Schlussbericht zum Projekt: Übertragbares Managementkonzept für Arnica montana - Teilvorhaben 2: Etablierung, Standardisierung und Qualitätskontrolle des genetischen Monitoring-Systems für Arnica montana

Für die gefährdete und im Rückgang begriffene Verantwortungsart Arnica montana wurde ein übertragbares genetisches Monitoring-System entwickelt. Hierfür wurden 14 hochvariable Mikrosatelliten-Marker für die Analyse optimiert sowie deren Variation molekular charakterisiert. Mittels Referenz-Proben und allelischen Leitern, das sind Mischproben der häufigsten Allele, ist eine Standardisierung und somit Verschneidung von genotypischen Daten aus verschiedenen Laboren jetzt möglich, was eine Grundlage für langfristiges Monitoring darstellt. Die so optimierten Marker wurden in Zusammenarbeit mit den Projektpartnern erfolgreich eingesetzt, um die Populationsqualität der hessischen Tieflandvorkommen von A. montana zu untersuchen. Hieraus ließ sich ableiten, dass der Rückgang der Bestände auch genetische Ursachen hat. Zahlreiche Bestände sind Reliktvorkommen, die nur noch aus einem oder wenigen Klonen bestehen, die sich kaum noch sexuell reproduzieren. Tendenziell bestand ein Zusammenhang zwischen der Bestandsgröße und der Neigung zu klonalen Strukturen. Dennoch sollte die Rosettenzahl nicht als alleiniger Indikator verwendet werden. Auch wenn A. montana durch ein Selbstinkompatibilitäts-System vor Inzucht-Effekten weitgehend geschützt ist, führt ein überwiegend klonales Wachstum dennoch zu einem fast vollständigen Verlust der genetischen Variabilität, die nur durch entsprechende Management-Maßnahmen zu beheben ist, wenn diese Bestände langfristig überleben sollen. Für entsprechende populationsstützende Maßnahmen zur Anreicherung der genetischen Vielfalt sind im Projektgebiet noch einige gut erhaltene Bestände mit ausreichend genetischer Vielfalt vorhanden. Diese könnten potentiell als Spenderpopulationen genutzt werden. Hierbei ist zu beachten, dass A. montana in Hessen eine klar Populationsstruktur ausbildet mit zwei deutlich unterschiedlichen genetischen Gruppen. Dabei gruppieren die nordwestlich gelegenen Bestände miteinander sowie die südöstlich gelegenen. Darüber hinaus zeigte sich, dass die zerstreut vorkommenden Bestände von A. montana nur zu einem geringen genetischen Austausch untereinander in der Lage sind, so dass eine genetische Anreicherung auf natürlichem Wege unwahrscheinlich ist. Für populationsstützende Maßnahmen wird daher zunächst empfohlen, nur Bestände aus der gleichen genetischen Gruppe zu mischen. Um zu prüfen, ob die Mischung von Pflanzen aus unterschiedlichen Beständen einen positiven Effekt hat, wurden kontrollierte Kreuzungsexperimente durchgeführt, die zeigten, dass A. montana bei Selbstbestäubung nur einen geringen Samenansatz hat, was die Selbstinkompatibilität bestätigt. Für eine Aussage hinsichtlich der Kreuzung genetisch unterschiedlicher Bestände bestehen zurzeit keine Hinweise auf negative Effekte, jedoch muss eine Untersuchung der zweiten Folgegeneration noch erfolgen, die eventuelle negative Auszuchteffekte anzeigen könnte. Dies konnte leider innerhalb der Projektlaufzeit aufgrund technischer Probleme nicht mehr untersucht werden, wird jedoch von uns weiterverfolgt. Insgesamt lieferten die genetischen Untersuchungen wertvolle Erkenntnisse und Hinweise für das Management von A. montana auch über Hessen hinaus. Diese konnten auf zwei Fachtagungen für praktische Anwender und in Form eines Praxisleitfaden neben den umfangreichen ökologischen und praktischen Erkenntnissen an die Zielgruppe vermittelt werden

Characterization of 23 polymorphic SSR markers in Salix humboldtiana (Salicaceae) using next‐generation sequencing and cross‐amplification from related species

Premise of the study: We present a set of 23 polymorphic nuclear microsatellite loci, 18 of which are identified for the first time within the riparian species Salix humboldtiana (Salicaceae) using next‐generation sequencing. Methods and Results: To characterize the 23 loci, up to 60 individuals were sampled and genotyped at each locus. The number of alleles ranged from two to eight, with an average of 4.43 alleles per locus. The effective number of alleles ranged from 1.15 to 3.09 per locus, and allelic richness ranged from 2.00 to 7.73 alleles per locus. Conclusions: The new marker set will be used for future studies of genetic diversity and differentiation as well as for unraveling spatial genetic structures in S. humboldtiana populations in northern Patagonia, Argentina.EEA BarilocheFil: Bozzi, Jorge Alfredo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche; ArgentinaFil: Liepelt, Sascha. University of Marburg. Faculty of Biology. Conservation Biology Group; AlemaniaFil: Ohneiser, Sebastian. University of Marburg. Faculty of Biology. Conservation Biology Group; AlemaniaFil: Gallo, Leonardo Ariel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche; ArgentinaFil: Marchelli, Paula. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bariloche; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Leyer, Ilona. University of Marburg. Faculty of Biology. Conservation Biology Group; Alemania. University of Geisenheim. Institute of Botany, Plant Ecology and Nature Conservation; AlemaniaFil: Ziegenhagen, Birgit. University of Marburg. Faculty of Biology. Conservation Biology Group; AlemaniaFil: Mengel, Christina. University of Marburg. Faculty of Biology. Conservation Biology Group; Alemani

Authenticated DNA from Ancient Wood Remains

• Background The reconstruction of biological processes and human activities during the last glacial cycle relies mainly on data from biological remains. Highly abundant tissues, such as wood, are candidates for a genetic analysis of past populations. While well-authenticated DNA has now been recovered from various fossil remains, the final ‘proof' is still missing for wood, despite some promising studies. • Scope The goal of this study was to determine if ancient wood can be analysed routinely in studies of archaeology and palaeogenetics. An experiment was designed which included blind testing, independent replicates, extensive contamination controls and rigorous statistical tests. Ten samples of ancient wood from major European forest tree genera were analysed with plastid DNA markers. • Conclusions Authentic DNA was retrieved from wood samples up to 1000 years of age. A new tool for real-time vegetation history and archaeology is ready to us

Age at onset as stratifier in idiopathic Parkinson’s disease – effect of ageing and polygenic risk score on clinical phenotypes

Several phenotypic differences observed in Parkinson’s disease (PD) patients have been linked to age at onset (AAO). We endeavoured to find out whether these differences are due to the ageing process itself by using a combined dataset of idiopathic PD (n = 430) and healthy controls (HC; n = 556) excluding carriers of known PD-linked genetic mutations in both groups. We found several significant effects of AAO on motor and non-motor symptoms in PD, but when comparing the effects of age on these symptoms with HC (using age at assessment, AAA), only positive associations of AAA with burden of motor symptoms and cognitive impairment were significantly different between PD vs HC. Furthermore, we explored a potential effect of polygenic risk score (PRS) on clinical phenotype and identified a significant inverse correlation of AAO and PRS in PD. No significant association between PRS and severity of clinical symptoms was found. We conclude that the observed non-motor phenotypic differences in PD based on AAO are largely driven by the ageing process itself and not by a specific profile of neurodegeneration linked to AAO in the idiopathic PD patients

Differential Gene Expression Reveals Candidate Genes for Drought Stress Response in Abies alba (Pinaceae)

Increasing drought periods as a result of global climate change pose a threat to many tree species by possibly outpacing their adaptive capabilities. Revealing the genetic basis of drought stress response is therefore implemental for future conservation strategies and risk assessment. Access to informative genomic regions is however challenging, especially for conifers, partially due to their large genomes, which puts constraints on the feasibility of whole genome scans. Candidate genes offer a valuable tool to reduce the complexity of the analysis and the amount of sequencing work and costs. For this study we combined an improved drought stress phenotyping of needles via a novel terahertz water monitoring technique with Massive Analysis of cDNA Ends to identify candidate genes for drought stress response in European silver fir (Abies alba Mill.). A pooled cDNA library was constructed from the cotyledons of six drought stressed and six well-watered silver fir seedlings, respectively. Differential expression analyses of these libraries revealed 296 candidate genes for drought stress response in silver fir (247 up- and 49 down-regulated) of which a subset was validated by RT-qPCR of the twelve individual cotyledons. A majority of these genes code for currently uncharacterized proteins and hint on new genomic resources to be explored in conifers. Furthermore, we could show that some traditional reference genes from model plant species (GAPDH and eIF4A2) are not suitable for differential analysis and we propose a new reference gene, TPC1, for drought stress expression profiling in needles of conifer seedlings

Data from: Within-range translocations and their consequences in European larch

In contrast to biological invasions, translocations of individuals within a species range are understudied, due to difficulties in systematically detecting them. This results in limited knowledge about the corresponding processes and uncertainties regarding the status of extant populations. European larch, a forest tree whose fragmented native distribution is restricted to the Alps and to other Central European mountains, has been massively planted for at least 300 years. Here we focus on the genetic characterization of translocations having taken place within its native range. Microsatellite variation at 13 nuclear loci and sequence data of two mitochondrial DNA fragments were analyzed on the basis of a comprehensive range-wide population sample. Two complementary methods (Geneclass and Structure) were used to infer translocation events based on nuclear data whereas mitochondrial data were used for validation of these inferences. Using Geneclass, we found translocation events in a majority of populations. Additional cases of translocation and many instances of admixture were identified using Structure, thanks to the clear-cut ancestral genetic structure detected in this species. In particular, a strong divide between Alpine and Central European populations, also apparent at mitochondrial markers, helped uncover details on translocation events and related processes. Translocations and associated admixture events were found to be heterogeneously distributed across the species range, with a particularly high frequency in Central Europe. Furthermore, translocations frequently involved multiple geographic sources, some of which were over-represented. Our study illustrates the importance of range-wide investigations for tracing translocations back to their origins and for revealing some of their consequences. It provides some first clues for developing suitable conservation and management strategies

Genetic insights into the hybrid origin of Abies × borisii-regis Mattf

Abies × borisii-regis Mattf. (King Boris fir) is a taxon endemic to the southern Balkan Peninsula, described as a hybrid between the widespread A. alba Mill. (Silver fir) and the Greek endemic A. cephalonica Loud (Greek fir). Even though A. × borisii-regis has attracted much research attention in the past, its origin, geographical distribution and taxonomic status are not fully elucidated and molecular evidence for hybridization is missing. To shed more light on this issue, we analyzed representative populations from these three Abies taxa using paternally inherited (chloroplast) and maternally inherited (mitochondrial) DNA markers. Both Silver and Greek fir could be clearly distinguished using mitochondrial markers, while we observed a mixture of maternal lineages in the A. × borisii-regis populations. In contrast, using chloroplast markers, we could not identify species-specific haplotypes, but a neighbor-joining analysis of population genetic distances revealed two separate clusters for the Silver fir and the Greek fir, while the A. × borisii-regis populations were placed in intermediate positions. Our results are in agreement with the hypothesis that the A. × borisii-regis populations investigated are a result of hybridization between A. cephalonica and A. alba

Annotation and re-sequencing of genes from de novo transcriptome assembly of abies alba (pinaceae)

Premise of the study: We present a protocol for the annotation of transcriptome sequence data and the identification of candidate genes therein using the example of the nonmodel conifer Abies alba. Methods and Results: A normalized cDNA library was built from an A. alba seedling. The sequencing on a 454 platform yielded more than 1.5 million reads that were de novo assembled into 25 149 contigs. Two complementary approaches were applied to annotate gene fragments that code for (1) well-known proteins and (2) proteins that are potentially adaptively relevant. Primer development and testing yielded 88 amplicons that could successfully be resequenced from genomic DNA. Conclusions: The annotation workflow offers an efficient way to identify potential adaptively relevant genes from the large quantity of transcriptome sequence data. The primer set presented should be prioritized for single-nucleotide polymorphism detection in adaptively relevant genes in A. alba

Log-log plot of up- and down-regulated transcripts in response to drought stress on GO-level 4 in silver fir seedlings.

<p>Transcripts are differentiated by their GO ancestor: metabolic process (GO:0008152), response to stimulus (GO:0050896) or other ancestor. Most obvious GO terms associated with drought stress, as well as photosynthesis, are highlighted and labeled specifically.</p