| Maize is one of the most important crops in China which can be used as food, feed and the raw materials of industry. With the development of agricultural technology and the breeding of high yield varieties, the production of maize goes up steadily. Maize diseases and insect pests have become a major limitation of the yield increasing. The maize ear rot has been widely concerned for wide range of incidence, great effects on grain yield and harmful secondary metabolites. There are many research groups working on this disease about the anti-disease mechanism and the QTL mapping. A number of domestic and foreign research team carried out the research of resistance analysis and QTL mapping of maize ear rot. However, the mechanism of resistance to maize ear rot is complex and this disease is greatly influenced by environmental factors, and there is no fine mapping population and high density linkage map, therefore, study of maize ear rot are still concentrated in the preliminary QTL mapping, which has a large confidence interval. For these, the major resistance gene has not been cloned. This restricts the breeding process of maize ear rot high resistant varieties. In this study, we got two resistant materials and a susceptible material from maize chromosome segment substitution lines based on a multi-years field artificial inoculation identification of the resistance. We sequenced these three materials and two parents by SLAF-seq technology, in order to obtain the sequencing results as a basis for developing specific resistance molecular markers and mining candidate genes, and qRT-PCR is used to validate the candidate gene. The results of this study are as follows:(1) Based on the simplified genome sequencing, we got 6866 polymorphic markers evenly distributing on 10 chromosomes of maize. These markers can represent the whole maize genome, so as to achieve the purpose of simplifying the genome. Through quality controlling and screening,1845 homozygous markers are different between the two parents, including 1642 SNP markers,157 InDel markers and 46 mutational restriction sites. Based on these 1845 markers, we confirmed the genotype source and drew the map of genotypic source distribution.(2) According to the source of genes of three chromosome segment substitution lines (CSSLs), one enrichment region of donor genotype was identified on chromosome 1, one on chromosome 3 and one on chromosome 6, respectively. All the CSSLs have the same genetic background with the recipient parent. The results showed that, the differences in these three sections among CSSLs are the critical factors, which resulted in different resistance to ear rot. It was suggested that there were resistant genes associating with maize ear rot in the three sections.(3) We developed specific molecular markers in candidate region on chromosome 3 and chromosome 6. We got ten polymorphic markers among parents, including seven known and three new markers. We validated these ten markers using genome DNA of parents and three CSSLs by PCR. The results are similar to sequencing results.(4) On the basis of analysis of candidate regions and the annotation of gene function, we got two candidate genes in the hot region on chromosome 3. We validated these two candidate genes using qRT-PCR. The results showed that the expression of two candidate genes were significantly up-regulated after artificial inoculation, indicating that the two candidate genes are related to the response against invasion of pathogen. |
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