Sherry Flint-Garcia USDA-ARS Columbia, MO Development and Characterization of Maize-Teosinte Introgression Libraries Sherry Flint-Garcia USDA-ARS Columbia, MO

Outline Introduction Development of Teo NIL Libraries Applications of Teo NIL Libraries Lofty Ideas

Evolutionary Genetics of Maize Selection Domesticated from Zea mays ssp. parviglumis, existed/exists in an intermediate form of landraces Single domestication event in Mexican highlands 6,000~9,000 years ago

Impact of Artificial Selection GENOMICS Nested Association Mapping (NAM) BREEDING Germplasm Enhancement of Maize (GEM) Teosinte Introgression Libraries GENETICS Teosintes Maize Landraces Inbred Lines Unselected (Neutral) Gene Domestication Gene Improvement Gene Artificial Selection Plant Breeding Domestication 98% (~49,000) maize genes 2% (~1,000) maize genes

Development of Teo NIL Libraries B73 × teosinte (parviglumis) 10 accessions F1 : B73 × teosinte teosinte BC1 BC2 BC3 BC4 B73 F1

Library Development 161 BC4S2 NILs: 2 libraries (done in 2009) 643 BC4S2 NILs: 8 libraries (done in 2008) 83 BC4DH NILs: duplicated library 887 Near Isogenic Lines (NILs) Z031E0035 Z035E0012 Illumina GoldenGate 768 SNP assay

Expected Genotype Ratios for BC4S2 (sibbed): Introgression Stats # Avg. # Avg. % Avg. % Avg. % Population Lines Regions Teosinte Het. Teozygous Coverage Ames 21785 96 2.5 4.5 2.9 1.6 4.3 Ames 21786 87 2.3 4.4 2.9 1.5 3.8 Ames 21789 93 2.5 3.9 2.9 1.1 3.6 Ames 21814 77 2.1 3.8 2.5 1.3 2.9 Ames 21889 91 2.7 4.7 3.3 1.3 4.2 Ames 21809 79 2.7 4.9 3.1 1.7 3.8 Ames 21812 82 2.1 4.6 3.1 1.5 3.8 PI 384065 53 2.5 4.2 2.4 1.7 2.2 PI 384066 64 2.2 4.0 2.5 1.5 2.6 PI 384071 82 2.3 4.1 2.6 1.5 3.3 PI 384071 - DH 83 1.4 1.9 0.1 1.8 1.6 Expected Genotype Ratios for BC4S2 (sibbed): 95% BB, 3% BT het., 1.5% TT

Library Coverage c1 c2 c3 c4 c5 c6 c7 c8 c9 c10 10 maize-teosinte libraries 804 BC4S2 NILs and 83 BC4DH NILs Each line: 2.3 chromosomal segments 4.1% of the teosinte genome 3.3X genome coverage

BC4S2 vs BC4DH Coverage c1 c2 c3 c4 c5 c6 c7 c8 c9 c10

Applications of Teosinte NILs

1. Empirical Genetics Questions 1000 Selected Genes What do these selected genes do? What traits were targeted by artificial selection during domestication/breeding? Are selected genes important? Tillering/branching? ? Auxin-mediated Protein Degradation Auxin response factor, ARF1 0.01 0.02 1000 2000 3000 1 (bp) Diversity (π) Inbreds Teosinte Could be any trait!

2. Evaluate Allele Series Trait Value B73 Maize Alleles Teosinte Examine the range of allele effects of maize versus teosinte. Validate QTL identified by NAM.

Nested Association Mapping (NAM) Flowering Time A Brief Digression Nested Association Mapping (NAM) Flowering Time Ed Buckler, Jim Holland, Mike McMullen, et al Buckler, et al. (2009) Science

Flowering Genetic Architecture At least 39 QTL explain flowering architecture Genetic architecture of maize is very complex and very different than Arabidopsis. Buckler, et al. (2009) Science

Many QTL with small Effects Significant QTL 24h 36h Increase Flowering Time Decrease Flowering Time Additive Allelic Effects 333 alleles significant at P = 0.05. Only 7 alleles had more than 24 hour effect. Buckler, et al. (2009) Science

……………….………………. QTL……………………….………. Allele Series 1 ……………….………………. QTL……………………….………. 39 Additive Allelic Effects (days) 69% of QTLs had both alleles with both positive and negative effects. Buckler, et al. (2009) Science

“Gene Stacking” Earliest Latest Large differences in parental lines (32 day span) are the product of stacking large numbers of modest effect QTL. Buckler, et al. (2009) Science

Back To Teosinte

Flowering Evaluation ~850 NILs from 10 libraries Days to Anthesis (DTA) from: Ithaca, NY (2 reps), Rebecca Nelson & Oliver Ott Ithaca, NY (2 reps), Buckler lab Raleigh, NC (8 reps), Peter Balint-Kurti Madison, WI (1 rep), Loren Trimble St. Paul, MN (1 rep), Nathan Springer Columbia, MO (4 reps) TOTAL 18 reps

Teosinte Introgressions vs. NAM Photo- period Gene? Teosinte NILs Additive Effect (days) -1.0 -1.1 -2.6 -4.1 0.7 -0.9 0.7 -2.9 -0.7 -2.3 -1.0 -1.2 1.0 0.7 1.6 3.2 4.2 2.2 1.1 2.3 1.9 1.3 2.2 2.4 2.8 0.8 3.8 4.5

3. Reintroduce Genetic Variation Sucrose UDP Glucose ADP Glucose Amylose Amylopectin sh1 : sucrose synthase sh2, bt2 : ADP-glucose pyrophosphorylase su1 : debranching enzyme ae1 : SBE-IIB wx1 : g.b starch synthase Selected Genes in Maize Starch Pathway; Whitt et al. 2002 Biological hypothesis: A loss of genetic variation results in a loss of phenotypic variation. Breeding hypothesis: We can improve modern traits.

Kernel Traits Zein Profile α family β γ δ seed size 10 20 30 40 50 60 70 80 Carbohydrate Protein Fat Percent of Kernel Weight Teosinte (N = 11) Landraces (N = 17) Inbred Lines (N = 27) β γ α family δ Zein Profile Flint-Garcia et al. (2009) TAG

Teosinte NIL Evaluations Hoekenga – Iron Bioavailability Tracy – Germination AgReliant – Agronomic Traits Buckler – Flowering Harmon – Circadian Clock Nelson – NCLB Brutnell – Shade Avoidance Response Dallo – Mal de Rio Cuarto Disease Balint-Kurti – SCLB and GLS Smith – Smut Hibbard – Corn Rootworm Resistance Flint-Garcia – Starch, Protein, Oil Zein Profile Amino Acid Profile

Lofty Ideas ? Agronomics Fertilizer Density Mechanization 7000 BC 2010 Select only on yield ideotype

Teosinte Synthetic 75% B73 (SS), 25% teosinte

NSF Maize Diversity Project Acknowledgements Syngenta AgReliant NSF Maize Diversity Project www.panzea.org