Working in Partnership to Attain Priority Crop Genetic Resource, Genomics, and Genetic Improvement Research Goals

1. Working in Partnership to Attain PriorityCrop Genetic Resource, Genomics, and Genetic Improvement Research Goals Peter Bretting USDA-Agricultural Research Service Office of National Programs Peter.bretting@ars.usda.gov http://www.ars.usda.gov/research/programs/programs.htm?NP_CODE=301

2. Outline for Presentation • American Seed Research Summit • USDA/ARS National Program in Plant Genetic Resources, Genomics, and Genetic Improvement • Plant Genetic Resources and Information Management • Plant Genomics, Genetic Analyses and Genome Databases • Maize and soybeans: genotyping and nested association mapping • Genetic Improvement of Crops • Maize: GEM Project • Conclusion

4. American Seed Research Summit Research, Education, and Policy Goals and Strategies • Strengthen public and private partnerships to accomplish national seed research priorities • Coordinate and engage industry stakeholders to support stable funding for seed and breeding education, research, and development • Attract and develop a pool of diverse, high-quality plant researchers • Ensure that the regulatory system governing the development and implementation of new technology is efficient, effective, and science-based. • Develop an education and advocacy program to communicate the value of seed and crop research to the public.

5. USDA/ARS National Program in Plant Genetic Resources, Genomics, and Genetic Improvement (Crop Genes R’ Us!) • Largest NP, with 125+ projects, and about $128 million gross annual budget. • Conducted by about 300 scientists at more than 50 ARS locations. • Extensive public and private sector partnerships. • Goal: deliver crop genetic, genomic and bioinformatic tools, information, genetic resources, and improved crop varieties to enhance U. S. agricultural productivity and security.

6. NP 301 Action Plan Research Components • Plant and Microbial Genetic Resource Management • Crop Informatics, Genomics, and Genetic Analyses • Genetic Improvement of Crops

7. One of the largest national genebank systems. More than 510,000 samples of more than 13,400 plant species. Large collections of the major staple crops important to U. S. and world agriculture. Large holdings of crops without major collections at international agricultural research centers, e.g., cotton, soybean, various horticultural and “specialty” crops. Germplasm Resources Information Network (GRIN): an international standard. The USDA/ARS National Plant Germplasm System (NPGS)

8. USDA National Plant Germplasm System (NPGS)

9. Plant Genetic Resource Management in Genebanks • Acquisition • Maintenance • Regeneration • Documentation and Data Management • Distribution • Characterization • Evaluation • Enhancement

10. Avoidance of Cross Pollination and Seed Mixing • Extensive quality assessment when the seed enters the NPGS • Procedures and protocols for regeneration • Physical isolation • Cages • Hand pollination • Care in storage and distribution to prevent mixing

11. Maize in the NPGS • Isolation from sources of out crossing • Regenerate ~400 accessions/year • Sometimes plantings yield as few as 5,000 seeds for some accessions

12. GRIN-Global • GRIN = Germplasm Resources Information Network.http://www.ars-grin.gov/ The genebank information management system for the NPGS, and for Canada’s genebank system (GRIN-Canada). • The Global Crop Diversity Trust asked ARS and Bioversity International (an International Agricultural Research Center) to enhance and expand GRIN to address global germplasm information management needs. • The Trust awarded ARS a 3-year, $1.4 million grant to develop GRIN-Global; ARS is devoting $900K in-kind support to the project. ARS effort is located in Beltsville, MD and Ames, IA.

13. GRIN-Global • Based on GRIN, but can be implemented in both a system-wide and “stand-alone” local management mode • Supports multiple users via a “user-friendly” interface • Maintains linkages with other databases and interoperates with existing systems • Advanced querying, custom and third-party applications

14. Presentation Tier (Web Browser) Presentation Tier (Windows Desktop Client) Business Tier (Web Service) Data Tier (MySQL, Oracle, SQL Server) Three Tier Architecture

15. GRIN-Global • On-line ordering/request capability • Database-flexible, free of recurrent licensing costs, with interface and database schema source code open and available without restriction to further development • Designed to serve as the global standard plant genebank information management system

16. Future Prospects

17. Trends in demand for NPGS germplasm and information vs. NPGS budget

18. Result?: mismatch between expanding demand for PGR and static NPGS capacity to manage it.

19. NP 301 Action Plan Research Components • Plant and Microbial Genetic Resource Management • Crop Informatics, Genomics, and Genetic Analyses • Genetic Improvement of Crops

20. Genomic Information and Research Tools • Genetic markers: polymorphic and heritable—simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) • Expressed sequence tags (ESTs) • Genetic maps • Quantitative trait loci (QTLs) • Physical maps • Complete and partial genome sequences

21. Application of Genomic Information to Genetic Analyses: The Nested Association Mapping (NAM) Genetic Map [E. Buckler, J. Holland, M. McMullen (USDA/ARS), and many university and private-sector collaborators]NAM is the most powerful tool for dissection of the genetic bases of quantitative traits for any species – period.

22. Linkage Mapping Nested Association Mapping Recent and ancient recombination High Power High resolution Analysis of many alleles Moderate genetic marker density High projected marker density Association Mapping Recent recombination High power Low resolution Analysis of 2 alleles Moderate marker density Genome scan Historic recombination Low power High resolution Analysis of many alleles High marker density Candidate gene testing

23.             SSD    1 2 NAM    200 Nested Association Analysis 25 DL B97 CML103 CML228 CML247 CML277 CML322 CML333 CML52 CML69 Hp301 Il14H Ki11 Ki3 Ky21 M162W M37W Mo18W MS71 NC350 NC358 Oh43 Oh7B P39 Tx303 Tzi8 ×B73 F1s Yu et al. (2008) Genetics 178: 539

24. NAM Genotyping and Genetic Map • Genotyping with more than 1500 single nucleotide polymorphism (SNP) genetic markers • Map consists of 1106 loci (38 composite loci) and ~1400 cM genetic distance, therefore an average marker density of 1.3 cM/marker. • It is a composite or consensus map and distances and potentially order can not be assumed to translate directly to individual family maps.

25. Maize Phenomics: Massively Parallel Phenotyping of The Nested Association Mapping Population THE MAIZE DIVERSITY PROJECT

26. Experimental Evaluation of NAM NAM lines evaluated in 2006 – 2007 at each of the following sites: Aurora, NY Champaign, IL Columbia, MO Clayton, NC Homestead, FL Ponce, PR 11 Total Environments

27. + Tassel main spike length Tassel branch angle Tassel branch no.

28. B73 X NC358 B73 X P39 B73 X M37W B73 X IL14H B73 X Tx303 B73 X B97 B73 X Mo18W B73 X Ki11 B73 X NC350 B73 X Ky21 B73 X Oh7B B73 X Ki3 B73 X CML103 B73 X Oh43 B73 X MS71 B737 xHp301 B73 X M162W B73 X Tzi8 B73 X CML52 B73 X CML228 B73 X CML277 B73 X CML247 B73 X CML69 … … RIL1 RIL2 RIL199 RIL200 RIL1 RIL2 RIL199 RIL200 σ2G(F)2 σ2G(F)1 Partitioning Genetic Variance Line-to-line variation within a family – due to differences between alleles from B73 and alleles from Diverse Line founder of that family. B73 X CML322 B73 x CML333 Among-Family Genetic Variance: σ2F Family-to-family variation – due to differences among different founders. Within-Family Genetic Variances

29. The Maize Genetics andGenomics Database (Maize GDB) (C. Lawrence et al.)USDA/ARS, AMES, IA www.maizegdb.org/ Phenotypes Integrating structural and genetic maps with maize genomic sequence Genome annotation Research community support

30. Soybean Nested Association Mapping (NAM) Project (P. Cregan, D. Hyten, ARS-Beltsville) Agricultural Research Service Collaborators: USDA/ARS Iowa St. Univ. Univ. of Nebraska Univ. of Illinois Univ. of Tennessee Univ. of Maryland Funding Support

31. Soybean NAM • Design focused on yield • Populations will be restricted to Maturity Group III • Hub parent is IA3023 • Lines crossed with IA3023 included both elite and exotic germplasm • 121 total lines selected by research community • Crosses made the summer of 2008 • Parents were genotyped with the Universal Soy Linkage Panel 1.0

32. What’s next with the Soybean NAM? • During population development, the number of families will be reduced to 40 based on field observations during the inbreeding process. • Each population will consist of 250 F5 RILs • Total size: 10,000 lines • Phenotyping will be done with a ‘connected’ incomplete block experimental design (Tested in 30 environments) • Best Linear Unbiased Predictions (BLUPs) of grain yield, i.e., breeding values for every RIL

33. The Universal Soy Linkage Panel 1.0 (USLP 1.0) With United Soybean Board and collaborator funding 180 sets of the USLP 1.0 (17,280 genotypes) were acquired for gene/QTL Discovery ARS Collaborators Ames, IA Beltsville, MD Columbia, MO Raleigh, NC Stoneville, MS Urbana, IL Wooster, OH State University Collaborators N. Carolina St. Univ. N. Dakota St. Univ. Ohio State Univ. So. Illinois Univ. S. Dakota St. Univ. Univ. of Arkansas Univ. of Georgia Univ. of Illinois Univ. of Minnesota Univ. of Missouri Univ. of Nebraska Virginia Tech

34. Universal Soy Linkage Panel (USLP 1.0) 1,536 SNPs selected from 3,110 SNPs mapped on the Soybean Consensus Map • SNPs have diverse allele frequencies • Average polymorphism in bi-parental crosses • Elite cultivars= 458 • PI landraces = 544 • Elite crossed with PI landrace = 590 • Spread throughout the genome • Assayed using the Illumina GoldenGate assay • 192 DNA samples run in three days • Large orders reduce cost from $11,000 per 96 DNA samples to $5,500 per 96 DNA samples

35. The Universal Soy Linkage Panel 1.0 (USLP 1.0)for Gene/QTL Discovery - Traits Under Study in Collaborative Projects - Enhanced Seed Composition Reduced linolenic acid oil Elevated oleic acid oil Lower saturates Higher protein Resistance to Biotic Stress Soybean Cyst Nematode Soybean Rust Phytophthora Root Rot Foliar Feeding Insects Soybean Aphid Resistance to Abiotic Stress Drought Iron Deficiency Chlorosis Seed Yield – Assessment and enhancement of genetic diversity

36. Source of Genetic Improvement for Soybean • USDA germplasm collection is the source for new genetic variation for soybean improvement for every important trait USDA Soybean Collection (21,000 accessions) 18,090 accessions collected mostly from Asia (Landraces) 1,116 wild soybeans 17 Introduced landraces (86% of the Germplasm base) 513 Public cultivars released after 1946 Hyten et al. 2006, PNAS 103: 16666-16671

37. Soy HapMap • The United Soybean Board recently agreed to fund a $2.9M project to characterize the entire USDA Soybean Germplasm Collection of 21,000+ wild and cultivated soybeans with 50,000 SNP DNA markers. • The Illumina’s Beadstation will allow genotyping of this collection with 50,000 SNPs within three years • New gene discovery through association analysis • Enables breeders to select germplasm with greatest potential for agronomic improvement • Decipher the signatures of selection (allele frequency changes) associated with soybean yield improvement over 75 years of soybean breeding to help understand yield Soybean Genomics and Improvement Lab USDA, ARS, BARC-West Beltsville, Maryland 60,800 SNP Infinium Chip

38. Controlling Iron Deficiency Chlorosis in Soybeans • Research by R. Shoemaker (ARS-Ames), C. Vance (ARS-St. Paul) and collaborators at N. Dakota State Univ. and Iowa State Univ. • Iron is a limiting growth factor on 30% of cropland • Iron is also a major nutritional deficiency in much of the world • Using global gene expression profiling to identify genes involved in iron metabolism • Currently evaluating how to control the genetic expression to enhance iron balance in commercial plant varieties. • Developing molecular markers to speed the public release of commercial varieties with improved iron efficiency.

39. Mapping & Cloning Genes Responsible for Soybean Protein • Research by R. Shoemaker (ARS-Ames), C. Vance (ARS-St. Paul), collaborators at the Univ. of Illinois and Univ. of Nebraska • The locus of a major trait that controls seed protein content was mapped to Chromosome 20 • High-throughput transcript sequencing and mapping identified several candidate genes • Currently identifying the specific genes and evaluating modifications to change the protein content

41. Haplotype and Allele States for varieties SNP Haplotypes and Linkage Disequilibrium (R. Shoemaker, ARS-Ames)

42. NP 301 Action Plan Research Components • Plant and Microbial Genetic Resource Management • Crop Informatics, Genomics, and Genetic Analyses • Genetic Improvement of Crops

43. Germplasm Enhancement of Maize (GEM) Project • A collaborative effort of public and private sector researchers to broaden and enhance the maize germplasm base. More than 60 collaborators. • Two permanent breeding sites: • Ames, IA for development of 25% tropical and temperate exotic • Raleigh, NC for development of 50% tropical • GEM is administered by the USDA-ARS Plant Introduction Research Unit (PIRU) located in Ames, IA; and the Plant Science Research Unit (PSRU) in Raleigh, NC • Technical Steering Group (TSG) provides guidelines for research, germplasm, and methods. Germplasm Enhancement of Maize

44. GEM Project Cooperators Germplasm Enhancement of Maize

45. GEM Objectives • Manage and coordinate a multi-site cooperative program for germplasm evaluation, development, and information sharing • Evaluate diverse maize germplasm for adaptation, yield, stress resistance, and key value-added traits (VATs) • Develop and release enhanced germplasm with key traits • Develop innovative means of managing and transferring information to the maize community Germplasm Enhancement of Maize

46. GEM Germplasm Releases Germplasm Enhancement of Maize * Crop Science registered and ** 20 of these 29 lines were Crop Science registered.

47. Number of Releases by Traits Germplasm Enhancement of Maize

48. New GEM Initiatives • Allelic diversity • New un-sampled races being tapped • Goal to assess ~300 races for adaptation in US • New elite exotic sources being acquired • More than 40 germplasm sources acquired from Thailand, Peru, Nigeria, Argentina, Chile, and France • Provide resistance to exotic diseases • Shade house to reduce photoperiod response • Make tropical introgressions in Ames, IA • 23 tropical sources (11 races) successful (so far) • Double haploid research • Explore feasibility with exotic germplasm Germplasm Enhancement of Maize

49. Fusarium Ear Rot Germplasm Enhancement of Maize Susceptible Line Resistant Line GEMS-0002 Public Release Bill Dolezal, Pioneer Hi-Bred Int’l, Woodland, CA (2005)

50. Integrated national programmatic approach, with extensive academic and private sector collaborations/partnerships Genetic Enhancementand Breeding Genome Databases Exploit untapped genetic diversity in genebanks, and breeding populations Plant genomics, gene discovery Genetic markers & Bioinformatics