Power and limitations of the chloroplast trnL (UAA) intron for plant DNA barcoding

Pierre Taberlet, Eric Coissac, François Pompanon, Ludovic Gielly, Christian Miquel, Alice Valentini, Thierry Vermat, Gérard Corthier, Christian Brochmann, Eske Willerslev, Pierre Taberlet, Eric Coissac, François Pompanon, Ludovic Gielly, Christian Miquel, Alice Valentini, Thierry Vermat, Gérard Corthier, Christian Brochmann, Eske Willerslev

Abstract

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.

Figures

Figure 1
Figure 1
Position of the primers c, d, g and h on the chloroplast trnL (UAA) gene. The P6 loop amplified with primer g and h is indicated in green.
Figure 2
Figure 2
Positions of the primers c and d on the secondary structure of the trnL (UAA) exon (A) and of the primers g and h on the secondary structure of the trnL (UAA) intron (B) for Nymphaea odorata [modified from Ref. (33)]. Highly conserved elements of the catalytic core (P, Q, R1, R2 and S) are located in grey boxes. The P6 loop, amplified with primers g and h, is identified by green letters. The 3′ ends of each of the four primers c, d, g and h are marked out by an arrow and their positions are identified by red letters.
Figure 3
Figure 3
Example of multi-peak profiles obtained after capillary electrophoresis of the fluorescent PCR products obtained using the g and h primers. (A) Permafrost sample drilled from Main River Ice Bluff (N.E. Siberia, 64.06N, 171.11E), between 21 050 and 25 440 years old (uncalibrated 14C years, based on AMS dating of plant macrofossils from the section); g fluorescent primer; each peak represents at least one arctic plant species. (B) Human feces sample; h fluorescent primer; three of the four main peaks have been identified after cloning and sequencing: peak 1, nonidentified; peak 2, banana (Musa acuminata); peak 3, lettuce (Lactuca sativa); and peak 4, cacao (Theobroma cacao).

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