Precision Genomics in Soybean

Justin Anderson

Advisor: Dr. Robert StuparUniversity of Minnesota

Department of Agronomy and Plant Genetics

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/

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

GOI

GOI

Normal soybean

ZFN transformed;Mutates GOI

Targeted Mutation

Curtin et al. 2012

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)

Target Region

ZFN/TALEN

NHEJrandom mutation

Donor template

Gene Targeting

Potential of a Double Strand Break

Modify Copy Number

Rhg1

+Rhg1

ZFN Rhg1

Rhg1

Rhg1

Rhg1 Rhg1

Rhg1

PROS• They work in soy

Designer NucleasesZinc Finger Nucleases CRISPR/ 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 http://taleffectors.com/wp-content/uploads/2011/12/TALENfig1.png

http://www.sigmaaldrich.com/content/dam/sigma-aldrich/life-science/functional-genomics/zinc-finger-nucleases.

http://www.google.com/imgres?imgurl=http://www.pnabio.com/products/image

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: 25-42.

Hairy roots: Initial testing

• Agrobacterium rhizogenes strain K599 is used for hairy root transformation

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)

Contact/Acknowledgments

• Justin Anderson (me) ande9112@umn.edu

• Advisor: Robert Stupar stup0004@umn.edu• Dan Voytas• Shaun Curtin• Jean-Michel Michno• Junqi Liu

Plug:UMN Plant Breeding Symposium

travel funding availablewww.plantbreedingsymposium.umn.edu

Gene Targeting in Plants

• ZFNs:– Shukla et al. 2009– Townsend et al. 2009– Cai et al. 2009

• TALENs– Baltes et al. 2014

• CRISPR/Cas9

Inducible promoter

Left ZFA 1 Right ZFA 1 Left ZFA 1 Right ZFA 2

Transformation Vector coding two ZFNs

R-gene cluster

12

34

5

Induce

ZFN 1

ZFN 2

12

34

5

ZFN 1

ZFN 2

R-gene cluster

Deletion

12345

Wild Type

12

34

5

ZFN 1

ZFN 2

R-gene cluster

Inversion

ZFN 1

ZFN 2

Inversion

12

34

5

Inversion

12345

Wild Type

1 2 3 4 5

12

34

5

ZFN 1

ZFN 2

R-gene cluster

12

34

5

Duplication

12

34

5

12

34

5

Duplication

12

34

5

12

34

5

12345 12345

Deletion

Duplication

12345

Wild Type