Isolation and characterization of microsatellite loci from the Japanese red pine, Pinus densiflora
Isolation and characterization of microsatellite loci from the Japanese red pine, Pinus densiflora
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DOI：
10.1046/j.1365-294x.2000.00954-10.x
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发表时间：
2000-08-01
影响因子：
4.9
通讯作者：
Hogetsu, T
中科院分区：
文献类型：
--
作者：
Lian, C;Miwa, M;Hogetsu, T
日本红松（Pinus densiflora Sieb. et Zucc.）是日本的主要树种之一。近年来，该物种受松材线虫危害严重，分布面积逐渐减少。为了保护和保存该物种的森林，了解密花松在自然界中的繁殖方式可能是必不可少的。在本文中，作为研究赤花松繁殖行为的第一步，开发了多态性微卫星标记。在 Miwa 等人（2000）修改的富集方法（Edwards 等人，1996）之后进行微卫星分离。使用改良的十六烷基三甲基溴化铵 (CTAB) 方法（Zhou 等人，1999）从白花松的新鲜叶子中提取总 DNA，并用 BamHI、EcoRI、EcoRV、XbaI 或 XhoI 限制性内切酶消化。 [0101] 将通过上述限制性消化产生的片段，除了直接形成平端末端的EcoRV之外，用DNA平端化试剂盒(Takara Shuzo Co.)平端化，然后用DNA连接试剂盒(Takara Shuzo Co.)与EcoRI/NotI/BamHI接头连接。然后通过聚合酶链式反应 (PCR) 扩增接头连接的片段。
Japanese red pine (Pinus densiflora Sieb. et Zucc.) is one of main tree species in Japan. In recent years, the species has been severely damaged by pine wood nematodes, and the distribution area has been gradually decreasing. In order to protect and preserve forests of this species, it may be indispensable to know how P. densiflora is reproduced in nature. In this paper and as a first step to investigate the reproduction behaviour of P. densiflora, polymorphic microsatellite markers were developed.Microsatellite isolation was performed after an enrichment method (Edwards et al. 1996) modified by Miwa et al.(2000). Total DNA was extracted from fresh leaves of P. densiflora with a modified cetyltrimethylammonium bromide (CTAB) method (Zhou et al. 1999), and digested with either BamHI, EcoRI, EcoRV, XbaI or XhoI restriction enzyme. Fragments produced by the above restriction digestion, except for EcoRV which directly made blunted ends, were blunted by a DNA Blunting Kit (Takara Shuzo Co.) and subsequently ligated with an EcoRI/NotI/BamHI adaptor by a DNA Ligation Kit (Takara Shuzo Co.). The adaptor-ligated fragments were then amplified by polymerase chain reaction (PCR).