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Optimization of DNA extraction and PCR protocols for phylogenetic analysis in Schinopsis spp. and related Anacardiaceae.

Mogni VY, Kahan MA, de Queiroz LP, Vesprini JL, Ortiz JP, Prado DE - Springerplus (2016)

Bottom Line: The modifications proposed allowed the extraction of 70-120 µg of non-degraded genomic DNA per gram of dry tissue that resulted useful for PCR amplification.Sequence analyses of amplicons showed similarity with the corresponding Schinopsis accessions available at GenBank.The methodology presented here can be routinely applied for molecular studies of the group aimed to clarify not only aspects on the molecular biology but also the taxonomy and phylogeny of this fascinating group of vascular plants.

View Article: PubMed Central - PubMed

Affiliation: Facultad de Ciencias Agrarias, Universidad Nacional de Rosario, Campo Experimental Villarino, S2125ZAA Zavalla, Santa Fe Argentina ; IICAR, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET-UNR), Zavalla, Argentina.

ABSTRACT
The Anacardiaceae is an important and worldwide distributed family of ecological and socio-economic relevance. Notwithstanding that, molecular studies in this family are scarce and problematic because of the particularly high concentration of secondary metabolites-i.e. tannins and oleoresins-that are present in almost all tissues of the many members of the group, which complicate the purification and amplification of the DNA. The objective of this work was to improve an available DNA isolation method for Schinopsis spp. and other related Anacardiaceae, as well as the PCR protocols for DNA amplification of the chloroplast trnL-F, rps16 and ndhF and nuclear ITS-ETS fragments. The modifications proposed allowed the extraction of 70-120 µg of non-degraded genomic DNA per gram of dry tissue that resulted useful for PCR amplification. PCR reactions produced the expected fragments that could be directly sequenced. Sequence analyses of amplicons showed similarity with the corresponding Schinopsis accessions available at GenBank. The methodology presented here can be routinely applied for molecular studies of the group aimed to clarify not only aspects on the molecular biology but also the taxonomy and phylogeny of this fascinating group of vascular plants.

No MeSH data available.


1.5 % Agarose gel electrophoresis of PCR products of several accessions of Schinopsis spp. and related species. atrnL-F amplified in two parts. Lanes1, 20, 27 molecular marker, 2–5S. boqueronensis, 6–7, 9S. cornuta, 10S. peruviana, 11–14S. heterophylla, 15–18, 21S. brasiliensis, 22–23S. marginata, 24S. lorentzii, 25–26S. brasiliensis, 28–29Astronium urundeuva and Apterokarpos gardneri, and 8, 19 without sample. brps16. Lanes1, 13 and 21 molecular marker, 2 S. peruviana, 3–6S. heterophylla, 5–9, 14–15S. brasiliensis, 10–11S. marginata, 12S. lorentzii, 16–17Astronium urundeuva and Apterokarpos gardneri, 18–19S. balansae, 20S. lorentzii, 22–24S. boqueronensis, 25–27S. cornuta. cndhF. Lanes1, 19 and 29 molecular marker, 11, 12, 17–19Astronium urundeuva, Apterokarpos gardneri, Lithraea molleoides, Loxopterygium grisebachii and Schinus areira; other lanes, Schinopsis spp. Arrows indicate the reference bands of the marker Lambda EcoRI/HindIII (564 bp in a, 947 and 831 in b and 831 and 564 in c) to estimate the amplicons size
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Fig2: 1.5 % Agarose gel electrophoresis of PCR products of several accessions of Schinopsis spp. and related species. atrnL-F amplified in two parts. Lanes1, 20, 27 molecular marker, 2–5S. boqueronensis, 6–7, 9S. cornuta, 10S. peruviana, 11–14S. heterophylla, 15–18, 21S. brasiliensis, 22–23S. marginata, 24S. lorentzii, 25–26S. brasiliensis, 28–29Astronium urundeuva and Apterokarpos gardneri, and 8, 19 without sample. brps16. Lanes1, 13 and 21 molecular marker, 2 S. peruviana, 3–6S. heterophylla, 5–9, 14–15S. brasiliensis, 10–11S. marginata, 12S. lorentzii, 16–17Astronium urundeuva and Apterokarpos gardneri, 18–19S. balansae, 20S. lorentzii, 22–24S. boqueronensis, 25–27S. cornuta. cndhF. Lanes1, 19 and 29 molecular marker, 11, 12, 17–19Astronium urundeuva, Apterokarpos gardneri, Lithraea molleoides, Loxopterygium grisebachii and Schinus areira; other lanes, Schinopsis spp. Arrows indicate the reference bands of the marker Lambda EcoRI/HindIII (564 bp in a, 947 and 831 in b and 831 and 564 in c) to estimate the amplicons size

Mentions: PCR assays performed using the DNA preparations diluted 1/10 allowed the generation of all fragments tested. The addition of BSA 1/1000; MgCl2 5 mM and DMSO 1 M (for ETS and ITS) in the PCR mixture was crucial for amplification success, as it was previously reported (Savolainen et al. 1995; Baldwin et al. 1995; Särkinen et al. 2012). This is probably because BSA has a high content of lysine; it joins phenolic compounds when added to the PCR mix, avoiding Taq polymerase inactivation (Kreader 1996). On the other hand, DMSO acts by relaxing the typical secondary structure of nuclear ribosomal regions during amplification (Álvarez and Wendel 2003). Chloroplast regions trnL-F, rps16 and ndhF resulted in amplicons of 400, 900 and 650 bp respectively (Fig. 2a–c). On the other hand, the amplification of the nuclear region ETS resulted in a fragment of approximately 300 bp (Fig. 3a), and ITS2 in 200–300 bp (Fig. 3b).Fig. 2


Optimization of DNA extraction and PCR protocols for phylogenetic analysis in Schinopsis spp. and related Anacardiaceae.

Mogni VY, Kahan MA, de Queiroz LP, Vesprini JL, Ortiz JP, Prado DE - Springerplus (2016)

1.5 % Agarose gel electrophoresis of PCR products of several accessions of Schinopsis spp. and related species. atrnL-F amplified in two parts. Lanes1, 20, 27 molecular marker, 2–5S. boqueronensis, 6–7, 9S. cornuta, 10S. peruviana, 11–14S. heterophylla, 15–18, 21S. brasiliensis, 22–23S. marginata, 24S. lorentzii, 25–26S. brasiliensis, 28–29Astronium urundeuva and Apterokarpos gardneri, and 8, 19 without sample. brps16. Lanes1, 13 and 21 molecular marker, 2 S. peruviana, 3–6S. heterophylla, 5–9, 14–15S. brasiliensis, 10–11S. marginata, 12S. lorentzii, 16–17Astronium urundeuva and Apterokarpos gardneri, 18–19S. balansae, 20S. lorentzii, 22–24S. boqueronensis, 25–27S. cornuta. cndhF. Lanes1, 19 and 29 molecular marker, 11, 12, 17–19Astronium urundeuva, Apterokarpos gardneri, Lithraea molleoides, Loxopterygium grisebachii and Schinus areira; other lanes, Schinopsis spp. Arrows indicate the reference bands of the marker Lambda EcoRI/HindIII (564 bp in a, 947 and 831 in b and 831 and 564 in c) to estimate the amplicons size
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835408&req=5

Fig2: 1.5 % Agarose gel electrophoresis of PCR products of several accessions of Schinopsis spp. and related species. atrnL-F amplified in two parts. Lanes1, 20, 27 molecular marker, 2–5S. boqueronensis, 6–7, 9S. cornuta, 10S. peruviana, 11–14S. heterophylla, 15–18, 21S. brasiliensis, 22–23S. marginata, 24S. lorentzii, 25–26S. brasiliensis, 28–29Astronium urundeuva and Apterokarpos gardneri, and 8, 19 without sample. brps16. Lanes1, 13 and 21 molecular marker, 2 S. peruviana, 3–6S. heterophylla, 5–9, 14–15S. brasiliensis, 10–11S. marginata, 12S. lorentzii, 16–17Astronium urundeuva and Apterokarpos gardneri, 18–19S. balansae, 20S. lorentzii, 22–24S. boqueronensis, 25–27S. cornuta. cndhF. Lanes1, 19 and 29 molecular marker, 11, 12, 17–19Astronium urundeuva, Apterokarpos gardneri, Lithraea molleoides, Loxopterygium grisebachii and Schinus areira; other lanes, Schinopsis spp. Arrows indicate the reference bands of the marker Lambda EcoRI/HindIII (564 bp in a, 947 and 831 in b and 831 and 564 in c) to estimate the amplicons size
Mentions: PCR assays performed using the DNA preparations diluted 1/10 allowed the generation of all fragments tested. The addition of BSA 1/1000; MgCl2 5 mM and DMSO 1 M (for ETS and ITS) in the PCR mixture was crucial for amplification success, as it was previously reported (Savolainen et al. 1995; Baldwin et al. 1995; Särkinen et al. 2012). This is probably because BSA has a high content of lysine; it joins phenolic compounds when added to the PCR mix, avoiding Taq polymerase inactivation (Kreader 1996). On the other hand, DMSO acts by relaxing the typical secondary structure of nuclear ribosomal regions during amplification (Álvarez and Wendel 2003). Chloroplast regions trnL-F, rps16 and ndhF resulted in amplicons of 400, 900 and 650 bp respectively (Fig. 2a–c). On the other hand, the amplification of the nuclear region ETS resulted in a fragment of approximately 300 bp (Fig. 3a), and ITS2 in 200–300 bp (Fig. 3b).Fig. 2

Bottom Line: The modifications proposed allowed the extraction of 70-120 µg of non-degraded genomic DNA per gram of dry tissue that resulted useful for PCR amplification.Sequence analyses of amplicons showed similarity with the corresponding Schinopsis accessions available at GenBank.The methodology presented here can be routinely applied for molecular studies of the group aimed to clarify not only aspects on the molecular biology but also the taxonomy and phylogeny of this fascinating group of vascular plants.

View Article: PubMed Central - PubMed

Affiliation: Facultad de Ciencias Agrarias, Universidad Nacional de Rosario, Campo Experimental Villarino, S2125ZAA Zavalla, Santa Fe Argentina ; IICAR, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET-UNR), Zavalla, Argentina.

ABSTRACT
The Anacardiaceae is an important and worldwide distributed family of ecological and socio-economic relevance. Notwithstanding that, molecular studies in this family are scarce and problematic because of the particularly high concentration of secondary metabolites-i.e. tannins and oleoresins-that are present in almost all tissues of the many members of the group, which complicate the purification and amplification of the DNA. The objective of this work was to improve an available DNA isolation method for Schinopsis spp. and other related Anacardiaceae, as well as the PCR protocols for DNA amplification of the chloroplast trnL-F, rps16 and ndhF and nuclear ITS-ETS fragments. The modifications proposed allowed the extraction of 70-120 µg of non-degraded genomic DNA per gram of dry tissue that resulted useful for PCR amplification. PCR reactions produced the expected fragments that could be directly sequenced. Sequence analyses of amplicons showed similarity with the corresponding Schinopsis accessions available at GenBank. The methodology presented here can be routinely applied for molecular studies of the group aimed to clarify not only aspects on the molecular biology but also the taxonomy and phylogeny of this fascinating group of vascular plants.

No MeSH data available.