Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, People's Republic of China.
PLoS One. 2012;7(6):e39256. doi: 10.1371/journal.pone.0039256. Epub 2012 Jun 14.
Universal nuclear protein-coding locus (NPCL) markers that are applicable across diverse taxa and show good phylogenetic discrimination have broad applications in molecular phylogenetic studies. For example, RAG1, a representative NPCL marker, has been successfully used to make phylogenetic inferences within all major osteichthyan groups. However, such markers with broad working range and high phylogenetic performance are still scarce. It is necessary to develop more universal NPCL markers comparable to RAG1 for osteichthyan phylogenetics.
METHODOLOGY/PRINCIPAL FINDINGS: We developed three long universal NPCL markers (>1.6 kb each) based on single-copy nuclear genes (KIAA1239, SACS and TTN) that possess large exons and exhibit the appropriate evolutionary rates. We then compared their phylogenetic utilities with that of the reference marker RAG1 in 47 jawed vertebrate species. In comparison with RAG1, each of the three long universal markers yielded similar topologies and branch supports, all in congruence with the currently accepted osteichthyan phylogeny. To compare their phylogenetic performance visually, we also estimated the phylogenetic informativeness (PI) profile for each of the four long universal NPCL markers. The PI curves indicated that SACS performed best over the whole timescale, while RAG1, KIAA1239 and TTN exhibited similar phylogenetic performances. In addition, we compared the success of nested PCR and standard PCR when amplifying NPCL marker fragments. The amplification success rate and efficiency of the nested PCR were overwhelmingly higher than those of standard PCR.
CONCLUSIONS/SIGNIFICANCE: Our work clearly demonstrates the superiority of nested PCR over the conventional PCR in phylogenetic studies and develops three long universal NPCL markers (KIAA1239, SACS and TTN) with the nested PCR strategy. The three markers exhibit high phylogenetic utilities in osteichthyan phylogenetics and can be widely used as pilot genes for phylogenetic questions of osteichthyans at different taxonomic levels.
通用核蛋白编码基因座（NPCL）标记物适用于多种分类群，具有良好的系统发育区分能力，在分子系统发育研究中具有广泛的应用。例如，RAG1 是一种代表性的 NPCL 标记物，已成功用于对所有主要硬骨鱼类群进行系统发育推断。然而，具有广泛工作范围和高系统发育性能的此类标记物仍然很少。有必要开发更多与 RAG1 相当的用于硬骨鱼系统发育的通用 NPCL 标记物。
方法/主要发现：我们基于具有大外显子且表现出适当进化率的单拷贝核基因（KIAA1239、SACS 和 TTN），开发了三个长的通用 NPCL 标记物（每个标记物均大于 1.6kb）。然后，我们将这三个长的通用标记物与参考标记物 RAG1 在 47 种有颌脊椎动物物种中的系统发育效用进行了比较。与 RAG1 相比，三个长的通用标记物中的每一个都产生了相似的拓扑结构和分支支持，与当前公认的硬骨鱼系统发育一致。为了直观比较它们的系统发育性能，我们还估计了四个长的通用 NPCL 标记物的系统发育信息量（PI）分布。PI 曲线表明，SACS 在整个时间尺度上表现最佳，而 RAG1、KIAA1239 和 TTN 表现出相似的系统发育性能。此外，我们比较了嵌套 PCR 和标准 PCR 扩增 NPCL 标记片段时的成功率。嵌套 PCR 的扩增成功率和效率远远高于标准 PCR。
结论/意义：我们的工作清楚地表明，在系统发育研究中，嵌套 PCR 优于传统 PCR，并且使用嵌套 PCR 策略开发了三个长的通用 NPCL 标记物（KIAA1239、SACS 和 TTN）。这三个标记物在硬骨鱼系统发育学中具有很高的系统发育效用，可以广泛用作不同分类水平硬骨鱼系统发育问题的先导基因。
