Improved shatter resistance of canola Plant Breeding by ... · CRISPR/Cas9 in resynthesized...

Improved shatter resistance of canola

by CRISPR/Cas9 and EMS mutagenesis

Janina Braatz

Canola Innovation Day, Saskatoon, 7 Dec 2017

Plant Breeding

Institute

Faculty of Agricultural and Nutritional Sciences

The natural seed dispersal mechanism of

Brassica napus troubles farmers

• Dry siliques

• Fragile

• Yield loss

• Volunteer plants

• Weather

• Animals

• Machines

The dehiscence zone promotes seed shattering

3

Silique cross-section Silique model Underlying gene network

(simplified)

Valves

Replum

Dehiscence

zone

Beak

Pedicel

IND

FUL

DELLA

ALC

DELLA

RPL

AG

PRX13

PID

WAG2

SPT

GA3OX1

FIL

JAG YAB3

PIN3

PRX55 PRX30 ?

?

NST1 ADPG1

?

IRX3 IRX4 IRX5

IRX10 IRX12 FRA8

OMT1

NST3 ADPG2

SHP1 SHP2

CRISPR/Cas9 system can induce frameshift

mutations at target locus

4

Cas9 target upstream of the bHLH domain

BnALC

ACGCCGCTTGTGCAGCCGCTGAAACT

ACGCCGCTTGTGCAGTCGCTGAAACT

BnaA.ALC.a

BnaC.ALC.a

exon

bHLH domain

Cas9 target site

intron

4.7 kb intron in A paralog *

*

0 550 1,100 bp

1A 1B 2 3 4 5

(Modified from Braatz et al. 2017)

© American Society of Plant Biologists

Model of CRISPR/Cas9-mediated

gene knock-out

(Modified from Agrotis and Ketteler 2015)

CRISPR/Cas9 construct was transformed into

rapeseed hypocotyl explants

5

Transformation:

• Hypocotyl explants of spring

cultivar ‘Haydn’

• Agrobacterium-mediated

transformation

• 1 transgenic T1 plant

(transformation rate: 0.9%)

Regenerating plantlets

pCas9-TPC construct (after Fauser et al. 2014)

PcUbi (promotor)

Cas9

PPT resistance RB

sgRNA specific BnALC target

AtU6-26 (promotor) Pea3A (terminator)

LB

pVS-1 ori

pUC19 ori

Spectinomycin

resistance

The T1 plant contained four Bnalc mutant alleles

6


Sanger sequencing of cloned BnALC PCR amplicons

of the double heterozygous T1 plant

A1 ACGCCGCTT-GTGCAGCCGCTGAAACT

ACGCCGCTT-GTGCAGTCGCTGAAACT

BnaA.ALC.a

BnaC.ALC.a

A2, -2 bp

A3, -7 bp

C2, +1 bp

C3, -1 bp

BnaA.ALC.a

BnaC.ALC.a

Allele

ACGCCGC---GTGCAGCCGCTGAAACT

ACG--------TGCAGCCGCTGAAACT

ACGCCGCTTGGTGCAGTCGCTGAAACT

ACGCCGCTT--TGCAGTCGCTGAAACT

C1

(Braatz et al. 2017)

T1 plant

The T2 progeny showed the expected Mendelian

segregation

7

Transgene genotypes alc genotypes

Trans-

genic

Non-

transgenic

Chi²

testb

A2A2

C2C2

A2A2

C2C3

A2A2

C3C3

A2A3

C2C2

A2A3

C2C3

A2A3

C3C3

A3A3

C2C2

A3A3

C2C3

A3A3

C3C3

Chi²

testc

O 27 9 0

3 4 0 3 14 2 1 6 3 8.52

Ea 27 9 2.25 4.25 2.25 4.25 9 4.25 2.25 4.25 2.25

a: under the assumption that the T1 parent CP1 was non-chimeric (A2A3/C2C3) b: 3:1 segregation, Chi2

(0.999;2) = 13.82 c: 1:2:1:2:4:2:1:2:1 segregation, Chi2

(0.999; 8) = 26.12

Inheritance of transgene and CRISPR/Cas9-induced BnALC mutations in 36 T2 plants.

O = observed, E = expected number of plants

(Braatz et al., 2017)

PCR test for T-DNA presence

1 2 3 4 5 6 7 8 9 10 11

No off-target effects were detected in two

homologous regions

8

Alignment of the CRISPR-Cas9 target sequence with two

potential off-target sites identified by a BLAST search

BnaA.ALC.a

BnaC.ALC.a

BnaC04g13390D

Non-coding region on chr. C02

CCGCTTGTGCAGCCGCTGAAACT

CCGCTTGTGCAGTCGCTGAAACT

CCGCTTGTGCAGTCTCTGAAACT

CCGCTTTTGCAGCCGCAGAAA--


Sanger sequencing of the T1 plant and five T2 progeny showed only

wild type sequences in the two potential off-target sites.


Whole genome shotgun sequence of T1 plant was

produced

9

gDNA of T1 plant

(www.illumina.com)

HiSeq 2500, 1 lane

Paired-end sequencing

412 mio. raw data reads

Quality trimming and

mapping against transformation vector

and Darmor-bzh reference

(BWA mem, SAMtools, Novosort, R)

Average 20x genome coverage

Information about inserted sequences

(Sandra Driesslein, Nils Stein, Axel Himmelbach and Martin Mascher, IPK, Gatersleben, Germany)

The T1 plant carried vector backbone insertions

10

Transgenic T1

0 10 15 5

1000

500

100

50

10

5

1

read

dep

th

pV

S-1

ori

pU

C1

9 o

ri

Sp

ecR

LB

Cas

9

Pea

3A

(T)

bar

cas

sett

e

RB

PcU

bi4

-2(P

)

position in vector sequence (kb) 0 10 15 5

2.6

2.4

2.2

2.0

1.8

1.6

read

dep

th

Negative control: Rapeseed cv. ‘Groß Lüsewitzer’ (Schmutzer et al. 2015)

AtU

6-2

6(P

)

targ

et,

sgR

NA

position in vector sequence (kb)


Mapping of genome sequences against the transformation vector sequence average genome coverage, half & double of the coverage


The general plant growth of T1 and T2 resembled

the wild type

11

T1 Haydn T2

alc mutants

Haydn

alc T1

(Modified from Braatz et al., 2017)


Bench-top phenotyping of single siliques assesses

shatter resistance

12

Measure: Peak tensile force

separating valves and replum

Tensile force measurement

F

Cas9-induced Bnalc shatter resistance was masked

by transformed genotype

13 Conditions: Greenhouse, 16 h light, 22 °C, Bnalc mutations in ‘Haydn’ background, 30 siliques/ 5 plants/ genotype

Statistics: Regression at SL 5.5 cm, standard error, ANCOVA, same letters no significant difference (p ≥ 0.05)

0

1

2

3

4

5

Ten

sile

forc

e (N

ma

x)

Tensile force measurements

‘Haydn’ Bnalc T2

Genotypes

Δ 0.4 N

Bnalc

double

mutant

BnALC

wild type

a a

‘Haydn’ shows high shatter resistance

14 Conditions: Greenhouse, 16 h light, 22 °C, 30 siliques/ 5 plants/ genotype


a a

a a a

a,b

b b

b


0

1

2

3

4

5

'Express' 'Apex' 'Artoga' 'Avatar'

Te

nsil

e f

orc

e (

Nm

ax)

Genotypes

Winter varieties

0

1

2

3

4

5

'Express' 'Drakkar' 'Haydn' 'Mozart' 'Westar'

Ten

sile

forc

e (N

ma

x)

Genotypes

Spring varieties

(Braatz et al., under review)

EMS mutants confirmed the Cas9-induced Bnalc

shatter resistance

15


Conditions: Greenhouse, 16 h light, 22 °C, Bnalc mutations in ‘Express’ background, 30 siliques/ 5 plants/ genotype


0

1

2

3

Ten

sile

forc

e (N

ma

x)

Genotypes

aacc aacc aacc AACC AACC AACC aaCC aaCC AAcc AAcc AACC

Δ 0.9 N

Bnalc

double

mutant

BnALC

wild type

Bnalc

single

mutant ‘Express’ alc-1/alc-4 alc-5

backcrossed F2

alc-2 alc-5

F2

alc-3 alc-5

backcrossed F2

a

b ab

ab ab

c

c c bc

abc abc Δ 0.7 N

Genotypes

(Braatz et al., under review)

Preliminary field data support

Bnind shatter resistance

16

0

1

2

3

4

Av

era

ge h

arv

est

per

plo

t (k

g)

Genotypes

Conditions: 2016/17, Rheinbach (Bonn), randomized block design, three repetitions, 3 x 3 m plots, 11 cm row distance,

55 seeds/m2, backcrossed F3 Bnind mutations in ‘Express’ background, two 1 x 0.1 m seed collection trays per plot

(28.06.17), seed loss until 31.07.17, harvest 02.08.17, direct cutting of 4.86 m² central plot

Statistics: n = 3 (harvest), n = 6 (loss), bars show std. dev./ median, t-tests, same letters no significant difference (p ≥ 0.05)

Seed collection tray Overview of field trial in full bloom

a

b c c,d d d

ind-2 ind-6

a,b a,b

b,c

c c

a

ind-2 ind-6

Bnind

double

mutant

BnIND

wild type

Bnind

single

mutant

Four BnNST1 homoeologs were targeted by

CRISPR/Cas9 in resynthesized rapeseed RS306

17 (Braatz, unpublished)

BnNST1

0 1,200 2,400 bp

1 2 3*

400 800 2,000 1,600 exon

NAC domain

intron

CRISPR/Cas9 target site

TAR motif iii

absent in BnaA.NST1.a *

Cas9 target upstream of the NAC domain

T1 plant

• Indel mutations in all four gene copies of primary transformant

• Multiple alleles per gene chimeric mosaic T1 plant

• Inheritance to T2 currently under investigation

• Phenotyping pending

This study provided

18

• Insights into the efficiency of

CRISPR/Cas9-mediated mutagenesis of

polyploid rapeseed

• Novel mutations for breeding shatter

resistant rapeseed

• Information on the effect of Bnalc and

Bnind mutations on shatter resistance

Future efforts will involve

19

• Phenotypic assessment of Bnnst1

mutants

• Marker-assisted backcross of mutant

alleles into elite material

• Establishment of DNA-free

transformation protocol to produce non-

GMO mutants for the European market

I would like to acknowledge

20

Prof. Dr. Christian Jung, Dr. Hans-Joachim Harloff, and all colleagues of the

Plant Breeding Institute, University of Kiel

Zoological Institute, Functional Morphology

and Biomechanics, University of Kiel

Prof. Dr. Stanislav Gorb

Dr. Lars Heepe

Leibniz Institute of Plant Genetics and

Crop Plant Research (IPK), Gatersleben

Dr. Martin Mascher

Dr. Nils Stein

Dr. Axel Himmelbach

Variation statistics, University of Kiel

Dr. Mario Hasler

IKMB, University of Kiel

Sanger sequencing lab

Formerly Saaten-Union BioTec, Leopoldshöhe

Dr. José Orsini

Institute of Crop Science and Resource Conservation, INRES,

University of Bonn

Prof. Dr. Jens Léon

Winfried Bungert

Karin Woitol

Karlsruhe Institute of Technology, Karlsruhe

Prof. Dr. Holger Puchta

NPZ Innovation, Hohenlieth

Dr. Gunhild Leckband

Dr. Amine Abbadi

Dr. Steffen Rietz

Funding

Stiftung Schleswig-Holsteinische Landschaft

And you for your attention!

Improved shatter resistance of canola Plant Breeding by ... · CRISPR/Cas9 in resynthesized...

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