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

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Presentation on theme: "Precision Genomics in Soybean Justin Anderson Advisor: Dr. Robert Stupar University of Minnesota Department of Agronomy and Plant Genetics."— Presentation transcript:

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 GOI Normal soybean ZFN transformed; Mutates GOI Targeted Mutation

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

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

7 Modify Copy Number + Rhg1 ZFN Rhg1

8 PROS They work in soy Designer Nucleases Zinc Finger NucleasesCRISPR/ Cas9TAL Effector Nucleases CONS  Low specificity compared to TALENS/CRISPR  Takes 2-3 weeks for assembly  A lot of molecular work involved PROS  More specificity when targeting then ZFN  You can design/assemble a TALEN in 7 Days CONS  They have yet to work in soy  Assembly can be difficult  Very Large PROS  You can design/assemble a CRISPR in 5 Days  Very easy to design/assemble  Potential for multiplexing  Smaller size than ZFN/TALEN CONS  Has not been tested with agrobacterium  Potential for off target mutations  Not as much specificity as TALENS content/uploads/2011/12/TALENfig1.png science/functional-genomics/zinc-finger-nucleases. w.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 Mol Biol 1069:

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) Advisor: Robert Stupar Dan Voytas Shaun Curtin Jean-Michel Michno Junqi Liu Plug: UMN Plant Breeding Symposium travel funding available

13 Gene Targeting in Plants ZFNs: – Shukla et al – Townsend et al – Cai et al TALENs – Baltes et al CRISPR/Cas9

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

15 R-gene cluster Induce ZFN 1 ZFN 2

16 ZFN 1 ZFN 2 R-gene cluster

17 Deletion Wild Type

18 ZFN 1 ZFN 2 R-gene cluster Inversion

19 ZFN 1 ZFN 2 Inversion

20 Wild Type

21 ZFN 1 ZFN 2 R-gene cluster Duplication

22

23 Deletion Duplication Wild Type


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