Using Genetics, Genomics, and Breeding to Understand Diverse Maize Germplasm

Transcription

1 Using Genetics, Genomics, and Breeding to Understand Diverse Maize Germplasm Sherry A. Flint-Garcia United States Department of Agriculture Agriculture Research Service (USDA ARS) Corn breeding and genetic analysis of agronomic traits relies on phenotypic variation and genetic variation in the genes controlling these traits. My research program focuses on understanding genetic diversity in maize so we can mine beneficial alleles from the appropriate germplasm sources for continued corn improvement. The process of domestication that began 9000 years ago has had profound consequences on maize, where modern corn has moderately reduced genetic diversity across nearly all genes in the genome relative to teosinte, and severely reduced levels of diversity for key genes targeted by domestication. The question that remains is whether these reductions in genetic diversity have impacted our ability to make progress in corn breeding today. As an outcrossing species, maize has tremendous genetic variation compared to most other crops. The complementary combination of genome-wide association mapping (GWAS) approaches, large HapMap datasets, and germplasm resources are leading to important discoveries of the relationship between genetic diversity and phenotypic variation in inbred lines. However, among the traits targeted during domestication and breeding are many yield component traits, including number of ears, kernel row number, seed size, and kernel composition. Therefore, we must reintroduce variation from landraces and/or teosinte if we hope to learn how domestication has impacted these yield component traits, and yield itself.

2 Using Genetics, Genomics, and Breeding to Understand Diverse Maize Germplasm Sherry Flint-Garcia USDA-ARS Columbia, MO

3 Outline Introduction to Maize Domestication & Diversity Inbred Lines Teosinte, the wild ancestor Breeding with Zea

4 Maize Domestication Teosinte (ssp parviglumis) Landraces Inbred Lines Artificial Selection Domesticated from Zea mays ssp. parviglumis Single domestication event ~9,000 years ago in Mexico Intermediate form of landraces; populations adapted across the Americas to specific microclimates and/or human uses

5 Consequences of Artificial Selection Zea Synthetic Teosinte NILs Teosinte Synthetic Unselected (Neutral) Gene Domestication Gene Improvement Gene Teosintes Domestication Artificial Selection Maize Landraces Plant Breeding Maize Inbred Lines 98% (~49,000) maize genes Nested Association Mapping (NAM) Maize ATLAS Project 2% (~1,000) maize genes Germplasm Enhancement of Maize (GEM)

6 Inbred Lines

7 Linkage-Based QTL Mapping Genome Scan Identify genomic regions that contribute to variation and estimate QTL effects Position (cm) Parent 1 F 1 Parent 2 F 2 population Genotype Phenotype Statistics for Mapping u1622 u1552 b2277 m231 b2248 b1225 b2077 b1520 LOD Score

8 Association Analysis Utilize natural populations Exploit extensive historical recombination Ancestral chromosomes Subject Population Candidate gene approach: Gene X Line 1 A C G A G 1.3m Line 2 A C G A T 1.4m Line 3 A T A A G 1.5m Line 4 C T A G T 1.8m A T G G T 2.0m Line N A C G G G 2.5m

9 Linkage vs. Association Analysis Linkage (QTL) Mapping Structured population BC, F2, RIL, etc Analysis of 2 alleles High power Low resolution cm (10-20 Mb in maize) Genome scan Don t know anything about the genetics underlying the trait Nested Association Mapping Association Mapping Unstructured population Unrelated or distantly-related Analysis of many alleles Low power High resolution bp in maize * * Candidate gene testing Pathway or candidates previously identified. Used as a validation method. * Genome Scan Don t know anything about the genetics underlying the trait. Used as a discovery method * Depends on the species/population

10 NAM Founders 0.1 NC33 B115 I137TN 81-1 MEF IL677A Ia5125 IA2132 IL101 F2 EP1 F7 CO255 NC366 B52 SC213R NC238 GT112 Mp339 GA209 D940Y T232 U267Y CI28A B2 F2834T Mo24W MS1334 IDS28 I-29 SA SG18 Sg1533 IDS91 IDS69 F6 F44 Ab28A CML328 Oh603 A441-5 CML323 SC55 NC264 NC370 NC320 NC318 NC334 NC332 CML92 CML220 CML218 A272 Tzi9 CML77 TZI10 CML311 CML349 CML158Q CML154Q CML281 CML91 parvi-14 parvi-36 parvi-03 parvi-49 parvi-30 NC356 TX601 NC340 NC304 NC338 NC302 NC354 TZI18 A6 Ki44 Ki43 Ki2007 Ki21 Ki2021 Ki14 CML238 CML321 CML157Q CML322 CML331 CML261 CML341 CML45 CML11 CML10 Q6199 CML314 CML258 CML5 CML254 CML61 CML264 CML9 CML108 CML287 Tzi11 CML38 CML14 SC357 L578 T234 N6 E2558W K64 CI64 CI44 CI31A NC230 CI66 K55 R109B MoG L317 NC232 VaW6 CH Va85 CI90C M14 H99 Va99 PA91 Ky226 H95 Oh40B VA26 Pa762 A619 Va22 Va17 Va14 Va59 Va102 Va35 T8 A654 Pa880 SD44 CH9 Pa875 H49 W64A WF9 Mo1W Os420 R NC260 Mo44 CI21E Hy A661 ND246 WD A554 CO125 CO109 B75 DE-3 DE-2 DE1 B57 C123 C103 NC360 NC364 NC362 NC342 NC290A NC262 NC344 NC258 CI91B CI187-2 A682 K4 NC222 NC236 CI3A K148 Mt42 MS153 W401 A556 B77 CM37 CMV3 W117HT CM7 CO106 B103 R4 Ky228 B164 Mo46 Mo47 Mo45 Hi27 Yu796-NS I205 Tzi16 Tzi25 B79 B105 N7A SD40 N28HT A641 DE811 H105W A214N H100 A635 A632 A634 B14A H91 B68 B64 CM174 CM105 B104 B84 NC250 H84 B76 B37 N192 A679 B109 NC294 NC368 NC326 NC314 NC328 R229 A680 NC310 NC324 NC322 NC330 NC372 B73Htrhm B73 NC308 NC312 NC306 NC268 B46 B10 A239 C49A C49 A188 W153R A659 R177 W22 W182B CI NC348 NC298 NC352 NC336 NC296 NC346 NC296A NC292 SWEET POPCORN TROPICAL- SUBTROPICAL STIFF STALK NON STIFF STALK MIXED Based on 89 SSR loci MO17 Oh43E ssp. parviglumis NC300 NC350 P39 M37W HP301 CML277 B97 CML103 CML69 CML52 CML228 CML247 CML332 IL14H Ky21 Ki11 Ki3 MS71 Mo18W OH7B M162W Tx303 TZI8 CML333 NC358 OH43 Flint-Garcia, et al. (2005) Plant J.

11 NAM Development Association Mapping Linkage Mapping Yu, et al. (2008) Genetics; McMullen, et al. (2009) Science

12 Corn Tracker

13 NAM Kernel Composition Fiber Amylopectin Starch Amylose Jason Cook Zeins Protein Amino Acid Profiles Oil Fatty Acid Profiles NIR conducted by Syngenta Seeds, Inc.

14 LOD Oil Composition QTL in NAM Chr Joint Linkage Mapping - Oil Genetic Distance (cm) Joint Linkage Mapping - Oil LOD ,000,000 1,000,000,000 1,500,000,000 2,000,000,000 Physical Distance (bp) Cook, et al. (2012) Plant Phys.

15 Chr. 6 Oil Candidate: DGAT1-2 Encodes acyl-coa:diacylglycerol acyltransferase Fine mapped by Pioneer-Dupont Zheng, et al. (2008) Nature Genetics High parent = 19% oil High allele = 0.29% additive effect High allele has Phenylalanine insertion in C-terminus Oil 4.4% 5.3% 3.6% 3.9% Phenylalanine insertion Cook, et al. (2012) Plant Phys.

16 NAM Genome Wide Association (GWAS) 1.6 Million HapMap.v1 SNPs projected onto NAM Bootstrap (80%) sampling to test robustness of models GWAS - Oil 70.0 RMIP ,000,000 1,000,000,000 1,500,000,000 2,000,000,000 Physical Distance (bp) Cook, et al. (2012) Plant Phys.

17 DGAT 1-2 (Chr 6: 105,013, ,020,258) NAM Population: 24 HapMap.v1 SNPs in DGAT 281 Association Panel: 2 55K SNPs in DGAT (plus the 3 bp indel) M2: Phe Insertion M1 M5 M4 M3 Marker Trait Population Analysis Method BPP P-Value Effect M1 Oil 282 Assn. MLM (Q+K) - 1.2E M2 Oil 282 Assn. MLM (Q+K) - 9.9E M3 Oil NAM GWAS - Bootstrap M4 Oil 282 Assn. MLM (Q+K) - 4.3E M4 Starch NAM GWAS - Bootstrap M5 Oil NAM GWAS - Bootstrap M5 Starch NAM GWAS - Bootstrap Cook, et al. (2012) Plant Phys.

18 DGAT 1-2 (Chr6: 105,013, ,020,258) M5 M4 M3 M2 (indel) M1 = B73 Genotype = Non-B73 Genotype Rare allele? Cook, et al. (2012) Plant Phys.

19 Summary NAM Kernel Composition Genetic Architecture of Kernel Composition Traits Governed by many QTL (N = 21-26) with small to moderate effects GWAS results confirm many QTL DGAT is our favorite gene, but we still don t have the complete story! NAM In General We can identify the common alleles in maize, but still have problems with rare alleles. Cook, et al. (2012) Plant Phys.

20 Ames Plant Introduction Station Inbreds 2,815 inbred lines from the Ames PI Station Genotyping-by-sequencing (GBS) - 681,257 SNPs Romay, et al. (2013) Genome Biology

21 Ames Plant Introduction Station Inbreds More than half of the SNPs in collection are rare! Expired PVPs 48% 77% 42% 302 Association panel = 75%, NAM founders = 57%. Romay, et al. (2013) Genome Biology

22 Teosinte - The Wild Ancestor

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

24 Library Development 10 libraries of 887 BC4S2/DH Near Isogenic Lines (NILs) Z031E0035 Illumina GoldenGate 768 SNP assay Z035E0012 BC4S4 NILs: GBS & RAD sequencing = 33, ,000 SNPs Lines to be released in 2013

25 Library Coverage c1 c2 c3 c4 c5 c6 c7 c8 c9 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 c10 KEY

26 BC4S2 vs BC4DH from PI donor c1 c2 c3 c4 c5 c6 c7 c8 c9 c10 S2 DH S2 DH S2 DH S2 DH S2 DH S2 DH S2 DH S2 DH S2 DH S2 DH KEY

27 Applications of Teosinte NILs

28 Application 1. Empirical Genetics 1000 Selected Genes What do these selected genes do? What traits were targeted by artificial selection during domestication/breeding? Are selected genes important? Auxin response factor, ARF1 Tillering/branching? Diversity (π) Inbreds Teosinte (bp)

29 Application 2. QTL Mapping Phenotyped at up to 18 reps: Maturity, plant & ear height, kernel row number, kernel weight, kernel shape, leaf length-width, Zhengbin Liu Grain composition (protein, starch, oil) Avi Karn

30 Days to Anthesis NAM RILs Teosinte NILs

31 Kernel Row Number Pop Add. Eff.(rows) Z Z Z Z Z Z Z Z Z Z Pop Add. Eff.(rows) Z Z Z Z Z Z Z Z Z Z

32 Pop Add.Eff.(g) Z Z Z Z Z Z Z Z Seed Weight (50 kernels) Pop Add.Eff.(g) Z Z Z Z Z Z Z Z Pop Add.Eff.(g) Z Z Z Z Z Z Z Z

33 Application 3. Reintroduce Variation Percent of Kernel Weight Teosinte (N = 11) Landraces (N = 17) Inbred Lines (N = 27) β seed size γ α family Zein Profile Zeins Endosperm δ Embryo 10 0 Carbohydrate Protein Fat Biological hypothesis: A loss of genetic variation results in a loss of phenotypic variation. Breeding hypothesis: We can improve modern traits. Flint-Garcia et al. (2009) TAG

34 Teosinte NIL Collaborations Hibbard Corn Rootworm Resistance Harmon Circadian Clock Tracy Germination AgReliant Agronomic Traits Hoekenga Iron Bioavailability Buckler Flowering Nelson NCLB Balint-Kurti SCLB & GLS Smith Corn Smut Brutnell Shade Avoidance Dallo Mal de Rio Cuarto Virus Baxter Ionomics Turlings Terpenes & Armyworms

35 Breeding With Zea

36 Synthetic Populations NAM Synthetic Teosinte Synthetic Zea Synthetic B73 & NAM parents are inbreds, teosinte has NEVER been inbred

37 Zea Synthetic Inbreeding Depression 38% B73, 2% each NAM parent, 12% teosinte S0 Male S0 Female Selfed (S1) F pairs Full Sib (FS) F 0 Ginnie Morrison

38 Zea Synthetic Doubled Haploids 38% B73, 2% each NAM parent, 12% teosinte Anna Selby Genotype by GBS Identity by Descent (IBD) Doubled Haploids (DH) Per se Hybrids Association Analysis & Genomic Selection Goal: 2000 DH First 800+ in 2013 Another 700 in Puerto Rico AgReliant producing more 2014 trial: MO, NC, NY, IA

39 Crazy? Idea 7000 BC 2014 Agronomics Fertilizer Density Mechanization? ideotype Select only on yield

40 75% B73 (SS), 25% teosinte Teosinte Synthetic

41 Acknowledgements NSF Maize Diversity Project Syngenta Seeds AgReliant Genetics Susan Melia-Hancock, Kate Guill, Jason Cook, Zhengbin Liu, Ginnie Morrison, Christopher Bottoms, Avi Karn, Anna Selby