An efficient method for purification of PCR products for sequencing

A high-throughput DNA sequencing method that generated high quality data was developed. A frame fashioned from a standard agarose gel combined with 0.1%–0.2% low-melting point (LMP) agarose gel was used to isolate the PCR product of interest. Collected PCR products were centrifuged without any reagents and the supernatants were directly used for a sequenc- ing reaction. This method is simple and labor efficient, provides high quality sequences at a low cost, and bypasses problems with impure PCR products. This technique has been used for single nucleotide polymorphism (SNP) discovery in Populus angustifolia trees

Predicting whole genome protein interaction networks from primary sequence data in model and non-model organisms using ENTS

Background The large-scale identification of physical protein-protein interactions (PPIs) is an important step toward understanding how biological networks evolve and generate emergent phenotypes. However, experimental identification of PPIs is a laborious and error-prone process, and current methods of PPI prediction tend to be highly conservative or require large amounts of functional data that may not be available for newly-sequenced organisms. Results In this study we demonstrate a random-forest based technique, ENTS, for the computational prediction of protein-protein interactions based only on primary sequence data. Our approach is able to efficiently predict interactions on a whole-genome scale for any eukaryotic organism, using pairwise combinations of conserved domains and predicted subcellular localization of proteins as input features. We present the first predicted interactome for the forest tree Populus trichocarpa in addition to the predicted interactomes for Saccharomyces cerevisiae, Homo sapiens, Mus musculus, and Arabidopsis thaliana. Comparing our approach to other PPI predictors, we find that ENTS performs comparably to or better than a number of existing approaches, including several that utilize a variety of functional information for their predictions. We also find that the predicted interactions are biologically meaningful, as indicated by similarity in functional annotations and enrichment of co-expressed genes in public microarray datasets. Furthermore, we demonstrate some of the biological insights that can be gained from these predicted interaction networks. We show that the predicted interactions yield informative groupings of P. trichocarpa metabolic pathways, literature-supported associations among human disease states, and theory-supported insight into the evolutionary dynamics of duplicated genes in paleopolyploid plants. Conclusion We conclude that the ENTS classifier will be a valuable tool for the de novoannotation of genome sequences, providing initial clues about regulatory and metabolic network topology, and revealing relationships that are not immediately obvious from traditional homology-based annotations

Genome-wide analysis of Aux/IAA and ARF gene families in Populus trichocarpa

<p>Abstract</p> <p>Background</p> <p>Auxin/Indole-3-Acetic Acid (Aux/IAA) and Auxin Response Factor (ARF) transcription factors are key regulators of auxin responses in plants. We identified the suites of genes in the two gene families in <it>Populus </it>and performed comparative genomic analysis with <it>Arabidopsis </it>and rice.</p> <p>Results</p> <p>A total of 35 <it>Aux/IAA </it>and 39 <it>ARF </it>genes were identified in the <it>Populus </it>genome. Comparative phylogenetic analysis revealed that several Aux/IAA and ARF subgroups have differentially expanded or contracted between the two dicotyledonous plants. Activator <it>ARF </it>genes were found to be two fold-overrepresented in the <it>Populus </it>genome. <it>PoptrIAA </it>and <it>PoptrARF </it>gene families appear to have expanded due to high segmental and low tandem duplication events. Furthermore, expression studies showed that genes in the expanded <it>PoptrIAA3 </it>subgroup display differential expression.</p> <p>Conclusion</p> <p>The present study examines the extent of conservation and divergence in the structure and evolution of <it>Populus Aux/IAA </it>and <it>ARF </it>gene families with respect to <it>Arabidopsis </it>and rice. The gene-family analysis reported here will be useful in conducting future functional genomics studies to understand how the molecular roles of these large gene families translate into a diversity of biologically meaningful auxin effects.</p

Mechanisms of cadmium toxicity and tolerance in Populus

Abstract ID#: 26642&#xd;&#xa;Background and Methods:&#xd;&#xa;&#xd;&#xa;Cadmium (Cd), ranked the 7th most hazardous substance, is one of the most widespread pollutants of soil and water in industrialized nations. Its increased movement in soil-plant systems is posing a serious threat to human health. Cd, without any known functions in plants, was found to be toxic even at minute concentrations, leading to the development of symptoms such as leaf roll, chlorosis and root and shoot growth reduction. Phytoremediation is an emerging cost-effective and environment friendly technology that utilizes high biomass producing plants including Populus plants to remove, transform or stabilize contaminants in soils. The objectives of our study were to record phenotypic variation in a Populus pedigree to Cd exposure, to identify Cd tolerant and susceptible genotypes of Populus, to map QTLs (Quantitative Trait Loci - genomic regions responsible) for Cd tolerance and accumulation in Populus and to predict a mechanism for Cd toxicity and tolerance in susceptible and tolerant Populus genotypes.&#xd;&#xa;&#xd;&#xa;QTL mapping was accomplished by conducting a greenhouse hydroponic study in which 252 genotypes of a Populus pseudo-backcross progeny were grown for 40 days and treated with 25 &#xb5;M Cd. Phenotypic variation in total dry weight was recorded on these genotypes and was used for identifying QTL for Cd tolerance. We identified genotypes with contrasting responses to the Cd treatment and conducted a microarray study to identify potential Cd tolerance mechanisms based on gene expression patterns.&#xd;&#xa;&#xd;&#xa; &#xd;&#xa;&#xd;&#xa;Results/Conclusions:&#xd;&#xa;&#xd;&#xa;Significant variation was observed among genotypes in response to Cd treatment based on changes in total dry weight. Cd tolerant and susceptible genotypes were identified based on the least square mean differences (Control-Cd treated for each genotype) among all the genotypes. Three QTLs were identified for Cd tolerance and they accounted for approximately 25% of the phenotypic variation in Cd tolerance measured as total dry weights.&#xd;&#xa;&#xd;&#xa;In the microarray study, the Cd-susceptible genotype had higher expression of Fe-transporters compared to the tolerant genotypes, and Cd and Fe levels were significantly different in foliage. Even the susceptible genotype controls had higher, though not significant, Fe levels than tolerant genotype controls. We therefore hypothesize that part of the mechanism for Cd tolerance is determined by the differences in the activity of Fe transporters in genotypes with differential Fe homeostasis

Pleiotropic and Epistatic Network-Based Discovery: Integrated Networks for Target Gene Discovery

Biological organisms are complex systems that are composed of functional networks of interacting molecules and macro-molecules. Complex phenotypes are the result of orchestrated, hierarchical, heterogeneous collections of expressed genomic variants. However, the effects of these variants are the result of historic selective pressure and current environmental and epigenetic signals, and, as such, their co-occurrence can be seen as genome-wide correlations in a number of different manners. Biomass recalcitrance (i.e., the resistance of plants to degradation or deconstruction, which ultimately enables access to a plant’s sugars) is a complex polygenic phenotype of high importance to biofuels initiatives. This study makes use of data derived from the re-sequenced genomes from over 800 different Populus trichocarpa genotypes in combination with metabolomic and pyMBMS data across this population, as well as co-expression and co-methylation networks in order to better understand the molecular interactions involved in recalcitrance, and identify target genes involved in lignin biosynthesis/degradation. A Lines Of Evidence (LOE) scoring system is developed to integrate the information in the different layers and quantify the number of lines of evidence linking genes to target functions. This new scoring system was applied to quantify the lines of evidence linking genes to lignin-related genes and phenotypes across the network layers, and allowed for the generation of new hypotheses surrounding potential new candidate genes involved in lignin biosynthesis in P. trichocarpa, including various AGAMOUS-LIKE genes. The resulting Genome Wide Association Study networks, integrated with Single Nucleotide Polymorphism (SNP) correlation, co-methylation, and co-expression networks through the LOE scores are proving to be a powerful approach to determine the pleiotropic and epistatic relationships underlying cellular functions and, as such, the molecular basis for complex phenotypes, such as recalcitrance

Characterization of a large sex determination region in Salix purpurea L. (Salicaceae).

Dioecy has evolved numerous times in plants, but heteromorphic sex chromosomes are apparently rare. Sex determination has been studied in multiple Salix and Populus (Salicaceae) species, and P. trichocarpa has an XY sex determination system on chromosome 19, while S. suchowensis and S. viminalis have a ZW system on chromosome 15. Here we use whole genome sequencing coupled with quantitative trait locus mapping and a genome-wide association study to characterize the genomic composition of the non-recombining portion of the sex determination region. We demonstrate that Salix purpurea also has a ZW system on chromosome 15. The sex determination region has reduced recombination, high structural polymorphism, an abundance of transposable elements, and contains genes that are involved in sex expression in other plants. We also show that chromosome 19 contains sex-associated markers in this S. purpurea assembly, along with other autosomes. This raises the intriguing possibility of a translocation of the sex determination region within the Salicaceae lineage, suggesting a common evolutionary origin of the Populus and Salix sex determination loci

Contrasting patterns of evolution following whole genome versus tandem duplication events in Populus

Comparative analysis of multiple angiosperm genomes has implicated gene duplication in the expansion and diversification of many gene families. However, empirical data and theory suggest that whole-genome and small-scale duplication events differ with respect to the types of genes preserved as duplicate pairs. We compared gene duplicates resulting from a recent whole genome duplication to a set of tandemly duplicated genes in the model forest tree Populus trichocarpa. We used a combination of microarray expression analyses of a diverse set of tissues and functional annotation to assess factors related to the preservation of duplicate genes of both types. Whole genome duplicates are 700 bp longer and are expressed in 20% more tissues than tandem duplicates. Furthermore, certain functional categories are over-represented in each class of duplicates. In particular, disease resistance genes and receptor-like kinases commonly occur in tandem but are significantly under-retained following whole genome duplication, while whole genome duplicate pairs are enriched for members of signal transduction cascades and transcription factors. The shape of the distribution of expression divergence for duplicated pairs suggests that nearly half of the whole genome duplicates have diverged in expression by a random degeneration process. The remaining pairs have more conserved gene expression than expected by chance, consistent with a role for selection under the constraints of gene balance. We hypothesize that duplicate gene preservation in Populus is driven by a combination of subfunctionalization of duplicate pairs and purifying selection favoring retention of genes encoding proteins with large numbers of interactions

Efficiency of gene silencing in \u3ci\u3eArabidopsis\u3c/i\u3e: direct inverted repeats vs. transitive RNAi vectors

We investigated the efficiency of RNA interference (RNAi) in Arabidopsis using transitive and homologous inverted repeat (hIR) vectors. hIR constructs carry self-complementary intron-spliced fragments of the target gene whereas transitive vectors have the target sequence fragment adjacent to an intron-spliced, inverted repeat of heterologous origin. Both transitive and hIR constructs facilitated specific and heritable silencing in the three genes studied (AP1 , ETTIN and TTG1 ). Both types of vectors produced a phenotypic series that phenocopied reduction of function mutants for the respective target gene. The hIR yielded up to fourfold higher proportions of events with strongly manifested reduction of function phenotypes compared to transitive RNAi. We further investigated the efficiency and potential off-target effects of AP1 silencing by both types of vectors using genome-scale microarrays and quantitative RT-PCR. The depletion of AP1 transcripts coincided with reduction of function phenotypic changes among both hIR and transitive lines and also showed similar expression patterns among differentially regulated genes. We did not detect significant silencing directed against homologous potential off-target genes when constructs were designed with minimal sequence similarity. Both hIR and transitive methods are useful tools in plant biotechnology and genomics. The choice of vector will depend on specific objectives such as cloning throughput, number of events and degree of suppression required

Finding New Cell Wall Regulatory Genes in Populus trichocarpa Using Multiple Lines of Evidence.

Understanding the regulatory network controlling cell wall biosynthesis is of great interest in Populus trichocarpa, both because of its status as a model woody perennial and its importance for lignocellulosic products. We searched for genes with putatively unknown roles in regulating cell wall biosynthesis using an extended network-based Lines of Evidence (LOE) pipeline to combine multiple omics data sets in P. trichocarpa, including gene coexpression, gene comethylation, population level pairwise SNP correlations, and two distinct SNP-metabolite Genome Wide Association Study (GWAS) layers. By incorporating validation, ranking, and filtering approaches we produced a list of nine high priority gene candidates for involvement in the regulation of cell wall biosynthesis. We subsequently performed a detailed investigation of candidate gene GROWTH-REGULATING FACTOR 9 (PtGRF9). To investigate the role of PtGRF9 in regulating cell wall biosynthesis, we assessed the genome-wide connections of PtGRF9 and a paralog across data layers with functional enrichment analyses, predictive transcription factor binding site analysis, and an independent comparison to eQTN data. Our findings indicate that PtGRF9 likely affects the cell wall by directly repressing genes involved in cell wall biosynthesis, such as PtCCoAOMT and PtMYB.41, and indirectly by regulating homeobox genes. Furthermore, evidence suggests that PtGRF9 paralogs may act as transcriptional co-regulators that direct the global energy usage of the plant. Using our extended pipeline, we show multiple lines of evidence implicating the involvement of these genes in cell wall regulatory functions and demonstrate the value of this method for prioritizing candidate genes for experimental validation