2.3.1 Concerns About the Use of Molecular Markers to Describe Varieties de novo 352.3.2 Concerns About the Use of Phenotypic Characteristics to Describe Varieties
de novo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362.4 Improving the DUS process: The rationale for Change to the Use of Molecular
Abstract Genetic gain is a critical means to improve crop production and will in-creasingly be relied upon to further improve agricultural productivity in ways that aremore sustainable. Partly through the use of molecular markers plant breeders havebeen able to increase the rate of genetic gain by increasing efficiencies in selectionfor improved performance of agronomic traits. Greater knowledge of the geneticbasis of agronomic traits will help breeders to more efficiently explore and harnessplant genetic resources including those that are currently exotic. Efficient processes
J. S. C. Smith () · B. K. Nelson · D. S. Phillips · R. A. WinelandDuPont Pioneer, 7300 NW 62nd Avenue, P.O. Box 1004, Johnston, Iowa 50131, USAe-mail: [email protected]
E. S. JonesSyngenta Biotechnology, Inc., 3054 East Cornwallis Road,Research Triangle Park, Raleigh, North Carolina 27709–2257
to obtain intellectual property protection (IPP) are important to allow the privatesector to invest in research and product development. Morphological data are cur-rently the criteria by which varieties are judged to meet the criteria for Plant VarietyProtection (PVP); similar data also form an important component of patent filings.Molecular markers that are based upon specific Single Nucleotide Polymorphisms,including those surveyed using whole genome sequence data, now provide the basisfor intellectual property (IP) systems that are more efficient, precise, cost effective,better supportive of IP, and with true potential for greater harmonization. We reporton how such a transition could be undertaken.
It is possible to roughly approximate the amount of genetic improvement that farmersin Syria have been able to achieve during 9,000 years that have elapsed since thedomestication of wheat (Triticum spp.). Araus et al. (2007) report yields of 5.78t/ha for today’s Syrian wheat landraces when grown in irrigated plots and 2.46 t/hawhen grown in rain-fed plots; none of the plots received fertilizer. These authors alsoreport historical yields calculated from archaeological specimens of (1.61 t/ha and1.51 t/ha) (mean 1.56 t/ha). Using these data then annual rates of yield increase dueto genetic gain were from (2.46 − 1.56) = 0.9/9000 t/ha/yr for rain-fed conditions to(5.78 − 1.56) = 4.22/9000 t/ha/yr for well-watered conditions (i.e., 0.1 kg/ha/yr to0.47 kg/ha/yr for rain-fed and for well-watered sites, respectively).
Achieving a more productive agriculture depends with increasing significanceupon the ability to maximize the net positive interactions of genotypic x environ-mental factors that contribute to harvestable yield. Average wheat yields in GreatBritain today are 8.5 t/ha and up to 10–14 t/ha. These yield levels far eclipse thosethat had been achieved in previous centuries and millennia in Great Britain (Fig. 2.1a).It was not until AD 1750 that wheat yields in Great Britain reached levels that hadalready been achieved nearly 8000 years earlier in the middle Euphrates area. Wheatyields in Great Britain rose at a faster rate over the next two centuries predominantlyas a result of changes in crop management practices. In contrast, additional capabil-ities to raise yields by increasing the rate of genetic gain beyond that which couldbe accounted for by mass selection only became possible after the rediscovery ofMendel’s laws of inheritance at the beginning of the 20th century. Garton Brothersdeveloped a succession of new wheat varieties adapted for Great Britain startingwith “White Monarch” in 1899. A significant increase in the rate of yield increase
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Fig. 2.1 a UK wheat yields (tones/ha) from AD 1200–2011 (Austin and Arnold 1989; Ogilvie andFarmer 1997) b US maize yields (tones/ha) from AD 1100–2010. (Smith 1989; FAOSTAT 2011)
30 J. S. C. Smith et al.
occurred during the 1960s due to continued changes in crop management practicescoupled with more effective plant breeding. It is estimated that since 1982, 88 %of the gains in cereal yields of UK winter wheat are now due purely to geneticchange brought by plant breeding and that “national yields could well be in declinein the absence of any variety improvement.” (Mackay et al. 2011). Following thecompletion of the UK 2012 wheat harvest yields, a farm survey estimated to be 14 %below the five year moving average due to the wettest growing season in a century(NFU 2012); consequently, wheat and food prices are expected to rise (Malik 2012).UK wheat yields in 2012 were equivalent to lower mean yields last seen as a resultof good harvests during the late 1980s (NFU 2012). These data help demonstrateadvances in yields that farmers and societies have grown accustomed to, but oftenwithout acknowledgement or any appreciation of their causalities, while once againproviding a reminder of the confounding effects of climate.
As an extreme example, consider instead that the rate of gain in wheat yields inGreat Britain had remained the range of 0.1–0.47 kg/ha/yr, a rate achieved in Syriaduring the millennia following domestication and without the participation of plantbreeders. With those rates of genetic gain it would require from 8,723 to 41,000years to reach just half the present day wheat yields in Great Britain (c. 8.5 t/ha)from their level in AD 1220 (0.3 t/ha) (assuming the other half of yield improvementto then equal today’s yields would come from improved crop and soil managementpractices). Under these circumstances inhabitants of Great Britain could anticipatetoday’s wheat yields would not be reached until the year AD 9943 at the earliest; andby AD 42220 at the latest (and assuming good weather)!
A similar story can be told for the history of maize agriculture in the United States.Maize was initially domesticated near Oaxaca, Mexico, some 8,000 years ago. USmaize yields (Fig. 2.1b) remained stagnant from the Civil War until the advent of hy-brids in the 1930s. Increases in US maize production prior to the 1930s were entirelydue to the taking of more land into agriculture. In contrast, yield changes during thelast eight decades have been due to changes in crop management (improved weedand pest control, fertilizer, mechanization, and increased planting density) with theselection of genotypes that are better adapted to yield in those changed managementconditions (Castleberry et al. 1984; Russell 1984; Duvick 2005). These examples arerepresentative of the broader global picture. Calderini and Slafer (1998) found thatyields achieved soon after the beginning of agriculture were very similar to globalaverage yields that were being attained, some 8,000–10,000 years later at the begin-ning of the 20th century, prior to the large-scale initiation of plant breeding. Thesedata indicate that global demand for cereals had, at least up to nearly one century ago,largely been met by taking more land into cultivation. To continue such an approachis not sustainable (Borlaug and Dowswell 2005). The need to develop new crop va-rieties optimally adapted to a productive agricultural environment can help reducepressures on the environment and maintain biodiversity. Regardless of whether oneespouses the “land sparing” (minimizing demand for farmland by increasing yield)or “land sharing” (wildlife friendly farming boosting densities of wild populationson farmland, which may decrease agricultural yields) (Green et al. 2005) strategiesfor maintaining or increasing biodiversity, it is imperative that crop varieties are bestadapted by virtue of their genetic potential to the habitats in which they are grown
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so as to maximize production in those environments (Green et al. 2005; Godfray2011; Phalan et al. 2011). The same argument for establishing the most appropriatefit of crop genotype with agricultural environment applies whether farming is con-ducted organically or with the aid of chemical pesticides and fertilizers. Indeed, itmay well be that under organic or low input farming conditions, development of thebest adapted crop genotype is especially critical as, in those circumstances, therewill likely need to be yet greater reliance on the use of genetic inputs to help counterpressures from pests, diseases, and weeds.
Sustainable increases in agricultural productivity while safeguarding biodiversitywill require diverse approaches such as biotechnology, and certain elements used in“organic” farming (Lal 2001; Marlander et al. 2003; Ammann 2008, 2009; Brookesand Barfoot 2008; Rudel et al. 2009; Ronald 2011; Raven 2010; Bennett et al. 2011;Godfray 2011; Foley et al. 2011; Phalan et al. 2011). The Royal Society (2009)has called for the “sustainable intensification of global agriculture in which yieldsare increased without the adverse environmental impact and without the cultivationof more land.” DEFRA (2009) estimate wheat yields in Great Britain for 2025 and2050 of 11.4 t/ha and 13.0 t/ha, respectively. “Protecting biodiversity and ensuringfood security are part of a single agenda” (Godfray 2011). And while the furtherintensification of agriculture alone may not ensure environmental sustainability, “itis an essential step in the process because crop and pasture lands comprise aboutone-third of Earth’s ice-free surface” (Rudel et al. 2009).
Future capabilities to further improve agricultural production will continue tobe dependent upon the more effective use of genetic resources to allow farmers toincrease productivity in the face of changes in climates, methods of crop husbandry,and to resist ever-evolving strains of pests and pathogens (Hoisington et al. 1999;Crookston 2006; Qin et al. 2006; Warburton et al. 2006; Glaszmann et al. 2010; Penget al. 2011). Not only will farmers be asked to produce more bountiful harvests underthese circumstances, they will also be expected to contribute to improved nutrition,new products for industry including biofuels, and to contribute as stewards of theenvironment (Foley et al. 2011). Faced with these demands, farmers will increasinglylook to a continual supply of new and better adapted varieties. As other avenues forimproving crop production plateau (e.g., weed and pest control) or genetic meansare found to achieve the same ends, future prospects for continuing to improveproductivity will increasingly rely upon the breeding of better adapted genotypes(Mackay et al. 2011).
2.2 Intellectual Property Protection
It is critical that both public and private resources be optimally invested into researchand product development required for plant breeding. A strong and effective publicsector is vital to provide a global foundation of more basic research, education andfor the conduct of plant breeding, most especially in circumstances where the privatesector is absent or inadequate to meet farmers’needs by region and/or by crop. Manypublic sector institutions seek IPP. The Bayh-DoleAct of 1980 allows US universities
32 J. S. C. Smith et al.
to obtain ownership of an invention in preference to the US government. Universitiesin many countries have established dedicated groups to facilitate obtaining IP andlicensing including in the field of plant breeding and agricultural biotechnology.The John Innes Centre, a research centre with charity status in the UK, states that“our research innovations will often require substantial further investment to reachapplications in the market place and that IP protection has an important role increating favorable conditions for the uptake and use of such research findings” (JIC2012). The Brazilian Agricultural Research Corporation (EMBRAPA) which is verylargely publicly funded uses IPP as do many other publicly funded research centersas a critical element to help them to achieve their goals to increase agriculturalproductivity (Cohen, 2000). In rare cases, centres of the CGIAR can utilize IP whenit can help achieve goals of increased public dissemination of improved varieties ortechnologies (SGRP 2010). The commercial basis of privately funded organizationsmakes the protection of IP mandatory; no commercially funded organisation canbe sustainable if it provides the fruits of its research free to its competitors withoutat least first having had the chance to recoup its investments. Our goal here is toaddress the technical aspects of describing and identifying varieties to help achievean IP environment that can optimally attract private investments into commerciallyfunded plant breeding with the goals to contribute to the increase of genetic gain andsocial welfare (Hayes et al. 2009).
2.2.1 Methods of IPP Used in Plant Breeding
There are at least four ways by which plant breeders can obtain IPP. These are (1)contracts, (2) trade secrets, (3) PVP or Plant Breeders’ Rights (PBR), and (4) UtilityPatents. In addition, the United States provides PVP-type protection for varieties ofasexually reproducing non-tuberous species (The US Plant Patent Act). Trade secretscan help provide protection for parent lines of hybrids. Inbreeding depression, whichoccurs as a result of pollination in fields of F1 hybrids, contributes to encouragingfarmers to purchase new hybrid seed annually. Contracts can include bag-tag “shrink-wrap” type protection including use in closed-loop systems where growers contractto return harvested seed to the owner of the variety. PVP is a sui generis form ofprotection prescribed by diplomatic conferences of the L’Union internationale pourla protection des obtentions végétales (International Union for the Protection of NewVarieties of Plants (UPOV)indexUPOV). Under the auspices of the 1995 Trade-Related Aspects of Intellec-tual Property Rights (TRIPS) within the World Trade Organisation, countries mayexclude plants and animals from patentability. However, any country that does ex-clude plant varieties from patent protection is obliged to provide an effective suigeneris system of plant protection. Patent laws are country specific, for exam-ple France and Germany provide exemptions to allow further breeding includingcommercialization of the non-patented germplasm background in circumstanceswhere specific traits are patented whereas US patent law has no such exemption.
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Table 2.1 Comparison of plant breeders rights and patent systems. (After Krattiger 2004)
Criteria UPOV 1978 UPOV 1991 Utility patents U.S. plant patentact
Protects Varieties of listedspecies
Varieties of allspecies
Plant genotype notnormally found innature
Asexuallyreproducednontuberousplants
Requires NoveltyDistinctnessUniformitystability
NoveltyDistinctnessUniformityStability
NoveltyUtilityNonobviousnessEnablement
NoveltyDistinctnessStability
Disclosure Fullmorphologicaldescription
Full morphologicaldescription
Enabling disclosureBest mode
disclosure depositof novel material
As complete aspossible
Photographs anddrawings
Claims Refer to specificpatents
Single varietalclaim
Exemption Farmer andbreederexemption
Farmer and breederexemption
Some country patentlaws includeexemptions
None
Rights Prevents othersfrom producingfor commercialpurpose
As UPOV 1978 plusprevents importand export andextension toessentiallyderived varieties
Prevent others frommaking, using orselling claimedinvention
Prevent othersfrom asexuallyreproducing,selling, or using
UPOV, Union internationale pour la protection des obtentions végétales (International Union forthe Protection of New Varieties of Plants) (http://www.upov.int/)
Reviews of IP methods are provided by Williams and Weber 1989; Fernandez-Cornejo 2004; Krattiger 2004; Le Buanec 2004; and by CAMBIA (undated) availableat http://www.patentlens.net/daisy/patentlens/1234.html. An outline comparison ofPVP and patent systems (after Krattiger 2004) is presented in Table 2.1.
The International Seed Federation recently completed a revision of its positionon IP (ISF 2012).
2.3 Technical Aspects of Obtaining IPP
Practical enforcement of IPP requires that individual varieties have a legally en-forceable grant of protection. In order to meet this requirement, varieties must becharacterized or described and subsequently be reliably identifiable from other vari-eties, including from among those that are closely related. It is very highly desirablethat variety identification is free from interference by environmental factors that canaffect phenotype, possible at any point in the life-cycle of the plant, be fast (minutesto hours) and relatively inexpensive. In practical terms, these demands can only bemet through the use of molecular marker data.
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Particularly during the last 3–5 years, there have been huge improvements in everyconceivable aspect of molecular marker profiling; the genome can be assayed in veryhigh detail (thousands of SNPs), very quickly (hours), with extremely high fidelity,and with direct output to databases linked to agronomic field data and pedigrees(Mackay TFC 2009; Lai et al. 2010; Yan et al. 2011). Whole genome sequencescans of inbred lines and varieties may soon be cost effective and the norm. Thesemethods are increasingly becoming an integral part of plant breeding to help betterunderstand and thus, to manage the genetic control and expression of importantagronomic traits. Just as modern sequencing technologies are being used to helpcharacterise the genetic basis of crop germplasm, one might also reasonably supposethat a similar source of data would be used to identify and characterise new varietiesfor registration, certification, evaluation of varietal purity, and the granting of IPP.
It must therefore seem unimaginably arcane and probably completely nonsensicalto most readers, when they discover that plant varieties are still primarily evaluatedfor eligibility for varietal status and awarding of IPP upon their morphological ap-pearance. For example, a newly developed genotype will be ineligible as a newvariety (and thus not be eligible for protection under PVP and rejected from use inagriculture where countries require variety registration) if it cannot be shown to bemorphologically distinct from all previously known varieties of that species. Mostall of the morphological characteristics that are used to determine distinctness werepurposely chosen so as not to be associated with agronomically important featuresof a cultivated variety.
Molecular marker data (isozymes and protein storage proteins) were first usedin the plant breeding industry to characterise inbred lines, hybrids and varieties, tomeasure varietal purity and to test pedigrees in the early 1980s. Then, as a result ofrapid, and enduring series of developments in molecular marker technologies UPOVestablished a special working group (Biochemical and Molecular Techniques) toprovide an ongoing review of the capabilities of molecular marker systems in respectof their potential usage to support the goals of UPOV. Areas where UPOV currentlystates that marker data can be employed are: (1) variety identification or validation,but only after making initial description; (2) as a perfect surrogate for an existingmorphological or resistance characteristic, (3) to help manage reference collectionsmore efficiently, and (4) as a means to help determine genetic conformity in respectof determining whether a variety is essentially derived from an initial variety.
To date, neither UPOV nor seed associations have accepted that marker data alonecan be used to help characterise or to describe a new variety de novo. Instead, deter-mination that a genotype meets the criteria established by UPOV to be afforded thestatus of a new variety still depends upon an examination of numerous morphologi-cal characteristics in regard to the criteria of Distinctness, Uniformity, and Stability(DUS). There is widespread acceptance that molecular marker data can be used toidentify varieties once a de novo description has already been made (MMEDV 1999;ESA 2011; Heckenberger et al. 2005a, b, c; ISF 2004a, b, 2005, 2006, 2007a, b,2008, 2009, 2012; Rodrigues et al. 2008; Kahler et al. 2010).
The very rapidity of marker system development during the past 30 years has itselfconfounded the ability of authorities to approve an internationally agreed standard-ized system. Moreover, each marker system has had some drawbacks which have
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undermined the desire to advocate, at least until now, for a change from the use ofmorphological characteristics to the use of molecular data. For example, RestrictionFragment Length Polymorphisms (RFLPs) first made a hundred or more molecularmarkers available per species with the ability to survey markers at known map posi-tions collectively sampling each chromosome arm. However, RFLPS were heavilyresource demanding and relatively slow to use. Simple Sequence Repeats (SSRs)provided a significant step forward in throughput with increased reliability of scor-ing. However, SSRs scored on one platform could not always readily be aligned withthose scored on a different platform and changes in the design of PCR primers hadto be very carefully monitored. More recently, Single Nucleotide Polymorphisms(SNPs) have become the marker system of choice due to their compatibility withultra-high throughput information, chemistry and robotic laboratory systems and tohave access to tens of thousands of markers collectively providing a very high powerof discrimination with very high repeatability. Another huge advantage of SNPs isthat, because they are sequence based, the same designated loci can still be scoredeven as DNA platform technologies continue to change and including when wholegenome sequence data are collected. SNPs therefore provide a culmination of themost efficient, cost effective, discriminatory and enduring marker data that can beused to characterise cultivated varieties of crop species.
Factors such as methylation, which are beyond the realm of characterisation orprediction of simply inherited Mendelian traits, including molecular markers, playas yet an imperfectly understood but important role in the expression of phenotype(Martienssen and Colot 2001). In addition, there is huge underlying complexity ofinteractive regulatory networks which condition phenotype (Kaufmann et al. 2010).These findings might suggest that an approach using molecular markers to charac-terize and to distinguish among plant varieties both qualitatively and quantitativelyis naıve, if not redundant. We would respond that whether the goals are to evaluateDUS or to identify or compare varieties then the source of data should be selectedthat most effectively and efficiently allows the relevant criteria to be determined. Atleast in our experience, it is clear that the continued use of a set of morphologicalattributes is neither optimal nor practical to most effectively help provide for andsupport IP for varieties of major field crop species.
2.3.1 Concerns About the Use of Molecular Markers to DescribeVarieties de novo
Primary concerns about the use of molecular marker data as evidence upon which todetermine varietal status de novo relate to (1) the ability to find numerous additionaland potentially additional discriminatory data due to even a very low level of residualheterogeneity (and so potentially undermine the protection vested in the initiallydeclared variety); (2) additional costs that would be born by breeders having to runmarker assays during the breeding process to also assure that varieties are uniform,and thus stable, for the same characteristics that are used to establish distinctness; (3)
36 J. S. C. Smith et al.
concerns that an unduly high level of uniformity would be required; and (4) the needto establish standardized protocols and to demonstrate that laboratories around theglobe can generate data of sufficient quality and consistency. Some have additionalconcerns which relate to the legal definition of how a variety is described “by theexpression of its characteristics” and who consequently argue that since molecularmarker data are not expressed, at least in respect of being the result of transcriptionof the genome, then they should be excluded from eligibility for this usage.
2.3.2 Concerns About the Use of Phenotypic Characteristicsto Describe Varieties de novo
Concerns about the continued use of phenotypic characteristics stem, in very largepart, from the large contribution that genotype x environmental interaction and exper-imental error play in undermining the precision, and thus the discriminative power,of descriptions based upon morphological characteristics. Morphological data wereselected as the characteristics to provide the basis for determining varietal statusbecause prior to the mid 1970s no other means had been conceived or developed forany crop species. UPOV understood the problems that genotype by environmental(G×E) interaction, a feature inherently associated with morphological expression,would contribute by undermining the precision of varietal descriptions and thus mak-ing harmonization a particularly difficult proposition. UPOV attempted to mitigatethese problems by categorizing morphological characteristics according to the pre-sumed level of complexity of their genetic control; characteristics believed to beunder simpler genetic control would be less likely to be influenced by interactionwith the environment. However, more recent studies of the genetic control for manyof these characteristics (that are only now possible due to the availability of numer-ous molecular markers) indicate that the genetic control of many, if not most, ofthese characteristics is quite complex ensuring that their expression is quantitative innature (Sourdille et al. 1996; Austin et al. 2001; Bredemeijer et al. 2002; Mickelsonet al. 2002; Enoki et al. 2006; Li et al. 2007; Tian et al. 2011). For example, Tianet al. (2011) have shown that in maize 30–36 quantitative trait loci (QTLs) are as-sociated with just three leaf characteristics (upper leaf angle, leaf length, and leafwidth). And, for example, even when identical protocols have been used to collectmorphological description data for the same genotypes in different locations, theneven those data can be quite dissimilar (Jones et al. 2003; Hof and Reid 2008).
Due in large part to the effects of G×E, significant dedicated outlays of personneland field resources are required to obtain morphological data that are statisticallymeaningful for DUS purposes. These issues become exasperated as the numbers ofknown varieties annually increases, as is the case for most major field crop species,thereby rendering it practically impossible to directly compare a prospective newvariety with all previously declared and known varieties. Consequently, additionalstrategic approaches have had to be developed in an attempt to fulfill the UPOVrequirement to compare new potential varieties with varieties of common knowledgein that species. These approaches seek to use molecular marker data or a combination
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of marker and morphological data to exclude from the need for further comparisonsvarieties that are super-distinct from a candidate variety. As a result, a short-list ofexisting varieties that would then need to be more closely examined to each candidatevariety using morphological characteristics is created. Such a pre-screening exercisecan help significantly reduce the amount of field testing that is required in a secondand final year of field evaluations. It is also a requirement that a new candidatevariety is compared to the most similar variety and the distinguishing characteristicsidentified. Countries or regions that utilize a centralized approach to generating andcomparing morphological data can make such comparisons using the database of allpreviously known varieties of that species available to them. However, in countries orregions that require individual breeders to be the source of morphological data (e.g.,the U.S.), then breeders can only report the closest variety comparison in respectof the varieties for which morphological data are present in their own database. Itis not realistic to expect that every breeding company should create and maintain amorphological database of all varieties of common knowledge. Indeed, with regardto inbred lines of a hybrid crop that are maintained as trade secrets then only theinbreds that are publicly known or which are proprietary to that individual breederwill be available to that breeder. Consequently, there is a lack of consistency in howthe most similar comparison variety is selected. In addition, the collection of data byindividual applicants can lead to a variety of specific protocols being developed forthe collection of the required morphological traits. In addition, large G×E effectsassociated with the expression of morphological characteristics serve to inflate vari-ance, which then diminishes the ability to discriminate between varieties. Finally,there are significant differences, for historical reasons, between the manner in whichmorphological data are collected, databased and compared across countries, mostsignificantly with regard to the U.S. compared to other UPOV member countries. TheU.S. requires actual metrical data with standard errors. In contrast, UPOV requiresdata be translated or normalized to scores. In our experience, while both methods areeffective at evaluating distinctness, it is impossible to meaningfully merge the datafrom different systems of measurement, even after employing a variety of approachesto adjust or normalize the data so that “it speaks the same language” as the othersystem (Law et al. 2011a, b, c).
2.4 Improving the DUS process: The rationale for Changeto the Use of Molecular Characteristics
Some reference collections of many crop varieties, especially with regard to themajor field crops, have already become very large therefore making it practicallyimpossible to satisfy the requirement that new varietal candidates should be com-pared to all other varieties of common knowledge in that species, at least usingmorphological data. It is not surprising, therefore, that there is growing interest inthe use of molecular markers to reduce workloads and possibly also the costs forPVP offices to manage reference collections. In that context, it would be logical todiscuss whether the use of such a tool could reasonably be extended to all aspects of
38 J. S. C. Smith et al.
DUS testing. The use of molecular markers has the potential to significantly reduceerror that is effectively caused by genotype × environment interaction and whicheffectively reduces the precision of a description that can be realized using mor-phological data. Marker data can also provide a more standardized approach to thedefinition of distinctness because the genetic control and map positions of markerloci can be understood. Marker data also allow implementation of the requirementthat new putative varieties should be compared to all publicly known varieties of thespecies. And, as already noted, there are considerable differences in the technicalimplementation of criteria to measure, record, and compare morphological descrip-tions. Selecting a standard set of molecular markers on a crop by crop basis has thepotential to achieve a significantly higher level of harmonization and a more unifiedapproach among national authorities; an aspiration that UPOV ranks highly.
2.4.1 Criteria Required for the Development of StandardizedProcedures for DUS
For variety testing on the basis of D, U and S for plant variety rights or for nationallisting it would seem reasonable that the process could be carried out entirely withdata provided by molecular markers, provided that the following basic tenets aremet:
• UPOV wide consensus on the use of DNA-based markers in the DUS examina-tion process in order to obtain international acceptance of the DUS examinationreports. UPOV members would need to agree on a harmonized approach be-fore implementing the use of DNA-based markers in the different national DUSexamination processes. A transitional period may be required.
• There can be no risk of decreasing the minimum distance, necessary for thedeclaration of distinctness. A standard set of markers would need to be describedwith thresholds for distinctness such that it would be impossible, in the extremecase, to declare Distinctness on the basis of only one base pair difference in thewhole genome.
• Implementation of a marker based system for DUS assessment might not neces-sarily remove the requirement for the breeder to achieve Uniformity and Stabilityof morphological characteristics that are important to the farmer, grower or seedcertification authorities. At the same time, the introduction of the use of molecularmarkers should not generate any additional practical constraints for the breeders,e.g., in the field of variety description.
• Implementation of a marker based system for DUS assessment would require acrop by crop approach.
Such implementation would require:
• The use of a set of markers able to provide the highest discrimination capacity(polymorphism information) and genome coverage. It will be necessary to evenlysample the genome.
2 Genomic Approaches and Intellectual Property Protection for Variety Release 39
• The DNA-based markers, the methods to produce and record the data and carryout statistical computations need to be publicly available.
• When used to determine Distinctness, then either the same full set or a subsetof these DNA-based markers also need to be used for assessing Uniformity andStability.
• Evidence shows that there is intra-variety variation for DNA-based markerswithin existing varieties that have been declared sufficiently uniform and sta-ble. Therefore tolerance levels for marker uniformity and stability would need tobe established on a crop by crop basis.
• There can be no significant increase of the application or examination costs dueto such implementation.
2.4.2 Evaluation of SNPs and Development of StandardizedProcedures for DUS, EDV, and Variety Identificationin Maize
Many publications show the advantages afforded by the use of SNPs; high-throughput, high map resolution, and high repeatability (very low error rates)(Tenaillon et al. 2001; Bhattramakki et al. 2002; Ching et al. 2002; Vroh Bi et al.2006; Jones et al. 2007). Jones et al. (2007) reported data repeatability of, from98.1 to 99.3 % depending upon platform technology. And while SNPs show, on av-erage, less polymorphism on an individual locus basis due to their bi-allelic nature(compared to SSR loci which are usually polyallelic in populations), this potentiallimitation can be countered either by (i) selecting SNPs that have relatively highdiscrimination ability (a high Polymorphic Index Content or PIC) or by (ii) assem-bling individual linked or adjacent SNP loci into haplotypes and then reporting thoseindividual haplotypes out as individual alleles of a polyallelic system. Some havereported that from 7–11 times the number of SNP markers are required to providean equivalent degree of discrimination as SSRs (Laval et al. 2002; Yu et al. 2009;Van Inghelandt et al. 2010). In contrast, Nelson et al. (2011) found that, “at leastwhen the issue is to examine genetic similarities among moderately or closely relatedgermplasm, then the appropriate number of SNP loci need may be in the range of300–400,. . . provided they are selected on the basis of maintaining relatively highHe (PIC) as well as even genome coverage”.
The American Seed Trade Association (ASTA) and French Maize Breeders As-sociation (UFS) have jointly embarked on a project using an Illumina 56,000 SNPchip to profile a set of maize inbred lines including those of historic and currentimportance. The results of this study will provide a list of thousands of publiclyavailable SNP loci from which can be selected a set to measure genetic similarities(or distances) between pairs of inbred lines for the purpose of resolving questionsin regard to status as an Essentially Derived Variety. Such a set of SNPs could alsobe used for determining Distinctness. A much smaller subset of SNP loci could alsobe selected that would be used to evaluate uniformity and stability. The results and
40 J. S. C. Smith et al.
conclusions of this research will be presented to the International Seed Federation(ISF) and consequently could become promulgated by the ISF as recommended pro-cedures. Similar strategic approaches to select SNP sets and subsets could be usedfor maize and for other crops in various regions of the world. It will be importantto select sets and subsets of SNPS based upon their proven ability to discriminateamong germplasm that is relevant for each region and thus to avoid ascertainmentbias in the selection of the SNPs.
It will be important to establish a minimum level of % SNP profile similarityas a threshold for Distinctness. Such a threshold must be based upon use of a setof SNPS that are identified as meeting the criteria of (1) collectively providing forfairly even genome coverage and (2) having a proven ability to discriminate amongvarieties, even among those that are very closely related by pedigree. Otherwise, itcould always be potentially possible to find at least one (of several million SNPs)that would have a different base, or be polymorphic for the presence or absenceof several SNPs in one or more genetic regions (Fu and Dooner 2002; Wang andDooner 2006). One could consider using two approaches, or an amalgam of the two:First, given repeatability estimates of from 98.1–99.3 % that have been reported todate for the use of SNP technology in maize, then a value of 98–99 % similaritycould be an appropriate minimum boundary for determining distinctness. Second, itis also possible to examine SNP similarities among pairs of inbred lines or varietiesthat have already been declared as distinct varieties on the basis of morphologicalcomparisons. Such comparisons provide an opportunity to declare a distinctnessthreshold using SNPs that is calibrated using existing varieties and according topreviously agreed morphologically based standards used to declare distinctness; i.e.to calibrate a SNP threshold in relation to customary standards using morphologicaldata. SNP data presented by Nelson et al. (2008) for a set of U.S. maize inbredlines for which PVP certificates have now expired (and including several importantwidely-used publicly bred lines) provide some interesting comparisons. All of theinbreds reported by Nelson et al. (2008) have been declared distinct on the basis ofmorphological comparisons conducted by the US PVP Office. Variation was observedamong different sources of the same inbred line ranging from 96.3 % (for Mo17) to99.7 % similar (for three replicate Pioneer bred inbred lines). The most similar pair ofinbred lines was for B73 compared to the inbred F42 (approximately 99 % similar).Several other pairs of inbred lines also had very similar SNP profiles: B73–DJ7(96.6 %), PH207–Q381 (approx. 98 %), Mo17–Seagull–17 (approximately 97 %),LH51–Mo17 (92.9 %), B73–NKW8304 88.8 %). These data raise cautionary issuesconcerning the prospect of introducing marker data into the PVP system that willneed to be taken into consideration. First, some of these pairs of different inbredsare so similar by marker data that the equivalent marker threshold for distinctnesswould have to be impractically high and overlap with typical levels of laboratoryerror. In these cases a re-examination of the criteria and evidence used to determinedistinctness may be warranted. Second, marker-based distances between differentseed sources of Mo17 are so high that, if they are to be considered in the developmentof uniformity thresholds using SNPs then this may lead to an unwarranted decrease inthe standards used to declare distinctness. The finding of high levels of heterogeneity
2 Genomic Approaches and Intellectual Property Protection for Variety Release 41
Fig. 2.2 The number of inbreds from a reference set of 438 which have 15/15 matching isozymeprofiles or 16/16 matching SNP profiles in relation to the number of missing isozyme or SNP scores.SNPs maintain a much higher power of discrimination even in the face of 50 % missing data
among different seed lots of older inbred lines comes as no surprise because mostof these lines were not thoroughly selfed to uniformity before public release. Inbredlines of maize are currently bred to higher standards of uniformity.
It is an important principal that either the same or a subset of characteristics, whichare used as the basis for determining distinctness, are also used to assess uniformity,and thus stability. Otherwise, it would be possible to select inbred lines or varietiesthat meet the threshold of distinctness solely by purification of a heterogeneous seedlot. It is well known that seed lots of inbred lines that have not been selected foruniformity using marker data can still be heterogeneous (Mauria et al. 2000; Mauriaet al. 2002; Nelson et al. 2008). Consequently, it will be necessary to develop apanel of SNP loci which can be used cost-effectively during the breeding process,and subsequently also in determining genetic purity of inbred and hybrid seed lot. Itis increasingly routine breeding practice in maize to use double-haploids for inbredline development, consequently an increasing proportion of inbred lines and varietieswill be completely homozygous and will remain so unless there is contaminationor physical mixing during subsequent generations of seed increase or hybrid seedproduction.
We have found that providing SNP loci are judiciously selected, it is possible toidentify a relatively small set of SNP loci that collectively have a very high powerof discrimination among maize inbred lines, and which therefore could be usedto measure uniformity and stability (genetic purity) e.g., as few as 16 SNPs candiscriminate among > 400 Pioneer proprietary inbreds. A set of 16 SNPs can give16 times the level of discrimination compared with the standard set of 15 isozymesthat have historically been used at Pioneer to assess uniformity, and SNPs are farmore robust in the face of missing or erroneous data (Fig. 2.2). In addition, whereinbreds cannot be discerned using sub-sets of the 16 SNP markers, then those inbredsare likely to be highly related. In contrast, some of the inbreds that could not bedistinguished by isozyme data were unrelated by pedigree. Consequently, a small
42 J. S. C. Smith et al.
panel of SNP markers can therefore be cost-effective for routine use during thebreeding process, and can provide, not only an initial test of the level of uniquenessof an inbred, but also an evaluation of uniformity and stability, and in subsequentstages of inbred increase and hybrid seed production, an evaluation of genetic purity.
Determination of varietal status de novo according to the criteria of DUS is cur-rently considered as different (and indeed a necessary prior determination) from anysubsequent determination of essential derivation. Nonetheless, there exists the po-tential to greatly increase the efficiency of the EDV process; and ultimately thereforeto improve the level of IP that is afforded to initial varieties. Quite simply, it shouldbe possible to use the same set of SNPs that would be used as an initial step in thedetermination of Distinctness to also be the same set of markers that is used to helpEssential Derivation. Individual PVP Offices could publish SNP profiles of all dis-tinct varieties and thus individual breeders could utilize those data to also help obtainan initial determination of potential EDV status. There would then be no need forindividual breeders to repeat the generation of these data and a current challenge inthe initial determination of potential EDV status, not having access to the pertinentSNP profile of a proprietary inbred line bred by another breeder, would be removed.
2.5 Conclusions
Plant breeders who are employed by well-resourced agencies or companies increas-ingly have at their disposal technological capabilities to more effectively sourceuseful genetics from a much broader base of diverse germplasm than was available,either to those who invented agriculture, or to generations of farmers or previous gen-erations of plant breeders who have provided stewardship and gradually improvedthe performance of crop varieties. Whether plant breeders will actually use thosegenetic resources will depend upon the level of innovative research and productdevelopment that they can bring to bear in their breeding programs. The range ofavailable IPP influences the range and type of research and product developmentthat can be accomplished, at least by a private commercially funded organization.To have accessible a full range of choice in the level or type of IPP that is availableallows breeders in turn a full range of choice to determine the level of innovativeness,risk taking, level and term of research investments that can be a sustainable businessproposition for their enterprise. Exclusions in the level and type of IPP will lead toexclusions in the amount of innovation and risk taking that breeders will be able toexercise in the research and product development programs. Lowering the availableceiling of IPP will limit advances in productivity and in the absence of publiclyfunded support, reduce, or at least slow, access to a broader base of genetic diversityin breeding and in agriculture.
SNP data can be used to characterise genetic resources and they can also be usedto establish and to validate the varietal status of new plant varieties. Using a SNPbased system to characterise varieties it will be possible to make comparisons to allpreviously recognized varieties without the need to manage reference collections in
2 Genomic Approaches and Intellectual Property Protection for Variety Release 43
the field. There are opportunities for true harmonization of data and databases on aglobal basis by escaping the constraints that inevitably emanate due to the large GxEinteraction effects associated with morphological characteristics. Most importantly,there are opportunities in the judicious use of sequence data to improve the level andefficiency by which IP is afforded through the PVP process.
The means to characterize and to determine eligibility of newly developed geno-types for varietal status and use in agriculture will one day finally catch up withthe methods breeders use to help create those new genotypes. Such a developmentshould result in a more efficient IP system, one that is simpler and more effectiveto police. The overall goals should be to help provide a business environment thatwill allow a greater breadth of genetic diversity to be surveyed with ever-increasingeffectiveness and so to continually improve abilities to select new genotypes that areoptimally able to perform in target agricultural environments. An appreciation of theurgency to radically and quickly, yet sustainably improve agricultural productivitycan be comprehended by thinking back once again to the dawn of agriculture itself.As quoted by Clive James, the founder of the International Service of the Acquisitionof Agri-biotech Applications, it has been estimated that “in the next 50 years, theglobal population will consume twice as much food as has ever been consumed sinceagriculture began 10,000 years ago.”(Arabic Knowledge @ Wharton 2012; Hoising-ton et al.1999). Nearly one-third of this 50 year forecast time span has already beenspent.
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