Antunes R S P, Gomes V N, Prioli S M A P, Prioli R A, Júlio H F, Prioli L M, Agostinho C S, Prioli A J
Departamento de Ciências, Universidade Estadual de Maringá, Maringá, PR, Brazil.
Genet Mol Res. 2010 Apr 13;9(2):674-84. doi: 10.4238/vol9-2gmr759.
Brycon is one of the main genera of Neotropical freshwater fish. In Brazil, Brycon species have been found in many hydrographic basins, such as the Amazon, Paraná, Paraguay, and Araguaia-Tocantins basins. We examined the phylogenetic relationships among the species Brycon orbignyanus, B. hilarii, B. cf. pesu, B. cephalus, B. falcatus, and B. gouldingi, using mitochondrial and nuclear molecular markers. Specimens of B. orbignyanus were collected in the Paraná River. Specimens of B. hilarii were collected in the Manso River. Specimens of B. cephalus were obtained from a fish farm, and specimens of B. cf. pesu, B. falcatus and B. gouldingi were sampled in the Araguaia-Tocantins basin. DNA extraction was carried out using the phenol/chloroform method. Molecular polymorphism studies of Brycon species were carried out with the inter-simple sequence repeat (ISSR) technique, using the total DNA of six specimens of each species. In DNA amplification of B. cf. pesu, eight specimens were used. The partial sequence of mitochondrial cytochrome b was amplified by PCR. The PCR products were used directly in sequencing reactions. Each ISSR primer produced from 7 to 14 scorable and reproducible bands. The (GGAC)(3)A and (GGAC)(3)C primers produced the greatest number of species-specific bands. A 264-bp fragment, corresponding to the partial region of mitochondrial DNA cytochrome b, was sequenced and used for analysis. According to the phylogenetic tree obtained from the data, these Brycon species can be divided into two clades: one comprised only B. cf. pesu, and the second comprised the remaining Brycon species. We conclude that ISSR primers can be used for the identification of species-specific bands in fish, such as Brycon spp.
魮脂鲤属是新热带淡水鱼的主要属之一。在巴西，魮脂鲤属物种已在许多水文流域被发现，如亚马逊河、巴拉那河、巴拉圭河和阿拉瓜亚 - 托坎廷斯河流域。我们使用线粒体和核分子标记，研究了魮脂鲤（Brycon orbignyanus）、希氏魮脂鲤（B. hilarii）、疑似佩苏魮脂鲤（B. cf. pesu）、头魮脂鲤（B. cephalus）、镰状魮脂鲤（B. falcatus）和古氏魮脂鲤（B. gouldingi）之间的系统发育关系。魮脂鲤的标本采集于巴拉那河。希氏魮脂鲤的标本采集于曼索河。头魮脂鲤的标本取自一个养鱼场，疑似佩苏魮脂鲤、镰状魮脂鲤和古氏魮脂鲤的标本在阿拉瓜亚 - 托坎廷斯河流域采集。使用酚/氯仿法进行DNA提取。采用简单序列重复区间（ISSR）技术，对每个物种的六个标本的总DNA进行魮脂鲤属物种的分子多态性研究。在疑似佩苏魮脂鲤的DNA扩增中，使用了八个标本。通过聚合酶链式反应（PCR）扩增线粒体细胞色素b的部分序列。PCR产物直接用于测序反应。每个ISSR引物产生7至14个可评分且可重复的条带。（GGAC）(3)A和（GGAC）(3)C引物产生的物种特异性条带数量最多。对一个对应线粒体DNA细胞色素b部分区域的264碱基对片段进行测序并用于分析。根据从数据中获得的系统发育树，这些魮脂鲤属物种可分为两个进化枝：一个仅包含疑似佩苏魮脂鲤，另一个包含其余的魮脂鲤属物种。我们得出结论，ISSR引物可用于鉴定鱼类中的物种特异性条带，如魮脂鲤属物种。
