Forensic validation of a SNP and INDEL panel for individualisation of timber from bigleaf maple (Acer macrophyllum Pursch).

Illegal logging is one of the largest illicit trades in the world, with high profits and generally low risks of detection and prosecution. Timber identification presents problems for law enforcement as traditionally used forensic methods such as wood anatomy and dendrochronology are often unable to confidently match wood evidence to the remains of illegally felled trees. Here we have developed and validated a set of genetic markers for individualisation in bigleaf maple (Acer macrophyllum), a high value timber species often felled illegally in the USA. Using 128 single nucleotide polymorphisms and three insertion/deletion markers developed through massively parallel sequencing, 394 individuals were genotyped on the MassARRAY® iPLEX™ platform (Agena Bio-science™, San Diego, USA) to produce a population reference database for the species. We demonstrate that the resulting DNA assay is reliable, species specific, effective at low DNA concentrations (<1 ng/µL) and suitable for application to timber samples. The PID for the most common profile, calculated using an overall dataset level FST-correction factor, was 1.785 × 10-25 and PID-SIB across all individuals (treated as a single population) was 2.496 × 10-22. The further development of forensic identification assays for timber species has the potential to deliver robust tools for improved detection and prosecution of illegal logging crimes as well as for the verification of legality in reputable supply chains.

Clonal dynamics in western North American aspen (Populus tremuloides).

Clonality is a common phenomenon in plants, allowing genets to persist asexually for much longer periods of time than ramets. The relative frequency of sexual vs. asexual reproduction determines long-term dominance and persistence of clonal plants at the landscape scale. One of the most familiar and valued clonal plants in North America is aspen (Populus tremuloides). Previous researchers have suggested that aspen in xeric landscapes of the intermountain west represent genets of great chronological age, maintained via clonal expansion in the near absence of sexual reproduction. We synthesized microsatellite data from 1371 ramets in two large sampling grids in Utah. We found a surprisingly large number of distinct genets, some covering large spatial areas, but most represented by only one to a few individual ramets at a sampling scale of 50 m. In general, multi-ramet genets were spatially cohesive, although some genets appear to be fragmented remnants of much larger clones. We conclude that recent sexual reproduction in these landscapes is a stronger contributor to standing genetic variation at the population level than the accumulation of somatic mutations, and that even some of the spatially large clones may not be as ancient as previously supposed. Further, a striking majority of the largest genets in both study areas had three alleles at one or more loci, suggesting triploidy or aneuploidy. These genets tended to be spatially clustered but not closely related. Together, these findings substantially advance our understanding of clonal dynamics in western North American aspen, and set the stage for a broad range of future studies.

A mutation hotspot in the chloroplast genome of a conifer (Douglas-fir: Pseudotsuga) is caused by variability in the number of direct repeats derived from a partially duplicated tRNA gene.

We determined the DNA sequence of a 2.7-kb cpDNA XbaI fragment from douglas-fir [Pseudotsuga menziesii (Mirb.) Franco]. RFLPs revealed by the 2.7-kb XbaI clone were observed to vary up to 1 kb among species within the genus Pseudotsuga and up to 200 bp among trees of P. menziesii. The polymerase chain reaction (PCR) allowed the locus of polymorphism to be identified, and the variable region was then sequenced in a second Douglas-fir tree, a single tree of a related species, Japanese Douglas-fir (P. japonica), and in a species lacking a mutation hotspot in the region, Pinus radiata (Monterey pine). The locus of polymorphism is characterized by hundreds of base pairs of imperfect, tandem direct repeats flanked by a partially duplicated and an intact trn Y-GUA gene. The duplication is direct in orientation and consists of 43 bp of the 3' end of trnY and 25 bp of its 3' flanking sequence. Tandem repeats show high sequence similarity to a 27-bp region of the trnY gene that overlaps one end of the duplication. The two trees of Douglas-fir sequenced differed by a single tandem repeat unit, whereas these trees differed from the Japanese Douglas-fir sequenced by approximately 34 repeat units. Repetitive DNA in the Pseudotsuga cpDNA hotspot was most likely generated at the time of the partial trnY gene duplication and these sequences expanded by slipped-strand mispairing and unequal crossing-over.

Chloroplast DNA diversity among trees, populations and species in the California closed-cone pines (Pinus radiata, Pinus muricata and Pinus attenuata).

The amount, distribution and mutational nature of chloroplast DNA polymorphisms were studied via analysis of restriction fragment length polymorphisms in three closely related species of conifers, the California closed-cone pines-knobcone pine: Pinus attenuata Lemm.; bishop pine: Pinus muricata D. Don; and Monterey pine: Pinus radiata D. Don. Genomic DNA from 384 trees representing 19 populations were digested with 9-20 restriction enzymes and probed with cloned cpDNA fragments from Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] that comprise 82% chloroplast genome. Up to 313 restriction sites were surveyed, and 25 of these were observed to be polymorphic among or within species. Differences among species accounted for the majority of genetic (haplotypic) diversity observed [Gst = 84(+/- 13)%]; nucleotide diversity among species was estimated to be 0.3(+/- 0.1)%. Knobcone pine and Monterey pine displayed almost no genetic variation within or among populations. Bishop pine also showed little variability within populations, but did display strong population differences [Gst = 87(+/- 8)%] that were a result of three distinct geographic groups. Mean nucleotide diversity within populations was 0.003(+/- 0.002)%; intrapopulation polymorphisms were found in only five populations. This pattern of genetic variation contrasts strongly with findings from study of nuclear genes (allozymes) in the group, where most genetic diversity resides within populations rather than among populations or species. Regions of the genome subject to frequent length mutations were identified; estimates of subdivision based on length variant frequencies in one region differed strikingly from those based on site mutations or allozymes. Two trees were identified with a major chloroplast DNA inversion that closely resembled one documented between Pinus and Pseudotsuga.

High levels of population differentiation for mitochondrial DNA haplotypes inPinus radiata, muricata, andattenuata.

We analyzed mitochondrial (mt) DNA restriction fragment length polymorphisms (RFLPs) associated with cytochrome oxidase, subunit I (coxI)-related gene sequences in 268 trees derived from 19 natural populations of three species of pines from California (USA): Monterey pine (Pinus radiata D. Don), bishop pine (P. Muricata D. Don), and knobcone pine (P. attenuata Lemm.). Total genomic DNA was digested with four restriction endonucleases and probed with a 750-bp fragment of the mitochondrialcoxI gene amplified fromP. attenuata via the polymerase chain reaction (PCR). ThecoxI gene is repeated at least 4 times in some populations, and all variants that we observed resulted from complex rearrangements rather than from point mutations. There was limited intrapopulation variation, but strong differentiation among populations. When applied to haplotype frequencies, Nei's gene diversity within populations (Hs) averaged 7% (±3), and Gst varied from 75% forP. Radiata to 96% forP. muricata. The high degree of population differentiation for mtDNA suggests that it can be a powerful marker of population differences, but its rapid rate of structural evolution appears to result from recombination among a limited number of repetitive elements-giving frequent homoplasious fragment phenotypes. The phylogenetic trees disagreed with results from chloroplast DNA, nuclear gene, and morphological studies.