Forward screening for“broad” leaf mutants
Moritz Jöst Lenhard Lab
University Potsdam
width
length
area
0
0.5
1
1.5
2
2.5
Leaf
wid
th in
cm
0
5
10
15
20
25
30
35
Leaf
leng
th in
cm
0
10
20
30
40
Leaf
are
a in
cm
2
10/0
01/2
Seba
stia
n
L3 L4 144/
001/
11
322/
001/
1
1663
/001
/13
1856
/001
/5
2036
/001
/1
5287
/001
/3
5462
/001
/4
312/
001/
6/1
2033
/001
/8/1
/1
2033
/001
/8/1
/2
283/
001/
7/1/
4
field
_142
_row
1-4
1039
/001
/1
3422
/001
/6
189/
001/
6/5/
8
439/
001/
3/1/
3
439/
001/
3/1/
4
669/
001/
2
5169
/001
/1
3623
/001
/7
Leaf size candidates HorTILLUS in the field in Katowice 2016
-60
-40
-20
0
20
40
Diffe
renc
e in
%
Leaf 3
Leaf 4
-25 -20 -15 -10
-5 0 5
10 15
Diffe
renc
e in
% Leaf 3
Leaf 4
-40 -30 -20 -10
0 10 20 30
Diffe
renc
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
-60
-40
-20
0
20
40
“Broad Leaf” mutants from the Nordgen collection - Phenotyping in the phytotron
-100
-80
-60
-40
-20
0
20
40
Max. width leaf 3:
Area leaf 3:
Relative to WT (%):
-30
-20
-10
0
10
20
30
leaf 3 width
-50
-40
-30
-20
-10
0
10
20
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:
-60
-40
-20
0
20
40
Task: select the “best” 3 “Broad Leaf” mutants for further analysis:
-100
-80
-60
-40
-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