Mitochondrial Genome of Fagus sylvatica L. as a Source for Taxonomic Marker Development in the Fagales.

European beech, Fagus sylvatica L., is one of the most important and widespread deciduous tree species in Central Europe and is widely managed for its hard wood. The complete DNA sequence of the mitochondrial genome of Fagus sylvatica L. was assembled and annotated based on Illumina MiSeq reads and validated using long reads from nanopore MinION sequencing. The genome assembled into a single DNA sequence of 504,715 bp in length containing 58 genes with predicted function, including 35 protein-coding, 20 tRNA and three rRNA genes. Additionally, 23 putative protein-coding genes were predicted supported by RNA-Seq data. Aiming at the development of taxon-specific mitochondrial genetic markers, the tool SNPtax was developed and applied to select genic SNPs potentially specific for different taxa within the Fagales. Further validation of a small SNP set resulted in the development of four CAPS markers specific for Fagus, Fagaceae, or Fagales, respectively, when considering over 100 individuals from a total of 69 species of deciduous trees and conifers from up to 15 families included in the marker validation. The CAPS marker set is suitable to identify the genus Fagus in DNA samples from tree tissues or wood products, including wood composite products.

A single gene underlies the dynamic evolution of poplar sex determination.

Although hundreds of plant lineages have independently evolved dioecy (that is, separation of the sexes), the underlying genetic basis remains largely elusive1. Here we show that diverse poplar species carry partial duplicates of the ARABIDOPSIS RESPONSE REGULATOR 17 (ARR17) orthologue in the male-specific region of the Y chromosome. These duplicates give rise to small RNAs apparently causing male-specific DNA methylation and silencing of the ARR17 gene. CRISPR-Cas9-induced mutations demonstrate that ARR17 functions as a sex switch, triggering female development when on and male development when off. Despite repeated turnover events, including a transition from the XY system to a ZW system, the sex-specific regulation of ARR17 is conserved across the poplar genus and probably beyond. Our data reveal how a single-gene-based mechanism of dioecy can enable highly dynamic sex-linked regions and contribute to maintaining recombination and integrity of sex chromosomes.

SNP Markers as a Successful Molecular Tool for Assessing Species Identity and Geographic Origin of Trees in the Economically Important South American Legume Genus Dipteryx.

Dipteryx timber has been heavily exploited in South America since 2000s due to the increasing international demand for hardwood. Developing tools for the genetic identification of Dipteryx species and their geographical origin can help to promote legal trading of timber. A collection of 800 individual trees, belonging to 6 different Dipteryx species, was genotyped based on 171 molecular markers. After the exclusion of markers out of Hardy-Weinberg equilibrium or with no polymorphism or low amplification, 83 nuclear, 29 chloroplast, 13 mitochondrial single nucleotide polymorphisms (SNPs), and 2 chloroplast and 5 mitochondrial INDELS remained. Six genetic groups were identified using Bayesian Structure analyses of the nuclear SNPs, which corresponded to the different Dipteryx species collected in the field. Seventeen highly informative markers were identified as suitable for species identification and obtained self-assignment success rates to species level of 78-96%. An additional set of 15 molecular markers was selected to determine the different genetic clusters found in Dipteryx odorata and Dipteryx ferrea, obtaining self-assignment success rates of 91-100%. The success to assign samples to the correct country of origin using all or only the informative markers improved when using the nearest neighbor approach (69-92%) compared to the Bayesian approach (33-80%). While nuclear and chloroplast SNPs were more suitable for differentiating the different Dipteryx species, mitochondrial SNPs were ideal for determining the genetic clusters of D. odorata and D. ferrea. These 32 selected SNPs will be invaluable genetic tools for the accurate identification of species and country of origin of Dipteryx timber.

Molecular evidence for three genetic species of Dipteryx in the Peruvian Amazon.

There is a high international demand for timber from the genus Dipteryx, or "shihuahuaco" as it is known in Peru. Developing tools that allow the identification and discrimination of Dipteryx species is therefore important for supporting management of natural populations and to underpin legal trade of its timber. The objective of this study was the molecular characterization of Dipteryx species in the Peruvian Amazonia. Two plastid regions (cpDNA: trnH-psbA and matK) were sequenced and 11 microsatellite markers (nDNA) were genotyped for 32 individuals identified as Dipteryx charapilla, D. micrantha morphotype 1 and D. micrantha morphotype 2. Using the concatenated sequences of the plastid genes, we identified ten haplotypes that were not shared between the species or between the D. micrantha morphotypes. Haplotypic diversity was greater in D. micrantha morphotype 2 and D. charapilla than in D. micrantha morphotype 1, which presented only one haplotype with a wide distribution in Peru. The microsatellites allowed the discrimination of the same three clades and identified diagnostic alleles for each clade. These results allowed us to demonstrate that the two morphotypes of D. micrantha are different at both the plastid and nuclear markers, which supports the existence of three genetically distinct species in Peru. This study provides information for the genetic discrimination of Dipteryx species and emphasises the importance of conserving the genetic variability of this genus in the Peruvian Amazonia.

Lignans and sesquiterpene lactones from Hypochaeris radicata subsp. neapolitana (Asteraceae, Cichorieae).

Four undescribed lignans and two undescribed sesquiterpenic acids, together with three known compounds (hypochoeroside C, hypochoeroside D, and 5-O-caffeoylshikimic acid) were isolated from the roots of Hypochaeris radicata subsp. neapolitana (Asteraceae, Cichorieae). The lignans were identified as 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranoside, 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranosyl-2'-O-methacrylate, (7S,8R,8'R)-7-(3,4-dihydroxyphenyl)-3',4'-dihydroxy-7,8,7',8'-tetrahydronaphtho [8,8'-c]furan-1(3H)-one, and (7S,8R,8'R)-7-(3,4-dihydroxyphenyl)-3',4'-dihydroxy-8'-(hydroxymethyl)-7,8,7',8'-tetrahydronaphthalen-8-carboxylic acid. The two sesquiterpenic acids were identified as the ring open precursors of hypochoerosides C and D. Structures were elucidated using NMR and HRMS. Absolute configurations of (7S,8R,8'R)-7-(3,4-dihydroxyphenyl)-3',4'-dihydroxy-7,8,7',8'-tetrahydronaphtho [8,8'-c]furan-1(3H)-one and (7S,8R,8'R)-7-(3,4-dihydroxyphenyl)-3',4'-dihydroxy-8'-(hydroxymethyl)-7,8,7',8'-tetrahydronaphthalen-8-carboxylic acid were determined using electronic circular dichroism (ECD) spectroscopy. 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranoside was evaluated for its anti-proliferative activity against myeloma cell lines MM1S, U266, and NCI-H929 and showed cytotoxicity at 100 mM against MM1S strain. No neurotoxicity was observed for major compounds 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranoside, hypochoeroside C, and hypochoeroside D in a fluorescence assay measuring neurite outgrowth in dorsal root ganglion (DRG) neurons. Additionally, compounds 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranoside, hypochoeroside C, hypochoeroside D, and hypochoerosidic acid D were quantified in unstressed and drought-stressed plants using HPLC-DAD. Drought-stressed plants were found to contain lower concentrations of the lignan 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy-O-ß-D-glucopyranoside and sesquiterpene lactone hypochoeroside C.

High Level of Conservation of Mitochondrial RNA Editing Sites Among Four Populus Species.

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.

Assessing the Ability of Chloroplast and Nuclear DNA Gene Markers to Verify the Geographic Origin of Jatoba (Hymenaea courbaril L.) Timber.

Deforestation-reinforced by illegal logging-is a serious problem in many tropical regions and causes pervasive environmental and economic damage. Existing laws that intend to reduce illegal logging need efficient, fraud resistant control methods. We developed a genetic reference database for Jatoba (Hymenaea courbaril), an important, high value timber species from the Neotropics. The data set can be used for controls on declarations of wood origin. Samples from 308 Hymenaea trees from 12 locations in Brazil, Bolivia, Peru, and French Guiana have been collected and genotyped on 10 nuclear microsatellites (nSSRs), 13 chloroplast SNPs (cpSNP), and 1 chloroplast indel marker. The chloroplast gene markers have been developed using Illumina DNA sequencing. Bayesian cluster analysis divided the individuals based on the nSSRs into 8 genetic groups. Using self-assignment tests, the power of the genetic reference database to judge on declarations on the location has been tested for 3 different assignment methods. We observed a strong genetic differentiation among locations leading to high and reliable self-assignment rates for the locations between 50% to 100% (average of 88%). Although all 3 assignment methods came up with similar mean self-assignment rates, there were differences for some locations linked to the level of genetic diversity, differentiation, and heterozygosity. Our results show that the nuclear and chloroplast gene markers are effective to be used for a genetic certification system and can provide national and international authorities with a robust tool to confirm legality of timber.

Complete Chloroplast Genome Sequences of Four Meliaceae Species and Comparative Analyses.

The Meliaceae family mainly consists of trees and shrubs with a pantropical distribution. In this study, the complete chloroplast genomes of four Meliaceae species were sequenced and compared with each other and with the previously published Azadirachta indica plastome. The five plastomes are circular and exhibit a quadripartite structure with high conservation of gene content and order. They include 130 genes encoding 85 proteins, 37 tRNAs and 8 rRNAs. Inverted repeat expansion resulted in a duplication of rps19 in the five Meliaceae species, which is consistent with that in many other Sapindales, but different from many other rosids. Compared to Azadirachta indica, the four newly sequenced Meliaceae individuals share several large deletions, which mainly contribute to the decreased genome sizes. A whole-plastome phylogeny supports previous findings that the four species form a monophyletic sister clade to Azadirachta indica within the Meliaceae. SNPs and indels identified in all complete Meliaceae plastomes might be suitable targets for the future development of genetic markers at different taxonomic levels. The extended analysis of SNPs in the matK gene led to the identification of four potential Meliaceae-specific SNPs as a basis for future validation and marker development.

Deletion lengthening at chromosomes 6q and 16q targets multiple tumor suppressor genes and is associated with an increasingly poor prognosis in prostate cancer.

Prostate cancer is characterized by recurrent deletions that can considerably vary in size. We hypothesized that large deletions develop from small deletions and that this "deletion lengthening" might have a "per se" carcinogenic role through a combinatorial effect of multiple down regulated genes. In vitro knockdown of 37 genes located inside the 6q12-q22 deletion region identified 4 genes with additive tumor suppressive effects, further supporting a role of the deletion size for cancer aggressiveness. Employing fluorescence in-situ hybridization analysis on prostate cancer tissue microarrays, we determined the deletion size at 6q and 16q in more than 3,000 tumors. 16q and 6q deletion length was strongly linked to poor clinical outcome and this effect was even stronger if the length of both deletions was combined. To study deletion lengthening in cancer progression we eventually analyzed the entire cancers from 317 patients for 6q and 16q deletion length heterogeneity and found that the deletion expanded within 50-60% of 6q and 16q deleted cancers. Taken together, these data suggest continuous "deletion lengthening" as a key mechanism for prostate cancer progression leading to parallel down regulation of genes with tumor suppressive properties, some of which act cooperatively.

Development of Molecular Markers for Determining Continental Origin of Wood from White Oaks (Quercus L. sect. Quercus).

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 indels 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 indels 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.

Genome Sequences of Populus tremula Chloroplast and Mitochondrion: Implications for Holistic Poplar Breeding.

Complete Populus genome sequences are available for the nucleus (P. trichocarpa; section Tacamahaca) and for chloroplasts (seven species), but not for mitochondria. Here, we provide the complete genome sequences of the chloroplast and the mitochondrion for the clones P. tremula W52 and P. tremula x P. alba 717-1B4 (section Populus). The organization of the chloroplast genomes of both Populus clones is described. A phylogenetic tree constructed from all available complete chloroplast DNA sequences of Populus was not congruent with the assignment of the related species to different Populus sections. In total, 3,024 variable nucleotide positions were identified among all compared Populus chloroplast DNA sequences. The 5-prime part of the LSC from trnH to atpA showed the highest frequency of variations. The variable positions included 163 positions with SNPs allowing for differentiating the two clones with P. tremula chloroplast genomes (W52, 717-1B4) from the other seven Populus individuals. These potential P. tremula-specific SNPs were displayed as a whole-plastome barcode on the P. tremula W52 chloroplast DNA sequence. Three of these SNPs and one InDel in the trnH-psbA linker were successfully validated by Sanger sequencing in an extended set of Populus individuals. The complete mitochondrial genome sequence of P. tremula is the first in the family of Salicaceae. The mitochondrial genomes of the two clones are 783,442 bp (W52) and 783,513 bp (717-1B4) in size, structurally very similar and organized as single circles. DNA sequence regions with high similarity to the W52 chloroplast sequence account for about 2% of the W52 mitochondrial genome. The mean SNP frequency was found to be nearly six fold higher in the chloroplast than in the mitochondrial genome when comparing 717-1B4 with W52. The availability of the genomic information of all three DNA-containing cell organelles will allow a holistic approach in poplar molecular breeding in the future.

FISH Oracle 2: a web server for integrative visualization of genomic data in cancer research.

BACKGROUND: A comprehensive view on all relevant genomic data is instrumental for understanding the complex patterns of molecular alterations typically found in cancer cells. One of the most effective ways to rapidly obtain an overview of genomic alterations in large amounts of genomic data is the integrative visualization of genomic events. RESULTS: We developed FISH Oracle 2, a web server for the interactive visualization of different kinds of downstream processed genomics data typically available in cancer research. A powerful search interface and a fast visualization engine provide a highly interactive visualization for such data. High quality image export enables the life scientist to easily communicate their results. A comprehensive data administration allows to keep track of the available data sets. We applied FISH Oracle 2 to published data and found evidence that, in colorectal cancer cells, the gene TTC28 may be inactivated in two different ways, a fact that has not been published before. CONCLUSIONS: The interactive nature of FISH Oracle 2 and the possibility to store, select and visualize large amounts of downstream processed data support life scientists in generating hypotheses. The export of high quality images supports explanatory data visualization, simplifying the communication of new biological findings. A FISH Oracle 2 demo server and the software is available at http://www.zbh.uni-hamburg.de/fishoracle.

Recurrent deletion of 3p13 targets multiple tumour suppressor genes and defines a distinct subgroup of aggressive ERG fusion-positive prostate cancers.

Deletion of 3p13 has been reported from about 20% of prostate cancers. The clinical significance of this alteration and the tumour suppressor gene(s) driving the deletion remain to be identified. We have mapped the 3p13 deletion locus using SNP array analysis and performed fluorescence in situ hybridization (FISH) analysis to search for associations between 3p13 deletion, prostate cancer phenotype and patient prognosis in a tissue microarray containing more than 3200 prostate cancers. SNP array analysis of 72 prostate cancers revealed a small deletion at 3p13 in 14 (19%) of the tumours, including the putative tumour suppressors FOXP1, RYBP and SHQ1. FISH analysis using FOXP1-specific probes revealed deletions in 16.5% and translocations in 1.2% of 1828 interpretable cancers. 3p13 deletions were linked to adverse features of prostate cancer, including advanced stage (p < 0.0001), high Gleason grade (p = 0.0125), and early PSA recurrence (p = 0.0015). In addition, 3p13 deletions were linked to ERG(+) cancers and to PTEN deletions (p < 0.0001 each). A subset analysis of ERG(+) tumours revealed that 3p13 deletions occurred independently from PTEN deletions (p = 0.3126), identifying tumours with 3p13 deletion as a distinct molecular subset of ERG(+) cancers. mRNA expression analysis confirmed that all 3p13 genes were down regulated by the deletion. Ectopic over-expression of FOXP1, RYBP and SHQ1 resulted in decreased colony-formation capabilities, corroborating a tumour suppressor function for all three genes. In summary, our data show that deletion of 3p13 defines a distinct and aggressive molecular subset of ERG(+) prostate cancers, which is possibly driven by inactivation of multiple tumour suppressors.

CHD1 is a 5q21 tumor suppressor required for ERG rearrangement in prostate cancer.

Deletions involving the chromosomal band 5q21 are among the most frequent alterations in prostate cancer. Using single-nucleotide polymorphism (SNP) arrays, we mapped a 1.3 megabase minimally deleted region including only the repulsive guidance molecule B (RGMB) and chromodomain helicase DNA-binding protein 1 (CHD1) genes. Functional analyses showed that CHD1 is an essential tumor suppressor. FISH analysis of 2,093 prostate cancers revealed a strong association between CHD1 deletion, prostate-specific antigen (PSA) biochemical failure (P = 0.0038), and absence of ERG fusion (P < 0.0001). We found that inactivation of CHD1 in vitro prevents formation of ERG rearrangements due to impairment of androgen receptor (AR)-dependent transcription, a prerequisite for ERG translocation. CHD1 is required for efficient recruitment of AR to responsive promoters and regulates expression of known AR-responsive tumor suppressor genes, including NKX3-1, FOXO1, and PPARγ. Our study establishes CHD1 as the 5q21 tumor suppressor gene in prostate cancer and shows a key role of this chromatin remodeling factor in prostate cancer biology.

Integrative genomic analyses reveal an androgen-driven somatic alteration landscape in early-onset prostate cancer.

Early-onset prostate cancer (EO-PCA) represents the earliest clinical manifestation of prostate cancer. To compare the genomic alteration landscapes of EO-PCA with "classical" (elderly-onset) PCA, we performed deep sequencing-based genomics analyses in 11 tumors diagnosed at young age, and pursued comparative assessments with seven elderly-onset PCA genomes. Remarkable age-related differences in structural rearrangement (SR) formation became evident, suggesting distinct disease pathomechanisms. Whereas EO-PCAs harbored a prevalence of balanced SRs, with a specific abundance of androgen-regulated ETS gene fusions including TMPRSS2:ERG, elderly-onset PCAs displayed primarily non-androgen-associated SRs. Data from a validation cohort of > 10,000 patients showed age-dependent androgen receptor levels and a prevalence of SRs affecting androgen-regulated genes, further substantiating the activity of a characteristic "androgen-type" pathomechanism in EO-PCA.

Genomic deletion of PTEN is associated with tumor progression and early PSA recurrence in ERG fusion-positive and fusion-negative prostate cancer.

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene is often altered in prostate cancer. To determine the prevalence and clinical significance of the different mechanisms of PTEN inactivation, we analyzed PTEN deletions in TMAs containing 4699 hormone-naïve and 57 hormone-refractory prostate cancers using fluorescence in situ hybridization analysis. PTEN mutations and methylation were analyzed in subsets of 149 and 34 tumors, respectively. PTEN deletions were present in 20.2% (458/2266) of prostate cancers, including 8.1% heterozygous and 12.1% homozygous deletions, and were linked to advanced tumor stage (P < 0.0001), high Gleason grade (P < 0.0001), presence of lymph node metastasis (P = 0.0002), hormone-refractory disease (P < 0.0001), presence of ERG gene fusion (P < 0.0001), and nuclear p53 accumulation (P < 0.0001). PTEN deletions were also associated with early prostate-specific antigen recurrence in univariate (P < 0.0001) and multivariate (P = 0.0158) analyses. The prognostic impact of PTEN deletion was seen in both ERG fusion-positive and ERG fusion-negative tumors. PTEN mutations were found in 4 (12.9%) of 31 cancers with heterozygous PTEN deletions but in only 1 (2%) of 59 cancers without PTEN deletion (P = 0.027). Aberrant PTEN promoter methylation was not detected in 34 tumors. The results of this study demonstrate that biallelic PTEN inactivation, by either homozygous deletion or deletion of one allele and mutation of the other, occurs in most PTEN-defective cancers and characterizes a particularly aggressive subset of metastatic and hormone-refractory prostate cancers.

FISH Oracle: a web server for flexible visualization of DNA copy number data in a genomic context.

BACKGROUND: The rapidly growing amount of array CGH data requires improved visualization software supporting the process of identifying candidate cancer genes. Optimally, such software should work across multiple microarray platforms, should be able to cope with data from different sources and should be easy to operate. RESULTS: We have developed a web-based software FISH Oracle to visualize data from multiple array CGH experiments in a genomic context. Its fast visualization engine and advanced web and database technology supports highly interactive use. FISH Oracle comes with a convenient data import mechanism, powerful search options for genomic elements (e.g. gene names or karyobands), quick navigation and zooming into interesting regions, and mechanisms to export the visualization into different high quality formats. These features make the software especially suitable for the needs of life scientists. CONCLUSIONS: FISH Oracle offers a fast and easy to use visualization tool for array CGH and SNP array data. It allows for the identification of genomic regions representing minimal common changes based on data from one or more experiments. FISH Oracle will be instrumental to identify candidate onco and tumor suppressor genes based on the frequency and genomic position of DNA copy number changes. The FISH Oracle application and an installed demo web server are available at http://www.zbh.uni-hamburg.de/fishoracle.

AnnotationSketch: a genome annotation drawing library.

SUMMARY: To analyse the vast amount of genome annotation data available today, a visual representation of genomic features in a given sequence range is required. We developed a C library which provides layout and drawing capabilities for annotation features. It supports several common input and output formats and can easily be integrated into custom C applications. To exemplify the use of AnnotationSketch in other languages, we provide bindings to the scripting languages Ruby, Python and Lua. AVAILABILITY: The software is available under an open-source license as part of GenomeTools (http://genometools.org/annotationsketch.html).