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GeneMax Services, 85410 Dudley, Chapel Hill, NC 27517
* Corresponding author (rita.mumm{at}genemaxservices.com).
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONFactors Affecting the Choice...Summary of the Implications...REFERENCES |
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ACKNOWLEDGMENTS |
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Received for publication April 7, 2007.
GeneMax Services, 85410 Dudley, Chapel Hill, NC 27517
* Corresponding author (rita.mumm{at}genemaxservices.com).
Key factors affecting the choice of breeding methods employed in the development of transgenic maize (Zea mays L.) hybrids are identified and evaluated, particularly as these pertain to the use and balance of backcross and forward breeding in the overall design of a breeding program. These factors are type of trait, population(s) under selection, the predicted response to selection, stewardship of transgenic events, costs, and risks. Analysis suggested that simultaneous hybrid improvement and event integration via a forward breeding approach is not practical, given that some level of backcrossing is basic to event integration and the need to contain events not fully authorized by the local government for cultivation, food, and feed. Considering event integration conducted in a stream separate from hybrid improvement, a forward breeding component to event integration may be warranted, particularly if a threshold level of expression of the transgenic trait of interest is dependent on endogenous alleles that interact with the event, some of which may be influenced by the environment. For this situation, directives are given for determining the number of generations of backcrossing to be conducted to create partial conversions from which to select for favorable endogenous alleles via forward breeding. In addition, ways to maximize the advantages of a backcross-only approach to event integration are discussed.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONFactors Affecting the Choice...Summary of the Implications...REFERENCES |
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This work aims to identify the key factors affecting the choice of breeding methods employed in the development of transgenic maize (Zea mays L.) hybrids, particularly as these pertain to the use and balance of backcross and forward breeding methods in the overall design of a breeding program. Backcross breeding involves repeated crossing with selection to an elite inbred, with the goal of recovering a derived line that essentially resembles this elite parent with the addition of one or a small number of favorable alleles from the nonrecurrent parent. Forward breeding refers to any system of inbred line development, irrespective of the number of loci involved or the balance of favorable alleles among the parents of the population, involving the creation of a source population followed by inbreeding with selection, with the goal of recovering an improved line for one or more traits (e.g., pedigree selection).
The scope of this work is defined in terms of a framework for an overall breeding program and a set of options for the design of such a program. A breeding program focused on the development of transgenic maize hybrids aims to combine key agronomic and quality characteristics with the most advantageous transgenic traits to create top-performing hybrids that meet customer needs at a local level. The design of such a program centers on the cornerstone relationship between hybrid improvement (which includes new line development) and event integration.
Options for program structure include (i) utilizing a forward breeding method in new line development aimed at both hybrid improvement and event integration; (ii) conducting hybrid improvement independent of event integration and utilizing backcross breeding to introduce events to elite parents of candidate hybrids; and (iii) implementing a balance between forward and backcross breeding for event integration, conducting the main thrust of hybrid improvement in a stream separate from a program aimed at introgressing events into elite parents of candidate hybrids, with final selection of elite lines after partial conversion.
Events targeted for integration may include the following types: (i) events authorized by the local government for cultivation, food, and feed; (ii) events that are not yet fully authorized by the local government but will be once these clear assessment for environmental and food safety; and (iii) events that are not authorized and never will become authorized, because in the course of trait development and event selection, these events will be discontinued as commercial candidates in the process of selecting the best event for commercial introduction. Events without full authorization are subject to containment guidelines and precautions must be taken to prevent their release into the food and feed chain.
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Factors Affecting the Choice of Breeding Method |
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TOPABSTRACTINTRODUCTIONFactors Affecting the Choice...Summary of the Implications...REFERENCES |
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Type of Trait
Through transformation, one or more genes are introduced to a targeted host species, become incorporated in the genome, and then function as fully heritable genetic factors. These genes encode for proteins (or in the case of antisense genes, lead to RNA transcripts) which produce a specific biochemical effect. In turn, this biochemical effect results in a specific phenotype typically referred to as the "transgenic trait." An event is a specific source of a given transgenic trait that is uniquely defined in terms of the actual DNA sequence that has been incorporated into the target genome via transformation as well as the chromosomal location(s) of the DNA insertion(s).
Transgenic traits represent novel traits or new levels of expression of common traits. The mechanisms involved reflect genetic engineering (e.g., high, sustained expression levels due to the promoter and enhancer sequences used in construction of the transformation vector). Novel traits may be due to one or more exogenous genes.
An example of a transgenic trait in maize is resistance to Lepidopteran insect pests (see Table 1 for a list of events commercialized to date in the United States that confer Lepidopteran resistance).
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Initial transgenic traits in corn have focused on input traits (i.e., traits of primary utility or value to the grower), mainly insect resistance and herbicide tolerance (see http://www.aphis.usda.gov/brs/not_reg.html for a list of petitions of nonregulated status granted or pending by USDA/APHIS for further information. Note that approval by APHIS represents one step toward obtaining full authorization for commercialization in the United States). Event selection during transgenic trait development has been heavily biased in favor of the ideal event profile. The next wave of transgenic traits includes grain quality or composition, stress tolerance, and yield enhancement (USDA, 2006), bringing the potential for events to interact with endogenous genes associated with metabolic pathways influencing phenotypic expression of quantitative traits (see Mackay, 2001, for an overview of interaction effects among genes affecting quantitative traits).
The degree of dominance dictates the number of copies of the event required in the commercial hybrid, with partial dominance or recessiveness potentially necessitating a broader breeding effort for event integration to bring in the event from both sides of the pedigree. At the extreme wherein all hybrids are targeted for a transgenic trait lacking complete dominance, comprehensive penetration of the breeding program with a given event could favor forward breeding aimed at simultaneous hybrid improvement and event integration. Events requiring additional support to achieve a given threshold for the trait of interest may call for pyramiding with endogenous genes to achieve commercial levels of trait expression. Integration of multiple factors to achieve trait expression would tend to favor backcross breeding. Epistatic gene action involving the event and other genes influencing trait expression may require selection at loci other than the event to ensure performance at or above a given threshold for the trait of interest. Epistasis could favor forward breeding, depending on the number of loci involved, the magnitude of the epistatic effects, the consistency of effects across germplasm, and the impact of the environment on interactive loci.
One concern with transgenic traits that represent a new level of expression of a common trait (rather than a novel trait) is that, with new line development aimed at simultaneous hybrid improvement and event integration, endogenous alleles associated with the common trait may be lost. For example, in developing new lines from a source population containing one of the events for Lepidopteran resistance listed in Table 1, endogenous alleles for resistance to first brood European corn borer [Ostrinia nubilalis (Hübner)] could be lost, especially since the event acts in a dominant fashion and the size of the effect could mask effects of other genetic factors. From the opposite perspective, the question arises of whether a new line developed in isolation of a transgenic trait with which it is likely to be coupled or have integrated is being selected under appropriate conditions. One option to deal with both issues involves the use of transgenic testers in new line development. In a breeding program where event integration is conducted independently of hybrid improvement, a nontransgenic line in development could be testcrossed to at least one inbred carrying events of broad usage (particularly authorized events) to create seed for performance trials. This would allow for evaluation of the line in pertinent transgenic hybrid combinations as well as facilitate line per se selection for similar traits that may impact production performance.
The current trend is to "stack" or bundle events in hybrids to create valued combinations of transgenic traits. This can be accomplished through breeding or molecular stacking. Breeding facilitates a mix-and-match approach to event combinations. Strategies devised to minimize the presence of linked chromosomal segments carrying unfavorable alleles from the event-donor source in the backcrossing process (see Frisch, 2005; Servin et al., 2004; Ribaut et al., 2002; Frisch and Melchinger, 2001; Hospital, 2001) can be useful for effectively pyramiding the desired events. A number of studies have demonstrated the feasibility of incorporating up to five genetic factors via backcrossing (Ribaut et al., 1999, 2002; Reddy and Comstock, 1976). Stacking through breeding would tend to favor backcrossing, particularly as the number of desired events increases and approaches five.
Molecular approaches to stacking through transformation aim to create a single insertion site containing various genes of interest, producing a single genetic unit for multiple trait expression. Molecular approaches to stacking offer advantages in event registration with predetermined combinations and can facilitate considerable cost savings in event integration, especially as transgenic lines are advanced through selfing stages to homozygosity. Although molecular stacking places some restrictions on event combinations and requires accurate long-term market forecasting to be successful, it offers more flexibility in choice of breeding methods because the bundle of transgenic traits can be manipulated as a single unit.
Population(s) under Selection
Government safety assessments and authorizations required for commercial release of transgenics are made on an event-by-event basis. The cost of developing and obtaining full authorization to commercialize a transgenic event (
US$100 million; Monsanto Company, 2005) results in a limited number of sources of a transgenic trait of interest in the marketplace. Typically, a biotech provider will target one or a few sources of a transgenic trait for commercialization, although there may be competing sources developed by different biotech providers. A "first generation" transgenic trait may be followed or replaced in the marketplace by an improved version of the particular trait (referred to as a "follow-on" trait). An example is the commercial launch of an event in 2002 that confers resistance to Lepidopteran insect pests of corn through the Cry1F Bt gene (Table 1), which extends activity to black cutworm [Agrotis ipsilon (Hufnagel)] and increases activity against fall armyworm [Spodoptera frugiperda (Smith)] over that available with Cry1A events introduced in the 1990s (Gianessi et al., 2002). Thus, a breeding program developing high-performance transgenic hybrids could be dealing with several sources of a particular type of transgenic trait.
On the other hand, because each event is uniquely defined in terms of the actual DNA sequence that has been integrated within the target genome via transformation as well as the chromosomal location(s) of the DNA insertion(s), each event originates as an individual line. Early events were created using nonelite germplasm (e.g., Hi-II which was derived from B73 x A188; Armstrong et al., 1991). The ability to transform and regenerate elite inbreds has increased, which may tend to reduce the emphasis on eliminating event-donor genomic contributions during event integration. Nonetheless, the initial donor source for starting event integration with a particular event limits broad exploitation of genes other than the event of interest in that donor due to the need to maximize each authorized event. Even if the event source is considered elite, expansive use of a particular line in hybrid improvement would drastically reduce the genetic diversity within the program overall.
This highlights the dynamic nature of trait source in a breeding program, with a number of events for a particular trait potentially accessible and possibly changing over time as follow-on traits are developed, coupled with a limited genetic base with each. The proportion of hybrids to feature the transgenic trait(s) and the degree of flexibility demanded in the breeding program (e.g., to access events from either side of the pedigree) will play into the choice of breeding method. If various transgenic versions of inbreds or hybrids are desired, backcross breeding is favored. A newly developed, improved inbred or hybrid can be converted in different backcross streams to several transgenic traits and even events within a trait. Events that become discontinued in development can be easily eliminated from the breeding program without jeopardizing new line development in hybrid improvement. However, with a forward breeding approach aimed at simultaneous hybrid improvement and event integration, it may be more difficult to expunge an event after it is introduced to a source population developed for new line development. For example, USDA/APHIS considers that all progeny derived from an unauthorized event are regulated, including nulls for the event, until determined otherwise through petitioning to USDA for deregulated status and providing the supporting molecular evidence that demonstrates that event expression is absent and that the locus representing the insertion site in the event of interest is not altered in the null segregant when compared to the regulated parent (APHIS Biotechnology Regulatory Services [BRS], oral and written communication, August 2006).
Of course, use of forward breeding assumes the presence of favorable alleles at loci other than the event of interest in the event-donor that could be recovered and that would contribute advantage in a breeding program. There is limited genetic diversity at least initially with a new event source, as discussed above, affecting the overall utility of the event-donor beyond providing a source of the event. Backcrossing could be utilized to introgress the event into multiple, diverse elite lines, which then could be used to produce breeding starts for the derivation of new lines, but that would take some time. With respect to any given cross, the probability of recovering lines with more favorable alleles than the best parent is based on several factors including the balance of favorable alleles between the event-donor and the other parent, the genetic variation present among the progeny, the number of loci involved, heritability of the trait, selection intensity, and the ability to identify superior progeny (Ribaut et al., 2002; Bailey, 1977). Use of a relatively nonelite event-donor favors backcross breeding. A regime consisting mainly of backcrossing does not preclude selection among versions of a converted line since lines derived from even several generations of backcrossing to an elite parent theoretically do not represent 100% recovery of the recurrent parent germplasm, and thus, may be considered partial conversions.
Predicted Response to Selection
With either forward or backcross breeding, selection is key to maintaining the event of interest throughout successive generations of inbreeding to ensure a high probability that improved homozygous lines derived from a population created using the event-donor as a parent will carry the transgenic trait. With backcrossing, for example, overall heterozygosity is halved each generation as loci become fixed for one parental allele or the other at unselected loci. However, with selection, favorable alleles contributed from the donor parent exerting large effects on the trait are more likely to be maintained in a heterozygous state (as are alleles at linked loci, whether favorable or unfavorable). The probability that a favorable allele from the donor parent will remain segregating is a function of the magnitude of its effect on the trait, the intensity of selection, and the number of generations of backcrossing (Hill, 1998). Selection for an event per se is generally easy, based on the size of the effect and the use of screening tools. These may detect the portions of the DNA sequence inserted through transformation (or DNA flanking the insertion site) or the presence of a unique protein produced by the event of interest. Additionally, some events can be selected through phenotypic screening for expression of genes characteristic of the event, such as herbicide tolerance. If adequate expression of the transgenic trait requires the presence of endogenous alleles in addition to the event of interest, these too could potentially be recovered from the event-donor. However, just as for the event, selection for these alleles must be conducted through successive generations of inbreeding to fixation. If selection is based strictly on phenotype, field testing must be conducted to effectively screen for the trait. For traits such as stress tolerance which can be complicated to screen, such testing may be difficult and costly. With a transgenic trait for increased yield or yield stability, field testing would be required at numerous locations which may be challenging, particularly with yet-unauthorized events that must be isolated from other materials to prevent pollen escape.
Use of genomic technologies such as molecular markers can significantly influence response to selection and enable solutions to issues like linkage drag (i.e., unfavorable alleles linked to the event of interest in the event-donor at hand) (Frisch, 2005; Hospital, 2001). Gene expression technologies can be utilized to analyze gene function and the molecular mechanisms underlying trait expression (Eisen and Brown, 1999). Such technologies could potentially be directed to facilitate selection for endogenous alleles that support transgenic trait expression (Seki et al., 2002; Bernardo, 2002; Johnson and Mumm, 1996; Dudley, 1993), including alleles involved in epistatic interactions with events.
Speed is an important feature of response to selection and factors prominently into the development of transgenic hybrids because speed to market with a new transgenic trait may provide the opportunity to set the standard for the trait of interest and to garner strength as the market leader. Speed can favor backcross breeding, which facilitates cycling at three to four generations per year in continuous nurseries or greenhouses without overall lag in development of improved hybrids (Mumm, 1996).
Stewardship of Transgenic Events
Consider the following categories of plant materials in a breeding program focused on the development of transgenic maize hybrids: (i) those containing events unauthorized by the local government, (ii) those containing events authorized by the local government, and (iii) nontransgenic. Government regulations pertaining to the way yet-unauthorized transgenic events are grown, handled, and shipped typically set these plant materials apart from other materials in the ways these are managed. Furthermore, even for events that have received authorization by the local government, plant materials may need to be managed differently than nontransgenic materials, particularly if the breeding program engages in germplasm exchange internationally. Events approved for cultivation, food, or feed in one country may not have these authorizations in other countries. Effective containment requires control of pollen flow, volunteer plants, seed mixing, and human error leading to mislabeling of seed or plant materials, which may make it impractical to have programs for these categories of materials integrated or even side by side. (Panetta, 2005; Mumm and Walters, 2001).
Another aspect to consider is trait life cycle. As improved transgenic traits are developed, events commercialized earlier may become obsolete. Some event authorizations may reach expiration and, if not extended, there will be the need to discard all seed materials of a particular event at some point in time, further emphasizing the need for some segregation of materials.
Costs and Risks
Clearly, costs factor into the choice of a breeding program. A cost–benefit analysis is the best means to determine the net return of research investments with a given breeding method and to compare relative cost-effectiveness among breeding methods. As pointed out by Hoisington and Melchinger (2005), key elements of such comparisons include relative costs, time savings, benefits of accelerated release of improved germplasm, and the amount of operating capital needed and available. The latter can be essential to use of high-tech strategies. Although the cost-benefit analysis may justify deployment of high-tech strategies, capital may be necessary to establish access to these. Relative costs will take into account significant support systems for the breeding program such as quality systems to ensure that transgenic stewardship objectives are met. Economic advantages of partitioning the breeding program by event category (as discussed above) may not only lead to substantial savings, but may reduce risks associated with regulatory breeches as well.
Perhaps the greatest risk in transgenic hybrid development is that of failing to provide adequate containment of unauthorized events. Effective stewardship is essential to protecting research investments, maintaining compliance with government regulations, and ensuring customer satisfaction (Mumm and Walters, 2001). Regulatory breaches compromise public trust and can result in costly fines or remediation efforts.
Freedom to operate is a risk that favors the separation of hybrid improvement from event integration. If, for example, any of the components used in the transformation vector come under patent protection during the course of transgenic hybrid development, product pipelines and revenues and research investments could be significantly compromised.
Overlap in hybrid development and event integration may present a potential risk to overall effectiveness of the breeding program. It is assumed that a breeding program with components of both hybrid improvement and event integration would engage specialists in each area. With the intersection of these activities, corn breeders become involved to a higher degree in transgenic trait evaluation, requiring them to be trait specialists and to take on additional responsibilities for event stewardship. Likewise, competition can arise for facilities. Given a finite number of field testing plots, phenotypic evaluation to facilitate selection of partial conversions could jeopardize the overall effort directed to hybrid improvement.
Speed, discussed earlier as influencing the choice of breeding method, also factors into risks. The flip side of the advantage of speed to market is the threat of competing hybrids featuring a transgenic trait of interest or a particular source of that trait introduced ahead in the marketplace, potentially setting a standard that may become a market challenge.
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Summary of the Implications of Factors |
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TOPABSTRACTINTRODUCTIONFactors Affecting the Choice...Summary of the Implications...REFERENCES |
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With event integration conducted independently from hybrid improvement, some degree of backcrossing is basic. However, a forward breeding component in event integration may be a necessity for transgenic events that interact with metabolic pathways depending on the number of loci involved, the magnitude of the epistatic effects, the consistency of effects across germplasm, and the impact of the environment on interactive loci.
To utilize forward breeding in event integration, a decision regarding the number of backcrossing generations to be conducted before line selection via forward breeding would be required. Ribaut et al. (2002) concluded that, considering various backcross generations, selection for favorable alleles from both parents is most efficient when initiated in later rather than earlier backcross generations (e.g., BC3 vs. BC1 or BC2), citing the increasing ratio of the standard deviation to the mean of the donor genome contribution. Bailey (1977) showed that the probability of obtaining a line superior to the recurrent parent is optimized at a particular backcross generation. Probabilities differ depending on the number of loci involved, the potential contribution from the nonrecurrent parent, the selection intensity, and trait heritability. For example, the probability of obtaining superior lines from parents with an 80/20% split for favorable alleles was optimized at the BC2 generation in the case of 20 loci and at the BC4 generation in the case of 60 loci (when a 10% selection intensity was applied at the specified backcross generation and trait heritability was 0.5) (Table 3 ). Formulae provided by Bailey can be used to compute probabilities to support a decision as to when to shift event integration into forward breeding in a specified circumstance.
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With the requirement for some backcrossing and the advantages of a backcross-only approach to event integration, another potential avenue to pursue in the situation involving interaction between the candidate transgene and the genome focuses on the identification of these interactions up front, so as to include all factors required for adequate trait expression in the transformation vector. This would necessitate additional testing in the proof-of-concept stage of transgenic trait development but could be most efficient and effective in the long term, particularly in light of the trend to stack transgenic traits and demand for more than one transgenic version of the same hybrid. With all factors required for adequate and dependable transgenic trait expression represented in the event, these can in essence be treated as a single genetic unit in breeding efforts.
Further discussion on event design and event selection is outside of the scope of this work but the relevance of these issues to reliable and consistent event expression could be addressed more fully.
In closing, it should be noted that this analysis is directed to maize and does not necessarily fit for other crops, especially self-pollinated crops wherein cultivars are varieties with some degree of heterogeneity. Although the factors identified as affecting choice of breeding method would hold, the impact of these and their implications could potentially be quite different.
Many thanks to John W. Dudley and G.R. (Dick) Johnson for their critical review, thoughtful insights, and helpful suggestions regarding this work. Thanks also go to two anonymous reviewers.
Received for publication April 7, 2007.
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TOPABSTRACTINTRODUCTIONFactors Affecting the Choice...Summary of the Implications...REFERENCES |
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