1. The American Chestnut Foundation’s Breeding Program for Blight Resistance Frederick V. Hebard, William Y. C. White & Shawn Yarnes Meadowview Research Farms 14005 Glenbrook Ave. Meadowview, VA 24361 USA http://www.acffarms.org TACF was created in 1983 as a private, non-profit organization. Mission: to restore the American chestnut to American forests through a scientific program of breeding and cooperative research.

2. C x A  F 1 x A  B 1 x A  B 2 x A  B 3 x B 3  B 3 -F 2 x B 3 -F 2  B 3 -F 3 Expected Proportion Chinese 1/2 1/4 1/8 1/16 Year Cross Made 1930s 1940s 1989-1995 1994-2002 1999-2009(estimate 2005- Backcrossing First, American and Chinese chestnut trees are crossed with each other. The progeny from this first cross are then backcrossed to American chestnut. Each cycle of backcrossing reduces the fraction of Chinese genes by a factor of one half. Finally, backcrosses are intercrossed to yield progeny that will breed true. Blight resistance is retained by inoculating progeny and selecting resistant ones. More than one American parent is used at each backcross to avoid inbreeding. One set of American parents constitutes a line, the different Americans are denoted A1 to A4 below. C x A1  F 1 x A2  B 1 x A3  B 2 x A4  B 3

3. Backcrossing More than one American chestnut line also is needed, also to avoid inbreeding, when B 3 s are intercrossed with each other. C x A13  F 1 4 x A14  B 1 4 x A15  B 2 4 x A16  B 3 4 C x A9  F 1 3 x A10  B 1 3 x A11  B 2 3 x A12  B 3 3 x C x A5  F 1 2 x A6  B 1 2 x A7  B 2 2 x A8  B 3 2 C x A1  F 1 1 x A2  B 1 1 x A3  B 2 1 x A4  B 3 1 x  B 3 -F 2 12  B 3 -F 2 34

4. Twenty B 3 lines (families) eliminates most inbreeding after intercrossing, enhancing the chance that a population will not collapse. Effect of Partial Diallel Size on Inbreeding Chapters add genetic diversity The effect of adding sets of 20 B 3 -F 2 progeny from our chapters on inbreeding effective population size, for one source of blight resistance. Restoration ecologists estimate that effective population size needs to be 50 to avoid immediate collapse from inbreeding depression and 500 for mutation to offset long-term erosion of genetic diversity by drift. Chapters also add adaptation to their local environment in addition to general genetic diversity. Number of Chapters Inbreeding Coefficient Inbreeding Effective Population Size 10.020772 20.0115130 30.0085176 40.0070214 50.0060248

5. SourceNumber Currently Used C. mollissima23 C. crenata2 C. sativa1 C. dentata19 C. henryi & sequinii????? Our primary source of blight resistance is Chinese chestnut, because that was identified as the most resistant species by the USDA in the 1930s. No resistance was known in American chestnut until the 1980s. We also have a few crosses with Japanese and European chestnut. C. henryi and C. sequinii also may have individuals with high resistance, based on recent observations in China. Sources of Blight Resistance Castanea henryi Castanea seguinii

6. Number of Trees at Meadowview in 2008 Type of TreeNumber of Nuts or Trees Sources of Resistance American Lines American 189669 Chinese 1813105 F1F1 5292369 B1B1 5311529 B2B2 20521324 B3B3 23921073 B4B4 51644 B 1 -F 2 47144 B 2 -F 2 22555 B 3 -F 2 32422349 B 3 -F 3 46415 F2F2 35555 F3F3 622 Other 5931 73116 Total 49603

7. TACF Meadowview Nut Harvest 2008 Cross TypeBag s BursNutsCbagsCbursCNuts AxA398612... B1B1 11527819721230 B2B2 39273161149442 B3B3 34065827939702 B4B4 82156699140 Chapter2664759524462907418 B 1 -F 2 4521073346112700 B 1 xC2254228718320 B 2 -F 2 2235472518420 B 2 xC13200110 CxC23356613125580 F1F1 29810021022345 LSA18203805248819331325 B 1 -F 3 612817271 B 2 -F 3 564412231 B 3 -F 2 1499924975 B 3 -F 3 24214092 American22732308 Chinese3159 Chinkapin4250 Total70987

8. B 3 -F 2 family size needs to be 9 or 10 selected trees to capture most alleles from each B 3. Obtaining fewer selections per parent would result in loss of genetic diversity. 0.00 0.25 0.50 0.75 1.00 051015 Family Size Probability of Capturing All Alleles Effect of Family Size on the Probability of Capturing Both Alleles from 20 Independently Assorting Loci B 3 -F 2 Selection B 3 -F 2 Seed Orchard

9. Selection for Recurrent Type Trichome density and type Stipule size and shape Leaf shape Tooth shape Lenticel density, size and color Stem color Form Time of leaf emergence Vigor Each backcross generation will vary in the remaining proportion of Chinese heritage. By picking trees with the fewest Chinese traits (except blight resistance), we accelerate the recovery of American type. At the present time, this selection is done using the following morphological traits: Price Farm viewed from Bryan Farm

10. Cutting disks of inoculumInserting disk in tree Taping inocu- lation point Elder Hostel Inoculation CrewCrew in Action Selection for resistance We determine the blight resistance of chestnut trees by giving them the disease, inoculating them with the blight fungus, and observing the resultant cankers.

11. Highly blight-resistant and blight-susceptible Clapper x Graves B 1 -F 2 progeny, characterized by small and large cankers, respectively.

12. Table 1. Number of trees in canker size classes in 1993. Cross Type Size Class (cm) _________ 1.0-2.62.6-4.24.2-5.85.8-7.47.4-9.09.0-10.610.6- ______________________________________________________________________________ American352 F 1 Nanking243 Seedling Chinese273 Meiling Chinese122 Nanking Chinese32 F 2 Mahogany052348482915 B 1 -F 2 ClapperxGraves 42584116112544 ______________________________________________________________________________ Table 2. Number of trees in canker size classes in 2003. Cross Type Size Class (cm) __________ 1.0-2.02.0-3.03.0-4.04.0-5.05.0-6.06.0-7.07.0-8.0 ________________________________________________________________________________ American 42221 F 1 Nanking 1231 Chinese3336 B 2 -F 2 Clapper311153716123 B 2 -F 2 Mahogany311213114141 ________________________________________________________________________________

13. Chestnut Blight Resistance Rating Scheme Ep155 SG1 2-3 smallmediumlargelargerlargest smallmediumlargesmall Strain of Cryphonectria parasitica Canker size Canker size Rating 12345

14. Number of Clapper B 3 -F 2 seedlings in resistance classes after inoculation with the blight fungus in 2004 and 2005.

15. Blight-resistant Chinese to American B 1 -F 2, ‘Clapper’ x ‘Graves.’ Obtaining trees like this suggests it should be possible to backcross the blight resistance of Chinese into American chestnut.

16. The goal.