Kashiwa Takeshi, Kozaki Toshinori, Ishii Kazuo, Turgeon B Gillian, Teraoka Tohru, Komatsu Ken, Arie Tsutomu
United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology (TUAT), Fuchu, Tokyo 183-8509, Japan; Present address: Chemical Biology Research Group, RIKEN Center for Sustainable Resource Science (CSRS), Wako, Saitama 351-0198, Japan.
Graduate School of Agriculture, Tokyo University of Agriculture and Technology (TUAT), Fuchu, Tokyo 183-8509, Japan.
Fungal Genet Biol. 2017 Jan;98:46-51. doi: 10.1016/j.fgb.2016.12.001. Epub 2016 Dec 2.
A small chromosome in reference isolate 4287 of F. oxysporum f. sp. lycopersici (Fol) has been designated as a 'pathogenicity chromosome' because it carries several pathogenicity related genes such as the Secreted In Xylem (SIX) genes. Sequence assembly of small chromosomes in other isolates, based on a reference genome template, is difficult because of karyotype variation among isolates and a high number of sequences associated with transposable elements. These factors often result in misassembly of sequences, making it unclear whether other isolates possess the same pathogenicity chromosome harboring SIX genes as in the reference isolate. To overcome this difficulty, single chromosome sequencing after Contour-clamped Homogeneous Electric Field (CHEF) separation of chromosomes was performed, followed by de novo assembly of sequences. The assembled sequences of individual chromosomes were consistent with results of probing gels of CHEF separated chromosomes with SIX genes. Individual chromosome sequencing revealed that several SIX genes are located on a single small chromosome in two pathogenic forms of F. oxysporum, beyond the reference isolate 4287, and in the cabbage yellows fungus F. oxysporum f. sp. conglutinans. The particular combination of SIX genes on each small chromosome varied. Moreover, not all SIX genes were found on small chromosomes; depending on the isolate, some were on big chromosomes. This suggests that recombination of chromosomes and/or translocation of SIX genes may occur frequently. Our method improves sequence comparison of small chromosomes among isolates.
尖孢镰刀菌番茄专化型（Fol）参考菌株4287中的一条小染色体已被指定为“致病染色体”，因为它携带了几个与致病性相关的基因，如木质部分泌蛋白（SIX）基因。基于参考基因组模板对其他菌株中的小染色体进行序列组装很困难，这是因为菌株之间的核型存在差异，并且存在大量与转座元件相关的序列。这些因素常常导致序列组装错误，使得我们不清楚其他菌株是否拥有与参考菌株中一样携带SIX基因的致病染色体。为了克服这一困难，我们在通过轮廓钳位均匀电场（CHEF）分离染色体后进行了单染色体测序，随后对序列进行了从头组装。单个染色体的组装序列与用SIX基因探测CHEF分离的染色体凝胶的结果一致。单个染色体测序显示，除了参考菌株4287之外，在尖孢镰刀菌的两种致病形式以及甘蓝黄萎病菌尖孢镰刀菌菜豆专化型中，几个SIX基因位于一条单个的小染色体上。每条小染色体上SIX基因的特定组合各不相同。此外，并非所有SIX基因都位于小染色体上；根据菌株的不同，有些位于大染色体上。这表明染色体的重组和/或SIX基因的易位可能经常发生。我们的方法改进了不同菌株间小染色体的序列比较。
