Molecular sexing of individual Ryukyu Robins Erithacus komadori using buccal cells as a non-invasive source of DNA
Molecular sexing of individual Ryukyu Robins Erithacus komadori using buccal cells as a non-invasive source of DNA
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使用口腔细胞作为非侵入性 DNA 来源对琉球知更鸟 Erithacus komadori 个体进行分子性别鉴定
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发表时间：
2003
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通讯作者：
Shin‐Ichi Seki
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作者：
Shin‐Ichi Seki
人类（Feigelson et al. 2001）、实验动物（Zimmerman et al. 2000）、牲畜（Kwon et al. 1993）和野生灵长类动物（Hashimoto et al. 1996） DNA的广泛来源，但尚未用于野生鸟类。血液是鸟类分子生态学研究中最常用的DNA来源，因为安全有效的采样技术已经发展得很好（见Wingfield 1999年的综述），因此认为没有必要采用其他非侵入性方法。然而，对于仅在日本西南部流行的琉球Robin Erithacus komadori （Kawaji & Higuchi 1989）来说，血液采样是困难的。由于其分布有限，在一些岛屿上数量急剧下降，已被指定为日本的“国家自然宝藏”和“国家濒危野生动植物物种”。因此，鹿儿岛县政府和国家文化厅都出于政治和文化的原因，反对从知更鸟，尤其是雏鸟身上采集血液样本。然而，为了揭示性别单一性雏鸟的死亡率和传播距离，需要进行分子性别测定（Seki 2002）。在这里，我报告了使用从口腔细胞中提取的DNA对成年知更鸟进行分子性别鉴定的结果，并将其与血液中的DNA进行了比较，并检验了口腔细胞作为鸟类性别鉴定DNA来源的可靠性。研究人员从20只雄性和20只雌性成年琉球罗宾斯身上获得了口腔细胞和血液样本，这些样本之前已经根据形态学特征进行了性别鉴定。2002年4月15日至6月30日，在日本鹿儿岛中之岛的繁殖季节（经许可使用雾网）捕获了知更鸟。用棉签在每只知更鸟的口腔和喉咙内部滚动五次，收集口腔细胞。将每个拭子放入微管中，浸泡在1ml保存缓冲液（150 mM NaCl, 10 mM Tris-HCl pH 8.0, 10 mM EDTA）中，在室温下保存一周，并将其运送到实验室，在5°C下保存长达一个月（如需更长的保存时间，请参见附录1）。加入10 ml 10%-SDS后，用蛋白酶K （5 ml 20mg/ml）在55℃下消化1小时，在37℃下再消化16小时。然后用常规的苯酚/氯仿法提取DNA。经臂静脉穿刺取血30 μ l，无不良反应。血样在室温下保存于GenTLE solution I （Takara）中，并在取样后10天内使用提取试剂盒（QIAamp DNA Blood Mini kit， QIAGEN）提取DNA。使用Ellegren & Fridolfsson（1997）的引物集3007F （5 -TACATACAGGCTCTACTCCT-3）和3112R (5 -CCCCTTCAGGTTCTTTAAAA-3)，参照Fridolfsson & Ellegren（1999）的方法，通过染色体解旋酶- dna结合（CHD）基因的长度多态性来确定每个个体的性别。所有PCR反应在25 m l体积的Perkin Elmer 9600热循环仪上进行，使用Taq DNA聚合酶（Takara Taq, Takara）。每个反应混合物含有0.625单位Taq， 200 mM dNTPs, 20 mM Tris-HCl pH 8.0, 50 mM KCl, 2 mM MgCl2, 5 pmol引物。热轮廓包括94°C的初始变性步骤，持续两分钟，然后是“触地”方案，其中退火温度每循环降低1°C，从60°C开始直到达到50°C的温度。然后在50°C的恒定退火温度下再运行35个循环。使用颊细胞作为非侵入性DNA来源，在94°C下变性，对个体的小komadori进行分子性别鉴定
vasive source of DNA for humans (Feigelson et al. 2001), experimental animals (Zimmerman et al. 2000), livestock (Kwon et al. 1993), and wild primates (Hashimoto et al. 1996), but have not been used for wild birds. Blood is the most commonly used source of DNA for avian molecular ecological studies because safe and effective sampling techniques have been well-developed (reviewed in Wingfield 1999), hence other non-invasive methods have not been deemed necessary. In the case of the Ryukyu Robin Erithacus komadori, endemic only to southwestern Japan (Kawaji & Higuchi 1989), however, blood sampling is difficult. Because of its limited distribution and the steep population declines on some islands, it has been designated as a “national natural treasure” and “national endangered species of wild fauna and flora” of Japan. Consequently, both the Kagoshima prefectural government and the national agency for cultural affairs are against taking blood samples from robins, especially from nestlings, for political and cultural reasons. However, in order to reveal sex-related mortality and dispersal distances of sexually monomorphic fledglings, molecular sexing is needed (Seki 2002). Here I report the results of molecular sexing of adult robins using DNA extracted from buccal cells, comparing them with those from blood, and examine the reliability of buccal cells as a source of DNA for avian sexing. Samples of both buccal cells and blood were obtained from 20 male and 20 female adult Ryukyu Robins, that had been previously sexed based on morphological characteristics. Robins were captured (under license by mist-net) during the breeding season, from 15 April to 30 June 2002, on Nakanoshima Island, Kagoshima, Japan. Buccal cells were collected by rolling cotton swabs against the inside of each robins’ mouth and throat five times. Each swab was put into a microtube and soaked in 1 ml of preservation buffer (150 mM NaCl, 10 mM Tris-HCl pH 8.0, 10 mM EDTA), and kept at room temperature during the one week it took to transport them to the laboratory, where they were stored at 5°C for up to one month (see also Appendix 1 for longer preservation). After adding 10 m l of 10%-SDS, the samples were digested with proteinase K (5 m l of 20mg/ml) at 55°C for one hour and an additional 16 hours at 37°C. DNA was then extracted by the conventional phenol/chloroform method. Up to 30 m l of blood samples were obtained by brachial vein puncture and no ill effects were recorded. Blood samples were stored in GenTLE solution I (Takara) at room temperature and DNA was extracted using an extraction kit (QIAamp DNA Blood Mini Kit, QIAGEN) within 10 days of sampling. The sex of each individual was determined by length polymorphism at the chromo-helicase-DNAbinding (CHD) gene, using Ellegren & Fridolfsson’s (1997) primer set 3007F (5 -TACATACAGGCTCTACTCCT-3 ) and 3112R (5 -CCCCTTCAGGTTCTTTAAAA-3 ), following the methods used by Fridolfsson & Ellegren (1999). All PCR reactions were performed in 25 m l volumes on a Perkin Elmer 9600 Thermal Cycler, using Taq DNA polymerase (Takara Taq, Takara). Each reaction mixture contained 0.625 unit of Taq, 200 mM dNTPs, 20 mM Tris-HCl pH 8.0, 50 mM KCl, 2 mM MgCl2, 5 pmol of primers. The thermal profile comprised an initial denaturing step of 94°C for two minutes, followed by a “touch-down” scheme where the annealing temperature was lowered by 1°C per cycle, starting from 60°C until a temperature of 50°C was reached. Then 35 additional cycles were run at a constant annealing temperature of 50°C. Denaturation was at 94°C for Molecular sexing of individual Ryukyu Robins Erithacus komadori using buccal cells as a non-invasive source of DNA