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DNA isolation is a fundamental technique for all molecular biology laboratories. Depending on the plant species, DNA isolation can be challenging. In particular, adapted protocols rarely exist for tree species which are not used as standard model organisms. Here, we describe a flexible DNA isolation protocol that works for 59 tree species in a modular system. It is based on an ATMAB-containing extraction buffer to which proteinase K and/or boric acid are added, depending on the plant species. Subsequent purification steps include one or two precipitations with dichloromethane and, depending on the tree species, an optional sodium acetate precipitation. Using leaf material of a hybrid poplar clone from in vitro culture, it was determined that higher amounts of DNA could be isolated with this material than from field leaves. Starting from leaf material, DNA isolation for difficult cases was achieved with cambium or root tissue. This protocol was used to extract DNA for subsequent PCR amplification. Markers for cpDNA, mtDNA, and genomic DNA were used for standardized testing.

Analysis of wolves dietary is a currently important theme because of the discussion about wolves preying on livestock as sheep or goats. We developed molecular markers to especially amplify the DNA of the prey out of wolf scat. For this purpose, we used the mitochondrial D-Loop using public available sequences for wolf and seven potential prey species (even-toed ungulates). We developed special primers amplifying either the wolves DNA or the prey DNA. In a fragment of 223-225 basepairs (bp) length we identified 21 SNPs, two 1-bp indels and one 3-bp indel, and three microsatellites to separate seven prey species from each other. Validation of the markers was performed by sequencing the PCR products of 12 fresh prey tissues and 20 wolf scat samples using the different primer pairs.

RNA editing occurs in the endosymbiont organelles of higher plants as C-to-U conversions of defined nucleotides. The availability of large quantities of RNA sequencing data makes it possible to identify RNA editing sites and to quantify their editing extent. We have investigated RNA editing in 34 protein-coding mitochondrial transcripts of four Populus species, a genus noteworthy for its remarkably small number of RNA editing sites compared to other angiosperms. 27 of these transcripts were subject to RNA editing in at least one species. In total, 355 RNA editing sites were identified with high confidence, their editing extents ranging from 10 to 100%. The most heavily edited transcripts were ccmB with the highest density of RNA editing sites (53.7 sites / kb) and ccmFn with the highest number of sites (39 sites). Most of the editing events are at position 1 or 2 of the codons, usually altering the encoded amino acid, and are highly conserved among the species, also with regard to their editing extent. However, one SNP was found in the newly sequenced and annotated mitochondrial genome of P. alba resulting in the loss of an RNA editing site compared to P. tremula and P. davidiana. This SNP causes a C-to-T transition and an amino acid exchange from Ser to Phe, highlighting the widely discussed role of RNA editing in compensating mutations.

The Swiss pine (Pinus cembra) is a montane tree in Central Europe and, therefore, known for its hardiness against severe winter colds. The seeds are harvested and eaten as pine nuts. We assembled and characterized the complete chloroplast genome of P. cembra to serve as a valuable resource in future genetic studies. The complete plastome sequence is 116,609 bp in length and contains 113 genes including 79 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. A phylogenetic analysis of 34 Pinus plastome sequences shows that Pinus sibirica is the nearest relative to P. cembra and that there is a distinct clustering together with the other members of the section Quinquefoliae.

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 insertion/deletion (indel) polymorphisms 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 indel 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.

A geographically isolated population of an herbivorous insect (Tortrix viridana, Lepidoptera: Tortricidae) in the Bashkir Transural region and 5 further populations were investigated for genetic variation using eight microsatellite markers. The sample size per population was between 48 and 62 individuals. The genetic variation was higher within the isolated population than within populations in the center of the distribution area. No bottleneck effects were discovered during analyses that could have formatted the gene pool of this population. Balancing or directed selection toward preservation of specific alleles or promotion of heterozygous individuals could be an explanation for the unexpected high genetic diversity within this small and isolated population.

Blowfly larvae found on human corpses are important for the estimation of the postmortem interval (PMI) and other questions of forensic relevance. Some of these species are difficult to differentiate morphologically, therefore a molecular method was elaborated for species identification. Specific fragments of the COI and COII region of the mitochondrial DNA (mtDNA) were amplified followed by digestion with different restriction enzymes. Using a 1.3 kb fragment, identification of Lucilia sericata, Calliphora vicina and Calliphora vomitoria was possible by digestion with only one restriction enzyme using either DraI or HinfI. Furthermore, we sequenced 349 bp (a part of the COI and COII regions) from the same three species and found 34 nucleotide distinctions between C. vicina and L. sericata, 30 between C. vomitoria and L. sericata and 15 between the two Calliphora species. These results aid in quick identification of species used for estimation of PMI.

Different species of the genus Eucalyptus, originally native to Australia, are being cultivated in different parts of the world due to their fast growth and beneficial wood properties. In Mexico, probably up to 25 different Eucalyptus species (many of them with unknown species declaration) were introduced early in the 20th century. Many Eucalyptus species are cross compatible and information about provenances of the single eucalypt species is rare. In this study, an experimental plantation established in 1984 and located in Northeast of Mexico was chosen as example to re-assign the species name of six randomly selected Eucalyptus trees growing in this plantation. First, a phylogenetic tree was constructed from complete chloroplast sequences of 31 Eucalyptus species available in the NCBI database. The phylogenetic tree includes three of the nine Eucalyptus species known to be introduced to Mexico, namely E. camaldulensis, E. saligna and E. grandis, which belong to a clade named “Symphyomyrts”. By employing combined BLASTN and UPGMA analyses of six chloroplast (cp) regions, three of the six unknown eucalypt samples (Euc4, 5, 6) cluster together with E. microtheca and E. cladocalyx, whereas the other three (Euc1, 2, 3) were more similar to a group containing E. camaldulensis, E. grandis and E. saligna. UPGMA analysis of the ITS region overall shows the same rough clustering, but provide more detailed information for two samples being most likely assigned to E. camaldulensis.

Background The interaction between insect pests and their host plants is a never-ending race of evolutionary adaption. Plants have developed an armament against insect herbivore attacks, and attackers continuously learn how to address it. Using a combined transcriptomic and metabolomic approach, we investigated the molecular and biochemical differences between Quercus robur L. trees that resisted (defined as resistant oak type) or were susceptible (defined as susceptible oak type) to infestation by the major oak pest, Tortrix viridana L. Results Next generation RNA sequencing revealed hundreds of genes that exhibited constitutive and/or inducible differential expression in the resistant oak compared to the susceptible oak. Distinct differences were found in the transcript levels and the metabolic content with regard to tannins, flavonoids, and terpenoids, which are compounds involved in the defence against insect pests. The results of our transcriptomic and metabolomic analyses are in agreement with those of a previous study in which we showed that female moths prefer susceptible oaks due to their specific profile of herbivore-induced volatiles. These data therefore define two oak genotypes that clearly differ on the transcriptomic and metabolomic levels, as reflected by their specific defensive compound profiles. Conclusions We conclude that the resistant oak type seem to prefer a strategy of constitutive defence responses in contrast to more induced defence responses of the susceptible oaks triggered by feeding. These results pave the way for the development of biomarkers for an early determination of potentially green oak leaf roller-resistant genotypes in natural pedunculate oak populations in Europe.