Elimination of chromosomes constitution by in situ hybridization in sterile Allium species

通过原位杂交消除不育葱属物种的染色体构成

• 批准号: 07660030
• 负责人: OHI Michio
• 金额: $ 1.28万
• 依托单位: Faculty of Agriculture, Shinshu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07660030/
• 关键词: Allium grayi; FISH; Sterile strain; PCR; Chromosomes; cDNAライブラリー; In situハイブリダイゼーション; FISH法

Deference of chromosomes constitution between fertile strain and sterile strain in Allium grayi was researched by in situ hybridization. Probes for FISH were produced from genomic DNA in A.grayi.. The genomic DNAs extracted from young leaves were amplified by PCR using forty primers (Operon 10-mer Kits, A Kit 1-20, B Kit 1-20). From the result, Polymorphism's between two strains appeared in twenty primers. The polymorphic bands by electrophoresis were cut out and refined. These DNA fragments were amplified by PCR using the same probes. Twelve probes were retrieved by electrophoresis from 400 bp to 1800 bp. Obtained probes were screening by southern hybridization using ECL system of Amersham. However, No probes which were identified as fluorescent signals on the nitrocellulose filter. We are, presently, attempting selection of probes radiated and trying in situ PCR method, amplify the probes directly on the sample of somatic chromosomes.

利用原位杂交技术研究了灰葱可育系和不育系染色体组成的差异。从灰曲霉的基因组DNA中产生FISH探针。使用40个引物（Operon 10-mer试剂盒，A试剂盒1-20，B试剂盒1-20）通过PCR扩增从幼叶提取的基因组DNA。结果表明，在20个引物中，两个菌株之间出现了多态性。切下电泳多态性条带并进行提纯。使用相同的探针通过PCR扩增这些DNA片段。通过电泳从400 bp至1800 bp回收12个探针。使用阿默舍姆的ECL系统通过Southern杂交筛选获得的探针。然而，在硝酸纤维素过滤器上没有被鉴定为荧光信号的探针。目前，我们正在尝试选择辐射探针，并尝试原位PCR方法，直接在体细胞染色体样品上扩增探针。