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Molecular Mapping of Downy Mildew Resistant Gene and Expression of Rs-AFPs in Radish (Raphanus sativus L.)

Author: JiangQiuZuo
Tutor: GongYiQin;LiuLiWang
School: Nanjing Agricultural College
Course: Olericulture
Keywords: radish (Raphanus sativus L.) downy mildew molecular makers Rs-AFPs RT-PCR
CLC: S631.1
Type: Master's thesis
Year: 2011
Downloads: 6
Quote: 0
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Abstract


Radish (Raphanus sativus L.), which was originated from China, was an important vegetable belonging to Brassica famiy. It had a long cultivation history and abundant germplasms in China. According to the statistics in 2006, the cultivation area of radish, which was valuable in edible and economy, was at the second place among all the vegetables’ cultivation area of our country. Downy mildew (DM) is a worldwide discontrustive disease of Brassicaceae vegetables induced by the (Oomycete Peronospora parasitica). Recently, the occurrence of radish downy mildew was seriously across the whole growing stage especially under the suitable temperature and in the humid environment, which affected the yield, quality and breeding of radish. So that, resistance breeding to downy mildew became an important aim in radish breeding work. Recently, the research of downy mildew in Brassicaceae vegetables was focused on brassica vegetables, such as cabbage, lettuce, Chinese cabbage, and Broccoli. The research of resistance to downy mildew in radish had just started and the study of physiological, inheritance, gene cloning were also just at the initial stage, which established the foundation for the development of resistance breeding to downy mildew and the new varieties selection of radish. To improve of breeding efficiency, we need to identification of markers tightly linked to downy mildew resistance gene in radish which we can select the genotype directly.F2 population derived from cross NAU-dhp08 (resistance parent)×NAU-qtbjq-06 (susceptibility parent) were inoculated at the seedling stage using an artificial inoculation method. The resistance is controlled by a single dominant gene and has no maternal effect. Bulked segregant analysis (BSA) was used to discover new candidate markers linked to the DM gene. Bulked DNAs were prepared from equal volumes of standardized DNA of 10 resistant and 10 susceptible F2 plants. RAPD primers, EST-SSR, SRAP, ISSR and RGA primer combinations were used to screen resistant and susceptible bulks. The most closed marker distance 2.3 cM (Em9/ga24370) from DM resistance marker.The genomic DNA and cDNA sequence was amplified by from DNA of the leaf of NAU-dhp08. The gene was designated as Rs-AFP1, Rs-AFP2, Rs-AFP3 and Rs-AFP4. Amipilificed fragment of genomic DNA sequences which contain of two exons and one introns. BLAST analysis in NCBI database suggested that Rs-AFPs is highly homologous with Brassica. Semi-quantitative RT-PCR analysis showed that expression of Rs-AFP 3 and Rs-AFP 4 was detectable in the leaf. The Rs-AFP3 was up-regulated at 24h after P. parasitica was inoculated, while it was down-regulated in the plant without inoculation and other phases post-inoculation. The Rs-AFP4 was up-regulated at 6h and 36h after P. parasitica was inoculated, while it was down-regulated in the plant without inoculation and other phases post-inoculation. This research can provide Theoretical Basis of biological preparation and using of Antimicrobial peptide.

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CLC: > Agricultural Sciences > Gardening > Vegetable gardening > Root vegetables ( taproot class ) > Radish
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