Discrimination of very closely-related insect species by molecular biological techniques

通过分子生物学技术区分非常密切相关的昆虫物种

• 批准号: 10556008
• 负责人: ISHIKAWA Yukio
• 金额: $ 8.51万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10556008/
• 关键词: Asian Corn Borer; RAPD-PCR; Species-specific primers; mitochondrial DNA; Ostrinia; ミトコンドリアDNA; 系統解析; 姉妹種; 種群

A handy molecular biological method to discriminate the Asian corn borer and its 7 allied species (Ostrinia furnacalis, O. orientalis, O. scapulalis, O. zealis, O. zaguliaevi, O. latipennis, O. palustralis and O. nubilalis) was sought, (1) Ostrinia spp. were collected from various localities in Japan, and CO II region of the mitochondrial DNA was sequenced for comparison. When the sample number was increased, more species shared the same mtDNA haplotypes, causing the phylogenetic tree of Ostrinia species more complex. No species-specific sequence was obtained. (2) Therefore, RAPD-PCR method was adopted instead. Eighty 10mer primers (Operon Inc., OPA01 - 20, OPB01 - 20, OPK02 - 20, OPL01 - 20, OPN03 - 09) were used to perform RAPD-PCR. Of the 80 primers examined, 73 amplified clear DNA bands. By using 20 individuals, sixteen primers were nominated as species-specific primers. A primer discriminated O. furnacalis, two primers discriminated O. palustralis, 10 primers discriminated O. latipennis, and one each primer discriminated O. orientalis and O. zealis, respectively. Regarding O. scapulalis and O. zaguliaevi, no species-specific primer has been found until now. By increasing the sample size to 52 individuals of 7 Ostrinia species, a primer, OPB-11, was confirmed to produce a species-specific band for O, palustralis and O. latipennis. With this primer, O. palustralis showed a 570bp band, while O. latipennis produced a 1250bp band.

寻求一种方便的分子生物学方法来区分亚洲玉米螟及其 7 个近缘物种（亚洲玉米螟、东方玉米螟、肩胛玉米螟、玉米螟、O. zaguliaevi、O. latipennis、沼泽螟和 O. nubilalis），(1) 玉米螟 spp。从日本各地收集，对线粒体DNA的CO II区进行测序以进行比较。当样本数量增加时，更多的物种共享相同的mtDNA单倍型，导致玉米螟物种的系统发育树更加复杂。没有获得物种特异性序列。 (2)因此改用RAPD-PCR方法。使用 80 个 10mer 引物（Operon Inc.、OPA01-20、OPB01-20、OPK02-20、OPL01-20、OPN03-09）进行 RAPD-PCR。在检查的 80 个引物中，73 个扩增出清晰的 DNA 带。通过使用 20 个人，16 个引物被提名为物种特异性引物。 1个引物可识别亚洲玉米螟，2个引物可识别沼泽玉米螟，10个引物可识别大叶螟，各引物分别可识别东方玉米螟和玉米螟。对于O. scapulalis和O. zaguliaevi，迄今为止尚未发现物种特异性引物。通过将样本量增加到 7 个玉米螟属物种的 52 个个体，引物 OPB-11 被证实能够产生 O、palustralis 和 O. latipennis 的物种特异性条带。使用该引物，O. palustralis 显示出 570bp 的条带，而 O. latipennis 则产生 1250bp 的条带。

项目成果

Kim,Hoshizaki,Huang,Tatsuki and lshikawa: "Usefulness of mitochondrial Coll gene sequences in examining phylogenetic relationships in the Asian corn borer, Ostrinia furnacalis, and allied species(Lepidoptera:Pyralidae)"Applied Entomology and Zoology. 34.

Kim、Hoshizaki、Huang、Tatsuki 和 lshikawa：“线粒体 Coll 基因序列在检查亚洲玉米螟、亚洲玉米螟和近缘物种（鳞翅目：螟虫科）系统发育关系中的有用性”应用昆虫学和动物学。

Kim, C., S.Hoshizaki, Y.Huang, S.Taisuki, Y.Ishikawa: "Usefulness of mitochondrial COII gene sequences in examining phylogenetic relationships in the Asian corn borer, Ostrinia furnacalis, and allied species (Lepidoptera : Pyralidae)"Applied Entomology an

Kim, C., S.Hoshizaki, Y.Huang, S.Taisuki, Y.Ishikawa：“线粒体 COII 基因序列在检查亚洲玉米螟、亚洲玉米螟和近缘物种（鳞翅目：螟虫科）系统发育关系中的有用性”

Ishikawa Y.,T.Takanashi and Y.Huang: "Comparative siudies on the sex pheromones of Ostrinia spp.in Japan the burdock borer Ostrinia zealis" Chemoecology. 9(印刷中). (1999)

Ishikawa Y.、T.Takanashi 和 Y.Huang：“日本玉米螟性信息素的比较研究”Chemoecology 9（出版中）。

Kim, C.-G., S. Hoshizaki, Y.-P. Huang, S. Tatsuki and Y. Ishikawa: "Usefulness of mitochondrial COII gene sequences in examining phylogenetic relationships in the Asian corn borer, Ostrinia furnacalis, and allied species (Lepidoptera: Pyralidae)."Appl. En

Kim，C.-G.，S. Hoshizaki，Y.-P。

Kim,C.,S.Hoshizaki,Y.Huang,S.Tatsuki and Y.Ishikawa: "Usefulness of mitochondrial COII gene sequences in examining phylogenetic relationships in the Asian corn borer, Ostrinia furnacalis, and allied species (Lepidoptera : Pyralidae)."Applied Entomology an

Kim,C.,S.Hoshizaki,Y.Huang,S.Tatsuki 和 Y.Ishikawa：“线粒体 COII 基因序列在检查亚洲玉米螟、亚洲玉米螟和近缘物种（鳞翅目：螟科）的系统发育关系中的有用性。