Is it Really American Chestnut?

There are still millions of American chestnut trees in eastern forests, though most are actually sprouts from roots of trees killed long ago by chestnut blight. In full sunlight, these sprouts can grow up to 30 feet tall, sometimes flowering and even producing nuts before the blight kills them again. To maintain genetic diversity in their breeding nurseries, TACF volunteers regularly search forests for flowering native chestnuts they can use as “mother trees.”

It’s important that the “mother trees” used to produce blight-resistant hybrids are pure American chestnut. Out in the forest, it can be hard to separate pure American chestnut from hybrids just by looking. European and Asian chestnut trees have been planted extensively in the East since European settlement, crossing with native trees to produce hybrids that look very similar to American chestnut.

Problems can also arise in the research fields themselves, where breeders produce hybrids by controlled pollination, bagging inoculated flowers to protect them from other pollen sources. Even though the flowers are protected, it’s still possible for pollen from a rogue source to fall onto an uncovered female flower. For the long process of backcrossing to recapture the desirable characteristics of an almost pure blight-resistant American chestnut, it’s particularly important that mistakes don’t get bred in and repeated in future crosses.

In some cases, the only way to tell for sure if a tree is pure American chestnut is by testing its DNA—in much the same way as the DNA of human children can be tested to establish paternity. Kubisiak and fellow researchers started working on the problem in the late 1990s, screening DNA from pure American chestnut, the foreign chestnut species, and various hybrids. For markers, they examined stretches of neutral DNA, fragments of genetic code that differ among individuals but haven’t been tied to a specific trait.

“We don’t know anything else about the markers, whether, for instance, they’re a gene or not,” says Kubisiak. “You can think of them as little monitors along a strand of DNA that allow you to look at variation in individuals. If you’re comparing two trees, you look at how many of these markers they share to see how alike they are.”

Kubisiak showed that markers can be used to distinguish between the different chestnut species and to quickly determine if a given tree is likely to be a pure American chestnut. “Fortunately, chestnut species are turning out to be different enough that each species harbors its own unique variation that can be used for identification purposes,” says Kubisiak. “The challenge we face now is to characterize this variation and determine which set of markers will be most useful for excluding hybrids. To be operationally feasible, the assay needs to be simple and inexpensive.”

So far, TACF has only used markers to look at seedlings from early generation backcrosses. But several of these progeny turned out to be products of contaminating pollen, so even these results have helped guide TACF in its breeding program. As more markers become available and easier and less expensive to use, they may be routinely used to determine such things as the percent of American chestnut in hybrids.

Diversity Issues

The markers Kubisiak is examining are also being used to ensure that the hybrids TACF develops are genetically diverse.

TACF’s ultimate goal is to restore a blight-resistant form of American chestnut throughout a native range that extends from Maine southwest to Mississippi, which means creating seedlings adapted to different climate and soil conditions. When they started their breeding program, TACF researchers already suspected that the seedlings they produced on research farms in Meadowview, VA, might not survive in northern Maine or southern Mississippi. They turned to the geneticists to find out how much genetic variation still existed in native American chestnut trees, and whether this information might be used to help TACF decide how many different breeding locations they needed to capture most of the genetic variation still present in the species.

In 2003, Kubisiak and SRS research geneticist James Roberds did a baseline study that analyzed DNA markers from 993 surviving American chestnut trees from 22 sites across the natural range. “We found that American chestnut acted like one big population,” says Kubisiak. “We showed that roughly 95 percent of the natural genetic variation in the species occurred in any one local population.”

That sounds like good news, but Kubisiak cautions that because researchers work with neutral DNA fragments, they’re not able to determine if the individual trees sampled have developed genetic adaptations to local environments. “Again, our study was based on neutral gene fragments, rather than on genes for traits such as bud break or cold hardiness that might show adaptations for specific locations,” says Kubisiak.

Kubisiak and Roberds suggested that breeding efforts collect material from a fairly large number of individuals (50 to 100 or more) from each of several geographic areas to capture variation associated with adaptive traits. They also suggested that at least three areas—northern, central, and southern parts of the range—be considered as locations for breeding efforts. There are now 17 State chapters, each with breeding programs that use surviving American chestnuts in their States, to ensure that there are materials adapted for much of the natural range.

Breeding Resistance

Kubisiak’s genetic markers really paid off for the hybrid breeding work when, in the late 1990s, he identified genetic markers linked with regions of the Chinese chestnut genome associated with resistance to chestnut blight. A DNA-based toolkit to test hybrid seedlings for resistance—as well as for how much unwanted Chinese chestnut still remains—is allowing researchers to know where they are in the quest to produce an almost pure American chestnut with full blight resistance.

To breed for blight resistance, TACF started off by simply crossing resistant Asian trees with American trees and testing for resistance by inoculating the resulting seedlings with chestnut blight. Resistant seedlings are then “backcrossed” with pure American chestnut three more times and then intercrossed with one another (see page 5). As backcrossing proceeded, TACF needed to know how many resistance genes from Chinese chestnut had to be present for crosses to produce elite trees with full blight resistance in later generations.

“Fortunately, there don’t seem to be that many genomic regions conditioning resistance,” says Kubisiak. “We were able to identify two regions that repeatedly show significant associations, which was very good news for TACF breeders. If more genes were involved, they might have to screen thousands to tens-of-thousands of plants to find elite seedlings for each cross.”

Meanwhile, out on the TACF research farms, the latest backcross seedlings are growing towards the revival of a great American tree. TACF projects that they’ll have tens-of-thousands ready for planting by 2015.

For more information:
Tom Kubisiak at 228–832–2747, x213 or tkubisiak@fs.fed.us

Recommended reading:

Kubisiak, T.L. 1999. Using DNA markers to distinguish among chestnut species and hybrids. The Journal of The American Chestnut Foundation: 13(1): 38–42.

Kubisiak, T.L.; Roberds, J.H. 2003. Genetic variation in natural populations of American chestnut. The Journal of The American Chestnut Foundation: 26(2): 42–48.

Kubisiak, T.L.; Roberds, J.H. 2006. Genetic structure of American chestnut populations based on neutral DNA markers. In: Restoration of American chestnut to forest lands: Proceedings of a conference and workshop. Natural Resour. Rep. NPS/NCR/CUE/NRR–2006/001. [Washington, DC]: U.S. Department of the Interior, National Park Service, National Capital Region, Center for Urban Ecology: 109–122.