Studies of population genetics on four mangrove tree species in Okinawa, Japan

本研究对四种红树（根茎科）的遗传变异进行了分析。分布于日本冲绳的candelia canddel （Rhizophoraceae）、Rhizophora stylosa （Rhizophoraceae）和sonneratialba (sonneratiacac)，并添加了一种Lumnitzera racemosa （combretacae）进行DNA分析。实验前，研究人员从四种树木上采集了树叶作为样本，并将其储存在一个冷冻库中。采用聚丙烯酰胺凝胶电泳法（PAGE）对4种红树的同工酶位点进行了分析。电泳后，有23种酶被染色。获得了茎尖根霉和木褐菌的5种酶的精细酶谱图。裸木布鲁氏菌的过氧化物酶（POD）存在多态性，而海桑的低脂酶（DIA）、酯酶（EST）和葡萄糖激酶（GR）存在多态性。从茎尖根hora stylosa叶片中提取DNA。采用CTAB（十六烷基三甲基溴化铵）法和Ice Cold Buffer法提取DNA。冰缓冲液法比CTAB法提取DNA效果更好。为了获得线粒体DNA片段，使用了18种限制性内切酶。扩增叶绿体DNA用了23条引物。线粒体和基因组DNA片段扩增并分析。由于冲绳红树林的遗传变异较小，引物的应用效率不高。结果表明，微卫星是分析琉球岛茎尖根hora stylosa遗传变异的有效方法之一。

In this studies, attempts have been made to analyze genetical variation of four mangrove tree species, Bruguiera gymnorrhiza (Rhizophoraceae). Kandelia candel (Rhizophoraceae), Rhizophora stylosa (Rhizophoraceae) and Sonneratia alba (Sonneratiaceac), distributed in Okinawa, Japan, and adding one species, Lumnitzera racemosa (Combretaceae) for DNA analysis. Leaves as samples were collected from trees of four species, and stocked in a deep-freezer before experiment. Polyacrylamide gel electrophoresis (PAGE) of a vertical slab-format method was used for analyzing isozyme loci of four mangroves. After the electrophoresis, 23 enzyme species were stained. Fine zymogrames were obtained five enzyme species for Rhizophora stylosa and five enzyme species for Bruguiera gymnorrhiza. Polymorphism was found only peroxidase (POD) for Bruguiera gymnorrhiza, but diaphorase (DIA), esterase (EST) and glucokinase GR) for Sonneratia alba.DNA was extracted from leaves of Rhizophora stylosa. Both CTAB (Cetyl Trimethyl Ammmonia Bromide) and Ice Cold Buffer methods were applied for DNA extraction. The Ice Cold Buffer method was better than the CTAB method for DNA extraction. To obtain mitochondrial DNA fragments, 18 restriction enzyme species were applied. For amplification of chloroplast DNA, 23 primers were used. Mitokondorial and genom DNA fragments were amplified and analyzed. Since genetical variations were small in studied mangrove species collected in Okinawa, primers applied were not so efficiently worked. However, we could obtain evidence that micro-satellites are one of efficient ways for gentical variation analysis of Rhizophora stylosa in Ryukyu islands.