Forward screening for“broad” leaf mutants

Moritz Jöst Lenhard Lab

University Potsdam

width

length

area

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Leaf

wid

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Leaf

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L3 L4 144/

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Leaf size candidates HorTILLUS in the field in Katowice 2016

-60

-40

-20

0

20

40

Diffe

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e in

%

Leaf 3

Leaf 4

-25 -20 -15 -10

-5 0 5

10 15

Diffe

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% Leaf 3

Leaf 4

-40 -30 -20 -10

0 10 20 30

Diffe

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e in

%

Leaf 3

Leaf 4

Leaf size candidates HorTILLUS in the Greenhouse in Potsdam 2017

width

length

area

Outlook

• Grow leaf size candidates from HorTILLUS under highly controlled conditions (Percival)

• If Leaf length difference can be verified: –> detailed growth analysis

• Backcross candidates to cv. Sebastian • Generate Mapping populations by crossing

candidates to different Barley cultivars

Phenotypic characterization of “broad” leaf mutants and identification of candidate

mutations

Moritz Jöst Lenhard Lab

University Potsdam

“Broad Leaf” mutants from the Nordgen collection Name „Parent cultivar“ Genotyping Mutagen

blf 1 Bonus Bonus OBS

blf2 Bonus Bonus colchicine, X-rays

blf3 Bonus Bonus X-rays

blf5 Bonus Bonus ethylene imine

blf6 Foma Bonus neutrons

blf7 Bonus Bonus gamma-rays

blf8 Foma Bonus ethylene imine

blf10 Foma Foma neutrons

blf11 Foma Foma ethylene imine

blf12 Foma Foma fast neutrons

blf13 Foma Bonus n-butyl methanesulfonate

blf14 Foma Kristina N-ethyl-N-nitrosourea

blf15 Foma Kristina N-ethyl-N-nitrosourea

blf16 Kristina Kristina neutrons

blf17 Kristina Kristina ethyl methanesulfonate

blf18 Kristina Kristina ethyl methanesulfonate

blf19 Kristina Kristina ethyl methanesulfonate

broad leaf 1-6

broad leaf 1-1

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-20

0

20

40

“Broad Leaf” mutants from the Nordgen collection - Phenotyping in the phytotron

-100

-80

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Max. width leaf 3:

Area leaf 3:

Relative to WT (%):

-30

-20

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10

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30

leaf 3 width

-50

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-30

-20

-10

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10

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30

leaf 3 area

Field trial in Katowice 2016 D

iffer

ence

in %

0

0.5

1

1.5

2

Kristina Foma Bonus Kristina Foma Bonus

Field 2016 Phytotron 2016

max. width leaf 3 a

b c

d d d

Leaf width of cultivars differs considerably comparing field and phytotron:

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Task: select the “best” 3 “Broad Leaf” mutants for further analysis:

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-80

-60

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-20

0

20

40

Max. width leaf 3:

Area leaf 3:

Relative to WT (%):

NG_blf13 NG_blf2 NG_blf11

WT mutant

Select the 3 best “Broad leaf”mutants and…:

• Mapping by sequencing: • Select F2 populations for mapping:

• X cv. Bowman • X cv. Barke • X cv. Golden Promise

• Exome capture + sequencing of WT and mutant

pools • Further fine mapping

0

5

10

15

20

Freq

uenc

y

F2 NG_blf13 x cv. Bowman

0

5

10

15

20

Freq

uenc

y

F2 NG_blf13 x cv. Golden Promise

0

5

10

15

20

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1

1.05

1.1

1.15

1.2

1.25

1.3

1.35

1.4

1.45

1.5

1.55

Freq

uenc

y

Classes leaf 3 width in cm

F2 NG_blf13 x cv. Barke

NG_blf13

cv. Bowman

cv. Golden Promise

cv. Barke

Selecting a mapping population: NG_blf13

Selecting a mapping population: NG_blf2

0

10

20

30

40

Freq

uenc

y

F2 NG_blf2 x cv. Bowman

0

5

10

15

20

25

30

35

0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 1.05 1.1 1.15 1.2 1.25 1.3 1.35

Freq

uenc

y

Classes leaf 3 width in cm

F2 NG_blf2 x cv. Golden Promise

NG_blf2

cv. Bowman

cv. Golden Promise

0

10

20

30

Freq

uenc

y F2 NG_blf11 x cv. Bowman

0

10

20

30

Freq

uenc

y

F2 NG_blf11 x cv. Golden Promise

0 5

10 15 20 25

0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 1.05 1.1 1.15 1.2 1.25

Freq

uenc

y

Classes leaf 3 width in cm

F2 NG_blf11 x cv. Barke

Selecting a mapping population: NG_blf11

NG_blf11

cv. Bowman

cv. Golden Promise

cv. Barke

Mapping by sequencing: • About 400 plants per F2 population were

phenotyped • Per population 50 phenotypically “WT” and 50

“mutant” plants were selected: • Leaf material sampled and pooled • DNA extraction -> Material will be send to Agnieszka

0 10 20 30 40 50 60

0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 1.05 1.1 1.15 1.2 1.25 1.3 1.35

NG_blf2 x Bowman

Outlook

• DNA pools will be subjected to exome capture and sequenced at the University of Silesia

-> Candidate mutations • Fine mapping using KASP Markers • Detailed phenotypic analysis

• Sections of mature leaf blades • 3D reconstructions of shoot apices

0

10

20

30

40

50

0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 1.05 1.1 1.15 1.2 1.25 1.3 1.35 1.4 1.45 1.5 1.55

Freq

uenc

y

NG_blf13 x cv. Barke

0 10 20 30 40 50 60

0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 1.05 1.1 1.15 1.2 1.25 1.3 1.35

Freq

uenc

y

NG_blf2 x Bowman

0

20

40

60

80

0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 1.05 1.1 1.15 1.2 1.25 1.3

Freq

uenc

y

Classes leaf 3 width in cm

NG_blf11 x cv. Barke