The Burnham plan

Based upon an analysis of published and unpublished data from the USDA and CAES programs, Burnham et al. (1986) reasoned that alleles for blight resistance could be transferred to the C. dentata genome by creating a hybrid with Chinese chestnut (C. mollissima Bl.) or other blight-resistant Castanea species and then repeatedly backcrossing to C. dentata while selecting for resistance alleles. Selection for resistance alleles would be possible, even when they are necessarily heterozygous after each backcross to C. dentata, because alleles for blight resistance were found to be partially dominant in the earlier programs, and there appeared to be two genes involved. Each generation of backcrossing to C. dentata would halve the C. mollissima fraction of the genome, on average, thus facilitating recovery of the C. dentata type in all respects except susceptibility to chestnut blight. The full breeding plan called for the production of a third backcross (B 3 ) that would be intercrossed to create a segregating B 3 F 2 population in which a fraction of the trees would be homozygous for all resistance alleles from the original Asian parent. Finally, following testing and selection for blight-resistance, the B 3 F 2 plantation would become a seed production orchard for a B 3 F 3 generation that would be essentially C. dentata in character but sufficiently blight-resistant to begin restoration of the species. Other details of the breeding plan are provided by Hebard (2006) and Jacobs et al. (2013).

As implemented at Meadowview beginning in 1989, the Burnham plan was expedited by beginning with two first-backcross (B 1 ) hybrid trees from the USDA and CAES breeding programs, called the ‘Clapper’ and ‘Graves’ trees, respectively. Each was derived from an F 1 hybrid between C. dentata and C. mollissima that was subsequently backcrossed to C. dentata (Hebard 2006). It was assumed that the Clapper and Graves trees might not contain identical alleles for blight resistance, and accordingly they have been bred in different breeding populations as distinct sources of resistance.

For the Clapper and Graves sources of blight resistance, a minimum of 25 sets of C. dentata trees have been used as parents in each backcross generation to enhance the capture of American allelic diversity and minimize inbreeding in the final, intercrossing generation (Namkoong 1991; Hebard 2002). An operational assumption was that as many as three unlinked loci carry resistance alleles, which meant that as few as one in 64 trees in the B 3 F 2 generation were expected to be homozygous for resistance at all loci. To ensure the highest, practical probability of recovering at least nine homozygous individuals per American line, the Clapper and Graves B 3 F 2 test plantations (before conversion to seed production orchards through selection) were designed to contain 1350 offspring from each open-pollinated B 3 line, or a total of 33,750 trees for a resistance source with 25 American lines (Hebard 2002). Because of the size of the task, these plantations have taken many years to build.

Progress in breeding the Clapper and Graves sources in the Meadowview breeding program

Breeding began by crossing the Clapper and Graves B 1 trees with 66 and 76 C. dentata individuals, respectively, with an average yield of 12 B 2 seedlings per cross. Selected Clapper and Graves B 2 s were crossed with 52 and 43 additional C. dentata individuals, with an average yield of 37 seedlings per cross. To evaluate resistance, B 2 and B 3 trees were inoculated in the spring of their fifth growing season with strains of C. parasitica with low (SG2-3) and high (Ep155) pathogenicity but both capable of killing C. dentata sprouts. We have found subsequently that resistance to the two strains is strongly correlated genetically (r g = 0.95 ± 0.07), suggesting that common loci confer resistance to both. However, using both strains extends the range of response levels detectable in a test. All individuals were culled except those with the smallest cankers for each strain. Additional selection was performed for C. dentata morphological traits among individuals with the smallest cankers (Hebard 1994), leaving approximately 4% of B 2 and B 3 trees to be used in further breeding. After selection in the B 3 , grand-progeny remained from 29 of the 66 C. dentata trees crossed with Clapper and 25 of the 76 trees crossed with Graves, exceeding the target of 20 American lines for each resistance source.

Assuming blight resistance is controlled by three incompletely dominant genes at segregating loci, and assuming successful selection for heterozygosity at all three loci, the B 2 and B 3 trees used for further breeding should have resembled F 1 hybrids in having intermediate levels of resistance. The average canker sizes or ratings of B 2 and B 3 selections met these expectations (Fig. 1). Furthermore, Diskin et al. (2006) found that the American phenotype was recovered in 96% of B 3 trees as judged by 24 scored or measured traits typically used to distinguish C. dentata from C. mollissima. These findings provided evidence that the breeding program was working as planned.

Fig. 1 Average blight canker areas (A) or average visual canker ratings (B) of selected Clapper and Graves B 2 s (A) and B 3 s (B) relative to C. mollissima and C. dentata controls and the F 1 hybrid between the two species. Each tree was inoculated in separate locations on the stem with both a weakly (SG2-3) and highly pathogenic (Ep155) strain of C. parasitica. Letters above bars within a panel indicate significant differences (P < 0.05, Tukey HSD tests) after accounting for inoculation year as a covariate Full size image

Intercrosses to generate B 3 F 2 s were carried out with open pollination among the B 3 selections. Creation of the Clapper and Graves B 3 F 2 test plantations at Meadowview began in 2002 and continues. As of 2015, 20 of 29 Clapper American lines and 13 of 25 Graves lines had been planted in all 9 blocks of their respective plantations (Table 1). Preliminary selection for blight resistance in the plantations is carried out at 2 years of age by artificially inoculating stems with the SG2-3 strain of C. parasitica. Typically, 80–85% of the trees are culled within a year after artificial inoculation because of significant canker expansion. At present, further selections are made after longer-term response to the initial inoculation, response to additional inoculations or naturally occurring infections, and progeny-testing.

Table 1 Status of planting and selection in Clapper and Graves B 3 F 2 test plantations at TACF’s Meadowview Research Farms in Virginia, USA Full size table

Approximately 10,000 seedlings remained in the Clapper and Graves B 3 F 2 test plantations at Meadowview in 2015, and 11,250 had not yet been planted (Table 1). The plantations will not become completed seed orchards until the Clapper plantation is reduced to approximately 260 trees with the highest resistance, and the Graves plantation similarly reduced to approximately 225 trees. Although B 3 F 3 seeds have been distributed from these plantations for testing purposes since 2008, their blight resistance has necessarily been limited by progress in selection (Table 1). In field trials in forest settings, progeny from B 3 F 2 trees have grown significantly faster than C. mollissima seedlings and not significantly slower than C. dentata (Clark et al. 2016). As of 2016, the trees in these trials are not yet large enough to support epidemics of blight, so their “field resistance” remains unknown.

Genetic variation in blight resistance after preliminary selection in the B 3 F 2

Experimental field tests of half- or full-sib progeny sets are the proof of parental genetic quality in a breeding program. Beginning in 2009, TACF has been progeny-testing B 3 F 2 trees that have passed the initial phenotypic selection for blight resistance, and to date we have completed evaluations of B 3 F 3 progenies from 293 Clapper and 131 Graves B 3 F 2 seed parents in experimental plantations at Meadowview. The seed parents were open-pollinated with a random and presumably average sample of other B 3 F 2 trees shedding pollen. Castanea dentata and C. mollissima, the progeny of intraspecific crosses, were also included in progeny tests as blight-susceptible and resistant controls. The progenies are challenged in their third growing season with weakly (SG2-3) and strongly (Ep155) pathogenic strains of the C. parasitica.

These progeny tests have provided the first estimates of heritability and the first experimental proof of progress toward the projected culmination of the plan presented by Burnham et al. (1986). Figure 2 shows the composite results of these tests using best linear unbiased predictions (BLUP) of mean responses to both strains of the pathogen.

Fig. 2 Severity of cankering in open-pollinated progeny sets from 293 Clapper and 131 Graves B 3 F 2 mother trees and C. mollissima and C. dentata controls following inoculation by weakly (SG2-3) and highly pathogenic (Ep155) strains of C. parasitica. Raw data for the Tukey box plots are best linear unbiased predicted mean scores for approximately 12 B 3 F 3 offspring per seed parent. Scores were scaled to a mean of 0 for C. mollissima and a mean of 100 for C. dentata. The dashed line shows the expected mean response to inoculation (49.8) for the offspring of a hypothetical seed orchard thinned to the most resistant of the 424 seed parents. Estimation of actual genetic gain following parental selection is based on the formula Gain = 2h 2 S, where h 2 is the heritability (narrow sense) for family means estimated from the progeny tests (0.45), and S is the selection differential. In this example, we assumed that the selected parents would be those whose progeny means were at least 1.6 standard deviations better than the mean for all B 3 F 3 trees Full size image

Based on the assumptions of the breeding program, the expected average resistance level of all B 3 F 3 offspring from unselected B 3 F 2 parents is that of an F 1 population, i.e., intermediate between C. dentata and C. mollissima as in Fig. 1. However, even after initial phenotypic selection for resistance among B 3 F 2 trees, the average level of cankering severity in B 3 F 3 offspring is more similar to C. dentata than to C. mollissima (Fig. 2). This result suggests that some selections in the B 2 or B 3 generations did not inherit the full set of blight resistance alleles from the source C. mollissima. Every offspring from backcrosses to C. dentata had at least a 50% complement of susceptibility alleles from the American parent, and most had more. The object, apparently not always achieved, was to identify those trees that had exactly a 50% complement of resistance alleles from C. mollissima. Additional evidence of imperfect selection is the fact that we found significant (P < 0.001) heritable variation in canker size among Clapper and Graves B 3 F 2 half-sib families. Assuming random pollination of the seed parents, there should have been no genetic variation in blight resistance among B 3 F 2 families if selected B 3 trees were heterozygous at all resistance loci.

The most likely explanation for these results is that some resistance alleles were lost through backcrossing to C. dentata. This would certainly have happened if resistance is controlled by more than three loci, in which case it would have been statistically improbable to recover all resistance alleles in every American line given the size of populations that could feasibly be generated and evaluated in field trials. Nonetheless, with the number of American lines across which the breeding program is replicated (25 and 29 for the two sources of resistance), we believe it is unlikely that the breeding program has completely lost any resistance alleles from the Clapper and Graves ancestors. If true, this means that resistance can be improved through further breeding.

Variation among B 3 F 3 progeny sets (Fig. 2) is the result of genetic differences among individual B 3 F 2 seed parents, some inevitably more blight-resistant than others, that were open-pollinated by a random host whose genetic quality was average. In other words, even the most resistant B 3 F 3 families were limited by the mediocrity (on average) of their many male parents. This limitation may have been further constrained by the tendency of heavily blighted trees to produce excessive numbers of male catkins. The average genetic resistance of B 3 F 3 s will increase until all but the most blight-resistant B 3 F 2 s are culled in the process of converting the test plantations to seed orchards, at which time both seed and pollen parents will be members of a selected population. Currently, the mean B 3 F 2 breeding values for canker severity are closer to C. dentata than to C. mollissima. Selection of the 5% most resistant B 3 F 2 from the 10,000 remaining trees is predicted to yield B 3 F 3 progeny with average canker severity that is almost exactly (by coincidence) intermediate in resistance between the average C. mollissima and the average C. dentata (Fig. 2). Actual gains may be less depending upon the level of genotype by environment interaction. Considering that the most resistant B 3 F 2 s have breeding values closer to C. mollissima than to C. dentata within pollen clouds from partially selected plantations, the maximum resistance of the B 3 F 3 progeny is expected to approach the mean resistance of C. mollissima upon completion of selection at B 3 F 2 . Restoration trials composed of B 3 F 3 progeny from selected B 3 F 2 parents will determine whether resistance after selection at B 3 F 2 is sufficient to generate reproductive, self-sustaining populations in the forest. To accelerate crown closure and help ensure success over competing vegetation, restoration plantings will typically have densities far greater than can survive to maturity (Nyland 1996), so it will not be necessary that all trees have high resistance.

Progress in breeding additional sources of resistance

In addition to the Clapper and Graves sources of resistance, TACF’s Meadowview program has advanced 22 other sources of Asian chestnut resistance variously from the F 1 through B 4 (Table 2). The purpose of this work is to increase the diversity of resistance alleles in the breeding program and to minimize vulnerability to evolution of virulence in the fungus (Hebard 2004). Castanea mollissima has been the preferred source of blight resistance because canker expansion after inoculation with Cryphonectria parasitica was slower on C. mollissima than on other Castanea species (Graves 1950; Clapper 1952). Two other Asian species of chestnut (C. crenata and C. seguinii Dode.) have also been utilized. Some of the C. mollissima sources of blight resistance (‘Nanking’, ‘Meiling’, and ‘Kuling’) were highly blight-resistant selections from the USDA’s breeding program for nut production (McKay and Jaynes 1969).

Table 2 Progress in breeding in additional sources of resistance at TACF’s Meadowview Research Farms Full size table

It is still not known whether different C. mollissima trees carry different alleles for resistance. This was investigated at Meadowview by test-crossing each of 16 C. mollissima trees with three others and inoculating the progeny at age 3 with the highly pathogenic Ep155 strain of C. parasitica. Additive genetic variance in the offspring—which would arise if individual parents differed in their average contribution to resistance—was not significantly greater than zero (likelihood ratio test P = 0.17). However, non-additive genetic variance associated with specific crosses was significant (P = 0.002). Non-additive genetic variance in blight resistance (e.g., variance resulting from dominance or epistasis) cannot be “fixed” to incrementally enhance resistance across generations, but it can contribute to the population mean level of resistance after selection in a given generation.

State chapter breeding programs

As a complement to the breeding program at Meadowview, the Burnham plan has been replicated by volunteers in most of TACF’s 16 state and multi-state chapters beginning with the Pennsylvania Chapter in 1994. In addition, members of the New York Chapter of TACF have been the principal sponsors of the SUNY-ESF work to enhance blight resistance through genetic transformation of C. dentata. The transformation project will be discussed in a subsequent section.

Most of the chapter activities are based on the Meadowview model and were started with pollen from selected B 2 s and B 3 s created at Meadowview using Graves or Clapper sources of resistance. There are 14 of these programs (Table 3). Chapter breeding efforts serve the important purpose of ensuring regional climatic adaptability in the future restoration program by using locally autochthonous C. dentata trees in backcross generations after the B 2 .

Table 3 Progress of TACF state chapters toward the goal of creating B 3 F 2 test plantations with various sources of C. mollissima resistance Full size table

A second, related purpose of the chapter-directed programs is to increase the diversity of the C. dentata genome captured by the breeding program. As shown by Hebard (2012), the contributions of the chapter breeding programs to the inbreeding effective population size of the anticipated founding population are expected to be substantial (>200 in aggregate). For these reasons, the chapter programs are regarded as critical to the task of restoring the species. These programs are assisted by four regional coordinators who are full-time employees of TACF, but the actual breeding work depends almost entirely on volunteer leadership and labor. Almost half of the state chapters have started creating B 3 F 2 or B 4 F 2 test plantations, and the earliest may be completely planted (though not culled to the final selections) as early as 2020. Chapter breeding programs manage 586 plantations of breeding material on approximately 335 ha of public and private land not owned by TACF, and volunteers log over 50,000 h annually in this effort (Table 4, also see Fig. 5 in Jacobs et al. 2013). Considering the scope, complexity, and duration (20+ years) of the chapter breeding projects, this application of citizen science and volunteer labor to the restoration of a species is exceptional and perhaps unique among conservation programs.