Development of a highly polymorphic chloroplast SSR set in Abies grandis with transferability to other conifer species-A promising toolkit for gene flow investigations.

The genus Abies is widely distributed across the world and is of high importance for forestry. Since chloroplasts are usually uniparentally inherited, they are an important tool for specific scientific issues like gene flow, parentage, migration and, in general, evolutionary analysis. Established genetic markers for organelles in conifers are rather limited to RFLP markers, which are more labour and time intensive, compared with SSR markers. Using QUIAGEN CLC Workbench 23.03, we aligned two chloroplast genomes from different Abies species (NCBI accessions: NC_039581, NC_042778, NC_039582, NC_042410, NC_035067, NC_062889, NC_042775, NC_057314, NC_041464, MH706706, MH047653 and MH510244) to identify potential SSR candidates. Further selection and development of forward and reverse primers was performed using the NCBI Primer Blast Server application. In this article, we introduce a remarkably polymorphic SSR marker set for various Abies species, which can be useful for other conifer genera, such as Cedrus, Pinus, Pseudotsuga or Picea. In total, 17 cpSSRs showed reliable amplification and polymorphisms in A. grandis with a total of 68 haplotypes detected. All 17 cpSSRs amplified in the tested Abies spp. In the other tested species, except for Taxus baccata, at least one primer was polymorphic.

Terpene Synthase Genes in Quercus robur - Gene Characterization, Expression and Resulting Terpenes Due to Cockchafer Feeding.

Root herbivory caused by larvae of the forest cockchafer (Melolontha hippocastani) enhances the impact of drought on trees, particularly in oak forest rejuvenations. In Germany, geographically distant oak stands show differences in infestation strength by the forest cockchafer. While in Southwestern Germany this insect causes severe damage, oak forests in northern Germany are rarely infested. It is known that root-released volatile organic compounds (VOCs) are perceived by soil herbivores, thus guiding the larvae toward the host roots. In this work, we exposed seedlings of two distant oak provenances to forest cockchafer larvae and studied their population genetic properties, their root-based VOC chemotypes, their attraction for larvae and terpene synthase gene expression. Based on nuclear and chloroplast marker analysis, we found both oak populations to be genetically highly variable while showing typical patterns of migration from different refugial regions. However, no clear association between genetic constitution of the different provenances and the abundance of cockchafer populations on site was observed. In contrast to observations in the field, bioassays revealed a preference of the larvae for the northeastern oak provenance. The behavior of larvae was most likely related to root-released volatile terpenes and benzenoids since their composition and quantity differed between oak populations. We assume repellent effects of these compounds because the populations attractive to insects showed low abundance of these compounds. Five different oak terpene synthase (TPS) genes were identified at the genomic level which can be responsible for biosynthesis of the released terpenes. TPS gene expression patterns in response to larval feeding revealed geographic variation rather than genotypic variation. Our results support the assumption that root-released VOC are influencing the perception of roots by herbivores.

Molecular genetic tools to infer the origin of forest plants and wood.

Most forest tree species exhibit high levels of genetic diversity that can be used to trace the origin of living plants or their products such as timber and processed wood. Recent progress to isolate DNA not only from living tissue but also from wood and wood products offers new opportunities to test the declared origin of material such as seedlings for plantation establishment or timber. However, since most forest tree populations are weakly differentiated, the identification of genetic markers to differentiate among spatially isolated populations is often difficult and time consuming. Two important fields of "forensic" applications are described: Molecular tools are applied to test the declared origin of forest reproductive material used for plantation establishment and of internationally traded timber and wood products. These applications are illustrated taking examples from Germany, where mechanisms have been developed to improve the control of the trade with forest seeds and seedlings, and from the trade with wood of the important Southeast Asian tree family Dipterocarpaceae. Prospects and limitations of the use of molecular genetic methods to conclude on the origin of forest plants, wood, and wood products are discussed.

Genetic and genomic approaches to assess adaptive genetic variation in plants: forest trees as a model.

With the increasing availability of sequence information at putatively important genes or regulatory regions, the characterization of adaptive genetic diversity and their association with phenotypic trait variation becomes feasible for many non-model organisms such as forest trees. Especially in predominantly outcrossing forest tree populations with large effective size, a high genetic variation in relevant genes is maintained, that is the raw material for the adaptation to changing and variable environments, and likewise for plant breeding. Oaks (Quercus spp.) are excellent model species to study the adaptation of forest trees to changing environments. They show a wide geographic distribution in Europe as dominant tree species in many forests and grow under a wide range of climatic and edaphic conditions. With the availability of a growing amount of functional and expressional candidate genes, we are now able to test the functional importance of single nucleotide polymorphisms (SNPs) by associating nucleotide variation in these genes with phenotypic variation in adaptive traits in segregating or natural populations. Here, we report on quantitative trait locus (QTL), candidate gene and association mapping approaches that are applicable to characterize gene markers and SNPs associated with variation in adaptive traits, such as bud burst, drought resistance and other traits showing selective responses to environmental change and stress. Because genome-wide association mapping studies are not feasible because of the enormous amount of SNP markers required in outcrossing trees with high recombination rates, the success of such an approach depends largely on the reasonable selection of candidate genes.

DNA from processed and unprocessed wood: factors influencing the isolation success.

Molecular genetic markers have numerous potential applications in environmental forensics if DNA can be isolated from 'difficult' non-human biological material such as hairs, feathers, or wood. The identification of the origin of wood is particularly important in order to identify illegally harvested and traded timber and wood products. We describe success rates of DNA isolation from wood based on a simple, previously published extraction protocol. The protocol was used to isolate DNA from a total of 406 wood samples, mainly of the important tropical tree family Dipterocarpaceae. The reliability of the extraction method was confirmed by comparing fragment sizes and sequences after isolation of DNA from leaves and wood of the same trees. We observed the success of amplification of chloroplast DNA (cpDNA) fragments of different lengths by means of PCR, investigated key factors influencing PCR, and conducted inhibitor tests for a subset of the samples. The average rate of successful PCR amplification was 75.7%. Main factors influencing the success of PCR amplification were the size of the amplified fragment and the processing status of the wood. Short fragments and unprocessed wood resulted in higher success rates. The success rate was also dependent on the age (storage duration) of the wood probe and on the investigated species. Amplification success was higher if DNA was isolated from outer sapwood (without cambium) in comparison to DNA isolated from the transition zone between sapwood and heartwood and the inner heartwood. However, inhibitor tests also indicated more PCR inhibitory substances in the outer sapwood in comparison to transition wood and heartwood. The addition of polyvinylpyrolidone (PVP) to the lysis buffer proved to be highly efficient to improve the amplification success if inhibitory substances were present.

Uptake and translocation of manganese in seedlings of two varieties of Douglas fir (Pseudotsuga menziesii var. viridis and glauca).

Douglas fir (Pseudotsuga menziesii) variety glauca (DFG) but not the variety viridis (DFV) showed symptoms of manganese (Mn) toxicity in some field sites. We hypothesized that these two varieties differed in Mn metabolism. To test this hypothesis, biomass partitioning, Mn concentrations, subcellular localization and 54Mn-transport were investigated. Total Mn uptake was three-times higher in DFG than in DFV. DFV retained > 90% of 54Mn in roots, whereas > 60% was transported to the shoot in DFG. The epidermis was probably the most efficient Mn barrier since DFV contained lower Mn concentrations in cortical cells and vacuoles of roots than DFG. In both varieties, xylem loading was restricted and phloem transport was low. However, sieve cells still contained high Mn concentrations. DFV displayed higher biomass production and higher shoot : root ratios than DFG. Our results clearly show that both varieties of Douglas fir differ significantly in Mn-uptake and allocation patterns rendering DFG more vulnerable to Mn toxicity.