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Leaf Epicuticular Wax Load Segregation in the Switchgrass (Panicum virgatum) Panicum virgatum 4WCR Population • Widespread distribution

• Outcrossing C4 perennial grass Jennifer Bragg, Lisa Chanbusarakum, Thomas Juenger, Christian Tobias USDA, ARS, WRRC • 2 main ecotypes: Upland and Lowland PAG Perennial Grasses Workshop • Polyploid (primarily 4x and 8x) January 16, 2018 • Adapted to diverse habitats

• Productive on marginal lands

• High yielding (7-10 tons/acre)

• DOE JGI genome sequencing

Switchgrass Adaptations: Upland Lowland Switchgrass Diversity Upland and Lowland Ecotypes • Highly polymorphic • Phenotypic diversity – Cold/heat tolerance Northern Uplands – Disease susceptibility Short growing seasons – Heading date / Flowering time Cold winters – Growth habit: bunchgrass vs rhizomatous Strong photoperiod & temperature signals – Tiller number, height, diameter – Leaf spectral qualities / Color Casler et al. 2011

Southern Lowlands Northern Upland Southern Lowland Long growing seasons Mild winters Variable precipitation

Investigating the Genetic Architecture of Upland/Lowland Traits in Switchgrass

4WCR Mapping Population Alamo Dacotah Summer West Austin

•Founders: 2 Upland and 2 Lowland tetraploids

•800 F2 progeny lines Can we discover the genes/genetic changes •Genotyped using ddRADseq responsible for adaptation and stress tolerance in natural populations of switchgrass? •Genetic linkage map (400 lines)

•Propagated in 2014

•Planted in 10 field locations 2015

Milano et al. G3 2016

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Plant Cuticle and Surface Waxes Genetic Improvement of Tallgrass Prairie Species •Identifying sequences to inform marker assisted selection in order to provide increased speed/flexibility to breeding efforts

Glaucousness / Epicuticular wax load

Tolerance to environmental stresses: •Heat and Drought Shepherd and 2006 Yeats and Rose 2013 •UV Radiation •Herbivory and Fungal pathogens

Increased fitness Higher yields

Hegebarth and Jetter 2017

Variation in Epicuticular Wax Between Ecotypes Switchgrass Wax Composition Lowland AP13 Upland VS16 Adaxial Lowland Upland Switchgrass wax % β-diketones 69

Hydroxy β-diketones 6 Hydrocarbons 4 Abaxial Esters 3 Free acids 2 Triterpene alcohols 2 Free alcohols 1 Unidentified species 13 Casler et al. 2011 10 µm Hegebarth and Jetter 2017 Tulloch and Hoffman 1980 Epicuticular Wax SEM

Quantitative Colorimetric Wax Assay Acidified Potassium Dichromate Can We Identify Genomic Regions Associated with Switchgrass Wax Load?

Leaf sample area measured Reaction of wax extract with K2Cr2O7 0 0.1 0.2 0.3 0.4 0.5 Switchgrass Field Sites sampled for this study

•Initiated sampling at emergence of panicles on AP13

Pv Wax Standards nd 0.6 •Collect 2 youngest, mature, 0.4

595 collared leaf

OD 0.2 R² = 0.989 0 •Leaves cold packed at field location Surface Wax Extracted in CHCl 0 0.2 0.4 0.6 3 mg swg wax and shipped to WRRC for wax Read absorbance at 595 nm measurements •Sampled from May to October 2017 •Austin, TX 30.26 °N •Columbia, MO 38.95 °N •Hickory Corners, MI 42.44 °N •Lincoln, NE 40.82 °N

Ebercon et al. 1977

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50 PKLE F Normal distribution 40 2 Wax measurements and Disease Severity Observed distribution Comparison of Wax Measurements 30 Between Field Sites 20

Leaf Sample Disease Ratings Frequency 10 Healthy Low Moderate Severe

100% 0

1.6 2.4 3.2 4.0 4.8 5.6 6.4 7.2 8.0 8.8 9.6

90% Healthy 0.8 Less 80 10.4 80% Wax (mg/dm2) 70 CLMB F2 70% F0 averages 60% 60 50 50% VS16 DAC6 40

% % total 40% 30 AP13 WBC3 30% Light Frequency 20 20% 10 10% 0

0%

0.6 1.2 1.8 2.4 3.0 3.6 4.2 4.8 5.4 6.0 6.6 7.2 7.8 8.4 9.0 9.6 Less PKLE CLMB KBSM LINC 30 Wax (mg/dm2) Disease rating LINC F Moderate 25 2 Average wax vs Disease Rating 20

Healthy Low Moderate Severe 15 2 5 Frequency 10 Broadsense heritability H 5 4 All sites 0.15 Severe 0

Narrow-sense heritability h2 SE

0.6 1.2 1.8 2.4 3.0 3.6 4.2 4.8 5.4 6.0 6.6 7.2 7.8 8.4 3 35 Less Wax (mg/dm2) 30 KBSM F2 PKLE 0.27 0.09 2 25 CLMB 0.19 0.08

20 Wax (mg/dm2)Wax 1 15 LINC 0.01 0.11 Excluded Frequency 10 KBSM 0.15 0.08 0 5 PKLE CLMB KBSM LINC

0 No cytoplasmic effects detected

1.6 2.4 3.2 4.0 4.8 5.6 6.4 7.2 8.0 8.8 9.6

Disease rating 0.8

Less 10.4 Wax (mg/dm2)

Average Wax/Cultivar Across Four Sites Preliminary QTL Analyses

AP13 WBC VS16 DAC qWAX-07K F0 Average Wax qWAX-05K 6.00 * Bars = SE 5.00

4.00 Wax 3.00

2.00 Wax (mg/dm2)Wax

1.00

0.00 Cluster of cytochrome P450 Long-chain-alcohol O-fatty- AP13 WBC VS16 DAC PKLE CLMB LINC KBSM oxidase family genes similar to acyltransferase / Wax synthase Site(ordered by latitude) barley CER-U Texas Texas CER2 acetyltransferase Very-long-chain beta-ketoacyl- Missouri CoA synthase genes Michigan CER6/CUT1 chalcone synthase

CER9 membrane assoc. ring finger

Alleles associated with wax content Conclusions Effect Plot for qWAX-07K Effect Plot for qWAX-05K Developed quantitative assay for measuring wax in switchgrass Austin Austin Identified differences in wax loads between ecotypes / cultivars with GxE effects

Identified two dominant QTL associated with wax load in the 4WCR population

Candidate genes identified within QTL regions AC BC AD BD AC BC AD BD Columbia Future Work

Allele assignments Refine QTL analysis when the additional 400 F2 lines are added to the map A : AP13 (Low) Repeat wax phenotyping for a second season B : Dacotah (Up) C : WBC (Low) Compositional analyses of wax in the F0 cultivars D : VS16 (Up)

Measure changes wax with developmental age of tissue in F0 plants AC BC AD BD

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Collaborators Thomas Juenger UT Austin Jason Bonnette UT Austin David Lowry Michigan State University Christian Tobias Lisa Vormwald Michigan State University Sheyla Aucar Felix Fritschi Mizzou Lisa Chanbusarakam Matthew Smith Mizzou Rob Mitchell USDA-ARS, Lincoln NB Prisca Cheng John Sanley USDA-ARS, Lincoln NB Toni Mohr Tina Williams

Jeremy Schmutz JGI/HudsonAlpha John Lovell Hudson Alpha Jerry Jenkins HudsonAlpha Avinash Sreedasyam HudsonAlpha Sujan Mamidi HudsonAlpha

Shiangqiang Shu LBL Dan Rokhsar DOE JGI Kerrie Barry DOE JGI

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