12 / SEEDWORLD.COM SEPTEMBER 2018 body, developed a forensic tool, called “Variety Tracer” to be applied in cases where infringement is suspected. As Naktuinbouw is the only organization in the Netherlands appointed by the government to assess varieties of nearly all crops for distinctness, uniformity and stability (DUS) for regis- tration and Plant Breeders’ Rights, morphological knowledge on many crops and on the PBR system is assured. Since 2004, Naktuinbouw also established a laboratory facility for variety identification using genotyping technologies. It is the combina- tion of these elements that guarantees the power of Variety Tracer. How Does Variety Tracer Work? To investigate a potential infringement of PBR, a Variety Tracer research project can be initiated. Often in a Variety Tracer project, a combination of morphological side-by-side growing trials and genetic research is applied, but just a growing trial or only a genetic analysis is also possible. Since every poten- tial infringement is unique, a custom-made research plan is needed. Together with the titleholder of the PBR, the particular infringement and special crop-specific information is discussed. For example, an outcrossing crop in which a lot of diversity is expected within a variety is treated differently than a vegeta- tively propagated crop for which all plants belonging to one variety have identical genotypes. After a final research plan is completed, sampling of all plant material to be included is col- lected. It is crucially important that the sampling is independent. To function as legal proof, a bailiff might be involved to take the suspected samples at the potential infringers growing site. For the genetic analysis, two main questions must be considered: “How many markers are needed?” and “Which reference frame- work is relevant?” To answer the latter question, Naktuinbouw developed guidelines for the selection of samples in a Variety Tracer analy- sis. Samples should be representative for the diversity within the species, meaning that all relevant morphological characteristics should be present in an unbiased way. Some closely related samples should be included, which can consist of parental lines with seedlings and, when present, known and accepted mutant varieties with the original variety. Choices on which technology to use highly depend on the crop involved and on what informa- tion is publically available. For many species, sequence informa- tion and crop-specific SNP or SSR markers are available. When no DNA information is available, a random marker technology like AFLP can be applied. As the interest in variety identity tests increases, the number of Variety Tracer projects grows every year. The projects vary from simple identity questions to detect mix-ups to complicated infringement cases involving many parties. In nearly all infringe- ment cases that were subject of the specialized PBR court in The Hague, Naktuinbouw was involved as independent expert and the Variety Tracer report served as evidence. Future Perspective With the release of next-generation sequencing (NGS) tech- nology in 2005, a real sequencing revolution has taken place. NGS machines enable massive parallel sequencing, increas- ing sequencing data output for decreasing costs. NGS is often referred to as high throughput sequencing. To increase the resolution and distinguishable power of DNA markers used in Variety Tracer projects, Naktuinbouw now invests to replace the old technologies like AFLP and SSR by SNP genotyping based on high throughput sequencing. Currently, the Variety Tracer 2.0 is being developed. There are three phases in the process of developing a SNP genotyping method for a particular species: the identification of SNPs and selection of a SNP panel that is representative for the genetic diversity within the species, development of a sequence-based method to determine the SNP genotype of multiple varieties in a high throughput and cost effective way and storage of all SNP genotypes in databases that can be used for variety identifica- tion purposes including infringements. Variety Tracer 2.0 will be better and cheaper to be applied for a plant world without piracy. SW In a Variety Tracer project often a combination of morphological side-by-side growing trials and genetic research is applied.