DNA marker development for plant genetic analysis

Als Molekulare Marker werden kurze DNA Abschnitte herangezogen, die man für die Untersuchung und Markierung einer bestimmten Stelle im Genom eines Individuums verwendet. Immer wenn in dieser Region zwischen zwei Individuen Unterschiede auftreten, dann kann die Region als molekularer Marker verwendet werden. In den letzten zwanzig Jahren hat die Entwicklung solcher genetischen Marker die biologische Forschung in Botanik, Forst- und Landwirtschaft revolutioniert. Molekulare Marker fanden Anwendung in Populationsgenetik und Genotypisierung, in Züchtung und Taxonomie. Die Untersuchung van Individuen mittels genetischem Fingerprint ergab wertvolle einblicke in Populationsstrukturen und die bestehende genetische Vielfalt. Je nachdem, welches Genom man untersucht (Organellen, Nucleus), haben die angewandten molekularen Marker unterschiedliche Aussagekraft und es können verschiedene Fragestellungen bearbeitet werden. Während mit Organellenmarkern beispielsweise postglaziale Wanderungsrouten von Spezies rekonstruiert werden können, dienen Kern-Marker unter anderem zur Untersuchung von Verwandschaftsverhältnissen bei Einzelindividuen. Die gegenständliche Arbeit trägt mit ihren Entwicklungen zur Identifizierung neuer Marker aus den drei verschiedenen in Pflanzen vertretenen Genomen bei, wodurch verschiedene Pflanzengenetische Fragestellungen behandelt werden können. Um die genetische Vielfalt im, in den Alpen vorkommenden, Almrausch (Rhododendron ferrugineum und Rhododendron hirsutum) untersuchen zu können, wurden Microsatellitenregionen mittels aus dem Kerngenom mittels, für Dinukleotide angereicherte Genbanken isoliert. Die so neu identifizierten Kern-Marker wurden in weiterer Folge für populationsgenetische Untersuchungen in Österreichischen und Italienischen Reliktbeständen der Alpenrose verwendet. Die gleiche Anreicherungsmethode wurde angewendet, um SSR Regionen für die Unterscheidung von Paeonia suffruticosa Sorten einer Sammlung in Schönbrunn zu entwickeln. Da die Anreicherungsmethode nicht die gewünschten Ergebnisse brachte, wurde für Paeonia die Datenbankanalyse von ESTs zur Anwendung gebracht, bei der bereits publizierte Gensequenzen für die Identifikation von SSRs in exprimierten Genen herangezogen werden. Dadurch konnten weitere SSRs identifiziert werden, mit denen eine Unterscheidung der Paeonia Individuen möglich war. Mit der gleichen Methode wurden auch 24 EST-SSRs für Picea abies charakterisiert, die durch den Umstand, dass sie in exprimierten Genen liegen, auch zusätzliche Information über funktionelle Variation in den untersuchen Probensets geben können. Für Quercus robur L. und Quercus petrea L. wurden mittels standard Labormethoden sowohl SSRs als auch VNTRs isoliert welche für die Untersuchung der genetischen Vielfalt Österreichischer Eichen als auch, gemeinsam mit anderen molekularen Markern, für die Herstellung einer genetischen Karte einer Französischen Eichenkreuzung verwendet wurden. Microsatelliten sind hypervariable DNA Regionen im Genom, die leicht mutieren. Durch die Untersuchung von Pflanzenmaterial, das mittels Gewebekultur vermehrt wurde, konnte gezeigt werden, dass sich Microsatelliten sehr gut für die Detetktion von somaklonaler Variation, hervorgerufen durch Stress, eignen. Maternal vererbte Markersysteme wurden sowohl für Pappel (Populus nigra L) als auch für Fichte (Picea abies) entwickelt. Um die bestehende genetische Vielfalt in Europäischen Schwarzpappelherkünften zu untersuchen, wurde ein Chloroplastenbasiertes Markersystem entwickelt, das dazu diente, das postglaziale Wanderungsverhalten der Pappel zu untersuchen. Bei der Fichte untersucht das neu entwickelte maternale Markersystem Unterschiede in der mitochondrialen DNA der zu untersuchenden Proben. Mit dem neuen Markersystem konnte für Fichte moderate genetische Variation in 37 österreichischen Herkünften identifiziert werden. Die neu entwickelten nukleären sowie Organellen-Marker stellen wertvolle Werkzeuge zur Untersuchung der genetischen Vielfalt in natürlichen Populationen sowie für Zucht- und Auswahlprogramme dar. Durch die Verwendung von DNA basierten molekularen Daten kann die genetische Struktur von Populationen und auch der Genfluss zwischen Populationen gemessen werden. Die Marker dienen auch, so wie im Fall der Pappel, dazu, natürlich auftretendes klonales Material zu identifizieren. Mit den neuen Markern ist es möglich, spezifische populationsgenetische als auch phylogeographische Fragestellungen zu beantworten, und, so wie bei Rhododendron, Entscheidungen über Konservierungsmaßnahmen getroffen werden.Genetic markers use short DNA sequences in order to investigate and label a particular locus in the genome of a given individual. Whenever this particular locus shows sequence differences between two individuals, this region can be used as molecular marker. The development of a variety of DNA-based markers in the past twenty years has revolutionized biological, forestry and agricultural science. Molecular markers have demonstrated their usefulness for plant genotyping and population genetic analysis. Genome characterization by DNA-fingerprinting has generated valuable information on population structure and molecular genetic diversity. Depending on the compartmental origin of the genomic information (organelle, nucleus), molecular markers can be used for answering different questions. While organellar markers could serve for postglacial migration studies, nuclear markers can be used for studying biodiversity and paternity analysis on the level of the single individual. With the work presented here, we contributed to the marker development in different areas of plant genomics, and proved the applicability of the different marker types targeting the three different genomes (nuclear, mitochondrial, chloroplast) available in plants. In order to investigate the genetic variation present in Alpine Rhododendron populations, nuclear SSRs were developed for Rhododendron ferrugineum and Rhododendron hirsutum by generating genomic libraries enriched for dinucleotide repeats. These newly generated markers subsequently were used to investigate relict populations of the alpine rose in Italy and Austria. The same method was applied to isolate SSR regions for the identification of Paeonia suffruticosa varieties available in a germ plasm collection in Schönbrunn. In the latter case, also an in silico approach was used in order to identify SSRs present in publicly available ESTs (sequence information on Expressed Sequence Tags). By this approach, 5 additional SSR markers for Paeonia suffruticosa as well as 24 EST-SSRs for Picea abies could be generated in relatively short time. These EST-SSRs have added value as they are part of expressed sequences and thus can be used for measuring the functional diversity in Norway spruce as well as Paeonia. For Quercus robur L. and Quercus petrea L. SSRs as well as VNTRs were isolated by a standard wet lab approaches and used for diversity measurements in Austrian populations of oak. In addition, these markers, together with RAPDs, RFLPs and SCARs were mapped on a genetic map of a French controlled oak cross. Due to their hypervariable nature and sensitivity to mutations, nuclear microsatellites for oak were used to test for somaclonal variation occurring in tissue cultue material over time. With this approach we could prove the usefulness of the SSRs for testing the genome stability under stress. Maternally inherited markers were developed for Populus nigra L. and Picea abies. In order to assess the genetic diversity in Populus nigra L. sampled throughout Europe, a chloroplast DNA based marker system was developed and applied. For Norway spruce on the other hand, the maternally inherited mitochondrial genome was targeted for marker development. Using these new mt markers for Norway spruce it could be shown that there is moderate genetic variation in Austrian Norway spruce strands, not detected before. The newly developed nuclear as well as organellar markers are valuable tools for the assessment of genetic diversity in natural populations as well as plant selection programs. Using this DNA based molecular information it is possible to analyze the genetic structure of populations, to estimate gene flow between populations and, as in the case of poplar, to identify naturally occurring clonal material. With these new markers, specific population genetic as well as phylogeographical questions can be answered, and, as in the case of rhododendron, decisions on conservational measures taken

The meristem-associated endosymbiont Methylorubrum extorquens DSM13060 reprograms development and stress responses of pine seedlings

Microbes living in plant tissues-endophytes-are mainly studied in crop plants where they typically colonize the root apoplast. Trees-a large carbon source with a high capacity for photosynthesis-provide a variety of niches for endophytic colonization. We have earlier identified a new type of plant-endophyte interaction in buds of adult Scots pine, where Methylorubrum species live inside the meristematic cells. The endosymbiont Methylorubrum extorquens DSM13060 significantly increases needle and root growth of pine seedlings without producing plant hormones, but by aggregating around host nuclei. Here, we studied gene expression and metabolites of the pine host induced by M. extorquens DSM13060 infection. Malic acid was produced by pine to potentially boost M. extorquens colonization and interaction. Based on gene expression, the endosymbiont activated the auxin- and ethylene (ET)-associated hormonal pathways through induction of CUL1 and HYL1, and suppressed salicylic and abscisic acid signaling of pine. Infection by the endosymbiont had an effect on pine meristem and leaf development through activation of GLP1-7 and ALE2, and suppressed flowering, root hair and lateral root formation by downregulation of AGL8, plantacyanin, GASA7, COW1 and RALFL34. Despite of systemic infection of pine seedlings by the endosymbiont, the pine genes CUL1, ETR2, ERF3, HYL, GLP1-7 and CYP71 were highly expressed in the shoot apical meristem, rarely in needles and not in stem or root tissues. Low expression of MERI5, CLH2, EULS3 and high quantities of ononitol suggest that endosymbiont promotes viability and protects pine seedlings against abiotic stress. Our results indicate that the endosymbiont positively affects host development and stress tolerance through mechanisms previously unknown for endophytic bacteria, manipulation of plant hormone signaling pathways, downregulation of senescence and cell death-associated genes and induction of ononitol biosynthesis.Peer reviewe

Characterization of variable EST SSR markers for Norway spruce (Picea abies L.)

<p>Abstract</p> <p>Background</p> <p>Norway spruce is widely distributed across Europe and the predominant tree of the Alpine region. Fast growth and the fact that timber can be harvested cost-effectively in relatively young populations define its status as one of the economically most important tree species of Northern Europe. In this study, EST derived simple sequence repeat (SSR) markers were developed for the assessment of putative functional diversity in Austrian Norway spruce stands.</p> <p>Results</p> <p>SSR sequences were identified by analyzing 14,022 publicly available EST sequences. Tri-nucleotide repeat motifs were most abundant in the data set followed by penta- and hexa-nucleotide repeats. Specific primer pairs were designed for sixty loci. Among these, 27 displayed polymorphism in a testing population of 16 <it>P. abies </it>individuals sampled across Austria and in an additional screening population of 96 <it>P. abies </it>individuals from two geographically distinct Austrian populations. Allele numbers per locus ranged from two to 17 with observed heterozygosity ranging from 0.075 to 0.99.</p> <p>Conclusions</p> <p>We have characterized variable EST SSR markers for Norway spruce detected in expressed genes. Due to their moderate to high degree of variability in the two tested screening populations, these newly developed SSR markers are well suited for the analysis of stress related functional variation present in Norway spruce populations.</p

Epigenetic regulation of adaptive responses of forest tree species to the environment

Epigenetic variation is likely to contribute to the phenotypic plasticity and adaptative capacity of plant species, and may be especially important for long-lived organisms with complex life cycles, including forest trees. Diverse environmental stresses and hybridization/polyploidization events can create reversible heritable epigenetic marks that can be transmitted to subsequent generations as a form of molecular “memory”. Epigenetic changes might also contribute to the ability of plants to colonize or persist in variable environments. In this review, we provide an overview of recent data on epigenetic mechanisms involved in developmental processes and responses to environmental cues in plant, with a focus on forest tree species. We consider the possible role of forest tree epigenetics as a new source of adaptive traits in plant breeding, biotechnology, and ecosystem conservation under rapid climate change.This is the publisher’s final pdf. The published article is copyrighted by the authors and published by Blackwell Publishing Ltd. and can be found at: http://onlinelibrary.wiley.com/journal/10.1002/%28ISSN%292045-7758.Keywords: forest trees, phenotypic plasticity, epigenetic memory of stressful conditions, adaptive response, epigenetics, environmental stres

Sequence Composition and Gene Content of the Short Arm of Rye (Secale cereale) Chromosome 1

BACKGROUND: The purpose of the study is to elucidate the sequence composition of the short arm of rye chromosome 1 (Secale cereale) with special focus on its gene content, because this portion of the rye genome is an integrated part of several hundreds of bread wheat varieties worldwide. METHODOLOGY/PRINCIPAL FINDINGS: Multiple Displacement Amplification of 1RS DNA, obtained from flow sorted 1RS chromosomes, using 1RS ditelosomic wheat-rye addition line, and subsequent Roche 454FLX sequencing of this DNA yielded 195,313,589 bp sequence information. This quantity of sequence information resulted in 0.43× sequence coverage of the 1RS chromosome arm, permitting the identification of genes with estimated probability of 95%. A detailed analysis revealed that more than 5% of the 1RS sequence consisted of gene space, identifying at least 3,121 gene loci representing 1,882 different gene functions. Repetitive elements comprised about 72% of the 1RS sequence, Gypsy/Sabrina (13.3%) being the most abundant. More than four thousand simple sequence repeat (SSR) sites mostly located in gene related sequence reads were identified for possible marker development. The existence of chloroplast insertions in 1RS has been verified by identifying chimeric chloroplast-genomic sequence reads. Synteny analysis of 1RS to the full genomes of Oryza sativa and Brachypodium distachyon revealed that about half of the genes of 1RS correspond to the distal end of the short arm of rice chromosome 5 and the proximal region of the long arm of Brachypodium distachyon chromosome 2. Comparison of the gene content of 1RS to 1HS barley chromosome arm revealed high conservation of genes related to chromosome 5 of rice. CONCLUSIONS: The present study revealed the gene content and potential gene functions on this chromosome arm and demonstrated numerous sequence elements like SSRs and gene-related sequences, which can be utilised for future research as well as in breeding of wheat and rye

Data from: Allele discovery of ten candidate drought-response genes in Austrian oak using a systematically informatics approach based on 454 amplicon sequencing

BACKGROUND: Rise of temperatures and shortening of available water as result of predicted climate change will impose significant pressure on long-lived forest tree species. Discovering allelic variation present in drought related genes of two Austrian oak species can be the key to understand mechanisms of natural selection and provide forestry with key tools to cope with future challenges. RESULTS: In the present study we have used Roche 454 sequencing and developed a bioinformatic pipeline to process multiplexed tagged amplicons in order to identify single nucleotide polymorphisms and allelic sequences of ten candidate genes related to drought/osmotic stress from sessile oak (Quercus robur) and pedunculate oak (Q. petraea) individuals. Out of these, eight genes of 336 oak individuals growing in Austria have been detected with a total number of 158 polymorphic sites. Allele numbers ranged from ten to 52 with observed heterozygosity ranging from 0.115 to 0.640. All loci deviated from Hardy-Weinberg equilibrium and linkage disequilibrium was found among six combinations of loci. CONCLUSIONS: We have characterized 183 alleles of drought related genes from oak species and detected first evidences of natural selection. Beside the potential for marker development, we have created an expandable bioinformatic pipeline for the analysis of next generation sequencing data

Elucidating Drought Stress Tolerance in European Oaks Through Cross-Species Transcriptomics

The impact of climate change that comes with a dramatic increase of long periods of extreme summer drought associated with heat is a fundamental challenge for European forests. As a result, forests are expected to shift their distribution patterns toward north-east, which may lead to a dramatic loss in value of European forest land. Consequently, unraveling key processes that underlie drought stress tolerance is not only of great scientific but also of utmost economic importance for forests to withstand future heat and drought wave scenarios. To reveal drought stress-related molecular patterns we applied cross-species comparative transcriptomics of three major European oak species: the less tolerant deciduous pedunculate oak (Quercus robur), the deciduous but quite tolerant pubescent oak (Q. pubescens), and the very tolerant evergreen holm oak (Q. ilex). We found 415, 79, and 222 differentially expressed genes during drought stress in Q. robur, Q. pubescens, and Q. ilex, respectively, indicating species-specific response mechanisms. Further, by comparative orthologous gene family analysis, 517 orthologous genes could be characterized that may play an important role in drought stress adaptation on the genus level. New regulatory candidate pathways and genes in the context of drought stress response were identified, highlighting the importance of the antioxidant capacity, the mitochondrial respiration machinery, the lignification of the water transport system, and the suppression of drought-induced senescence – providing a valuable knowledge base that could be integrated in breeding programs in the face of climate change