Reproducibility of next-generation-sequencing-based analysis of a CRISPR/Cas9 genome edited oil seed rape.

Next-generation-sequencing (NGS) becomes increasingly important for laboratories tasked with the detection of genetically modified organisms (GMOs) in food, feed and seeds. Its implementation into standardized workflows demands reliable intra- and inter-laboratory reproducibility. Here, we analyze the reproducibility of short- and long-read targeted NGS and long-read whole genome sequencing (WGS) data between three independent laboratories. Replicate samples were submitted for sequencing and comparatively analyzed. The targeted-NGS-samples consisted of oil seed rape (OSR) sampled from a commodity shipment spiked with a genome edited (GE) OSR and the WGS-samples consisted of leaf material from the GMOs' parental line. All laboratories delivered highly reproducible high-quality targeted NGS data with little variation. The detection of GMO-related sequences works well regardless of the facility, while the mapping to the complex genome is superior using long read data. Long read WGS is currently not suitable for routine use in enforcement laboratories, due to a large inter-laboratory variation.

GMO Genetic Elements Thesaurus (GMO-GET): a controlled vocabulary for the consensus designation of introduced or modified genetic elements in genetically modified organisms.

BACKGROUND: Various databases on genetically modified organisms (GMOs) exist, all with their specific focus to facilitate access to information needed for, e. g., the assistance in risk assessment, the development of detection and identification strategies or inspection and control activities. Each database has its unique approach towards the subject. Often these databases use different terminology to describe the GMOs. For adequate GMO addressing and identification and exchange of GMO-related information it is necessary to use commonly agreed upon concepts and terminology. RESULT: A hierarchically structured controlled vocabulary describing the genetic elements inserted into conventional GMOs, and GMOs developed by the use of gen(om)e-editing is presented: the GMO genetic element thesaurus (GMO-GET). GMO-GET can be used for GMO-related documentation, including GMO-related databases. It has initially been developed on the basis of two GMO databases, i.e. the Biosafety Clearing-House and the EUginius database. CONCLUSION: The use of GMO-GET will enable consistent and compatible information (harmonisation), also allowing an accurate exchange of information between the different data systems and thereby facilitating their interoperability. GMO-GET can also be used to describe genetic elements that are altered in organisms obtained through current targeted genome-editing techniques.

Molecular characterization of an unauthorized genetically modified Bacillus subtilis production strain identified in a vitamin B2 feed additive.

Many food and feed additives result from fermentation of genetically modified (GM) microorganisms. For vitamin B2 (riboflavin), GM Bacillus subtilis production strains have been developed and are often used. The presence of neither the GM strain nor its recombinant DNA is allowed for fermentation products placed on the EU market as food or feed additive. A vitamin B2 product (80% feed grade) imported from China was analysed. Viable B. subtilis cells were identified and DNAs of two bacterial isolates (LHL and LGL) were subjected to three whole genome sequencing (WGS) runs with different devices (MiSeq, 454 or HiSeq system). WGS data revealed the integration of a chloramphenicol resistance gene, the deletion of the endogenous riboflavin (rib) operon and presence of four putative plasmids harbouring rib operons. Event- and construct-specific real-time PCR methods for detection of the GM strain and its putative plasmids in food and feed products have been developed.

Down-regulation of small rubber particle protein expression affects integrity of rubber particles and rubber content in Taraxacum brevicorniculatum.

The biosynthesis of rubber is thought to take place on the surface of rubber particles in laticifers, highly specialized cells that are present in more than 40 plant families. The small rubber particle protein (SRPP) has been supposed to be involved in rubber biosynthesis, and recently five SRPPs (TbSRPP1-5) were identified in the rubber-producing dandelion species Taraxacum brevicorniculatum. Here, we demonstrate by immunogold labeling that TbSRPPs are localized to rubber particles, and that rubber particles mainly consist of TbSRPP3, 4 and 5 as shown by high-resolution two-dimensional gel electrophoresis and mass spectrometric analysis. We also carried out an RNA-interference approach in transgenic plants to address the function of TbSRPPs in rubber biosynthesis as well as rubber particle morphology and stability. TbSRPP-RNAi transgenic T. brevicorniculatum plants showed a 40-50% reduction in the dry rubber content, but neither the rubber weight average molecular mass nor the polydispersity of the rubber were affected. Although no phenotypical differences to wild-type particles could be observed in vivo, rubber particles from the TbSRPP-RNAi transgenic lines were less stable and tend to rapidly aggregate in expelling latex after wounding of laticifers. Our results prove that TbSRPPs are very crucial for rubber production in T. brevicorniculatum, probably by contributing to a most favourable and stable rubber particle architecture for efficient rubber biosynthesis and eventually storage.

Proteomic analysis of latex from the rubber-producing plant Taraxacum brevicorniculatum.

Many plants produce latex, a specialized, metabolically active cytoplasm. This is generally regarded as a defensive trait but latex may also possess additional functions. We investigated the role of latex in the dandelion species Taraxacum brevicorniculatum that contains considerable amounts of high-quality natural rubber by carrying out a comprehensive analysis of the latex proteome. We developed reliable protocols for the preparation of protein samples for one-dimensional gel electrophoresis, two-dimensional gel electrophoresis, and subsequent mass spectrometry analysis, which led to 278 unique identifications. A gene ontology classification system based on comparisons with known Arabidopsis thaliana root proteins showed that dandelion proteins involved in lipid metabolism and transport were enriched in the latex proteome, whereas those involved in stress responses were not. We also found that proteins involved in rubber biosynthesis were distributed among different fractions of the latex proteome.

Polyphenoloxidase silencing affects latex coagulation in Taraxacum species.

Latex is the milky sap that is found in many different plants. It is produced by specialized cells known as laticifers and can comprise a mixture of proteins, carbohydrates, oils, secondary metabolites, and rubber that may help to prevent herbivory and protect wound sites against infection. The wound-induced browning of latex suggests that it contains one or more phenol-oxidizing enzymes. Here, we present a comprehensive analysis of the major latex proteins from two dandelion species, Taraxacum officinale and Taraxacum kok-saghyz, and enzymatic studies showing that polyphenoloxidase (PPO) is responsible for latex browning. Electrophoretic analysis and amino-terminal sequencing of the most abundant proteins in the aqueous latex fraction revealed the presence of three PPO-related proteins generated by the proteolytic cleavage of a single precursor (pre-PPO). The laticifer-specific pre-PPO protein contains a transit peptide that can target reporter proteins into chloroplasts when constitutively expressed in dandelion protoplasts, perhaps indicating the presence of structures similar to plastids in laticifers, which lack genuine chloroplasts. Silencing the PPO gene by constitutive RNA interference in transgenic plants reduced PPO activity compared with wild-type controls, allowing T. kok-saghyz RNA interference lines to expel four to five times more latex than controls. Latex fluidity analysis in silenced plants showed a strong correlation between residual PPO activity and the coagulation rate, indicating that laticifer-specific PPO plays a major role in latex coagulation and wound sealing in dandelions. In contrast, very little PPO activity is found in the latex of the rubber tree Hevea brasiliensis, suggesting functional divergence of latex proteins during plant evolution.