The American Chestnut Foundation’s Breeding Program for Blight Resistance Frederick V. Hebard, William Y. C. White & Shawn Yarnes Meadowview Research Farms Glenbrook Ave. Meadowview, VA USA 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.

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 (estimate 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

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

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

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

Number of Trees at Meadowview in 2008 Type of TreeNumber of Nuts or Trees Sources of Resistance American Lines American Chinese F1F B1B B2B B3B B4B B 1 -F B 2 -F B 3 -F B 3 -F F2F F3F3 622 Other Total 49603

TACF Meadowview Nut Harvest 2008 Cross TypeBag s BursNutsCbagsCbursCNuts AxA B1B B2B B3B B4B Chapter B 1 -F B 1 xC B 2 -F B 2 xC CxC F1F LSA B 1 -F B 2 -F B 3 -F B 3 -F American Chinese3159 Chinkapin4250 Total70987

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 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

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

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.

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

Table 1. Number of trees in canker size classes in Cross Type Size Class (cm) _________ ______________________________________________________________________________ American352 F 1 Nanking243 Seedling Chinese273 Meiling Chinese122 Nanking Chinese32 F 2 Mahogany B 1 -F 2 ClapperxGraves ______________________________________________________________________________ Table 2. Number of trees in canker size classes in Cross Type Size Class (cm) __________ ________________________________________________________________________________ American F 1 Nanking 1231 Chinese3336 B 2 -F 2 Clapper B 2 -F 2 Mahogany ________________________________________________________________________________

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

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

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.

The goal.