Zhu H, Choi S, Johnston A K, Wing R A, Dean R A
Department of Plant Pathology and Physiology, Clemson University, South Carolina 29634, USA.
Fungal Genet Biol. 1997 Jun;21(3):337-47. doi: 10.1006/fgbi.1997.0996.
Magnaporthe grisea (Hebert) Barr causes rice blast, one of the most devastating diseases of rice (Oryza sativa) worldwide. This fungus is an ideal organism for studying a number of aspects of plant-pathogen interactions, including infection-related morphogenesis, avirulence, and pathogen evolution. To facilitate M. grisea genome analysis, physical mapping, and positional cloning, we have constructed a bacterial artificial chromosome (BAC) library from the rice infecting strain 70-15. A new method was developed for separation of partially digested large-molecular-weight DNA fragments that facilitated library construction with large inserts. The library contains 9216 clones, with an average insert size of 130 kbp (> 25 genome equivalents) stored in 384-well microtiter plates that can be double spotted robotically on to a single nylon membrane. Several unlinked single-copy DNA probes were used to screen 4608 clones in the library and an average of 13 (minimum of 6) overlapping BAC clones was found in each case. Hybridization of total genomic DNA to the library and analysis of individual clones indicated that approximately 26% of the clones contain single-copy DNA. Approximately 35% of BAC clones contained the retrotransposon MAGGY. The library was used to identify BAC clones containing a adenylate cyclase gene (mac1). In addition, a 550-kbp contig composed of 6 BAC clones was constructed that encompassed two adjacent RFLP markers on chromosome 2. These data show that the BAC library is suitable for genome analysis of M. grisea. Copies of colony hybridization membranes are available upon request.
稻瘟病菌（Magnaporthe grisea (Hebert) Barr）引发稻瘟病，这是全球水稻（Oryza sativa）最具毁灭性的病害之一。这种真菌是研究植物 - 病原体相互作用多个方面的理想生物体，包括与感染相关的形态发生、无毒力以及病原体进化。为便于稻瘟病菌的基因组分析、物理图谱构建和定位克隆，我们从水稻感染菌株70 - 15构建了一个细菌人工染色体（BAC）文库。开发了一种新方法用于分离部分消化的大分子DNA片段，这有助于构建大插入片段文库。该文库包含9216个克隆，平均插入片段大小为130 kbp（大于25个基因组当量），储存在384孔微量滴定板中，可通过机器人操作在单个尼龙膜上进行双点样。使用几个不连锁的单拷贝DNA探针筛选文库中的4608个克隆，每种情况下平均发现13个（最少6个）重叠的BAC克隆。全基因组DNA与文库杂交以及对单个克隆的分析表明，约26%的克隆含有单拷贝DNA。约35%的BAC克隆含有反转录转座子MAGGY。该文库用于鉴定含有腺苷酸环化酶基因（mac1）的BAC克隆。此外，构建了一个由6个BAC克隆组成的550 kbp重叠群，其涵盖了第2号染色体上两个相邻的RFLP标记。这些数据表明该BAC文库适用于稻瘟病菌的基因组分析。如有需要，可提供菌落杂交膜的副本。
