1 INTRODUCTION

American chestnut (Castanea dentata) was once one of the most economically and ecologically important hardwood species in the United States (Diamond, Giles, Kirkpatrick, & Griffin, 2000; Jaynes & Graves, 1963). In the first half of the 20th century, an exotic fungal pathogen—Cryphonectria parasitica—devastated the species, killing approximately four billion trees. While chestnut survives in the wild through re‐sprouting from uninfected roots, these new stems are invariably re‐infected with blight. Restoring chestnut to its former role as a keystone species of Appalachian forests has been the goal of a variety of federal, state, and private breeding programs since shortly after C. parasitica was introduced into North America.

Two approaches to developing blight‐tolerant populations of American chestnut are currently in progress. First, The American Chestnut Foundation (TACF) is introgressing alleles that confer blight tolerance from Chinese chestnut (Castanea mollissima) into American chestnut, followed by repeated backcrossing to American chestnut. The American chestnut backcross‐breeding program has largely been carried out by volunteer citizen scientists who have applied pollen from backcross trees selected for blight tolerance to rare flowering American chestnut trees ranging from Maine to Alabama. This backcross‐breeding program is expected to yield a population of hybrids that are morphologically indistinguishable from WT American chestnuts and that have blight tolerance that is intermediate between Chinese chestnut and American chestnut (Diskin, Steiner, & Hebard, 2006; Steiner et al., 2017). Second, authors of this study from the State University of New York‐College of Environmental Science and Forestry (ESF) have used Agrobacterium‐mediated transformation to insert a wheat oxalate oxidase (OxO) gene into the genome of American chestnut (Zhang et al., 2013). The transgenic lines with the highest expression of OxO have high levels of blight tolerance in early trials (Steiner et al., 2017). The oxalate oxidase transgene reduces the pathogenicity of C. parasitica on American chestnut by converting oxalic acid that is secreted by the fungus into hydrogen peroxide and carbon dioxide. These byproducts are commonly present in nontransgenic plants and hydrogen peroxide may enhance the plant's endogenous defense response (Newhouse et al., 2014).

Regulatory approval is currently being sought to allow distribution of transgenic American chestnut trees for horticultural and restoration purposes. In 2019, we anticipate that regulatory petitions on transgenic American chestnut trees will be submitted to the U.S. Department of Agriculture, the U.S. Environmental Protection Agency, and the U.S. Food and Drug Administration. These petitions will include molecular characterization, nutritional data, and possible ecological impacts. Research to date indicates no significant differences between transgenic and WT American chestnuts in growth, metabolism, colonization of roots by mycorrhizal fungi, leaf litter decomposition rates, germination of seeds from other plants in chestnut leaf litter, bee feeding on pollen, tadpole feeding on chestnut leaf litter, and nut nutrition (D' Amico et al., 2015; Goldspiel, Newhouse, Powell, & Gibbs, 2018; Newhouse et al., 2014; Newhouse et al., 2018). Regulators are expected to decide within a few years if these data are sufficient to grant nonregulated status to allow distribution of transgenic trees outside confined field trials.

Outcrossing transgenic trees to WT trees enables efficient rescue of the genetic diversity and adaptive capacity remaining in extant C. dentata populations. The natural range of American chestnut is highly climatically diverse, spanning approximately 15° latitude, from central Mississippi to coastal Maine, which corresponds to mean annual temperature variation of ~12°C. When a transgenic American chestnut with a single copy of OxO is bred with WT American chestnuts, approximately 50% of the progeny from these crosses are expected to inherit the OxO gene (Figure 1). The OxO gene has a dominant effect in that trees that inherit a single copy of the gene have blight tolerance that is similar to the transgenic parent (Newhouse et al., 2014). Trees that inherit OxO are readily identifiable with an enzymatic assay of oxalate oxidase activity or with PCR (Zhang et al., 2013). In contrast, selection for blight tolerance is laborious in TACF’s backcross program. Multiple independently segregating alleles from Chinese chestnut with incompletely dominant effects confer blight tolerance to backcross trees (Kubisiak et al., 1997, 2013). Due to multi‐genic inheritance, large populations of backcross trees must be inoculated with C. parasitica and currently only 1 in 150 backcross trees with the highest blight tolerance are selected (Steiner et al., 2017).

Figure 1 Open in figure viewer PowerPoint First generation (T 1 ) progeny of a cross between the “Darling58” transgenic American chestnut founder containing oxalate oxidase gene and blight‐susceptible wild‐type American chestnut. Both progeny were inoculated with a highly virulent strain of the fungus that causes chestnut blight (Cryphonectria parasitica) approximately 6 months before the photos were taken. The progeny on the left inherited the oxalate oxidase gene, whereas the progeny on the right did not inherit the gene (Photo courtesy of Andrew Newhouse)

To date, the OxO gene has been inserted into a single American chestnut background, the Ellis1 tree from New York, to produce the transgenic “Darling 58” founder being reviewed by the federal regulators. More than one generation of outcrossing this transgenic founder tree to WT American chestnut trees will be required to dilute out the founder genome and to increase the genetic diversity and adaptive capacity of the blight‐tolerant population. In the first generation of outcrossing, the progeny will inherit 50% of their genome from the transgenic founder. If those first generation progeny were to intercross, their progeny would be inbred due to the large proportion of their genomes inherited from the single transgenic founder. Inbreeding can lead to mortality or reduced vigor due to the large number of deleterious mutations that outcrossing tree species such as American chestnut carry in their genomes (Charlesworth & Willis, 2009; Wright, Ness, Foxe, & Barrett, 2008).

We currently plan to create up to three additional transgenic founders through Agrobacterium‐mediated insertion of OxO into three American chestnut trees' genomes. Creating more transgenic founders will alleviate the founder bottleneck on effective population size once transgenic founders are outcrossed to WT trees. Having additional founders also mitigates the risk that deleterious mutations in linkage disequilibrium with OxO in a single founder tree's genome will have negative effects on fitness among progeny that inherit the transgene in a homozygous state. However, outcrossing with multiple transgenic founders also carries the risk that the OxO transgene could be silenced in progeny that inherit multiple copies of OxO at different locations in the genome. To mitigate the risk of silencing, we intend to express OxO with different promoters in different transgenic founders. In the “Darling 58” founder, OxO is expressed with the constitutive CaMV 35s promoter. Oxalate oxidase in new transgenic lines will be expressed from a wound‐inducible promoter (win3.12; Yevtushenko, Sidorov, Romero, Kay, & Misra, 2004) or a different constitutive promoter (UBQ11; Norris, Meyer, & Callis, 1993). Expressing the OxO transgene with different promoters reduces the risk of silencing due to methylation of a specific promoter region (Rajeevkumar, Anunanthini, & Sathishkumar, 2015), though we acknowledge that posttranscriptional gene silencing would not be affected by specific promoters. In a forest setting, blight infection will eventually kill trees that have the OxO gene silenced; therefore, natural selection could maintain transgenic blight tolerance even if silencing occurs.

We have started the process of outcrossing transgenic trees to WT American chestnut trees. In permitted field trial plots, four WT parents have been used as parents in first‐generation crosses. Transgenic progeny from two of these WT parents have been crossed with 15 additional WT trees to generate second‐generation transgenic progeny. Offspring from early crosses like these will be available for personal and small‐scale plantings almost immediately after regulatory permission is granted for distribution. These transgenic progeny may also be used as parents for subsequent large‐scale outcrossing. The next step is to estimate how many additional outcrosses are needed to produce a diverse and regionally adapted restoration population.

We aim to increase the effective population size (N e ) of the transgenic blight‐tolerant population to more than 500. This does not mean that only 500 individual trees will be produced, but that there will be 500 sufficiently unique genetic backgrounds from which restoration can potentially proceed. A target effective population size of 500 is based on the hypothesis that populations of this size experience minimal genetic drift and are therefore at low long‐term risk of extinction (Jamieson & Allendorf, 2012). Like many forest trees, American chestnut is susceptible to inbreeding depression. We therefore also aim to reduce the average inbreeding coefficient (F) in the intercrossed population to less than 0.05 (Hedrick & Kalinowski, 2000). The inbreeding coefficient refers to the probability of inheriting two copies of the same allele from both parents averaged across the entire genome. Finally, we aim to reduce the extent of transgenic founder genome, especially on the carrier chromosome containing OxO, through multiple generations of selection against the founder genome using molecular markers. While the total length of the founder genome is expected to be halved with each generation of outcrossing, a portion of the founder genome is expected to persist on the carrier chromosome due to selection for the transgene (Hospital, 2001; Hospital, Chevalet, & Muslant, 1992; Peng, Sun, & Mumm, 2014). Marker‐assisted selection for recombination events near the transgene reduces the potential for making transgene‐linked deleterious mutations homozygous when transgenic progeny intercross. After outcrossing is complete, we intend to intercross the transgenic progeny and plant only the subset of progeny that inherit OxO in homozygous state in seed orchards. Large numbers of OxO‐homozygous seeds may then be generated in the second intercross generation with open‐pollination.