and Fendou56; between Jinda74and Zhongzuo229were not at same locus.3. Fine mapping for SMV resistance genesIn order to fine mapping the resistance gene to SMV strain SC8in Kefeng No.1, F2population （156individuals）, recombined inbred lines （RIL） population （184families）, F2 of secondary population （SP-F2,181individuals） of Kefeng No.1×Nannongl138-2were evaluated following inoculation with SC8and analyzed by simple sequence repeats （SSR）, single nucleotide polymorphisms （SNP） and genomic-SSR markers. Results indicated that the resistance gene （designated as RSC8） in Kefeng No.l was located on soybean chromosome2（MLG D1b）, closest markers locating two sides of RSC8respectively were BARCSOYSSR₀2₀610and BARCSOYSSR₀2₀616. The genetic distance of the two markers was0.32cM. Sequence analysis of soybean genome indicted that interval between the two genomic-SSR markers was less than200kb.By applying bulked segregate analysis （BSA）, two genomic-SSR markers （BARCSOYSSR₁4₁413and BARCSOYSSR₁4₁416） on soybean chromosome14（MLG B2） closely linked to SMV strain SC4resistant gene （designated as Rsc4） were identified in1047plants F2population of Dabaima×Nannongl138-2. The Rsc4was located at a distance of0.17cM from BARCSOYSSR141413and0.27cM from BARCSOYSSR₁4₁416（on the other side）. Sequence analysis of soybean genome indicted that interval between the two genomic-SSR markers was less than110kb.4. QRT-PCR analysis of the resistance candidate genesThe analysis of bioinformatics and QRT-PCR of the candidate genes in the RSC8region indicated that the expression level of the eleven genes were different at some time points in the post inoculated with SC8. The eight genes maybe involved in resistance to SMV strain SC8in soybean. Seven candidate genes for Rsc4were identified by QRT-PCR and their expression levels were different between the two parents （Dabaima and Nannong1138-2）. Based on the analysis of above, we inferred that candidate genes Glyma02g13310,13320,13400,13460,13470and Glymal4g38510,38560,38580, and39300could be key genes to activating plant defense pathways in response to SMV infection.5. Pyramiding for SMV resistance genesThe purpose of the research was to pyramid resistance genes RSC14Q, RSC8and RSC4to SMV using marker assisted selection （MAS） and traditional breeding methods. Qihuang No.1, Kefeng No.1and Dabaima were used as the donor of resistance genes RSC14Q, RSC8and RSC4, respectively. A set of F2, F3and F4populations derived from multi-cross and self-cross of （Qihuang No.1×Kefeng No.1）x（DabaimaxNannong1138-2） were developed for selecting individuals carrying three resistant genes. Nine molecular markers linked to the three SMV resistance genes were used for MAS. The results indicated that twenty F4plants carry all three SMV resistance genes were confirmed are resistant to three SMV strains （SC14, SC8and SC4） by inoculation. The results confirmed that the feasibility of pyramiding of resistance genes for SMV in soybean by MAS.