Restoration of American chestnut: A marriage of breeding and biotechnology

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1 Restoration of American chestnut: A marriage of breeding and biotechnology Jared Westbrook The American Chestnut Foundation National Academy of Science Forest Health Webinar December 12, 2017

2 Early breeding programs to introduce blight resistance from Asian Castanea spp. into American chestnut American chestnut Not resistant to blight Dominant canopy tree Chinese chestnut Resistant to blight Orchard tree F 1 Intermediate blight resistance, not competitive in the forest

3 The American Chestnut Foundation s Backcross breeding program Three generations of backcrossing to recover American chestnut form Charles Burnham Two generations of intercrossing to enhance blight resistance

4 Sources of resistance and backcross lines Graves Clapper BC 1 x 20 Americans 20 BC 2 x 20 Americans Clapper and Graves are BC1 trees from early breeding programs have been bred as distinct sources of resistance Fred Hebard TACF Emeritus

5 TACF s chapters breed backcross hybrids with American chestnuts throughout the range

6 Backcross trees intercrossed to enhance blight resistance BC 3 BC 3 BC 3 F 2 X = 1 of 64 of the BC 3 F 2 progeny expected to inherit 3 genes for blight resistance from both parents

7 Selection against blight susceptibility in seed orchards Artificially inoculate stems at age two with C. parasitica

8 Progress of selection in seed orchards at TACF s Meadowview Research Farms Clapper Graves N (%) trees planted N trees remaining 36,000 (100%) 25,000 (85%)

9 Progeny testing to make final selections for blight resistance after initial culling Unselected BC 3 F 2 seed orchard Partially selected seed orchard Ar0ficially inoculate & cull suscep0ble Open-pollination among BC 3 F 2 survivors Average canker severity Artificially inoculate BC 3 F 3 progeny from each selection candidate

10 Progeny testing is too slow to finish selection for blight resistance Meadowview breeding program Clapper Graves Number BC 3 F 2 trees remaining N BC 3 F 2 parents progeny tested N BC 3 F 2 to select N trees remaining after selection based on canker size >> N trees progeny tested

11 Development/validation of genomic prediction models for disease resistance 9/10 th of training population µ + Χβ = + Experimental design Canker size/root rot severity 1/10 th of training population Estimated marker effects Genome-wide markers Repeat 10 x Accuracy = correlation N markers Σ i = Marker effects x Marker genotypes = Predicted pathogen resistance

12 Proof-of-concept Genomic prediction of blight resistance of BC 3 F 3 progeny Weak strain Strong strain N individuals = 47 BC 3 F 2 N SNPs = 22,397 Response variable: BLUPs es0mated from canker ra0ng and canker lengths of open pollinated progeny of each BC3- F2 mother

13 Intermediate blight resistance expected after selection is complete in seed orchards Scale 100 = American chestnut average 0 = Chinese chestnut average Dashed line prediction of average canker severity of BC 3 -F 3 s after selection is complete in BC 3 -F 2 seed orchards

14 American chestnut BC 3 F 2 trees with improved blight resistance BC 3 F 2 tree American chestnut

15 Incorporating transgenic blight resistance into TACF s breeding program Transgenic pollen Bill Powell SUNY-ESF Chuck Maynard SUNY-ESF ~50% of offspring inherit the oxalate detoxifying gene Pollinate wild-type mother trees Harvest nuts and test for inheritance of OxO

16 Transgenic outcross simulation: N generations & N American parents/generation N outcross generations N American parents per generation Ne 2 nd Intercross Avg. F 2 nd intercross 2 2, , , 500, , 500, , 250, 250, Effective pop. size (Ne) greater and inbreeding (F) less with: Equal N American parents per generation than with bottleneck Increasing number of outcross generations Ne > 500 with 3 outcross generations & 500 American parents per generation

17 Outcrossing transgenic founder to diversify, minimize inbreeding, and stack resistance 3 generations of outcrossing to dilute the founder genome Backcross + OxO resistance stacking Outcross to 200 backcross selections/ generation x 3 generations, 1 resistance source/generation Transgenic American chestnut Create diverse pure American lines Outcross to 300 Americans/generation x 3 generations Clapper Nanking Graves Intercross to generate large quantities of seed for restoration x 500 American chestnuts per generation

18 American chestnut genetic diversity conservation Collecting pollen and seed from rare flowering wild trees Transplanting the wild trees and conserving the trees in orchards Objective: 2500 new sources of American chestnut planted in germplasm conservation orchards by 2020

19 Chestnut blight is not the only impediment to American chestnut restoration Phytophthora root rot caused by soil-borne pathogen Phytophthora cinnamomi Imported to N. America from S.E. Asia in 1800 s The North American and European Castanea species are susceptible, while the Asian species are resistant

20 Range of Phytophthora cinnamomi predicted to expand northward as climate warms Predicted range of P. cinnamomi by 2080 (Burgess et al. 2017) Range of P. cinnamomi as of 1945 (Crandall et al. 1945)

21 Screening backcross populations for resistance to Phytophthora cinnamomi Artificial inoculation with P. cinnamomi Severity ra0ng of root lesions Photos by S.N. Jeffers Measurement of above ground wilting and mortality

22 Some Graves families have high levels of root rot resistance Scaled breeding values PRR resistance American chestnut Clapper B3 F2s Graves B3 F2s Chinese chestnut

23 Resistance to chestnut blight and Phytophthora root rot uncorrelated PRR mortality BLUPs Blight canker severity BLUPs

24 Additional breeding required to combine blight and PRR resistance PRR resistant BC3-F2 selections X X Blight resistant transgenic outcross progeny Intercross to increase PRR resistance Select for PRR resistance and deploy X 2 generations of breeding and selection

25 Restoration of American chestnut depends on: Enhancing blight resistance so trees can reproduce in native range Combining blight and Phytophthora resistance in southern forests Having sufficient genetic diversity to adapt to a changing world

26 Questions?

27 The future: distribution of suitable habitat expected to shift North with climate change Current distribution of suitable habitat Predicted distribution of suitable habitat in 2080 (lower greenhouse gas emission scenario) Jessica Cavin Barnes NCSU Predicted distribution of suitable habitat in 2080 (higher greenhouse gas emission scenario)