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The development of three long universal nuclear protein-coding locus markers and their application to osteichthyan phylogenetics with nested PCR.

Shen XX, Liang D, Zhang P - PLoS ONE (2012)

Bottom Line: For example, RAG1, a representative NPCL marker, has been successfully used to make phylogenetic inferences within all major osteichthyan groups.We then compared their phylogenetic utilities with that of the reference marker RAG1 in 47 jawed vertebrate species.In addition, we compared the success of nested PCR and standard PCR when amplifying NPCL marker fragments.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT

Background: 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.

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Related in: MedlinePlus

Agarose gel electrophoretic analysis of the PCR products.The first fragments of the long NPCL marker SACS (SACS-F1) were amplified in 32 taxa using nested PCR and standard PCR, respectively. The upper image shows the results of nested PCR amplifications, and the lower image shows the results of standard PCR amplifications. Lanes 1–32 show identical PCR amplifications performed in different species. “-”: negative controls, “M”: DNA ladder.
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pone-0039256-g002: Agarose gel electrophoretic analysis of the PCR products.The first fragments of the long NPCL marker SACS (SACS-F1) were amplified in 32 taxa using nested PCR and standard PCR, respectively. The upper image shows the results of nested PCR amplifications, and the lower image shows the results of standard PCR amplifications. Lanes 1–32 show identical PCR amplifications performed in different species. “-”: negative controls, “M”: DNA ladder.

Mentions: To compare the nested PCR and standard PCR strategies, we separately amplified two overlapping fragments for each of three long NPCL markers using both PCR methods. The results of these PCR experiments are summarized in Figure 1. We categorized the agarose electrophoretic images of the PCR products into three groups: no target band or smear, weak target band with non-specific amplification and strong target band with non-specific amplification. Overall, the amplification success rate and efficiency of the nested PCR were overwhelmingly higher than that of the standard PCR. The PCR success rate was 100% for the nested PCR method but only 70.3% for the standard PCR method. Furthermore, the proportion of reactions yielding strong target bands with non-specific amplification in the nested PCR was notably higher than that in the standard PCR (96.4% versus 28.6%). Finally, some species (e.g., Batrachuperus yenyuanensis, Protopterus annectens) are somewhat refractory to target band amplification using standard PCR, but nested PCR was able to produce strong or weak target bands for these difficult samples. The experimental differences between the two PCR methods are demonstrated visually in Figure 2, which shows an agarose gel used to separate the products of amplification of the first fragments of SACS.


The development of three long universal nuclear protein-coding locus markers and their application to osteichthyan phylogenetics with nested PCR.

Shen XX, Liang D, Zhang P - PLoS ONE (2012)

Agarose gel electrophoretic analysis of the PCR products.The first fragments of the long NPCL marker SACS (SACS-F1) were amplified in 32 taxa using nested PCR and standard PCR, respectively. The upper image shows the results of nested PCR amplifications, and the lower image shows the results of standard PCR amplifications. Lanes 1–32 show identical PCR amplifications performed in different species. “-”: negative controls, “M”: DNA ladder.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375249&req=5

pone-0039256-g002: Agarose gel electrophoretic analysis of the PCR products.The first fragments of the long NPCL marker SACS (SACS-F1) were amplified in 32 taxa using nested PCR and standard PCR, respectively. The upper image shows the results of nested PCR amplifications, and the lower image shows the results of standard PCR amplifications. Lanes 1–32 show identical PCR amplifications performed in different species. “-”: negative controls, “M”: DNA ladder.
Mentions: To compare the nested PCR and standard PCR strategies, we separately amplified two overlapping fragments for each of three long NPCL markers using both PCR methods. The results of these PCR experiments are summarized in Figure 1. We categorized the agarose electrophoretic images of the PCR products into three groups: no target band or smear, weak target band with non-specific amplification and strong target band with non-specific amplification. Overall, the amplification success rate and efficiency of the nested PCR were overwhelmingly higher than that of the standard PCR. The PCR success rate was 100% for the nested PCR method but only 70.3% for the standard PCR method. Furthermore, the proportion of reactions yielding strong target bands with non-specific amplification in the nested PCR was notably higher than that in the standard PCR (96.4% versus 28.6%). Finally, some species (e.g., Batrachuperus yenyuanensis, Protopterus annectens) are somewhat refractory to target band amplification using standard PCR, but nested PCR was able to produce strong or weak target bands for these difficult samples. The experimental differences between the two PCR methods are demonstrated visually in Figure 2, which shows an agarose gel used to separate the products of amplification of the first fragments of SACS.

Bottom Line: For example, RAG1, a representative NPCL marker, has been successfully used to make phylogenetic inferences within all major osteichthyan groups.We then compared their phylogenetic utilities with that of the reference marker RAG1 in 47 jawed vertebrate species.In addition, we compared the success of nested PCR and standard PCR when amplifying NPCL marker fragments.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT

Background: 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.

Show MeSH
Related in: MedlinePlus