Fine Mapping And Pyramiding Of Resistance Genes And Expression Analysis Of The Resistance Candidate Genes To Soybean Mosaic 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 method for controlling this disease. The success and failure of breeding resistant varieties depend on resistant resources for local popular strains and inheritance of resistance. In China, SMV has been re-classified into21strains based on the reactions of SMV isolates in a set of soybean differentials. Therefore, the objectives of this study were to identify resistant resources, study the inheritance of resistance to the SMV SC8strains, fine mapping the resistance genes. For the candidate genes we will clone and sequence to get the resistance genes finally. In addition, by using quantitative real-time polymerase chain reaction （QRT-PCR） to analyze in order to obtain the final target genes and to lay the foundation for transgenic technology to improve soybean resistance.The main results were as follows:1. Analysis of inheritance of resistance genes:crosses of Kefeng No.1xNannong1138-2parents were made and selfed to construct F1, F2and V2.2populations. The individuals and populations were used to determine the inheritance for resistance to SMV widely distributed strain SC8. The results indicated that:after inoculation F1generation plants showed disease-resistant; the F2generation segregated to3resistant:1susceptible ratio. F2.3family segregated to1resistant:2segregate:I susceptible ratio, The results showed that the resistance of Kefeng No.1to SC8was controlled by a single dominant gene.2. Fine mapping of resistance genes:elected seven pairs of primers which contain BARCSOYSSR₀2₀596, BARCSOYSSR020602, BARCSOYSSR020610, BARCSOYSSR020616, BARCSOYSSR02₀618, BARCSOYSSR020621, BARCSOYSSR₀2₀727by a population of282F2plants was verified. Results indicated that the closest markers locating two sides of RSC8respectively were BARCSOYSSR020610and BARCSOYSSR020618. On this basis, SSR markers were used for fine mapping, a total of forty-three pairs of SSR primers were designed, thirteen of forty-three pairs of primers were polymorphic and ultimately select from four pairs of primers such as ZL-24, ZL-39, ZL-42and ZL-52for fine mapping research. Finally, the resistance gene RSC8was located between ZL-42and ZL-52, the genetic distance of the two marker was2.6cM, Sequence analysis of soybean genome indicted that interval between the two genomic-SSR markers was40kb.3. Clone and expression analysis of the resistance candidate genes:By using the bioinformatics and QRT-PCR, we found that there are five candidate genes in the RSC8region, two of them are not transcribed or encoded proteins which function are unknown. This study focuses on the three genes by bioinformatic analysis and quantitative real-time polymerase chain reaction to conduct. By bioinformatic analysis, we found that:these genes have speculated domains and functions, mainly contain zinc finger protein, MADS box protein and K-box region. The closest relationship with Glyma02g13400was Glyma01g08130which located in No.1chromosome and related genes in Kidney bean. Glyma02g13420and related genes in William82had closest genetic relatives, followed by related genes in beans, and related genes in alfalfa, the final spend for the related genes in monkey face, these were divided into a subgroup, members of the other subgroups of the gene had a distantly kinship; Glyma02g13430gene genetic relationship with the related genes in castor bean, followed by related genes in bean assigned to a subgroup, in the soybean genome database found that the gene belonged to the C3HC4type zinc structure. Using the QRT-PCR, we found Glyma02g13400, Glyma02g13420and Glyma02g13430genes had different levels of response induced by mechanical damage, Glyma02g13400and Glyma02g13420genes involved in resistance to SMV strain SC8in soybean.4. Study the pyramiding for SMV resistance genes:SMV strain SC4resistance gene RSC4in Dabaima, SC8resistance gene RSC8in Kefeng No.1and SC14resistance gene RSC14Q in Qihuang No.1were used as the donor parents for gene pyramiding. A series of F2, F3and F4lines derived from multi-cross and self-cross. On the basis of previous study, in this research we studied the F5lines derived from22F4lines by investigating the agronomic traits and marker-assisted selection, The results demonstrated that74F5plants which had favorable agronomic traits presumably carry all three SMV resistance genes for multiple and durable resistance to SMV, These may be useful in soybean resistance breeding.