Restoration of American chestnut: A marriage of breeding and - PowerPoint PPT Presentation

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

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

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

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

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

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 ¡

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

Progress of selection in seed orchards at TACF’s Meadowview Research Farms Clapper Graves N (%) trees 36,000 25,000 planted (100%) (85%) N trees 3297 2244 remaining

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

Progeny testing is too slow to finish selection for blight resistance Meadowview breeding program Clapper Graves Number BC 3 F 2 trees remaining 3200 2200 N BC 3 F 2 parents progeny tested 300 300 2009-2017 N BC 3 F 2 to select 300 300 ¡ ¡ N trees remaining after selection based on canker size >> N trees progeny tested

Development/validation of genomic prediction models for disease resistance 9/10 th of training population -0.1 µ + Χβ = +0.5 + +0.9 Experimental design Estimated marker Genome-wide markers effects Canker size/root rot severity Repeat 10 x Accuracy = correlation 1/10 th of training population N markers -0.1 Σ x = +0.5 i = 1 +0.9 Predicted pathogen resistance Marker effects Marker genotypes

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 ¡11 ¡– ¡30 ¡open ¡pollinated ¡progeny ¡of ¡each ¡BC3-­‑F2 ¡mother ¡

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

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

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

Transgenic outcross simulation: N generations & N American parents/generation N outcross N American Ne 2 nd Avg. F 2 nd generations parents per Intercross intercross generation 2 2, 1000 154 0.08 2 500, 500 297 0.03 3 2, 500, 500 190 0.02 3 500, 500, 500 530 0.009 4 250, 250, 250, 250 736 0.004 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

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

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

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

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)

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

Some Graves families have high levels of root rot resistance 100 90 Scaled breeding values 80 ● ● PRR resistance 70 ● ● ● ● 60 ● ● ● ● 50 ● ● ● 40 ● ● ● 30 ● 20 ● 10 0 − 10 American chestnut Chinese chestnut Clapper B3 − F2s Graves B3 − F2s

Resistance to chestnut blight and Phytophthora root rot uncorrelated 0.3 ● ● ● ● ● ● ● ● ● ● 0.2 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 0.1 ● ● ● ● ● ● PRR mortality BLUPs ● ● ● ● ● ● ● ● ● ● ● ● 0.0 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● − 0.1 ● ● ● ● ● ● ● ● ● ● ● ● − 0.2 ● ● ● ● ● ● ● ● − 0.3 ● ● ● ● ● ● ● ● ● − 0.4 ● − 0.5 ● − 0.6 − 2.00 − 1.75 − 1.50 − 1.25 − 1.00 − 0.75 − 0.50 − 0.25 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 Blight canker severity BLUPs

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

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

Questions? www.acf.org

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) Predicted distribution of suitable habitat in 2080 Jessica Cavin Barnes (higher greenhouse gas NCSU emission scenario)