Applying genomic tools to loblolly pine Adding value to our germplasm and our products

W. Patrick Cumbie, Dudley A. Huber, Salvador Gezan, Victor Steel, and Michael Cunningham

January 13, 2020

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Summerville, SC –Global HQ

Whakatane, NZ –Australasia HQ

Australasia• 20MM in Sales• 40% of NZ Pine Mkt• 20% of Aus Pine Mkt

North America• 340 MM in Sales• ~1/3 of Pine Market• 80% of MCP

Leading seedling producer with 450 million trees per year

Global operations• Southern U.S.• Brazil• New Zealand & Australia

Providing step-changes in tree productivity

• Faster growth • Improved log & wood quality• Disease resistance• Biomass production

ArborGen OverviewGlobal Leader in Seedling Genetics & Production

Campinas, SP, BR –S. America HQ

Australasia• 22MM in Sales• ~40% of NZ Pine Market• 20% of Australian Pine

Market

Brazil• 65 MM in Sales• Eucalyptus• 10 M Elite Pine

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ArborGen Nursery Platforms

Bare Root Nursery Containerized Nursery

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Three Major Categories of Loblolly Genetics Today

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MCP ® MCP-Select MCP-Elite Varieties1, 2, 3, 4….

Mass Controlled PollinatedOpen

Pollinated Varietals

• Multiple copies of best MCP seedlings, selected from extensive trials

• With the acquisition of CellFor, ArborGen is the only company in the world with the ability to produce varieties at scale

• Produced from best mother & fertilizedwith pollen of an unknown father tree

• Seedlings produced from best mother and father

• ArborGen’s has the most advanced and most broadly adapted MCP pipeline in the industry

OP Advanced, Select & Elite

Elite Genetics Products

ADVANCING GENETICS THROUGH BREEDING

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MCP® ProductionOver 900K pollination bags installed in 2019

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What type of tree do we seek?

Growth Stem Straightness

Rust Resistance

ReducedForking

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Why pursue genomic prediction?

• Breeding cycle: 12+ years

• Seed production: 8-10 years

• Seed development after pollination: 2 years

• Seed orchard lifespan: 25-30 years

• Crop rotation: 25 years

Breeding, Testing, Selection

Orch.Estab.

Orchard Development Seed Nursery

12 yr 2 yr8-10 yr2 yr 1 yr

2019 2031 2043 2046

Genomic Prediction/MAS

QCFingerprinting

QCPurity Testing

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Objectives for ArborGen genomics

Examine variation and marker-trait relationships with genomic approaches in ArborGen’s populations of loblolly pine.

• Develop a SNP resource• Explore Genomic Selection (GS)• Develop and evaluate Marker Assisted Selection (MAS)

opportunities• Pilot scale MAS deployment• Implement DNA fingerprinting to correct errors

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Where to apply genomic technology?

Test

Select

Breed

Test

Select

Breed

Test

Select

Breed

1st

2nd

3rd

Population Improvement Deployment

Elite Parents Superior crosses & varietals

Marker Assisted Selection

Marker Assisted Selection

Marker Assisted Selection

Genomic Selection

Genomic Selection

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SNP Resource Development

• Consortium Axiom 50K SNP array now available• Custom 220 SNP AgriSeq panel for fingerprinting/QC

SNP Discovery

• Internal SNP discovery

• Internal populations

• 320K SNPs

Addition of public SNPs

• PINEMAP (200K SNPs)

• UCD – Neale Lab (10K SNPs)

Axiom Screening Array

• 192 genotypes

• Parents representing 5 populations

• 360K SNPs

Axiom 50K Array

• 3 populations• 2

provenances• 25-30K SNPs

in each population

0

200

400

600

1 88 175

262

349

436

523

610

697

784

871

958

1045

1132

1219

1306

1393

1480

1567

1654

1741

DNA Conc. (ng/uL)

99% sample success

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Coastal Varietal Population & Experimental Design

• 3938 varieties (somatic embryogenesis)• 63 Full-sib families from 23 parents• Varieties within families: 1 to 225• 78 trials in 24 series• Experimental designs: Row-column, RCB• Established in the field: 2001 – 2013• Common checklots: 2-4 across all trials• Pedigree connections across series• Number of crosses & varieties within

cross varied by company

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Varietals are produced through somatic embryogenesis

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Preparing the analysis

• 1920 samples genotyped out of 3938 in the dataset• 1572 genotypes kept in the analysis

• Incorrect pedigree, planned duplicates, unplanned duplicates• H-BLUP performed for all traits in ASReml• 𝑌𝑌𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 = 𝜇𝜇 + 𝑆𝑆𝑖𝑖 + 𝑟𝑟𝑖𝑖 𝑖𝑖 + 𝑔𝑔𝑔𝑔𝑔𝑔𝑖𝑖 + 𝑔𝑔𝑔𝑔𝑔𝑔𝑖𝑖 + 𝑠𝑠𝑔𝑔𝑔𝑔𝑖𝑖𝑖𝑖 + 𝑔𝑔𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑖𝑖 𝑖𝑖𝑖𝑖 + 𝑆𝑆 × 𝑔𝑔𝑟𝑟𝑐𝑐𝑠𝑠𝑠𝑠𝑖𝑖𝑖𝑖𝑖𝑖 +

𝑆𝑆 × 𝑔𝑔𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑖𝑖𝑖𝑖(𝑖𝑖𝑖𝑖) + 𝑟𝑟 × 𝑔𝑔𝑟𝑟𝑐𝑐𝑠𝑠𝑠𝑠𝑖𝑖 𝑖𝑖 𝑖𝑖𝑖𝑖 + 𝑐𝑐𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖• Individual predictions were deregressed for SNP analysis

0.00

0.10

0.20

0.30

0.40

0.50

Height DBH Volume Rust Straightness Forking

h2 H2

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Markers selection for MAS

Trait # Significant SNPsCorrelation True-

Predicted Comment

Height 201 0.81 58 SNPs in common Ht, DBH, VolDBH 103 0.76

Volume 327 0.83

Rust 63 0.84 3 large effect SNPs

Straightness 147 0.89

Forking 300 0.90

• MAS models developed from varietal population can be applied to families including the same parents.• Screen new varieties from the same parents• Screen seedlings from MCP families for RC production

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Marker Assisted SelectionPredictions for volume within specific families• The population was subsampled 10 times for cross-validation

• Each run had 30 varieties removed by random selection.• Model was generated on 1523 samples

Model results for 1553 individuals included in the analysis (no missing data)

r = 0.824

SNPs screened using BayesCpi model and selected based on probability of inclusion. Selected subset of SNPs then run in Bayesian LASSO (GS3)

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Subsampling Results

Sampling Run r

1 0.815

2 0.894

3 0.836

4 0.737

5 0.638

6 0.773

7 0.863

8 0.972

9 0.858

10 0.852

Mean 0.824

Std Dev 0.087

Correlations ranged from 0.638 up to 0.97 with random sampling, but the average of the 10 runs is nearly identical to the correlation from the whole population true vs predicted results with no missing data.

R = 0.97

R = 0.85R = 0.63

R = 0.81

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Application of MAS • Less than 5% of varieties move forward in

testing pipeline.• Pre-screen untested trees

• Genotype larger sets of varietals in the lab prior to field trials

• Develop cost-effective low-density SNP arrays to screen more individuals

• Reduce testing footprint and costs • Test fewer genotypes across more sites

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Improving fusiform rust resistance

Phenotypes scored

Resistance marker “CC” = 11%

Susceptible marker “CT” = 57%

00.10.20.30.40.50.60.70.80.9

1

110

921

732

543

354

164

975

786

597

310

8111

8912

9714

0515

1316

2117

2918

3719

4520

5321

6122

6923

7724

8525

9327

0128

0929

1730

2531

3332

41

Baye

sCp

post

erio

r pro

babi

lity

SNP Marker

DominantSNP AdditiveSNP

Seed from 2 families was sent to the USDA Forest Service Resistance Screening Center

Chart1

CCL123x1

CTL123x1

CCL127x1

CTL127x1

CCL73x1

CTL73x1

CCL77x1

CTL77x1

CCL9 &103x1

CTL9 &103x1

CCL9 &107x1

CTL9 &107x1

Coastal South Carolina Inocula

Lower Gulf Coastal Plain

S Georgia/ N Florida

Mean

% Rust Incidence

0.0701754

0.5901639

0.1111111

0.6857143

0.1481481

0.530303

0.1176471

0.5294118

0.1071429

0.453125

0.1176471

0.6470588

Sheet1

Analysis Variable : Gall2

RSC-CodeInoculumSNPN ObsNMeanStd DevMinimumMaximum

M-5273x1L12CC57577%0.257713101

M-5273x1L12CT616159%0.495884701

M-5287x1L12CC363611%0.318727601

M-5287x1L12CT353569%0.471008201

M-5273x1L7CC545415%0.358582501

M-5273x1L7CT666653%0.502905301

M-5287x1L7CC686812%0.324585201

M-5287x1L7CT515153%0.504100801

M-5273x1L9 &10CC565611%0.312093901

M-5273x1L9 &10CT646445%0.501733101

M-5287x1L9 &10CC515112%0.325395701

M-5287x1L9 &10CT686865%0.481437701

3x1

Sheet1

Mean

Sheet2

Sheet3

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Establishing pilot plantations of marker-selected rust resistant seedlings

In the states of AL, FL, GA, MS, NC, & SC: 78% of the counties are at a 30% rust hazard or higher

Pilot plantations established Winter 2019

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Pedigree & Identity Corrections

14%

2%

• In this population there were 913 varieties genotyped.• We found 124 errors where family relationships did not match up.• We were able to correct all but 19 samples with the markers.

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Orchard DNA fingerprinting

192 SNP markers used to compare tree samples

2 Genotypes with the same name – Orchard staff noticed morphological & phenologicaldifferences

A grafted seed orchard ramet has a lifespan of 25+ years for seed productionExample 1 Example 2

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Summary

• Prediction models for marker assisted selection approaches using varietal populations to predict within families show promise. • Cross validation efforts suggest the ability to prescreen and

select before field trials• Successful identification of SNPs associated with fusiform rust

resistance• Application of fingerprinting has allowed corrections in seed

orchards at establishment - very valuable!• Near term opportunity to pre-screen trees in progeny trials• May require a change in the seed production pipeline to capture

gain and value

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Thank You

AcknowledgementsRoss Whetten, NCSCarol Loopstra, TAM

ArborGen:Chris JudyDavid Brown Hugo Palm-Leis Robert Moore Jimmy SeckingerChris RosierChristina CannistraJay CokeLes PearsonBernard FrazierJohn Mills

Thermo Fisher Scientific and its affiliates are not endorsing, recommending, or promoting any use or application o Thermo Fisher Scientific products presented by third parties during this seminar. Information and materials presented or provided by third parties are provided as-is and without warranty of any kind, including regarding intellectual property rights and reported results. Parties presenting images, text and material represent they have the rights to do so.

Applying genomic tools to loblolly pine �Adding value to our germplasm and our products���W. Patrick Cumbie, Dudley A. Huber, Salvador Gezan, Victor Steel, and Michael Cunningham��January 13, 2020Slide Number 2ArborGen Nursery PlatformsThree Major Categories of Loblolly Genetics TodayMCP® Production�Over 900K pollination bags installed in 2019�What type of tree do we seek?Why pursue genomic prediction?Objectives for ArborGen genomicsWhere to apply genomic technology?SNP Resource DevelopmentCoastal Varietal Population & Experimental DesignVarietals are produced through somatic embryogenesisPreparing the analysisMarkers selection for MASMarker Assisted Selection�Predictions for volume within specific familiesSubsampling ResultsApplication of MAS Improving fusiform rust resistanceEstablishing pilot plantations of marker-selected rust resistant seedlingsPedigree & Identity CorrectionsOrchard DNA fingerprintingSummarySlide Number 23