20 I EUROPEAN SEED I EUROPEAN-SEED.COM ers are not that far off from the same questions being asked in DUS testing. Breeding organizations have been using markers to answer D, U, and S questions for research and seed multiplication to maintain high levels of quality assurance for decades. As with any characterization system, there have to be clear and defined pro- tocols for the use of markers. It is going to take some time to develop these pro- tocols for use in DUS examination, but clearly, it can be accomplished for the many species covered by PBR. Official guidelines are in development for soy- bean and maize, at least in the U.S., and also for soybean in South America. ES: NOT EVERYBODY IS CONVINCED THAT WE SHOULD BE INTRODUCING SUCH TECHNIQUES IN DUS TESTING. WHAT IS THE REASON FOR THESE CONCERNS? BN: At the genesis of UPOV, morphology was the only characterization method that existed, so, for some, it is the firm belief that morphology should be maintained because it was the foundation upon which UPOV was founded. Some also insist that the definition of “expressed” in “expressed traits” requires that characteristics be used that have a genetic basis made visible through expression of RNA and protein to specific traits. Given where the breeding industry has moved technologically with DNA marker characterization and widely applied usage, this change should be pos- itive. If the UPOV system was being devel- oped today, it would utilize DNA markers or DNA sequence data. Furthermore, over the last 30 years there has been rapid evolution of marker technology making it a challenge to deter- mine which marker system to choose. Now however, molecular characterization has reached the single base pair level or the smallest unit of the DNA code. It is likely that technological advances will continue, but assays will remain at the base pair level allowing consistent data- bases of marker profiles to be developed. PN: I believe much of the concern stems from the unfamiliarity with the new tech- niques. Additionally, there is concern over the abuse or misapplication of the tech- nology. It is important that those who are exploring DNA marker methods, for the purpose of DUS, are aligned with stake- holders and provide transparency into the process. I mean, transparency into the prescribed applications of the technology and into the scientific process for arriv- ing at that prescription. Further, publicly available protocols should be established and adopted, which would minimize the opportunity for abusing the technology. ES: HOW CAN THESE BIOMOLECULAR TECHNIQUES HELP IMPROVE THE QUALITY AND EFFICIENCY OF DUS TESTING? PN: These techniques can improve the DUS examination process because DNA markers provide additional data points that improve the precision of the assess- ment. Molecular techniques can be highly informative and extremely precise. Furthermore, DNA marker techniques are cost-effective, and the cost contin- ues to fall; they come at a fraction of the cost of a standard field trial, and costs for these techniques continue to fall. BN: Since you are reading the DNA itself, the need to continually re-characterize varieties for comparison is eliminated by the simple fact that the Genotype X Environment interaction is gone. You can create the molecular profile of a line once in a matter of a few days and you’re done, versus repeated growing every season. This saves huge amounts of time and resources. In terms of accuracy, marker profil- ing typically has five per cent or less in genotyping error depending on the tech- nology used. That said, most breeding organizations and third-party labs are at two per cent or less and getting more accurate as time goes on. Determining DUS based on a molecular / genetic sam- pling of the genome provides a more appropriate means to compare varie- ties. Most morphological traits have an unknown basis of genetic control and many characteristics e.g. maize tassel characteristics, are under complex genetic control, genetic control that is influenced by the environment in its expression. Hence a morphological metric comparing varieties is not only less pre- cise than a DNA marker or sequence based metric, it is also biased by exam- ining the genetic differences between varieties through the lens of morpholog- ical expression of non-randomly selected islands of the genome rather than a more thorough sampling of the total genome. ES: WHICH OTHER FUNCTIONS COULD SUCH TECHNIQUES HAVE IN THE REALM OF PLANT BREEDERS' RIGHTS? BN: DNA marker data have been used for several years to identify varieties and to test pedigrees in cases of suspected misappropriation or counterfeiting. However, some wrongly argue that vari- eties established on the basis of morpho- logical distinctness cannot be identified by their unique marker profiles. The abil- ity to uniquely identify a variety and to test it’s pedigree after grant of the PVP are separate activities and the fact that morphology data were used to provide protection for a variety is irrelevant for enforcement. Markers have been used for decades to assure genetic identity of inbred lines and hybrids and to assure varietal iden- tity in lots of self-pollinated crops. DNA markers are used in the breeding process to help increase selection efficiencies. They are a very important tool to deter- mine the status of varieties in respect of their derivation according to the provi- sion of Essential Derivation according to the 1991 UPOV Convention. PN: Right now, most efforts are focused on the “D” in DUS, so in the future more can be done on the “U and S” portions. These techniques are already frequently used in IP enforcement. ES: HOW WILL DUS TESTING LOOK LIKE IN 10 OR 20 YEARS FROM NOW? WILL THERE BE AN INCREASED USE OF BIOMOLECULAR TECHNIQUES? BN: With the focus and energy dedi- cated to assessing markers in PVP by IMMODUS, the ASTA/US PVP Office Working Group, and other working groups around the globe, I am confident that DUS examination will make greater use of DNA markers in the future. PN: There is tremendous interest around the globe in the application of molecular techniques in DUS examination, so there is great positive momentum. One can refer to the discussions that are taking place in the UPOV BMT (Biochemical and Molecular Techniques); in the CPVO IMODDUS (Community Plant Variety Office, Integration of Molecular Data into DUS); innovative aspects devel- oped by China, and South Korea; efforts of the SAA working group (Seed Association of the Americas); and the US ASTA/PVPO working group. There are many bright, well-inten- tioned minds excited and working on this issue, and we are likely to have suc- cessful implementation of one or more of the techniques in some form in the near future. Morphology is the bedrock of DUS examination and will remain as such for the foreseeable future. But, molec- ular techniques can have clear value in DUS determination as a supplement to morphology. Molecular techniques can bring value to the DUS process by improving precision and collaboration. They augment morphology, in addition to the already approved and accepted methods. When properly applied through scientifically vetted methods, they serve as a common language by which breeders and PVP authorities around the globe can communicate.