Hybridization Partners Research Articles

Uncovering Molecular Quencher Effects on FRET Phenomena in Microsphere-Immobilized Probe Systems.

Double-stranded (ds) oligonucleotide probes composed of quencher-dye sequence pairs outperform analogous single-stranded (ss) probes due to their superior target sequence specificity without any prerequisite target labeling. Optimizing sequence combinations for dsprobe design requires promoting a fast, accurate response to a specific target sequence while minimizing spontaneous dsprobe dissociation events. Here, flow cytometry is used to rapidly interrogate the stability and selective responsiveness of 20 candidate LNA and DNA dsprobes to a 24 base-long segment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and ∼243 degenerate RNA sequences serving as model variants. Importantly, in contrast to quantifying binding events of dye-labeled targets via flow cytometry, the current work employs the Förster resonance energy transfer (FRET)-based detection of unlabeled RNA targets. One DNA dsprobe with a 15-base-long hybridization partner containing a central abasic site emerged as very stable yet responsive only to the SARS-CoV-2 RNA segment. Separate displacement experiments, however, indicated that ∼12% of these quencher-capped hybridization partners remain bound, even in the presence of an excess SARS-CoV-2 RNA target. To examine their quenching range, additional titration studies varied the ratios and spatial placement of nonquencher and quencher-capped hybridization partners in the dsprobes. These titration studies indicate that these residual, bound quencher-capped partners, even at low percentages, act as nodes, enabling both static quenching effects within each residual dsprobe as well as longer-range quenching effects on neighboring FAM moieties. Overall, these studies provide insight into practical implications for rapid dsprobe screening and target detection by combining flow cytometry with FRET-based detection.

Molecular Hybridization as a Strategy for Developing Artemisinin-Derived Anticancer Candidates.

Artemisinin is a natural compound extracted from Artemisia species belonging to the Asteraceae family. Currently, artemisinin and its derivatives are considered among the most significant small-molecule antimalarial drugs. Artemisinin and its derivatives have also been shown to possess selective anticancer properties, however, there are several limitations and gaps in knowledge that retard their repurposing as effective anticancer agents. Hybridization resulting from a covalent combination of artemisinin with one or more active pharmacophores has emerged as a promising approach to overcome several issues. The variety of hybridization partners allows improvement in artemisinin activity by tuning the ability of conjugated artemisinin to interact with various molecule targets involved in multiple biological pathways. This review highlights the current scenario of artemisinin-derived hybrids with potential anticancer activity. The synthetic approaches to achieve the corresponding hybrids and the structure-activity relationships are discussed to facilitate further rational design of more effective candidates.

Metal-dependent base pairing of bifacial iminodiacetic acid-modified uracil bases for switching DNA hybridization partner.

Dynamic control of DNA assembly by external stimuli has received increasing attention in recent years. Dynamic ligand exchange in metal complexes can be a central element in the structural and functional transformation of DNA assemblies. In this study, N,N-dicarboxymethyl-5-aminouracil (dcaU) nucleoside with an iminodiacetic acid (IDA) ligand at the 5-position of the uracil base has been developed as a bifacial nucleoside that can form both hydrogen-bonded and metal-mediated base pairs. Metal complexation study of dcaU nucleosides revealed their ability to form a 2:1 complex with a GdIII ion at the monomeric level. The characteristics of base pairing of dcaU nucleosides were then examined inside DNA duplexes. The results revealed that the formation of the metal-mediated dcaU-GdIII-dcaU pair significantly stabilized the DNA duplex containing one dcaU-dcaU mismatch (ΔT m = +16.1 °C). In contrast, a duplex containing a hydrogen-bonded dcaU-A pair was destabilized in the presence of GdIII (ΔT m = -3.5 °C). The GdIII-dependent base pairing of dcaU bases was applied to control the hybridization preference of DNA in response to metal ions. The hybridization partner of a dcaU-containing strand was reversibly exchanged by the addition and removal of GdIII ions. Since the incorporation of a single dcaU base can switch the hybridization behavior of DNA, the bifacial dcaU base would be a versatile building block for imparting metal responsiveness to DNA assemblies, allowing the rational design of dynamic DNA systems.

Intra-individual qualitative and quantitative comparison of [68Ga]Ga-DOTATATE PET/CT and PET/MRI.

Somatostatin receptor (SSTR) positron emission tomography (PET) is a cornerstone of neuroendocrine tumor (NET) management. Hybrid PET/magnetic resonance imaging (MRI) is now available for NET-imaging, next to PET/computed tomography (CT). To determine whether CT or MRI is the best hybrid partner for [68Ga]Ga-DOTATATE PET. Monocentric, prospective study. Patients received a same-day [68Ga]Ga-DOTATATE PET/CT and subsequent PET/MRI, for suspicion of NET, (re)staging or peptide receptor radionuclide therapy-selection. The union (PETunion) of malignant lesions detected on PETCT and PETMRI was the reference standard. Concordance of detection of malignant lesions in an organ was measured between PETunion and CT and PETunion and MRI. Seven bins were used to categorize the number of malignant lesions, containing following ordinal variables: 0, 1, 2-5, 6-10, 11-20, >20 countable and diffuse/uncountable. The difference in number of malignant lesions was obtained as the difference in bin level ('Δbin') between PETunion and CT and PETunion and MRI with a Δbin closer to zero implying a higher concordance rate. Twenty-nine patients were included. Primary tumors included 17 gastroenteropancreatic-NETs, 1 colon neuroendocrine carcinoma, 7 lung-NETs and 2 meningiomas. Patient level concordance with PETunion was 96% for MRI and 67% for CT (p = 0.039). Organ level concordance with PETunion was 74% for MRI and 40% for CT (p < 0.0001). In bone, there was a higher concordance rate for MRI compared to CT, 92% and 33%, respectively (p = 0.016). Overall, a mean Δbin of 0.5 ± 1.1 for PETunion/MRI and 1.4 ± 1.2 for PETunion/CT (p < 0.0001) was noted. In liver, a mean Δbin of 0.0 ± 1.1 for PETunion/MRI and 1.7 ± 1.2 for PETunion/CT was observed (p = 0.0078). In bone, a mean Δbin closer to zero was observed for PETunion/MRI compared to PETunion/CT, 0.6 ± 1.4 and 2.0 ± 1.5, respectively (p = 0.0098). Compared to SSTR PET/CT, SSTR PET/MRI had a higher patient and organ level concordance for malignant tumoral involvement and number of malignant lesions, with a clear added value in bone and liver specifically.

Seed Germination of Raspberry (Rubus idaeus L.) Depending on the Age of Seeds and Hybridization Partners

Abstract Research was carried out on the germination of seeds obtained from crossings between the cultivars of red raspberry (Rubus idaeus L.) ‘Sokolica’, ‘Willamette’, ‘Veten’, ‘Canby’ and ‘Glen Ample’ depending on the age of the seeds. The crossings were performed in the winter–spring of 2015–2018 in a heated greenhouse. The obtained seeds were stored dry in paper bags at a temperature of 4–5 °C. In January 2019, seeds were scarified and then cold stratified for 50 days and seeded in pots in a greenhouse. The seedling emergence was assessed 60 days after sowing. The seeds produced in 2015 had the lowest germination percentage (14% on average for all combinations of crosses), while the seeds produced in 2018 had a germination rate of 44.9%. Significant differences were also observed in the emergence of seedlings from different combinations. On average, the fewest seedlings were obtained from the combination ‘Glen Ample’ × ‘Willamette’ and the most from ‘Sokolica’ × ‘Willamette’.

Quantitative Analysis of In Situ Locked Nucleic Acid and DNA Competitive Displacement Events on Microspheres.

Synthetic analogues of natural oligonucleotides known as locked nucleic acids (LNAs) offer superior nuclease resistance and cytocompatibility for numerous scenarios ranging from in vitro detection to intracellular imaging of nucleic acids. While recognized as stronger hybridization partners than equivalent DNA residues, quantitative analysis of LNA hybridization activity is lacking, especially with respect to competitive displacement of the original hybridization partner by another oligonucleotide. In the current study, we perform in situ measurements of toehold-mediated competitive displacement of soluble, fluorescently labeled primary targets from probe strands immobilized on microspheres using high throughput flow cytometry. Both LNA-DNA hybrid sequences and pure DNA sequences are employed as the immobilized strands, as soluble, fluorescently labeled 9-base-long primary targets, and as unlabeled 15-base-long secondary or competitive targets. In addition to comparing chemically substituted and unsubstituted sequences, we explore the effects of mismatched primary targets and the location of the toehold segment within the primary duplexes on the resulting displacement profiles. The primary duplex or double-stranded probe (dsprobe) systems implemented here exhibited varying responses to unlabeled secondary targets ranging from surprisingly modest primary target displacement activity despite the presence of a six base-long nucleotide toehold segment at the dsprobe free end to distinctive displacement profiles sensitive to LNA substitutions and the placement of the toehold segment closer to the microsphere surface.

A Dynamic Relational View of Cross-Sector Collaborations

In this paper, we question how some of the taken-for-granted assumptions from the interorganizational alliance literature apply for collaborations between hybrid partners – i.e., actors with distinctive institutional logics – and that inherently generate positive externalities. Specifically, we look at how the dynamic relational view (Dyer, Singh, & Hesterly, 2018) applies to cross-sector collaborations (CSCs) – i.e., associations between nonprofit, for-profit, and/or public organizations. We offer a framework that details how these hybrid associations’ intrinsic characteristics affect some assumptions of the dynamic model. First, we show that, besides the interdependence of the partners’ resources, both the actors’ different institutional logics and the collaborations’ emphasis on tackling social issues also influence the collaborations’ governance choices. Second, we propose that, for many CSCs, the interdependence and the nature of the partners’ assets will not be the critical factor in deciding the initial investments on either relation-specific assets or knowledge-sharing routines. Instead, the prevalence of an institutional logic will be more relevant to determine these investments. To conclude, we argue that the cross-sector collaborations’ intrinsic characteristics can also accelerate, delay, or compromise the future investments in these sources of competitive advantages and the development of informal governance mechanisms, both critical to creating relational rents. Altogether, we develop a model that provides a temporal explanation for how the nature of cross-sector collaborations affects the development of their drivers of competitive advantages and the generation of relational rents.

Metal-Dependent DNA Base Pairing of 5-Carboxyuracil with Itself and All Four Canonical Nucleobases.

A 5-carboxyuracil (caU) nucleobase was found to pair not only with A (caU-A) by hydrogen bonding but also with other DNA nucleobases by metal coordination bonding. Metal-dependent formation of caU-CuII-caU, caU-HgII-T, caU-AgI-C, and caU-CuII-G pairs was demonstrated by duplex melting analysis and mass spectrometry. The duplexes containing caU-X (X = caU, T, C, and G) were significantly stabilized in the presence of the corresponding metal ions, while the DNA duplexes containing the caU-A pairs were destabilized by the addition of CuII. These results suggest that the hybridization partner of caU-containing DNA strands can be altered by metal complexation. As a result, this study provides a new direction to integrate caU nucleobases to construct diverse metallo-DNA supramolecules and metal-responsive DNA devices.

Nesting of the black-faced spoonbill (Platalea minor) (Pelecaniformes: Threskiornithidae) and the hybrid partner on the coasts of Khanka Lake (the Russian Federation)

At the east coast of Khanka Lake (Russian Federation) on May 15, 2020, an unusual pair of spoonbill was observed. One of the partners was a black-faced spoonbill; the second partner included features of both the black-faced spoonbill and the Eurasian spoonbill, which can be regarded as a hybrid. This pair had a nest with two eggs, which later hatched one chick with signs of a black-faced spoonbill.

A hybrid algorithm of ABC variant and enhanced EGS local search technique for enhanced optimization performance

A hybrid algorithm for optimizing a complex power system-based problem, economic environmental dispatch (EED) is proposed. The algorithm hybridizes a recently proposed artificial bee colony (ABC) variant referred to as JA-ABC3 and a local search technique, evolutionary gradient search (EGS) which has been enhanced i.e., augmented. The enhanced EGS has been inserted into JA-ABC3’s framework and the resulting hybrid algorithm, known as EGSJAABC3 is expected to exhibit robust optimization performance by showing the capability to reach the global optimum in less number of generations. JA-ABC3 which is generated through few modifications towards the standard ABC algorithm is the best candidate as it has exhibited better performance than the standard ABC and other ABC variants. Since JA-ABC3 is a global search algorithm, a local search technique, EGS that has been augmented is selected to be its hybrid partner as it also exhibits better or same performance than its kind. The task of the augmented EGS is to enhance the exploitation capability of the algorithm and thus, guides the solution faster towards the global optimum. In other word, the enhanced EGS is taking part in the exploitation process while JA-ABC3 takes role in exploration and some parts of the exploitation processes. Then, a number of benchmark functions are used to evaluate the robustness of EGSJAABC3 in terms of convergence speed and global optimum achievement. Next, the main significant output of this research is a robust optimization algorithm (i.e., EGSJAABC3) later applied to solve complex real-world problems that is known for their uncertainty. In this paper, EGSJAABC3 is tested to minimize EED on three test generator systems; 6, 10 and 40 units. The acquired outcome on both benchmark functions and EED application demonstrate the robustness of EGSJAABC3 as an optimization algorithm and therefore, provide other researchers and engineers a tool for solving optimization problems.

Topology of syngameons.

Syngameons are sets of species linked by interspecific hybridization. Common observations regarding the structure of syngameons are that hybridization propensity is not uniform across species and that patterns of hybridization are dominated by a few species. I use computer simulations to test these claims in naturally occurring syngameons selected from the literature and from personal observation. Natural syngameons, especially those involving plants, typically exhibit nonrandom structure: The first three order statistics for the number of hybrid partners and the variance in the number of hybrid partners are larger than chance alone would predict. The structure of two insect syngameons examined is not significantly different from random. To test a hypothesis that variation in hybridization propensity across species in natural syngameons is simply an artifact of hybridization opportunity, I examine the structure of four artificial syngameons (fertility relationships) produced by full diallel crosses. Three of four artificial syngameons exhibit nonrandom structure, as the observed variation in number of successful crosses is larger than chance alone would predict. In general, there are no significant results involving the order statistics. Finally, I discuss biogeographic, ecological, and phylogenetic hypotheses for variation in hybridization propensity across species in natural syngameons.

Climate as possible reproductive barrier in Pinus radiata (D. Don) interspecific hybridisation

Historically, interspecific hybridisation with Pinus radiata D. Don had limited success. The effect of environmental conditions and position of pollination bags in the tree were investigated as possible hybridisation barriers. The study was conducted in a P. radiata seed orchard in the Southern Cape (South Africa). Field data were compared to the climatic conditions at natural and commercial provenances of seven Mesoamerican Pinus species identified as possible hybrid partners. In vitro pollen studies were used to confirm whether interspecific crosses with P. radiata might be feasible within predefined climatic parameters. The temperature ranges for both top and northern side of P. radiata pine trees in the seed orchard was similar to the natural distribution of P. radiata, P. elliottii Engelm. and P. taeda L. in the USA. Results suggested that pollen of P. elliottii and P. taeda might be more suited to result in the successful pollination of P. radiata than the other Mesoamerican pine species tested in this study. Furthermore, the combination of minimum temperature and precipitation also showed a closer correlation to successful hybridisation with P. radiata for both P. elliotii and P. taeda. However, pollen tube elongation studies did not support these results, suggesting that mean temperature might not be the only determining factor of hybridisation success. Three circadian temperature models that mimic natural conditions were developed for Karatara and Sabie (Tweefontein, Witklip and Spitskop). These models will be tested in future in vitro studies to further evaluate temperature fluctuations between day and night regimes as a possible reproductive barrier limiting hybridisation success between P. radiata and other Mesoamerican pine species.

An ontology-based multi-agent virtual enterprise system (OMAVE): part 2: partner selection

A virtual enterprise (VE) is a collaboration model between multiple business partners in a value chain. The VE model is particularly feasible and appropriate for small- and medium-sized enterprises (SMEs) and industrial parks containing multiple SMEs that have different vertical competencies. The VE consortium’s success highly depends on its members. Therefore, it is crucial to select the most appropriate enterprises when forming a VE consortium. In this study, a new multi-agent hybrid partner selection algorithm is developed for application in the development of an ontology-based multi-agent virtual enterprise (OMAVE) system. In this platform, the agent’s interactions are supported by agent ontology, which provides concepts, properties and all message formats for the agents. Different types of agents collaborate and compete with each other so that unqualified or inefficient enterprises are eliminated from the enterprise pool. Only the remaining enterprises would be allowed to enter the negotiation process and propose in the bidding. The agent-based auctioning platform is coupled with a fuzzy–AHP–TOPSIS algorithm to evaluate partners based on their proposals and background. Accordingly, the winning enterprise for each task is identified and the whole project can be accomplished by assigning tasks to the responsible partners. To test and verify the functionality of the developed OMAVE system, a sample module using OMAVE applications and tools was manufactured. The last section of this paper presents the results of this case study, which validate the applicability of the proposed technique.

Sumitomo, US Hybrid partner for hydrogen power in public transit

Sumitomo, US Hybrid partner for hydrogen power in public transit

Genetic evidence of peripheral isolation and low diversity in marginal populations of the Acropora hyacinthus complex

Zooxanthellate corals are found throughout the tropics, but also extend into subtropical and marginal locations due to the presence of warm ocean currents. The population history of corals in marginal locations is of great interest in relation to changing global climatic conditions, as species edge zones might play an important role in evolutionary innovation. Here, we examine the genetic structure of a widely distributed coral species complex, Acropora hyacinthus, from tropical to high subtropical regions along the Kuroshio Current in Taiwan and Japan. Population genetic analysis of 307 specimens from 18 locations (7 reefal and 11 marginal) identified at least four genetic lineages within the A. hyacinthus complex: HyaA, HyaB, HyaC (dominating reefal locations) and HyaD dominating marginal locations in mainland Japan and Taiwan, except the upper Penghu Islands, which were dominated by HyaC. Crossing experiments suggested semi-incompatibility and hybridization between HyaC and D from reefal locations, implying that the existence of hybridization partners enhances diversification and genetic diversity. An incomplete barrier between the HyaC and HyaD dominations was found along the two straits in the Ryukyu Islands, where Kuroshio Current flows constantly. Despite geographical distance, the genetic composition of populations in mainland Japan was comparable to that in mainland Taiwan, which may reflect a region-specific connectivity around the northern limit of A. hyacinthus in the Pacific. In contrast, populations in the Ryukyu Islands were not significantly different from those of Palau and the Great Barrier Reef. While the precise taxonomic nature of the lineages found around the Kuroshio Current remains to be elucidated, these results indicate that, despite the presence of four lineages in the Kuroshio triangle, low genetic diversity populations of the two main lines might be isolating and differentiating in the marginal region.

Atomistic Simulations and Interfacial Morphology of Graphene Grown on SiC(0001) and SiC(000-1) Substrates

A Kinetic Monte Carlo scheme is applied to simulate with atomic resolution the synthesis of mono (few) layer(s) graphene (Gr) from a silicon carbide (SiC) substrate by selective evaporation of silicon (Si) atoms. The simulation computes the individual dynamics of the residual carbon (C) atoms which diffuse and reconfigure starting from the positions occupied in the SiC hexagonal lattice to the final Gr honeycomb structure. During the transition they gradually modify hybridization (from sp3 to sp2) and bond partners (from Si-C to C-C). We demonstrate that our method is able to recover the complex evolution steps of the epitaxial Gr on SiC in large systems for large time intervals. Moreover, the simulation results can be validated directly by means of comparison with experimental data when varying the material (e.g. initial surface configuration or polarity) or process (e.g. temperature and pressure) conditions.

Towards development of new ornamental plants: status and progress in wide hybridization.

The present review provides insights into the key findings of the hybridization process, crucial factors affecting the adaptation of new technologies within wide hybridization of ornamental plants and presents perspectives of further development of this strategy. Wide hybridization is one of the oldest breeding techniques that contributed enormously to the development of modern plant cultivars. Within ornamental breeding, it represents the main source of genetic variation. During the long history of wide hybridization, a number of methods were implemented allowing the evolution from a conventional breeding tool into a modern methodology. Nowadays, the research on model plants and crop species increases our understanding of reproductive isolation among distant species and partly explains the background of the traditional approaches previously used for overcoming hybridization barriers. Characterization of parental plants and hybrids is performed using molecular and cytological techniques that strongly facilitate breeding processes. Molecular markers and sequencing technologies are used for the assessment of genetic relationships among plants, as the genetic distance is typically depicted as one of the most important factors influencing cross-compatibility in hybridization processes. Furthermore, molecular marker systems are frequently applied for verification of hybrid state of the progeny. The flow cytometry and genomic in situ hybridization are used in the assessment of hybridization partners and characterization of hybrid progeny in relation to genome stabilization as well as genome recombination and introgression. In the future, new research and technologies are likely to provide more detailed information about genes and pathways responsible for interspecific reproductive isolation. Ultimately, this knowledge will enable development of strategies for obtaining compatible lines for hybrid production. Recent development in sequencing technologies and availability of sequence data will also facilitate creation of new molecular markers that will advance marker-assisted selection in hybridization process.

Interspecific hybridization among cultivars of hardy Hibiscus species section Muenchhusia.

Rose mallows belong to the Muenchhusia section of the Hibiscus genus. They represent a small group of cold tolerant North American plants and are popular ornamentals mainly because of their abundant, large and colorful flowers. Due to their geographical origin they are well suited for garden use in temperate regions worldwide. The aim of the study was to investigate hybridization barriers in crosses among cultivars of Hibiscus species from the Muenchhusia section: H. coccineus, H. laevis and H. moscheutos. Crossing barriers were identified as both pre- and post-zygotic. The analysis of pollen tube growth revealed inhibition of pollen tubes and their abnormal growth. In specific crosses the fertilization success was low. The pre-fertilization barriers did not cause a complete reproductive isolation between the hybridization partners. In relation to post-fertilization barriers, the occurrence of hybrid incompatibilities such as unviability, chlorosis, necrosis, stunted growth and albinism were the main drawback in production of hybrids. The appearance of symptoms of hybrid incompatibilities was dependent upon specific parental plants. The obtained progeny had intermediate leaf morphology and flower morphology compared to parental plants. Hybridity state was verified by morphological analysis and RAPD markers. Based on the overall plant morphology, 472 hybrid progenies were obtained.

Evaluation of reproductive barriers contributes to the development of novel interspecific hybrids in the Kalanchoë genus.

BackgroundInterspecific hybridization is a useful tool in ornamental breeding to increase genetic variability and introduce new valuable traits into existing cultivars. The successful formation of interspecific hybrids is frequently limited by the presence of pre- and post-fertilization barriers. In the present study, we investigated the nature of hybridization barriers occurring in crosses between Kalanchoë species and evaluated possibilities of obtaining interspecific hybrids.ResultsThe qualitative and quantitative analyses of pollen tube growth in situ were performed following intra- and interspecific pollinations. They revealed occurrence of pre-fertilization barriers associated with inhibition of pollen germination on the stigma and abnormal growth of pollen tubes. Unilateral incongruity related to differences in pistil length was also observed. The pollen quality was identified as a strong factor influencing the number of pollen tubes germinating in the stigma. In relation to post-fertilization barriers, endosperm degeneration was a probable barrier hampering production of interspecific hybrids. Moreover, our results demonstrate the relation of genetic distance estimated by AFLP marker analysis of hybridization partners with cross-compatibility of Kalanchoë species. At the same time, differences in ploidy did not influence the success of interspecific crosses.ConclusionsOur study presents the first comprehensive analysis of hybridization barriers occurring within Kalanchoë genus. Reproductive barriers were detected on both, pre- and post-fertilization levels. This new knowledge will contribute to further understanding of reproductive isolation of Kalanchoë species and facilitate breeding of new cultivars. For the first time, interspecific hybrids between K. nyikae as maternal plant and K. blossfeldiana as well as K. blossfeldiana and K. marnieriana were generated.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0394-0) contains supplementary material, which is available to authorized users.

Unexpected diversity of feral genetically modified oilseed rape (Brassica napus L.) despite a cultivation and import ban in Switzerland.

Despite cultivation and seed import bans of genetically modified (GM) oilseed rape (Brassica napus L.), feral GM plants were found growing along railway lines and in port areas at four sites in Switzerland in 2011 and 2012. All GM plants were identified as glyphosate-resistant GM event GT73 (Roundup Ready, Monsanto). The most affected sites were the Rhine port of Basel and the St. Johann freight railway station in Basel. To assess the distribution and intra- and interspecific outcrossing of GM oilseed rape in more detail, we monitored these two sites in 2013. Leaves and seed pods of feral oilseed rape plants, their possible hybridization partners and putative hybrid plants were sampled in monthly intervals and analysed for the presence of transgenes by real-time PCR. Using flow cytometry, we measured DNA contents of cell nuclei to confirm putative hybrids. In total, 2787 plants were sampled. The presence of GT73 oilseed rape could be confirmed at all previously documented sampling locations and was additionally detected at one new sampling location within the Rhine port. Furthermore, we found the glufosinate-resistant GM events MS8xRF3, MS8 and RF3 (all traded as InVigor, Bayer) at five sampling locations in the Rhine port. To our knowledge, this is the first time that feral MS8xRF3, MS8 or RF3 plants were detected in Europe. Real-time PCR analyses of seeds showed outcrossing of GT73 into two non-GM oilseed rape plants, but no outcrossing of transgenes into related wild species was observed. We found no hybrids between oilseed rape and related species. GM plants most frequently occurred at unloading sites for ships, indicating that ship cargo traffic is the main entry pathway for GM oilseed rape. In the future, it will be of major interest to determine the source of GM oilseed rape seeds.