Download Natural variation in Arabidopsis, a tool to identify genetic bases of

Natural variation in Arabidopsis,
a tool to identify genetic bases of
nitrogen use efficiency
S. Chaillou, F. Chardon, J. Laurette, S.
Ikram, O. Loudet and F. Daniel-Vedele
INRA Versailles
Plants have different nutrient requirements:
some species are very exigent, others are
less demanding.
Our objective is to find the genetic bases
of the nutrient demand by the plant. We
take the case of Nitrogen Use Efficiency in
the model plant Arabidopsis thaliana.
Searching for QTL of nitrogen use efficiency in Arabidopsis
- Creation of an Arabidopsis recombinant inbred lines
population from two genetically distant ecotypes (Bay 0 and
Shahdara)
- Obtaining a genetic map of the population with markers
(genotypical data)
- Growing plants on two types of nitrogen nutrition, normal
(10 mM) or limiting (3mM) and measuring several
parameters (phenotypical data)
- Correlation between genotypical and phenotypical data for
QTL detection
Recombinant Inbred Lines - RILs
Shahdara
Bay-0
Heterozygous
Bay-0 from
Germany
Shahdara from
Central Asia
Single Seed Descent
420 RILs
Bay-0 x Shahdara RIL population
Genetic map of the population of 420 F6
Recombinant Inbred Lines
I
II
T1G11
III
MSAT2-5
IV
NGA172
F21M12
V
MSAT4-39
MSAT4-8
NGA225
NGA249
ATHCHIB2
MSAT2-38
NGA8
MSAT1-10
MSAT5-14
MSAT3-19
NGA248
MSAT2-36
MSAT2-41
T27K12
MSAT4-35
NGA139
MSAT4-15
MSAT5-22
MSAT3-32
MSAT2-7
MSAT4-18
MSAT3-21
MSAT5-9
MSAT4-9
MSAT2-10
NGA128
MSAT2-22
F5I14
MSAT5-12
MSAT3-18
MSAT4-37
MSAT1-13
MSAT5-19
MSAT1-5
38 microsatellites markers
Loudet et al, TAG 2002
Phenotyping of the population
10 mM
3 mM
View of a set of 24 Arabidopsis lines
after 34 days of culture
N- (3 mM)
N+ (10 mM)
(Loudet et al., 2001)
Isolation of several QTL linked to biomass, total N, amino-acid or
sulfate content
SO3.1
After isolation of the QTL, we must validate
the QTL. The use of Heterozygous Inbred
Families (HIF) allows the validation of QTL.
Heterogeneous Inbred Family - HIF
Shahdara
Bay-0
RILXXX (F6)
Heterozygous
HIF - Sha
HIF - Het
HIF - Bay
Example of QTL validation using HIFs
AA content nmole/mg of DM
Amino acid content
220
200
180
160
X
X
140
120
B
S
¾ Mean of ILs 421 B = 173 nmole aa /mg of DM
¾ Mean of ILs 421 S = 155 nmole aa /mg of DM
Validation of a QTL for sulfate content
Shahdara
Bay-0
Heterozygous
068S: 110 nmol SO4/mgDW
HIF068 - Sha
068
068B: 80 nmol SO4/mgDW
HIF068 - Bay
Then, you may be lucky if a candidate
gene is present in the chromosome area
where the QTL is located.
That was the case for the QTL of sulfate
content. A gene coding for a key enzyme
of sulfate assimilatory pathway (APR2)
was present in the interval of interest on
the chromosome.
QTL SO3.1 confirmed
Let us have a look at sulfate assimilation….
ATP
sulphurylase
SO4--
Sulfite
APS
O-acetyl-L-serine
reductase
sulphydrylase
reductase
Cysteine
SO3-S-APS
2e-
ATP
2 GSH
6eFerredoxin
Glutathione
Proteins
OAS
Methionine
Serine acetyl
transferase
APR1
APS reductase
APR2
APR3
QTL SO3.1
Confirmation of the candidate gene APR2
APR2 sequenced on Bay-0, Shahdara and 24 ecotypes of Arabidopsis :
one Single Nucleotide Polymorphism resulting in one amino acid
change in the protein .
***
***
cTP
aa position
(P)APS reductase
V
V
V
A
V
V
V
V
V
D
D
D
D
D
D
G
D
D
T
T
T
T
T
T
T
T
N
R
R
R
R
R
R
L
R
R
M
M
M
M
M
M
I
M
M
S
S
S
S
S
S
S
S
T
T
T
T
T
T
T
T
T
L
G
G
G
G
G
G
E
E
E
R
R
S
R
R
R
K
K
K
5
38
40
46
49 56.1 58
65
79
107 111 349 385 399 430
Loudet et al 2007
S
Trx-like
Q
Q
Q
Q
Q
Q
E
E
E
K
K
K
R
R
R
R
R
R
I
I
I
I
V
V
I
V
V
A
A
A
A
A
E
A
A
A
A
S
A
A
A
A
A
A
A
hapl1
Col-0
hapl2
Edi-0
hapl3
Bur-0
hapl4
Kn-0
hapl5
Bay-0
hapl6
Shahdara
hapl7
Cvi-0
hapl8
Bla-1
hapl9
Ct-1
Representative
accession
To test the candidate gene APR2, we
performed a complementation test
• Test of dominance
SO4-- content
HIF068-B
a
HIF068-BXS
a
b
HIF068-S
0
20
40
60
80
100
Sulfate Content (nmol/mgDM)
120
• Dominance of Bay-0 allele => suggesting that Shahdara allele is less
active => accumulation of sulfate
• In the complementation test : the Bay-0 allele will be used to
complement a HIF-Shahdara
Loudet et al 2007
Transgenic complementation test in Shahdara:
restoration of the Bay-0 sulfate content level
140
120
b
Sulfate Content (nmol/mgDM)
100
a
80
a
a
a
a
a
60
a
40
20
0
-B
-S
8
8
6
6
F0
F0
I
I
H
H
HIF068-S (APR2-B)/T3
Loudet et al 2007
Thus, the QTL linked to sulfate content is
fully explained by APR2 allelic variation,
which is a successful candidate gene
approach.
If no remarkable gene is mapped in
the region of interest, we have to go
through a fine mapping approach
which allows to restrict the length of
the interval on the chromosome.
The use of rHIF lines for fine mapping
I
III
II III IV V
Line 404 (F6)
isheterozygous
at
theL4 locus
Shahdara
allele
allele
Heterozygous
allele
allele
L4 locus
F8 Self-fertilization
fertilization
Bay-0 allele
Self-fertilisation
fertilisation
F9
BBB SSSS
BB SSSSS
SSS BBBB
SS
BBBBBBSBBBBBSSBBBB SSS
B SSSSSSBSSSSSBBSSSS BBB
BBBBB
S
marker 1
I
II III IVV
I
II III IV V
I
II III IV V
marker 2
marker 3
marker 4
marker 5
404-2 (F7)
Heterozygous(F7)
(F7)
404-7 (F7)
marker 6
Self-fertilization
fertilization
MULTIPLICATION
F10
We compare the phenotypes of plants which are recombinant in the area of interest.
FINE MAPPING of a QTL
3.3
3.4
M1
M2
3.5
3.65
3.3
3.4
M1
M2
3.5
3.65
S
S
B
B
S
3.3
3.4
M2
3.5
3.65
S
S
B
S
RESULT
44-7
M1
147-8
S
The QTL interval is restricted from the distance 3.4 – 3.5 to the distance M1 – M2.
This fine mapping approach has to be
repeated until the QTL interval is
reduced to a portion containing about
20 genes in order to be able to find a
candidate gene.
So, it is a school for patience !
We can go further by observing the variability
among the Arabidopsis genotypes, which are
named « accessions ».
Arabidopsis thaliana accessions
Oy-0
St-0
Kn-0
Mh-1
Edi-0
Gre-0
Bur-0
N13
Stw-0
Blh-1
Pyl-1
Alc-0
Can-0
Ita-0
Ge-0
Shakata
Bl-1
Tsu
Jea
Ct1
Cvi-0
Akita
Sha
Mt-0
Core collection
Restricted set of accessions that encompasses the range of diversity of
the full collection. (Exist core collections of 8, 16, 24, 48 accessions)
(Mc khan et al., 2004)
4 mM
Bay-0 N13
0.2 mM
Sakata
Akita Shahdara Oy-0
Mh-1 Gre-0
Natural variation in Arabidopsis thaliana (core collection of 24 accessions)
28 days of culture on 4 mM nitrate (Sobia Ikram, 2007)
Hydroponic culture of
Arabidopsis plants
Can-0 from Spain
Root Mass / length
Primary Root Length
Phyllochron
20%
0%
-20%
-40%
-60%
-80%
-100%
Root Growth Rate
Root Fresh Matter
control
Bay-0 from Germany
N Limited
Leaf Number
Chlorophyll content
Shoot Fresh Matter
Shoot Root ratio
Root Mass / length
Primary Root Length
Phyllochron
20%
0%
-20%
-40%
-60%
-80%
-100%
Root Growth Rate
Root Fresh Matter
Leaf Number
Chlorophyll content
Shoot Fresh Matter
Shoot Root ratio
Phenotypic responses profiling of two contrasted ecotypes
faced to N limitation
We were able to distinguish three
classes of accessions:
- Class 1, plants which are very
affected by N starvation
- Class 2, plants which are less
affected
- Class 3, plants which sustain
quite well N starvation
After 7 days N starvation, the different genotypes of the core
collection can be sorted in three classes.
Shoot FW
Root FW
Class 1
- 45 %
+ 38 %
Class 2
- 36 %
+ 78 %
Class 3
- 23 %
+ 137 %
Values correspond to the decrease or increase in percentage of the control.
Dissimilarity
50000
45000
40000
35000
30000
25000
20000
15000
10000
Class 1
Dendrogram
col0
Oy0
N13
BL1
Gre0
Tsu0
Akita
Blh1
stw0
pyl1
Bur0
Sto
CtI
Mt0
Cvi0
Edi0
Bay0
shahdra
Jea
Can0
sakata
Mh1
ler
Ge0
Alc0
Kn0
5000
0
The three
classes of
accessions
Class 2
Class 3
Perspectives
• In a model plant, discovery of genes
involved in the capacity of plants to cope
with N deficiency in the soil
• In crop species, finding of new genes or
markers associated with N demand
• Select new cultivars having higher
nitrogen use efficiency
Olivier Loudet
Researcher
Françoise Vedele Fabien Chardon
Researcher
Head of NAP unit
And the Palace of Versailles !
Sobia Ikram
PhD student