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Molecular characterization of genetically modified plants can provide crucial information for the development of detection and identification methods, to comply with traceability, and labeling requirements prior to commercialization. Detailed description of the genetic modification was previously a challenging step in the safety assessment, since it required the use of laborious and time-consuming techniques. In this study an accurate, simple, and fast method was developed for molecular characterization of genetically modified (GM) plants, following a user-friendly workflow for researchers with limited bioinformatic capabilities. Three GM events from a diverse array of crop species—perennial ryegrass, white clover, and canola—were used to test the approach that exploits long-read sequencing by the MinION device, from Oxford Nanopore Technologies. The method delivered a higher degree of resolution of the transgenic events within the host genome than has previously been possible with the standard Illumina short-range sequencing strategies. The flanking sequences, copy number, and presence of backbone sequences, and overall transgene insertion structure were determined for each of the plant genomes, with the additional identification of moderate-sized secondary insertions that would have previously been missed. The proposed workflow takes only about 1 week from DNA extraction to analyzed result, and the method will complement the existing approaches for molecular characterization of GM plants, since it makes the process faster, simpler, and more cost-effective.

Food security is one of major concerns for the growing global population. Modern agricultural biotechnologies, such as genetic modification, are a possible solution through enabling an increase of production, more efficient use of natural resources, and reduced environmental impacts. However, new crop varieties with altered genetic materials may be subjected to safety assessments to fulfil the regulatory requirements, prior to marketing. The aim of the assessment is to evaluate the impact of products from the new crop variety on human, animal, and the environmental health. Although, many studies on the risk assessment of genetically modified (GM) food have been published, little consideration to GM feedstuff has been given, despite that between 70 to 90% of all GM crops and their biomass are used as animal feed. In addition, in some GM plants such as forages that are only used for animal feeds, the assessment of the genetic modification may be of relevance only to livestock feeding. In this article, the regulatory framework of GM crops intended for animal feed is reviewed using the available information on GM food as the baseline. Although, the majority of techniques used for the safety assessment of GM food can be used in GM feed, many plant parts used for livestock feeding are inedible to humans. Therefore, the concentration of novel proteins in different plant tissues and level of exposure to GM feedstuff in the diet of target animals should be considered. A further development of specific methodologies for the assessment of GM crops intended for animal consumption is required, in order to provide a more accurate and standardized assessment to the GM feed safety.

Breeding schemes that utilize modern breeding methods like genomic selection (GS) and speed breeding (SB) have the potential to accelerate genetic gain for different crops. We investigated through stochastic computer simulation the advantages and disadvantages of adopting both GS and SB (SpeedGS) into commercial breeding programs for allogamous crops. In addition, we studied the effect of omitting one or two selection stages from the conventional phenotypic scheme on GS accuracy, genetic gain, and inbreeding. As an example, we simulated GS and SB for five traits (heading date, forage yield, seed yield, persistency, and quality) with different genetic architectures and heritabilities (0.7, 0.3, 0.4, 0.1, and 0.3; respectively) for a tall fescue breeding program. We developed a new method to simulate correlated traits with complex architectures of which effects can be sampled from multiple distributions, e.g. to simulate the presence of both minor and major genes. The phenotypic selection scheme required 11 years, while the proposed SpeedGS schemes required four to nine years per cycle. Generally, SpeedGS schemes resulted in higher genetic gain per year for all traits especially for traits with low heritability such as persistency. Our results showed that running more SB rounds resulted in higher genetic gain per cycle when compared to phenotypic or GS only schemes and this increase was more pronounced per year when cycle time was shortened by omitting cycle stages. While GS accuracy declined with additional SB rounds, the decline was less in round three than in round two, and it stabilized after the fourth SB round. However, more SB rounds resulted in higher inbreeding rate, which could limit long-term genetic gain. The inbreeding rate was reduced by approximately 30% when generating the initial population for each cycle through random crosses instead of generating half-sib families. Our study demonstrated a large potential for additional genetic gain from combining GS and SB. Nevertheless, methods to mitigate inbreeding should be considered for optimal utilization of these highly accelerated breeding programs.

Implementation of molecular biotechnology, such as transgenic technologies, in forage species can improve agricultural profitability through achievement of higher productivity, better use of resources such as soil nutrients, water, or light, and reduced environmental impact. Development of detection and quantification techniques for genetically modified plants are necessary to comply with traceability and labeling requirements prior to regulatory approval for release. Real-time PCR has been the standard method used for detection and quantification of genetically modified events, and droplet digital PCR is a recent alternative technology that offers a higher accuracy. Evaluation of both technologies was performed using a transgenic high-energy forage grass as a case study. Two methods for detection and quantification of the transgenic cassette, containing modified fructan biosynthesis genes, and a selectable marker gene, hygromycin B phosphotransferase used for transformation, were developed. Real-time PCR was assessed using two detection techniques, SYBR Green I and fluorescent probe-based methods. A range of different agricultural commodities were tested including fresh leaves, tillers, seeds, pollen, silage and hay, simulating a broad range of processed agricultural commodities that are relevant in the commercial use of genetically modified pastures. The real-time and droplet digital PCR methods were able to detect both exogenous constructs in all agricultural products. However, a higher sensitivity and repeatability in transgene detection was observed with the droplet digital PCR technology. Taking these results more broadly, it can be concluded that the droplet digital PCR technology provides the necessary resolution for quantitative analysis and detection, allowing absolute quantification of the target sequence at the required limits of detection across all jurisdictions globally. The information presented here provides guidance and resources for pasture-based biotechnology applications that are required to comply with traceability requirements.