Editor’s Choice: Crop Genome Plasticity and Its Relevance to Food and Feed Safety of Genetically Engineered Breeding Stacks

Genetically engineered (GE) stacks, combinations of two or more single transgenic events (i.e. single-locus insertions) that have been produced by crossing sexually compatible parents, are an important and growing sector of the crop seed market. Stacked traits covered 26% of the global transgenic crop area in 2011 and were the fastest growing trait group, with a 31% increase in the area planted compared with 2010 (James, 2011). Stacked traits already dominate the market in some regions. For example, 95% of the cotton (Gossypium spp.) grown in Australia during 2011 had both herbicide tolerance and insect resistance traits (James, 2011). Worldwide, at least 12 countries are now growing stacked varieties, of which nine are developing countries (James, 2011). The rapid adoption of GE stacks has focused attention on whether the safety of such products differs from that of the individual events. At issue is whether combining two or more events via conventional breeding creates changes that require additional safety assessment, even though the safety of each event in the stack has been assessed previously. The two main concerns are (1) whether combining two or more events into a plant by conventional breeding increases genomic instability and (2) whether potential interactions between the products of the transgenes in GE stacks impact safety. This paper draws on insights from plant breeding, describes the plasticity of conventional plant genomes over generations of crossing and selection, and considers the implications of event stacking on food and feed safety in the context of the normal plant genome. The term GE is used here to distinguish the process of specific, intentional, and directed physical modification of the genome of a plant from random genetic modifications that occur in conventional breeding or by mutagenesis. The term GE is preferred over the term “genetically modified” (commonly referred to as GM) for these reasons. The term “event” refers to a single-locus insertion of recombinant DNA into the host genome regardless of the number of genes contained on the inserted piece of DNA. The term “conventional breeding” refers to methods of crossing plants with desired characteristics to generate offspring combining those desirable characteristics. These characteristics may include both non-GE and GE traits. This paper focuses on (1) the potential of transgenes to alter genome stability and (2) the potential risks to food and feed safety associated with genome instability. A companion paper focuses on potential interactions that can occur from transgene stacking (H.Y. Steiner, C. Halpin, J.M. Jez, J. Kough, W. Parrott, L. Underhill, N. Weber, and L.C. Hannah, unpublished data). The potential environmental impact that may arise from the cultivation of crops with GE stacks is outside the scope of these articles. Crops containing a single event, that carry multiple traits that are introduced simultaneously (i.e. molecular stacks resulting from cotransformation), or that are produced by retransformation of an event require a de novo safety assessment, as is customary for all new events, and, hence, are also outside the scope of this paper. Plant breeding is a major underpinning of modern agriculture, as it creates varieties containing multiple desirable traits through the stacking of both known and many unknown genes. While increasing yield potential is a major objective, protecting yield potential (i.e. breeding for resistance to biotic and abiotic stresses) is also critical. Many stress resistance genes have come from related species such as wild relatives of crop plants. Hajjar and Hodgkin (2007) reported that conventional breeding efforts in 19 of the world’s major crops had incorporated 111 genes from wild relatives into new varieties over the previous 20 years. Eighty percent of these genes confer disease resistance; the remainder control abiotic stress resistance or quality traits (Hajjar and Hodgkin, 2007). Modern non-GE crop varieties differ mainly from their predecessors by the incorporation and stacking of genes from distant relatives. For example, IR8 rice (Oryza sativa), released in 1966, is resistant to the green leafhopper (Nephotettix spp.) and moderately resistant to salinity, rice blast, and phosphorus deficiency. Just 11 years later, IR42 was released, which possessed resistance to multiple diseases (rice blast, Grassy stunt virus, Rice tungro virus, Ragged stunt virus, and bacterial blight), pests (brown planthopper [Nilaparvata lugens], stem borer), and abiotic stresses (nitrogen deficiency, zinc deficiency, iron deficiency, alkalinity, and iron toxicity; International Rice Research Institute, 1981). Today, many seed catalogs list the multiple resistance traits present in each variety. Although the genetic and biochemical bases for these traits are usually not well understood, conventional breeding has successfully stacked traits to create stable crop varieties that are considered safe and that, in most cases, were not subjected to safety assessments prior to commercialization. Gene introgression from one species into another via sexual cross-breeding is inherently imprecise: additional genes contained in the DNA linked to the desired gene are also incorporated. For example, transfer of the Tm-2 gene from Solanum peruvianum to tomato (Solanum lycopersicum) provides resistance to Tobacco mosaic virus. Four to 51 centimorgans of S. peruvianum DNA accompanied the Tm-2 gene when introgressed into different tomato varieties (Young and Tanksley, 1989). One centimorgan of DNA can contain up to several hundred thousand bases of DNA sequence and include multiple genes. Similarly, modern wheat (Triticum aestivum) varieties contain resistance genes from dozens of species representing six related genera. In some cases, entire chromosomes or chromosome arms representing millions of base pairs of DNA were transferred along with the desired resistance gene (Jones et al., 1995). Despite this imprecision, plant breeding has a remarkable record of safety; no newly released variety has had any novel or previously unknown food or feed hazard (H.Y. Steiner, C. Halpin, J.M. Jez, J. Kough, W. Parrott, L. Underhill, N. Weber, and L.C. Hannah, unpublished data). The primary way through which the presence of two or more transgenes could give rise to genetic instability in a GE stack, but not in the two transgenic parents, is through homologous recombination between sequences repeated in the transgenes. Therefore, it is necessary to first evaluate the natural stability of non-GE plant genomes and the role played by repeated sequences in creating genomic changes. Genomic diversity is caused by plasticity: the capacity of the genome to reorganize itself (Nevo, 2005). These changes involve alterations in both sequence (McClintock, 1984) and chromatin state (Brink, 1960; Hale et al., 2007), providing genetic variation for selection by breeders (Vyskot et al., 1991; Morgante et al., 2007). A large measure of the plasticity is the direct result of genome organization at the DNA level (Fig. 1). As discussed in the following sections, plant genomes contain substantial numbers of mobile (transposable) DNA elements and other repetitive coding and noncoding DNA sequences, all of which lead to genome diversity. Overview of the types of DNA in plants and their genomic arrangement. DNA sequences present in multiple copies, including mobile elements such as DNA transposons and retrotransposons, are important contributors to genomic plasticity. Even coding genes are seldom unique in a plant. A myriad of biochemical, physiological, and genomic investigations definitively establish that a high percentage of genes are present in duplicated forms or as members of multigene families. Multigene families have arisen through duplication of whole genomes, as seen in rapeseed (Brassicanapus), or of smaller genomic regions (Moore and Purugganan, 2005; Cheung et al., 2009). Other types of repetitive DNA include ribosomal genes and telomeres, both of which consist of tandem repeats. Tandem DNA repeats are particularly prone to changes in copy number resulting from unequal crossing over, gene conversion, intrastrand recombination, or replication slippage (Flavell, 1985; Strand et al., 1993). For example, such mechanisms generate new alleles of disease resistance genes (Kuang et al., 2004), which are commonly found in groups of related sequences called resistance gene clusters. Short stretches of repeated DNA sequences, called microsatellites, simple tandem repeats, or simple sequence repeats, are another type of repetitive DNA with a tandem arrangement. The mutation rate for copy number of dinucleotide repeats in maize (Zea mays) is almost eight for every 10,000 meioses (Vigouroux et al., 2002), or one in every the mutation rate for in wheat (Triticum is 10,000 meioses et al., linked duplicated DNA sequences have the potential to homologous recombination, to the of the DNA For example, many have sequences The genome many that sequences, that of repeats and homologous recombination the sequence et al., the DNA can as DNA and into other regions of the genome et al., et al., In some cases, of the DNA between two linked of a gene in in gene et al., the is that the DNA between the two genes is of the to One of such was found at the rice et al., between duplicated genes can create novel One is the of which seed et al., is the of in which of both tandem and of genes coding for from to seed is associated with of a whole the et al., 2004), through homologous The of homologous recombination that changes at recombination is by that to of homologous sequences between homologous chromosomes or et al., recombination also can occur in with recombination between homologous alleles from in to in and In the of homologous recombination in is between and genome et al., In recombination are no one recombination for every chromosome and the rate of recombination between alleles of one gene is usually one events. between homologous sequences in a genome is such recombination of homologous sequences on chromosomes may an of both and (Fig. for et al., 2007). changes such as or result in and, in some cases, from between homologous DNA sequences has been as a of both and between homologous on different between homologous in direct on one and between homologous in on one are and their are are in plants and are necessary for recombination and and can caused by or of DNA a mechanisms other DNA as for DNA for 2005; et al., 2011). 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