**2. Fundamentals**

Determining whether a plant variety is "essentially derived" has largely been left to scientists and plant breeders who favor a quantitative approach that assesses genetic similarity using "genetic thresholds," "genetic distances" and "genetic coefficients." Unfortunately, science is not settled on the most appropriate method of assessing whether a given plant variety is essentially derived [2]. As such, a key challenge facing plant IP is to traverse the gap between science and law as it relates to the essential derivation of plant varieties. Clarity is necessary to ensure the effectiveness of essential derivation as a legal concept and to instill confidence in the plant breeders' rights scheme. The importation of notions of quality into the assessment of essential derivation by the judiciary has a number of advantages, including the ability to meet the needs of developments in plant breeding techniques and providing plant breeders with greater certainty in relation to whether new varieties are essentially derived [2].

Who decides whether a plant variety is essentially derived from another? In case of dispute, the holder of the initial variety has the initial burden of demonstrating that another variety is derived from their variety. In an application procedure, there authorities granting plant variety rights are not in the best position to determine whether a given variety is an EDV. The IP rights protection system is generally noninterventionalist; an EDV need not be protected *per se*, and may, therefore, entirely elude the attention of the authority granting plant variety rights. Since EDVs concern the scope of the protection, it is the responsibility of the holder of the IV plant breeder's rights to enforce the right, and/or settle with the breeder of the variety which is essentially derived from the initial one. In case of disputes related to EDVs, if the parties cannot agree, then the courts will have the final word. However, there is little by way of case law available, which might indicate that either in most cases the parties settle the matters amongst themselves, and/or the anticipated costs of legal action do not justify a possible profit that such an action might provide.

In Article 14(5)(c), the UPOV convention provides some guidance on how EDVs might be obtained. Exemplary modes of obtaining EDVs are the selection of a natural or induced mutant, or of a somaclonal variant, the selection of a variant individual from plants of the initial variety, backcrossing, or transformation by genetic engineering [1]. The EDV need not be directly derived from the initial variety; it may also be bred by using one or more other varieties predominantly derived from the initial variety. Two principal criteria need to be met by an EDV: (i) there must have been an act of derivation, and (ii) there must be a significant extent of similarity at the phenotypical level. The latter is relatively easier to establish; the former—not so much.

The phenotype-based assessments of eventual essential derivation are timeconsuming, laborious, and costly processes that involve field and greenhouse comparisons. As well, various factors limit the expression and use of phenotypic traits. The measurement of morphological traits may be influenced by environmental factors, resulting in reduced precision and discriminatory power. This is especially

disadvantageous for the evaluation of disease resistance or complex quantitative traits, such as flowering time.

One of the key advantages of molecular marker-assisted breeding is that it increases the breeding efficiency compared to conventional breeding based on phenotypic selection. Molecular markers thus allow for the identification and selection of individual plants with high reliability—in any context. The use of molecular marker data to assist in the determination of EDVs has already received significant attention, as molecular markers are an important means to help determine EDV status [3]. In a few instances, crop-specific experimental guidelines have even been developed; see *infra*. Because the comparison of two varieties is by definition a relative endeavor, some of the relevant questions include: how deep should the marker analysis be? What should be the reference framework? What should be a representative sample of markers for a given phenotypic trait? Is the choice of marker technology important? What about the number of markers used? How general vs. species-specific should the marker analysis be? Fortunately, some of the above points have been addressed in the literature.

The choice of marker technology does not appear to be crucial. In several cases, the analysis of the same dataset with different technologies led to same conclusions. Any DNA marker technology can do the job, as long as the technology and performance have proven to be accurate. Furthermore, an increased number of markers will give a more accurate representation of the genome. Due to the ever-increasing abundance of reference genomes and advances in molecular technologies, functional markers that target specific alleles responsible for phenotype variation can and will be developed.

In disputes relating to plant breeders' rights, the initial burden is upon the owner of the IV who should provide *prima facie* proof of essential derivation and consequently claim the dependence of the new variety. This is precisely where molecular marker data can provide an indication that can be interpreted in relation to a trigger point to reverse the burden of proof [4].

Genetic distance estimates based on molecular markers are a preferred approach to estimating genetic conformity between putative EDVs and their corresponding initial varieties (IVs). For example, genetic similarity between two varieties can be suitably measured using both amplified fragment length polymorphisms (AFLPs) and simple sequence repeats (SSRs) [3]. To counterbalance the advantages and disadvantages of AFLP and SSRs, a complementary application of the two marker systems is suggested for discrimination between EDVs and independently derived varieties (IDVs) [5].

Critics of this approach and of these rights (the use of molecular markers for determination of EDVs in particular, and EDVs in general) point to the relatively dominant position of a very small number of corporations in the global seed market, the impact of this domination on farmers, and the role of IP in promoting these perceived corporate interests, often at the expense of environmental and social justice concerns [6]. Critics argue that the extension of plant breeders' rights to EDVs is controversial as it creates tension with the breeders' exemption [7]. This chapter submits that the determination of whether a given plant variety is an EDV is best approached as a holistic endeavor: both genetic comparisons and morphological differences between the varieties in question should be considered.

The extension of the protection for initial varieties to derived varieties was intended as an exceptional provision, which should be interpreted restrictively by its nature. In a time when plant gene editing is becoming routine, it is possible to use a single locus conversion and to introduce a single transgene into a variety. Such a modification could even be precisely introduced into the IV via new breeding

#### *Molecular Techniques for Essentially Derived Varieties DOI: http://dx.doi.org/10.5772/intechopen.105532*

techniques (NBTS, e.g., CRISPR/Cas-mediated genome editing, or mutagenesis). The newly obtained variety would thus have only a single genetic difference (single locus) vs. the initial variety, but a cleverly introduced single transgene could result in multiple (i.e., more than a few) downstream gene and protein expression modifications, for example, changes to a metabolic pathway, regulation of a chosen key transcription factor, etc. It could thus result in a clearly distinct phenotype (e.g., flower color, fruit size, etc.). Therefore, the determination of whether a derived variety differs significantly from the initial should be conducted on a case basis, on a crop basis, and on a species basis. Conversely, in instances where there are major morphological differences established between the varieties in question, there may not be a need to conduct a genetic comparison; a phenotypic comparison might suffice.

### **3. Particulars**

Possible starting points for the assessment of EDVs include the establishment of predominant derivation (e.g., evidence of genetic conformity with the IV), and conformity of the essential characteristics (evidence of conformity in the expression of the essential characteristics of the IV). Essential characteristics means heritable traits that contribute to the principal features, performance, or value of the variety, and/or characteristics that are important from the perspective of the producer, seller, supplier, buyer, recipient, or user. These essential characteristics may be different in different crops and species. They also may or may not be phenotypic characteristics used for the examination of distinctness, uniformity, and stability (DUS) [1]. The assessment of conformity is essentially a judgment call that is based on essential characteristics. The number of differences that results from the act of derivation should be minimal (one or very few). The derived variety must retain almost the totality of the genotype of the initial variety and be different from the IV by a very limited number of characteristics.

Numerous studies have demonstrated the utility of molecular genetic markers for the determination of EDVs. For example, DNA fingerprinting has been used to illustrate the possibilities and limitations in disputes on alleged fraud and essential derivation of ornamental plants, including roses and *Phalaenopsis* orchids. Following AFLP analysis of putative parents, the parties (breeders) settled the dispute(s) [8]. AFLP, as a multilocus marker technology, enables quick random screening of the plant genome. The effectiveness of a primer combination to reveal good AFLP patterns for a certain plant species depends on three factors: (i) the occurrence of the restriction sites recognized by the used enzymes; (ii) the number of selective bases added to the primer in the function of the genome size (five to six selective bases); and (iii) the GC content of the DNA [8].

To invest in research and product development, companies value the certainty afforded by IP protection. Property rights with respect to plant varieties are not an exception. Accordingly, a thorough review of the issues important to the private sector with respect to genomic approaches and IP protection for EDVs was recently provided [9]. Notably, the American Seed Trade Association (ASTA) and the French Seed Association (UFS, Union Française des Semenciers) jointly embarked on a project using an Illumina® 56,000 SNP (single nucleotide polymorphism) chip to profile a set of maize inbred lines including those of historic and current importance. The results of this study provided a list of thousands of publicly available SNP loci, as a repository for the selection of desired sets of markers to measure genetic similarities (or distances) between pairs of inbred lines. These loci can find utility, particularly for the purpose of resolving questions in regard to the possible status as an EDV [9]. These molecular tools can be very robust, and their utility has been demonstrated in numerous case studies. For example, by providing markers (e.g., SNP loci) that are judiciously selected, it is possible to identify a relatively small set of SNP loci that collectively have a very high power of discrimination among maize inbred lines, and which, therefore, could be used to measure uniformity and stability (genetic purity) for example, as few as 16 SNPs can discriminate among more than 400 pioneer proprietary inbred lines. Indeed, marker-based distances are more appropriate than pedigree records to assess genetic relationships among maize inbreds [9].

The international seed federation (ISF) works to represent the interests of the seed industry at a global level, including farmers, growers, industry, and consumers. ISF promotes the establishment and protection of IP rights for seeds, plant varieties, and associated technologies. ISF has adopted arbitration procedure rules tailored to the technical and legal aspects of essential derivation in plant breeding, using plant species-specific protocols and markers. Because the determination of genetic similarity thresholds varies between species depending on the genetic variability, the breeding procedures, and the used molecular tools, distinct guidelines and experimental protocols for handling disputes on essential derivation in various crops (e.g., cotton, lettuce, maize, oilseed rape, and perennial ryegrass) have been developed by the ISF [10]. For example, the ISF has supplied a list of 3072 SNP markers used in the guidelines provided for essential derivation in maize [11]. Furthermore, the key issues that should be addressed by technical experts to define molecular marker sets for establishing thresholds for EDV arbitration by the ISF have also been published [12].

Perhaps due to its economic significance, much of the studies on the characterization of the essential derivation of plant varieties have so far been conducted in maize [13–15]. These include the study of inbreds developed without and with a selection from F2 populations [13], the selection and evaluation of an optimal panel of SSR loci in maize [14], and a comparison of SSR and SNP data in different maize genotypes, using European and North American germplasm, with the help of a set of 163 SSR loci previously identified to help determine EDV status [15].

The increased availability and cost-efficiency of DNA-based markers in recent years make them attractive options to explore their use to supplement or even ultimately replace, existing morphological-, phenotype-, and protein-based approaches. Indeed, exploring such options is the focus of UPOV's working group on "biochemical and molecular techniques and DNA profiling in particular" (BMT). A recent review argues for the adoption of a DNA-based system which is endorsed by the UPOV, and highlights that the efficiency of phenotype-based assessments of plant variety protection and registration could be improved by the integration of DNA-based testing [16]. So far, two competing models, namely "characteristic-specific molecular markers" and "combining phenotypic and molecular distances in the management of variety collections" have been favored [16].

Plant variety protection is also of high relevance for the horticultural community. In cases of plant varieties with a relatively narrow gene pool, it is possible to use molecular markers as a technique for the identification of EDVs in highly identical genotypes. For example, in a case involving *Calluna vulgaris* (common heather), a successful marker comparison was performed by means of random amplified polymorphic DNA (RAPD) and intersimple sequence repeats (ISSR) fingerprinting while using 168 mono- and polymorphisms. Accordingly, this methodology has been

#### *Molecular Techniques for Essentially Derived Varieties DOI: http://dx.doi.org/10.5772/intechopen.105532*

recommended for the future establishment of proof-of-essential-derivation, not only for *C. vulgaris*, but also generally for other vegetatively propagated crops [17].

The concept of protecting the rights of breeders through EDVs has itself been the subject of criticism, as being directed to narrowing the breeder's exemption while expanding the exclusive rights of first-generation breeders [2]. Furthermore, the methodology of using molecular markers for the determination of EDVs has also specifically been attacked. It has been argued that utilizing molecular markers to assess essential derivation could actually provide a mechanism to undermine the intention of essential derivation—which is to discourage copying and free riding. Ironically, a breeder may use marker-assisted breeding to evade a declaration of essential derivation, through a clever selection of a molecular marker profile that is 'sufficiently different' from the initial variety. For instance, if the genetic threshold for a given variety is 90%, it may be possible to ensure that subsequent varieties will show genetic thresholds of 85% or less, despite there only being minor changes made to the initial variety. While the new variety may be quantitatively outside the boundary established for essential derivation, it may still draw on the qualitative features of the existing variety for its commercial appeal to the industry [2].

### **4. Discussion and prospects**

The UPOV system introduced in 1991 the concept of essential derivation and EDV. Some non-UPOV member countries (e.g., India, Malaysia, and Thailand) have also independently introduced the concept of essential derivation. China, a UPOV member operating under the 1978 Convention, is introducing EDVs via seed laws [18]. The EDV concept is alive and well. Due to the relative complexity associated with its definition, the implementation of the EDV concept has sometimes been challenging.

The use of molecular genetic markers can bolster the science-preferred quantitative approach to the determination of EDVs. Threshold values for genetic conformity can be determined by using similarities that are calculated from molecular marker characterizations of varieties [19]. The use of marker-based (dis)similarities in essential derivation cases raises statistical questions, such as how to define a reference population of varieties within which potential essential derivation disputes could occur, how many marker loci to use for a required precision, and how to define a threshold value on the basis of the observed distribution of similarity values. Initial results from special studies undertaken to answer these questions in lettuce and barley have been published [19]. It has already been shown that, at least for a subset of DUS phenotypic traits, molecular markers can be robustly used as a tool for the determination of all these three components of the DUS testing in the diploid crop species barley [20]. This is important since crop-specific DUS test guidelines have been developed and followed for plant variety assessment, to register a new variety. Plant varieties can differ significantly, and the phenotypic differences depend on the genetic constitution but also on the sample size, levels of confidence, reject numbers, etc. [21].

The study and applications of genomics continue to drive our understanding and characterization of germplasm collections, gene function, phenotypic expression and adaptability, the identification of agronomically favorable alleles, and the creation of improved cultivars. Expanding our knowledge of the genetic basis of agronomic traits will help breeders to more efficiently explore and harness plant genetic resources. It is becoming clear that criteria based solely on plant morphology and phenotype are

insufficient or even not applicable for the determination of EDVs. Molecular markers should help quantify the differences between similar and/or related plant varieties, increase the certainty of essential plant derivation, and assuage the concerns with respect to the scope of IP protection afforded by the concept of EDV. Accordingly, it would be reasonable to expect that the use of molecular markers in EDV determination and EDV-related disputes will increase in the future.

Crop production plays a key role in our society's needed and expanding sustainability goals. The development of new plant varieties is not only a scientific endeavor, but very much a social, political, and economic one as well. As well, IP rights regimes are established to achieve societal goals with a broad impact. The ability to obtain adequate protection of newly developed plant varieties as IP can encourage investments into agricultural biotechnology and specifically plant breeding. It is thus of paramount importance that innovations in plant breeding receive the appropriate innovation incentives. The advent of new molecular breeding techniques has reignited discussions about the scope of afforded plant breeders' rights. This might require modernization and redefining of the concept and definition of EDVs [22]. Technological advances that facilitate the simultaneous introduction of several transgenes might push the boundaries and fundamentally challenge the concept of essential derivation; perhaps a breeder exception should be formulated on a crop-by-crop basis [18]. Decisions about plant breeders' rights should be considered in conjunction with broader policy support, to ensure to the coexistence of a responsive public agricultural research system with a diverse and competitive private seed sector. They also depend on a well-functioning legal system with enforceable contracts between plant growers and merchants, the promotion of responsible business practices, and the encouragement of engagement of professional associations in agribusiness [23].

Refinements of the used molecular techniques should be expected in the future. To increase sensitivity and accuracy, simultaneous use of various and/or multiple types of markers/assays could be performed. It will be beneficial for the community of plant breeders to reach a consensus on the use of (sub)sets of plant species-specific markers that would be deemed necessary and sufficient for determining genetic resources [4]. It will also be important to establish a minimum level of relative marker profile similarity as a threshold for distinctness. Such a threshold must be based upon the use of a set of markers (e.g., SNPs) that are identified as meeting the criteria of (i) collectively providing for fairly even genome coverage, and (ii) having a proven ability to discriminate among varieties, even among those that are very closely related by pedigree [8].

Molecular genetic markers are ever-improving tools that are making an impact in the ongoing debate on the scope of IP rights protection for EDVs. They introduce a higher degree of scientific certainty. This is good because critics argue that protection for EDVs brings uncertainty in assessing what is an EDV, which then becomes problematic for the plant-breeding businesses globally. Breeders need simple rules, which are easy to understand and enforce. Some have even argued that small farmers in the developing world are in an inferior position, as they are not capable of making such comparisons and assessments [22]. Furthermore, the technological and economic environment in which plant breeders operate will likely continue to change and will become more complex. In that context, these changes may well cause plant breeders and policymakers to seek to further adjust the *sui generis* IP system for plants (and particularly for EDVs), which system can optimally contribute to—and complement—other forms of IP protection for plants [3].

Exciting days lie ahead for plant breeders.
