|Budget Amount *help
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1996: ¥6,900,000 (Direct Cost: ¥6,900,000)
Leaf samples immediately dried at the natural habitat of following species were used as samples to extract total DNA : Crawfurdia maculaticaulis, Gentiana algida, G.classicauris, G.rhodantha, G.yunnanense, Gentianopsis grandis, Tripterospermum distylum, T.hirticalyx, T.japonicum, T.japonicum var. involubile, T.lanceolatum. At the initial stage of this study rbcL or matK gene was intended to analyze but actually the inter transcribed spacer (ITS) regions of 18S-25S nuclear ribosomal DNA was analyzed. The extraction of total DNA,amplification of total ITS region, clcaning of the PCR products, sequencing of ITS region were carried out in the laboratory of Makino Herbarium, Tokyo Metropolitan University. For sequencing, the autosequensor ALFexpress (Pharmacia) , newly purchesed by this grant, was used. The sequences obtained were analyzed with PAUP 3.0s to construct the most parsimonious phylogenetic trees. As a result a consensus tree of the 32 equally parsimonious trees was obtained. The tree topology is generally conglueut with corrent understanding of the relationships between genera, excepting that Gentiana section Stenogyne joined to form a monophyletic clade with Tripterospermum and Crawfurdia with bootstrap probabilities 84%, which suggests that the genus Gentiana under present circumscription is not a monophyletic group. The triquetrous seeds commonly found in Gentiana section Stenogyne, Tripterospermum and Crawfurdiathe may be a synapomorphy of them. The recurved stamens might be gaind at the same time as triquetrous seeds but disappeared in Crawfurdia. Tripterospermum and Crawfurdia joined a monophyletic clade and the twining stem may be a synapomorphy of them. The monophyly of the species with berries was not significantly supported.