The American Chestnut Foundation

1
The American Chestnut FoundationsBreeding
Program

• F. V. Hebard
• Research Farms
• Meadowview, VA
• Fred_at_acf.org
• www.acffarms.org

2
Requirements for breeding

• Materials sources of resistance and flowering
  American chestnut trees.
• Crossing Method for making the next generation
  of progeny.
• Breeding Method the sequence of crosses to
  perform.
• Cultivation Method for growing the trees.
• Screening Methods for determining the blight
  resistance of progeny, and other traits.

3
Materials

• American chestnut trees flower in clearcuts

4
MaterialsSources of blight resistance

• The old USDA breeding program determined that
  Chinese chestnut had the most blight resistance
  of all the chestnut species. In American
  forests, the Chinese chestnut itself cannot reach
  the canopy when competing with other tree
  species. We were able to jumpstart our program
  by using two first backcrosses from the USDA
  Connecticut programs.
• The low levels of blight resistance in some of
  the large, surviving American chestnut trees
  might also be useful in a breeding program, which
  we are exploring.
• Additionally, we use Japanese chestnut as a
  source of resistance.

5
Crossing Method
Flower being pollinated
6
Pollination bag in place
7
2006 Harvest in Meadowview
Pollination Type Cross Number of Nuts Number of Nuts
Controlled Pollinations American x American 225
B1 483
B2 652
B3 83
B4 23
F1 245
Chinese x Chinese 1920
Crosses between European, Japanese Chinese 409
Large American x Chinese or Japanese 72
Large American x Small American 401
Large American x Large American 739
Open Pollinations B1-F3 2657
B2-F2 182
B2-F3 6601
B3-F2 17168
B3-F3 97
Total 31957
8
Backcrossing
Proportion Chinese
C x A F1 x A
B1 x A B2 x A
B3 x B3

B3-F2 x B3-F2
B3-F3
1/2 1/4 1/8 1/16 1/16 1/16
9
Frequency Distribution of Percent
American-Chestnut RAPD Alleles at First Backcross
10
Backcrossing
Proportion Resistant
C x A F1 x A
B1 x A B2 x A
B3 x B3

B3-F2 x B3-F2
B3-F3
all 1/4 1/4 1/4 1/16 all
11
Expected distribution of canker phenotypes and
genotypes in Chinese x American F2 progeny, based
on observation and interpolation.
12
BackcrossingNeed more than one American parent
C x A1 F1 x A2 B1 x A3 B2 x A4 B3
13
BackcrossingNeed more than one American chestnut
background
C x A13 F14 x A14 B14 x A15 B24 x A16 B34
C x A9 F13 x A10 B13 x A11 B23 x A12 B33

C x A5 F12 x A6 B12 x A7 B22 x A8 B32
C x A1 F11 x A2 B11 x A3 B21 x A4 B31
14
Intercrossing of Third BackcrossesHow do we do
it?
C x A13 F14 x A14 B14 x A15 B24 x A16 B34
C x A9 F13 x A10 B13 x A11 B23 x A12 B33
x
C x A5 F12 x A6 B12 x A7 B22 x A8 B32
C x A1 F11 x A2 B11 x A3 B21 x A4 B31 x

B3-F212 B3-F234
15
Intercrossing of Third BackcrossesB3-F2 family
size needs to be 9 or 10 to capture most alleles
from each B3
16
Mean RAPD Score in Individual Trees by Linkage
Group
Linkage Group Tree ID Tree ID Tree ID Tree ID Tree ID Tree ID Tree ID Tree ID Tree ID Tree ID Tree ID Tree ID Tree ID Tree ID Tree ID Tree ID
Linkage Group 8 21 26 30 35 41 43 45 46 48 49 62 64 74 299 252
A 2.0 1.2 1.1 1.8 2.0 2.0 2.0 1.0 2.0 1.9 2.0 1.2 1.1 2.0 2.0 1.3
B/E 1.9 1.3 2.0 1.3 1.0 1.3 1.4 1.9 1.5 1.0 1.2 2.0 1.6 2.0 1.0 2.0
C 1.5 1.0 1.9 1.9 2.0 1.8 1.8 2.0 1.0 1.0 1.9 2.0 1.1 1.1 1.0 1.5
D 2.0 2.0 1.0 1.0 1.2 2.0 1.0 2.0 1.7 2.0 2.0 1.0 1.0 1.0 2.0 2.0
F 1.6 1.0 2.0 1.2 1.0 1.0 1.0 1.1 2.0 1.0 2.0 2.0 1.0 1.7 1.0 2.0
G 1.0 1.3 2.0 2.0 1.0 1.9 1.0 1.0 1.3 1.1 1.0 2.0 2.0 1.0 1.9 1.9
H 1.7 1.0 1.0 2.0 1.0 2.0 2.0 1.0 1.0 1.5 1.0 2.0 2.0 1.0 1.0 2.0
I 1.0 1.0 1.0 1.9 1.3 1.6 2.0 1.1 1.9 2.0 1.1 1.9 1.8 2.0 1.7 2.0
J 2.0 1.0 1.0 1.1 2.0 1.8 1.0 2.0 1.1 1.1 2.0 2.0 1.0 1.0 1.9 2.0
L 1.1 2.0 2.0 1.2 1.9 1.0 1.0 2.0 1.0 1.0 2.0 2.0 2.0 2.0 1.0 2.0
M 2.0 2.0 2.0 1.8 2.0 2.0 2.0 1.7 1.8 2.0 2.0 2.0 2.0 2.0 2.0 2.0
Am. Non-Recomb. LGs 2 5 4 1 3 2 5 3 3 4 2 1 3 4 5 0
Chin. Non-Recomb. LGs 4 3 4 2 3 4 4 4 2 3 6 8 4 4 3 8
Total Non-Recomb. LGs 6 8 8 3 6 6 9 7 5 7 8 9 7 8 8 8
17
Frequency Distribution of Number of
Non-Recombinant Linkage Groups in Chestnut B1
18
Statistics for Markers on Linkage Groups in
Chestnut B1
Linkage Group Number of Markers Total cM Number of Trees with American Linkage Groups Number of Trees with Chinese Linkage Groups Number of Trees with Recombinant Linkage Groups Number of Trees with Recombinant Linkage Groups per cM
A 25 71.0 8 14 35 0.5
B/E 30 64.7 11 7 39 0.6
C 18 38.7 15 16 26 0.7
D 7 12.2 24 26 7 0.6
F 17 38.8 17 13 27 0.7
G 14 39.4 16 20 21 0.5
H 11 19.8 18 23 16 0.8
I 16 27.0 12 17 28 1.0
J 12 18.0 19 21 17 0.9
L 7 9.1 22 25 10 1.1
M 7 10.8 10 40 7 0.6
19
Intercrossing of Third BackcrossesWhat mating
design do we use?
C x A13 F14 x A14 B14 x A15 B24 x A16 B34
C x A9 F13 x A10 B13 x A11 B23 x A12 B33
x
C x A5 F12 x A6 B12 x A7 B22 x A8 B32
C x A1 F11 x A2 B11 x A3 B21 x A4 B31 x

B3-F212 B3-F234
20
Intercrossing of Third BackcrossesPossible
Mating Designs Complete Diallel
B3s 1 2 3 4 5 6 7
8 B3-F2s 1 11 12 13 14 15
16 17 18 2 21 22 23 24 25
26 27 28 3 31 32 33 34 35
36 37 38 4 41 42 43 44 45
46 47 48 5 51 52 53 54 55
56 57 58 6 61 62 63 64 65
66 67 68 7 71 72 73 74 75
76 77 78 8 81 82 83 84 85
86 87 88
21
Intercrossing of Third BackcrossesComplete
Diallel, No Selfing
B3s 1 2 3 4 5 6 7
8 B3-F2s 1 11 12 13 14 15
16 17 18 2 21 22 23 24 25
26 27 28 3 31 32 33 34 35
36 37 38 4 41 42 43 44 45
46 47 48 5 51 52 53 54 55
56 57 58 6 61 62 63 64 65
66 67 68 7 71 72 73 74 75
76 77 78 8 81 82 83 84 85
86 87 88
22
Intercrossing of Third BackcrossesHalf Diallel,
No Selfing
B3s 1 2 3 4 5 6 7
8 B3-F2s 1 12 13 14 15
16 17 18 2 23 24 25
26 27 28 3 34 35
36 37 38 4 45
46 47 48 5
56 57 58 6
67 68 7
78 8
23
Intercrossing of Third Backcrosses Disconnected
Partial Half Diallels, No Selfing
B3s 1 2 3 4 5 6 7
8 B3-F2s 1 12 13 14 2
23 24 3
34 4 5 56 57
58 6 67
68 7
78 8
24
Intercrossing of Third Backcrosses Disconnected
Partial Half Diallels, No SelfingTwo Parents per
Diallel
B3s 1 2 3 4 5 6 7
8 B3-F2s 1 12 2
3 34 4 5
56 6
7
78 8
25
Intercrossing of Third Backcrosses Connected
Partial Half Diallels, No SelfingTwo Parents
Circular Mating Design
B3s 1 2 3 4 5 6 7
8 B3-F2s 1 12 2
23 3 34 4
45 5
56 6
67
7
78 8 81
26
Intercrossing of Third Backcrosses One parameter
important to assessing which mating design is
best is inbreedingTo look at inbreeding
effects, we have toexamine B3-F4s, two
generations further thanB3-F2
27
Intercrossing of Third Backcross F2sHalf
Diallel, No Selfing, Two Parents per Diallel
B3-F2s 12 34 56
78 B3-F3s 12 1234
1256 1278 34
3456 3478 56
5678
78

28
Intercrossing of Third Backcross F2sHalf
Diallel, No Selfing, Two Parents per Diallel
B3-F3s 1234 1256 1278 3456 3478
5678 B3-F4s 1234 1234-1256
1234-1278 1234-3456 1234-3478
1234-5678 1256
1256-1278 1256-3456 1256-3478
1256-5678 1278
1278-3456 1278-3478 1278-5678 3456
3456-3478
3456-5678 3478
3478-5678 5678
29
Intercrossing of Third Backcross F3s Half
Diallel, No Selfing, Two Parents per Diallel
Inbreeding Coefficients
B3-F3s 1234 1256 1278 3456 3478
5678 B3-F4s 1234 1234-1256
1234-1278 1234-3456 1234-3478
1234-5678 1/8 1/8 1/8
1/8 0 1256
1256-1278 1256-3456 1256-3478
1256-5678 1/8 1/8
0 1/8 1278
1278-3456 1278-3478 1278-5678
0 1/8 1/8 3456
3456-3478
3456-5678
1/8 1/8 3478
3478-5678
1/8 5678
30
Intercrossing of Third Backcross F3s So its
fairly straightforward to calculate inbreeding
coefficients at B3-F4, except that if we increase
the number of B3 families from 8 to 20 the number
of B3-F4 cross combinations increases toward the
billions!
31
Intercrossing of Third BackcrossesDiallel size
does not affect inbreeding when pollination is
random, and when we limit the number of B3-F2
families per B3 family.
Effect of Partial Diallel Size on Inbreeding
32
Intercrossing of Third BackcrossesDiallel size
does affect inbreeding when only one male
pollinates, but not severely below diallel sizes
of 5 or 6, when the chance that pollinations will
be dominated by one male starts to diminish
sharply
Effect of Partial Diallel Size on Inbreeding When
Only One Male Pollinates
33
BackcrossingOne can select more than one
individual per B3 family without increasing
inbreeding under open pollination
Effect of B3 Family Size on Inbreeding
Inbreeding Coefficient at B3-F4
One Tree per B3 Family
Two Trees per B3 Family
Number of B3 Families
34
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35
Conclusions

• Two progeny per B3 family gives lower inbreeding
  than one
• At least nine to ten B3-F2 progeny are needed per
  B3 family to capture most alleles
• If each of those nine to ten sets of B3-F2
  progeny is segregated in a separate orchard,
  inbreeding at F4 is reduced
• Inbreeding is not strongly affected by available
  mating designs, in part because we use open
  pollination
• The American Chestnut Foundations breeding
  program will yield adequate effective population
  sizes (although the projections have a number of
  assumptions that will not hold true, decreasing
  the projected Ne)

36
Cultivation Method

• We use standard horticultural techniques adapted
  to screening chestnut trees for blight
  resistance. These are high-input horticultural
  methods, not silvicultural methods.
• Our orchard spacings are designed to grow trees
  as fast as possible until they can survive being
  screened for blight resistance long enough to
  produce a crop.
• Trees with intermediate levels of blight
  resistance need to be 3-4 years old, about 2 to 3
  inches in diameter at breast height, to survive
  inoculation.
• Trees with high levels of blight resistance can
  survive inoculation when they are younger.
• This requires a lot of land and effort compared
  to breeding field crops, and more time, of course.

37
After one growing season, the trees are about 18
inches tall.
38
Trees after one growing season.
39
After two growing seasons, trees are about 5 feet
tall.
40
And about 8 feet tall after three growing seasons.
41
About 12 feet tall after four growing seasons.
42
Number of Trees at Meadowview in 2006
Type of Tree Number of Number of Number of
Nuts or Trees Sources of Resistance American Backgrounds
American 2162 235
Chinese 692 51
F1 523 22 90
B1 425 15 33
B2 1559 10 91
B3 3818 9 77
B4 9 1 1
B1-F2 769 4 8
B2-F2 341 5 9
B3-F2 12376 2 29
B3-F3 121 1 2
F2 710 6 6
F3 6 1 1
Other 2502

Total 26013
43
Number of B3s by State in 2006
State Number of Number of Number of
Nuts or Trees Sources of Resistance American Backgrounds
Maine 1962 2 39
Vermont 110 1 2
Massachusetts 4502 2 46
Pennsylvania 4837 2 38
Maryland 689 1 11
Indiana 3931 1 19
Kentucky 802 2 7
Carolinas 1064 2 14
Tennessee 1287 1 10
Alabama 267 1 8

Total 19451
44
Resistance
Observation and Theory
45
Chestnut blight cankers are characterized also by
the presence of buff-colored mycelial fans in the
middle of the bark.
46
A photomicrograph of a mycelial fan growing
through chestnut bark.
myclelial fan
47
In American chestnut, mycelial fans form more
rapidly, in greater numbers, and grow more
quickly than in Chinese chestnut, which is
resistant to blight.
myclelial fan
myclelial fan
myclelial fan
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49
Resistance
Observation and Theory Conclusion from
Histopathology
American chestnut is recognizing and responding
to the blight fungus equally as fast as Chinese
chestnut. This implies that chestnut has a
different mechanism of resistance to blight than
occurs in biotrophic and hemibiotrophic
pathosystems, where all resistance appears to be
based on recognition of the pathogen and where
avirulence involves suppression of antigen
display. Chestnut blight resistance may or may
not be more stable than resistance to biotrophs,
but it is hard to imagine it being less
stable. Histopathology on older cankers was also
helpful to me in understanding what is occurring
inside cankers with various morphologies,
facilitating interpretation.
50
Demonstration of statistically significant
differences in canker length on American chestnut
for virulent isolates of Cryphonectria parasitica
51
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52
Resistance
Observation and Theory
Conclusion from Experiment of Previous Slide
Other Canker Measurements

• Canker size is a good metric of resistance to
  colonization. We have been unable to measure
  resistance to infection, which may be a defect in
  the program
• Canker size is strongly dependent upon
  environmental conditions, which can be
  manipulated experimentally
• Canker size can differ depending upon tree age
• This study gave results opposite of what was
  expected. It was easier to distinguish the
  intermediate blight resistance of Chinese x
  American F1s from susceptible American chestnut
  when trees were young, and easier to distinguish
  intermediate resistance from blight-resistant
  Chinese chestnut when trees were old.
• However, trees with low to intermediate levels of
  blight resistance do not survive inoculation
  until they are three years old, so need to be
  screened when older, while more resistant trees
  can be screened when young, occupying less space
  and time, but not as young as I had thought.

53
Resistance
Practical Screening
54
Blight-resistant and blight-susceptible Clapper x
Graves B1-F2 progeny, characterized by small and
large cankers, respectively, during third growing
season.
55
Blight-resistant Chinese to American B2-F2, 5
years old, 2 years after inoculation with
Cryphonectria parasitica.
56
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57
Blight-Resistant Clapper B3-F2
58
Chestnut Blight Resistance Rating Scheme
Rating
Strain of Cryphonectria parasitica
1
2
3
4
5
Ep155
Canker size
small
medium
large
larger
largest
SG1 2-3
Canker size
small
medium
large
small
small
59
Number of Clapper B3-F2 seedlings in resistance
classes after inoculation with the blight fungus
in 2004 and 2005.
60
Number of Clapper B3-F2 seedlings in resistance
classes after inoculation with the blight fungus
in 2006.
61
Number of Graves B3-F2 seedlings in resistance
classes after inoculation with the blight fungus
in 2006.
62

• Blight-resistant Clapper x Graves B2-F2, 15
  years old, inoculated 13 years ago.
• Obtaining trees like this suggests it should be
  possible to backcross the blight resistance of
  Chinese into American chestnut.