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Precision Genomics in Soybean

Precision Genomics in Soybean. Justin Anderson Advisor: Dr. Robert Stupar University of Minnesota Department of Agronomy and Plant Genetics. Stupar lab. Natural Variation Copy number variation Deleterious mutations Fast Neutron Induced Mutation

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Precision Genomics in Soybean

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  1. Precision Genomics in Soybean Justin Anderson Advisor: Dr. Robert Stupar University of Minnesota Department of Agronomy and Plant Genetics

  2. Stupar lab • Natural Variation • Copy number variation • Deleterious mutations • Fast Neutron Induced Mutation • Evaluating unique and marketable traits such as oil content, protein content, and plant structure • Precision Genomics • Implementation of engineered nuclease technology to target genes of interest stuparlab.cfans.umn.edu/

  3. Working with Soybean • Grown for protein and oil • National and Global production • Fixes nitrogen • Paleopolyploid • 12 mya and 50 mya • 60-85% of genes maintain a paralog from these genome duplications • Leads to genetic redundancy

  4. Targeted Mutation Normal soybean GOI ZFN transformed; Mutates GOI GOI

  5. Gene Targeting Nucleotide Binding (Zinc Finger) Endonuclease (Fok1) • Similar process with other designable nucleases • Zinc Finger Nucleases (ZFN) • Transcription activator-like effector nucleases (TALEN) • CRISPR/Cas9 • Meganuclease Curtin et al. 2012

  6. Potential of a Double Strand Break Target Region ZFN/TALEN NHEJ random mutation Gene Targeting Donor template

  7. Modify Copy Number Rhg1 + Rhg1 ZFN Rhg1 Rhg1 Rhg1 Rhg1 Rhg1 Rhg1

  8. Designer Nucleases Zinc Finger Nucleases TAL Effector Nucleases CRISPR/ Cas9 PROS • They work in soy PROS • More specificity when targeting then ZFN • You can design/assemble a TALEN in 7 Days PROS • You can design/assemble a CRISPR in 5 Days • Very easy to design/assemble • Potential for multiplexing • Smaller size than ZFN/TALEN CONS • Low specificity compared to TALENS/CRISPR • Takes 2-3 weeks for assembly • A lot of molecular work involved CONS • They have yet to work in soy • Assembly can be difficult • Very Large CONS • Has not been tested with agrobacterium • Potential for off target mutations • Not as much specificity as TALENS http://www.sigmaaldrich.com/content/dam/sigma-aldrich/life-science/functional-genomics/zinc-finger-nucleases. http://taleffectors.com/wp-content/uploads/2011/12/TALENfig1.png http://www.google.com/imgres?imgurl=http://www.pnabio.com/products/image

  9. Technique • ZFN assembly method published in Legume Genomics • TALEN and CRISPR/Cas9 widely available • Implementation • Hairy Root (somatic) • Whole plant (germline) • Curtin SJ, Anderson JE, Starker CG, Baltes NJ, Mani D, Voytas DF, Stupar RM. (2013) Targeted mutagenesis for functional analysis of gene duplication in legumes. Methods MolBiol 1069: 25-42.

  10. Hairy roots: Initial testing Agrobacterium rhizogenes strain K599 is used for hairy root transformation

  11. Delivery of nucleases to whole plants Co-Cultivation with strain 18r12 (Day 5) Shoot Induction (Day 19) Selection Medium (Day 33) Shoot Elongation (Day 60) Root Elongation (Day 90) Planting (Day 104) Screening and Testing (Day 120)

  12. Contact/Acknowledgments • Justin Anderson (me) ande9112@umn.edu • Advisor: Robert Stupar stup0004@umn.edu • Dan Voytas • Shaun Curtin • Jean-Michel Michno • JunqiLiu Plug: UMN Plant Breeding Symposium travel funding available www.plantbreedingsymposium.umn.edu

  13. Gene Targeting in Plants • ZFNs: • Shukla et al. 2009 • Townsend et al. 2009 • Cai et al. 2009 • TALENs • Baltes et al. 2014 • CRISPR/Cas9

  14. Transformation Vector coding two ZFNs Left ZFA 1 Right ZFA 1 Right ZFA 2 Inducible promoter Left ZFA 1

  15. Induce 1 2 ZFN 1 3 R-gene cluster 4 5 ZFN 2

  16. 1 2 ZFN 1 3 R-gene cluster 4 5 ZFN 2

  17. Wild Type 1 2 3 Deletion 4 5

  18. 1 2 ZFN 1 3 R-gene cluster 4 5 ZFN 2 Inversion

  19. 1 2 ZFN 1 3 4 5 ZFN 2 Inversion

  20. Wild Type 1 1 2 2 3 3 Inversion 4 4 5 5

  21. 1 1 2 2 ZFN 1 3 3 R-gene cluster 4 4 5 5 ZFN 2 Duplication

  22. 1 1 1 1 2 2 2 2 3 3 3 3 4 4 4 4 5 5 5 5 Duplication

  23. Wild Type 1 1 1 2 2 2 Deletion 3 3 3 4 4 4 Duplication 5 5 5

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