Phylogeny of extinct wolves based on nucleotide saguence

本研究旨在对保存在不同机构的灭绝日本狼和野狗的骨骼和皮肤标本进行形态学检查。我们试图利用这些被毛样本进行线粒体DNA部分核苷酸序列的测定，以获得保存在荷兰莱顿博物馆的标本的形态学数据和一张皮肤样本。我们也可以从东京国立科学博物馆的北海道大学农学部制备皮肤样本。收集了近40个形态学数据和9个皮肤样本，对测量结果进行多变量分析，发现亚洲大陆的狼与北海道的Ezo狼接近，以皮肤样品DNA为模板，以线粒体DNA的细胞色素B基因为引物，用聚合酶链反应（PCR）对5个样品中的2个样品进行扩增，结果表明，5个样品中的2个样品均扩增出了DNA片段考察我们无法扩增其他3个样本的任何DNA。一些淡褐色物质可抑制DNA聚合酶。计算机分析表明，其中一个是未知真核生物的细胞色素B基因，另一个是人类的细胞色素B基因。我们将继续在PCR前通过各种方法纯化DNA样本。

The present study aimed morphological examination of skeletal and skin specimens of extinct Japanese wolves and wild dogs which have been kept in various institutions. We attempted to determine thenucleotide sequence of part of mitrochonrial DNA using those pelage samples.We could get morphological data and a piece of skin samples from the specimens kept at Leiden museum in the Netherlands. We could also prepare the skin samples from, Faculty of Agriculture of Hokkaido University, National Science Museum, Tokyo. Neary 40 morphological data and 9 skin samples were collected.Multivariate analyzes of the measurement revealed wolves in Asian continent are close to Ezo-wolves in Hokkaido, next is domestic dogs and these species are rather distant relationships with the Japanese wolves.Polymerase chain reaction using DNA from skin samples as template and primer set for cytochrome b gene of mitochondrial DNA could amplify DNA fragment from two specimens out of 5 examined. We could not amplify any DNA for other 3 samples. Some pale brownish substance (s) may inhibit DNA polymerase. Computer analyzes of the sequences revealed that one is cytochrome b gene of unkown eukaryote and the other is cyto-chrome b of human. We will continue to purify the DNA samples before PCR by various methods.