Development of Molecular Markers for Determining Continental Origin of Wood from White Oaks (Quercus L. sect. Quercus)

Abstract

To detect and avoid illegal logging of valuable tree species, identification methods for the origin of timber are necessary. We used next-generation sequencing to identify chloroplast genome regions that differentiate the origin of white oaks from the three continents; Asia, Europe, and North America. By using the chloroplast genome of Asian Q. mongolica as a reference, we identified 861 variant sites (672 single nucleotide polymorphisms (SNPs); 189 insertion/deletion (indel) polymorphism) from representative species of three continents (Q. mongolica from Asia; Q. petraea and Q. robur from Europe; Q. alba from North America), and we identified additional chloroplast polymorphisms in pools of 20 individuals each from Q. mongolica (789 variant sites) and Q. robur (346 variant sites). Genome sequences were screened for indels to develop markers that identify continental origin of oak species, and that can be easily evaluated using a variety of detection methods. We identified five indels and one SNP that reliably identify continent-of-origin, based on evaluations of up to 1078 individuals representing 13 white oak species and three continents. Due to the size of length polymorphisms revealed, this marker set can be visualized using capillary electrophoresis or high resolution gel (acrylamide or agarose) electrophoresis. With these markers, we provide the wood trading market with an instrument to comply with the U.S. and European laws that require timber companies to avoid the trade of illegally harvested timber.

Fig 1. Phylogenetic relationship among chloroplast genomes of white oak species representing Old World and New World lineages.

The best maximum likelihood tree is shown for four white oak chloroplast genomes (Q. mongolica; Q. robur; Q. petraea; Q. alba) and one outgroup genome (Q. rubra). Inferred branch lengths in maximum likelihood substitutions are shown in bold, and bootstrap support values are show in italics. The phylogenetic resolution of informative indel markers are shown in black inverted triangles, and the resolution of the diagnostic PCR-RFLP marker is shown as a grey triangle.

Fig 2. Fragment patterns of the five markers for individuals from Asia (top), North America (middle) and Europe (bottom).

The sequence sizes for each peak as given in Table 3 are shown beneath the peaks. The first blue peaks appear smaller (112, 120) than the sequenced length (115, 123) given in Table 3. The color code of the peaks is as described in Table 2.

Fig 3. Marker trnDT visualized on a polyacrylamide gel.

Lane 1: 50 bp ladder, lane 8: zero control, lane 2–7 and 13–14: analysis of wood-derived DNA, its location is inferred from genotypes, lane 9–12: references from North America (US), Europe (EU) or Asia (AS), respectively.