BACKGROUND Identification of species via DNA sequences is the basis for DNA taxonomy and DNA barcoding. Currently there is a strong focus on using a mitochondrial marker for this purpose, in particular a fragment from the cytochrome oxidase I gene (COI). While there is ample evidence that this marker is indeed suitable across a broad taxonomic range to delineate species, it has also become clear that a complementation by a nuclear marker system could be advantageous. Ribosomal RNA genes could be suitable for this purpose, because of their global occurrence and the possibility to design universal primers. However, it has so far been assumed that these genes are too highly conserved to allow resolution at, or even beyond the species level. On the other hand, it is known that ribosomal gene regions harbour also highly divergent parts. We explore here the information content of two adjacent divergence regions of the large subunit ribosomal gene, the D1-D2 region. RESULTS Universal primers were designed to amplify the D1-D2 region from all metazoa. We show that amplification products in the size between 800-1300 bp can be obtained across a broad range of animal taxa, provided some optimizations of the PCR procedure are implemented. Although the ribosomal genes occur in multiple copies in the genomes, we find generally very little intra-individual polymorphism (<< 0.1% on average) indicating that concerted evolution is very effective in most cases. Studies in two fish taxa (genus Cottus and genus Aphyosemion) show that the D1-D2 LSU sequence can resolve even very closely related species with the same fidelity as COI sequences. In one case we can even show that a mitochondrial transfer must have occurred, since the nuclear sequence confirms the taxonomic assignment, while the mitochondrial sequence would have led to the wrong classification. We have further explored whether hybrids between species can be detected with the nuclear sequence and we show for a test case of natural hybrids among cyprinid fish species (Alburnus alburnus and Rutilus rutilus) that this is indeed possible. CONCLUSION The D1-D2 LSU region is a suitable marker region for applications in DNA based species identification and should be considered to be routinely used as a marker complementing broad scale studies based on mitochondrial markers.

中文翻译：

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对LSU rDNA D1-D2序列用于物种鉴定的评估。

背景技术通过DNA序列鉴定物种是DNA分类学和DNA条形码的基础。当前，非常关注使用线粒体标记物，特别是来自细胞色素氧化酶I基因（COI）的片段。尽管有充分的证据表明该标志物确实确实适用于广泛的分类学范围来描述物种，但也清楚的是，核标志物系统的互补可能是有利的。核糖体RNA基因可能适合于此目的，因为它们普遍存在并且可以设计通用引物。但是，到目前为止，已经假设这些基因过于保守，无法在物种水平上甚至在物种水平之外进行解析。另一方面，已知核糖体基因区域也具有高度不同的部分。我们在这里探索大亚基核糖体基因两个相邻的发散区域D1-D2区域的信息内容。结果通用引物经设计可扩增所有后生动物的D1-D2区域。我们显示，只要对PCR程序进行一些优化，就可以在广泛的动物分类中获得800-1300 bp之间大小的扩增产物。尽管核糖体基因在基因组中以多个拷贝出现，但我们发现个体内多态性很少（平均<0.1％），这表明在大多数情况下协同进化非常有效。对两个鱼类分类群（Cottus属和Aphyosemion属）的研究表明，D1-D2 LSU序列甚至可以解析与COI序列具有相同保真度的非常紧密相关的物种。在一种情况下，我们甚至可以证明必须发生线粒体转移，因为核序列确认了分类学分配，而线粒体序列将导致错误的分类。我们进一步探索了是否可以通过核序列检测出物种之间的杂种，并且对于塞浦路斯鱼类（Alburnus alburnus和Rutilus rut​​ilus）之间的自然杂种的测试案例表明，这确实是可能的。结论D1-D2 LSU区是适用于基于DNA的物种鉴定的合适的标记区，应被认为是常规用作补充基于线粒体标记物的大规模研究的标记物。而线粒体序列将导致错误的分类。我们进一步探索了是否可以通过核序列检测出物种之间的杂种，并且对于塞浦路斯鱼类（Alburnus alburnus和Rutilus rut​​ilus）之间的自然杂种的测试案例表明，这确实是可能的。结论D1-D2 LSU区是适用于基于DNA的物种鉴定的合适的标记区，应被认为是常规用作补充基于线粒体标记物的大规模研究的标记物。而线粒体序列将导致错误的分类。我们进一步探索了是否可以通过核序列检测出物种之间的杂种，并且对于塞浦路斯鱼类（Alburnus alburnus和Rutilus rut​​ilus）之间的自然杂种的测试案例表明，这确实是可能的。结论D1-D2 LSU区是适用于基于DNA的物种鉴定的合适的标记区，应被认为是常规用作补充基于线粒体标记物的大规模研究的标记物。