Power and limitations of the chloroplast trnL(UAA) intron for plant DNA barcoding. Nucleic Acid Res 35:e14

Laboratoire d'Ecologie Alpine, CNRS UMR 5553, Université Joseph Fourier, BP 53, 38041 Grenoble Cedex 9, France.
Nucleic Acids Research (Impact Factor: 9.11). 02/2007; 35(3):e14. DOI: 10.1093/nar/gkl938
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DNA barcoding should provide rapid, accurate and automatable species identifications by using a standardized DNA region as a tag. Based on sequences available in GenBank and sequences produced for this study, we evaluated the resolution power of the whole chloroplast trnL (UAA) intron (254-767 bp) and of a shorter fragment of this intron (the P6 loop, 10-143 bp) amplified with highly conserved primers. The main limitation of the whole trnL intron for DNA barcoding remains its relatively low resolution (67.3% of the species from GenBank unambiguously identified). The resolution of the P6 loop is lower (19.5% identified) but remains higher than those of existing alternative systems. The resolution is much higher in specific contexts such as species originating from a single ecosystem, or commonly eaten plants. Despite the relatively low resolution, the whole trnL intron and its P6 loop have many advantages: the primers are highly conserved, and the amplification system is very robust. The P6 loop can even be amplified when using highly degraded DNA from processed food or from permafrost samples, and has the potential to be extensively used in food industry, in forensic science, in diet analyses based on feces and in ancient DNA studies.

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    • "trnL loci have also been proposed for species identification. Although trnL loci provide a relatively low resolution, its primer is highly conserved and the amplification system is very robust (Taberlet et al., 2007). Furthermore, the combined use of rbcL and trnL as a two-locus cpDNA barcode has shown a high potential for identifying NW-European fern species (de Groot et al., 2011). "
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    ABSTRACT: The Phyllanthus genus, a plant used in traditional Thai medicine, has according to several pharmacopeias hepatoprotective properties. Not only is the anatomical morphology of these species relatively similar but they also share the Thai common names Look-Tai-Bai (ลูกใต้ใบ) and Yah-Tai-Bai (หญ้าใต้ใบ), which might cause confusion for laypersons. This study attempted to develop a method for accurate identification of Phyllanthus species, especially P. amarus, and to detect contaminants in P. amarus products by using DNA barcoding coupled with High Resolution Melting (HRM) analysis (bar-HRM). Two plastid loci (rbcL and trnL) were chosen for DNA barcoding to generate a suitable primer for distinguishing Phyllanthus species by HRM analysis. The five species of Phyllanthus were subjected to amplification for testing the specificity and discrimination power of the designed primers derived from rbcL and trnL regions. Sensitivity of the method (DNA barcoding conjugated with HRM) to detect adulterant in P. amarus samples was evaluated. The commercial P. amarus products obtained from a local market were authenticated. The primer pair derived from trnL DNA barcoding (PhylltrnL) had more specificity and power of discrimination for Phyllanthus species than that derived from rbcL DNA barcoding (PhyllrbcL). The result showed that Tm of P. amarus, P. urinaria, P. debillis, P. airy-shawii, and P. virgatus was 74.3±0.08, 73.04±0.07, 73.36±0.05, 72.21±0.06, 72.77±0.15 °C, respectively. This method proved to be a very sensitive tool that can be used for rapid detection of contamination as low as 1% of other Phyllanthus species in P. amarus admixtures. All commercial products of P. amarus obtained from a local market in Thailand were found to contain pure raw materials of P. amarus without any substitution or contamination. Our results indicated that the use of DNA barcoding coupled to HRM was an efficient molecular tool for correct species identification. This molecular tool provides a noteworthy benefit for quality control of medicinal plants industry plants for pharmacological prospects. Copyright © 2015. Published by Elsevier B.V.
    Gene 07/2015; 573(1). DOI:10.1016/j.gene.2015.07.046 · 2.14 Impact Factor
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    • "For example, the P6 loop sequence in three analyzed Solanum species , differed by only one SNP, the length of the loop being the same. In the similar way, the length of the corresponding loop sequences in eight cereal species differed by not more than 5 nucleotides, with the nucleotide sequences of the loop in the Tritii cum genus and the Secale accessions being absolutely identical and a single nucleotide deletion being detected in the Zea mays accession compared to the Panicum accessions (Taberlet et al., 2007). "
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    ABSTRACT: The intron located between the first and the second nucleotide of the leucine tRNA anticodon is the only representative of the group I introns in higher plants. In this work, the intron sequence of the plastid trnL gene was for the first time described in 16 legumes species and putative secondary structures of the full-length intron and its functional domains were reconstructed. A high level of polymorphism was detected for this intron in the Fabaceae genera and in the species within a single genus. Single nucleotide polymorphisms, have been found in the sequences of the catalytic center which are considered to be highly conservative.
    Russian Journal of Genetics: Applied Research 05/2015; 5(3):220-226. DOI:10.1134/S2079059715030053
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    • "The insect bodies were subjected to DNA extraction using a NucleoSpin Tissue kit (Macherey-Nagel, Dü ren, Germany). DNA isolates were used for amplification of chloroplast barcode—the intron trnL (UAA)—using the following primers: A49325 and B49863 (Taberlet et al. 1991) for basic amplification and sequencing of standard barcode , and A49425 and B49466 (minibarcodes; Taberlet et al. 2007) only for brief verification of presence of highly degraded plant DNA in isolates that failed to get PCR products using the previous primer pair. TrnL intron was selected as barcode for this research because several previous studies have shown its great utility in ecological applications, including host plant barcoding in insects, particularly beetles (e.g., Jurado- Rivera et al. 2009, Pinzó n-Navarro et al. 2010, Kubisz et al. 2012, Kajtoch et al. 2013, Kitson et al. 2013, Kishimoto et al. 2013, Montagna et al. 2013). "
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    ABSTRACT: Recently, several papers were published dealing with host plant identification for selected species of insects, including beetles. These studies took advantage of the DNA barcoding approach and generally showed that it is possible to identify diet composition from plant DNA present in insect guts. However, none of these studies considered how the impact of environmental conditions affected the likelihood of insect feeding and, therefore, the presence of host plant DNA that could be amplified and sequenced. In the present study, individuals of the polyphagous weevil Centricnemus leucogrammus (Germar, 1824) (Curculionidae: Entiminae) were used to test the hypothesis that harsh environmental conditions limited its feeding activity. The diet of 50 specimens collected during favourable conditions in the middle of the species reproductive period was compared against the diet of 50 specimens collected during harsh environmental conditions. Results clearly showed that almost no weevils fed during rainy and cold conditions and only a minority of individuals (20%) fed during the drought condition (on drought-resistant plants). It is important to consider such factors in any studies dealing with host plant identification and feeding behaviour. Results of ecological studies could lead to erroneous conclusions, e.g., underestimation of number and composition of host plants in the diet of studies species. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
    Environmental Entomology 03/2015; 44(2):1-5. DOI:10.1093/ee/nvv019 · 1.30 Impact Factor
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