Graham Moore
To recombine, or not recombinethat is the question??
October 2014
Hexaploid (Bread) wheatTriticum aestivum
2n = 6x =42
1 2 3 4 5 6 7
A
B
D
abcd
abcd
abcd
homologues
homoeologues
To be fertile, true homologues must pair at meiosis
Diploid – Homologues distinguished from non-homologous chromosomes
Wheat - Homologues, homoeologues and non-homologous chromosomes
Meiosis
1 Diploid cell
4 Haploid cells
Telophase II
Prophase I
DiploteneDiakinesis
PachyteneZygotene
LeptoteneMetaphase
I
Anaphase I
Telophase IProphase IIMetaphase
II
Anaphase II
Homologues seen as paired via Crossovers
at metaphase I
Homologues
separateand
segregatedSister chromatids
are separated
Homologous chromosomes must recognise each other, pair correctly and recombine
Both observed that deleting chromosome 5B in wheat hybrids induced crossovers between homoeologues
What controls “polyploid” chromosome pairing in wheat??
Sir Ralph Riley UK
Dr Ernie Sears US
• Reasoned a 5B locus was the major regulator of pairing and recombination in wheat
• Termed the locus, Ph1, (Pairing homoeologous 1)• And started 50 years of intense rivalry between wheat
researchers..
Infamous Wheat genetics meetingchaired by Sir Ralph-two Ph1 speakers
XGenome A
Genome B
Genome D
Wheat 2n= 42
1 2 43 5 6 7
Rye 2n= 14
1 2 43 5 6 7
Genome AGenome B
Genome D
Rye
Wheat-rye hybrid Ph1-
Wheat- rye hybrid n =
28
No homologues
Wheat-rye hybrid Ph1+
Wheat-rye hybrid n = 28
What they saw in a wheat hybrid
Up to one crossover Up to 7 crossovers
1) Does Ph1 actually block homoeologues pairing?
Leptotene Zygotene Pachytene
homologues
Chromosome Synapsis
Basics of chromosome pairing
The power of a cell biology experiment
“ primer added”Lateral element
“Glue added”Central Element
Early meiosis
After 50 years the key antibodies to meiotic proteins are available to answer two questions
Central Element“Glue”
Lateral Element“primer”
Synaptonemal complex formed
homoeologues
In wheat-wild relatives hybrids, experimented on by Riley and Sears, there are no homologues, only homoeologues
“Pachytene”Wheat-rye hybrid Ph1-
Martin et al Nature Communications 2014
Lateral element- “primer” greenCentral element-”glue” magenta
DNA-blue
Wheat-rye hybrid Ph1+
Lateral element- “primer” greenCentral element-”Glue” magenta
DNA-blue
“P” (µm) “G” (µm) Synapsis %
Wheat-Rye - 1662.95 378.50 26%
Wheat-Rye + 1594.00 403.95 27%
Homoeologue pairing is not reduced in wheat-rye hybrid by the presence of Ph1.
Ph1 has been named incorrectly for
50 years!
Martin et al Nature Communications 2014
Meiotic progression
“Pachytene”
Genome A
Genome B
Genome D
Wheat 2n= 42
1 2 43 5 6 7
Genome AGenome B
Genome D
Rye
Wheat- rye hybrid n =
28
No homologues Ph1 can’t block the homoeologues pairing in the
hybrid
Ph1’s effect on chromosome pairing
But Ph1 can block/reduce homoeologues pairing in wheatPh1 action is to promote homologues
to pair
telomeres
homologoussegments
What happens with pairing of homologues in wheat itself??
The identical chromosomes zip up from their telomere regions
Pilar Prieto et al 2004 Nat Cell Biol
Rye
segment
homologues
telomeres
‘Elongation” ofchromatin
This conformation change now reported in C elegans
Telomeres
Ph1+ Ph1+Ph1+
Ph1-
Ph1+
In wheat- homologues can elongate asynchronously without Ph1
Interstitial segments- 15% of the wheat chromosome
Pilar Prieto et al 2004 Nat Cell BiolColas et al. 2008 PNAS 2008
XGenome A
Genome B
Genome D
Wheat 2n= 42
1 2 43 5 6 7
Rye 2n= 14
1 2 43 5 6 7
Genome AGenome B
Genome D
Rye
Wheat-rye hybrid Ph1-
Wheat- rye hybrid n =
28
Wheat-rye hybrid Ph1+
Up to one crossover Up to 7 crossovers
2) Ph1 blocks recombination between homoeologues- When?
DOUBLE HOLLIDAY JUNCTION
5’3’5’3’
Non Crossover
5’3’5’3’
5’3’5’3’
Alternative paths
DNA STRANDS CUT AT ARROWS
Crossovers
5’3’5’3’
5’3’5’3’
ONE CHROMOSOME CUT
PAIR OF HOMOLOGUES orHOMOEOLOGUES
5’3’5’3’
STRAND INVASION5’3’5’3’
DNA synthesis
EXPOSED SINGLE STRANDED 3’ END
5’3’5’3’
5’
3’5
’3’
Rad51
spo11
MLH1 complex required for resolution of Double Holliday Junctions as crossovers
# MLH1 sites at
diplotene
# sites that will crossover=
- Wheat-rye hybrid Ph1+
Metaphase I
MLH1CDK2EXO1
MLH3
leptotene
diplotene
Ph1 blocks recombination between homoeologues- When?
Robin Holliday was at JIC
MLH1- greenDAPI- blue
Wheat-rye Ph1+ - one crossover- MLH1 number expected ≈ one foci
- MLH1 number observed = 22 + 3 foci_
Wheat-rye Ph1-- 7 crossovers- MLH1 number expected ≈ 7-8 foci
- MLH1 number observed = 20 + 3 foci_
Martin et al Nature Communications 2014
Summary of data in hybrids
homoeologues
Mlh1Cdk2
• Homoeologues pair (synapse) where Ph1 is present or absent.
• Recombination machinery loads but stalls with the loading of the last complex- the MLH1 complex.
• Stalling is partially alleviated by deleting Ph1
• The MLH1 complex is composed of MLH1, MLH3, EXO1 and CDK2, and regulated by CDK2
• So what is Ph1??
Strategy for delineating the Ph1 locus
Implementation
• Screened 10 irradiated wheat populations = 15 years
• Mapping deletion breakpoints and gene content revealed by cereal synteny (our development)
Wheat mutants
• Ph1 is defined as deletion effect• Define Ph1 with overlapping deletions • Deletions scored for presence/absence of Ph1
W h e a tc h r 5 B
Ph1 region in B rachypodium (to sca le )
B rachypodium region 1 genes
B rachypodium region 2 genes
B rachypodium B A C s
W h e a tc h r 5 A
W h e a tc h r 5 D
D ele tionbreakpoints
D ele tionbreakpoints
clk1
(cl
v)
stk1
vmp1
hyp
4 (n
ew)
rdr1
zip1
(zin
c)
hsp
20-1
raf1
(ra
8)
wdb1
(wrk
y)
ps1
v
h51
l (hap
5)
at1(
AtH
ypIII
)
hsp
90
gtl1
(chic
k)
sbp1
(sel
en)
cmt1
(cc
om
)
ugg1
(udp)
hyp
6 (A
tHyp
2)
fim
2
slp1
(sub)
hyp
3 (O
sHyp
II)
bna
1236
H11
Rev a
1236
H11
Rev b
1677
L4 C
33
1417
M11
350
OsH
ypI
916G
3 R
ev
1390
E11
Uni
pep
1
fim
1
mic
1 (A
tHyp
7)
hyp
5 (m
arck
s)
R ice chr 8 genes
Ph1 region in rice chr 9 (to sca le)
R ice chr 9 genes
rlk2
hyp
scf
OsH
ypI CS379
3
CS977
raf
wdb
ps1
v
h5l
1
atat el
p
gtl
cmt
hyp
clk
scf
hsp
90
raf
wdb
h5l
gtl
cmt
pdp
gtl2
cmt2
fim
1
hyp
slp
fim
2
C84
6
CS
100
2 &
455-9
-1
CS
1010
pfk
hyp
hyp
plp
1
1 2 F 16
1 7 8 3A 2 2
1 3 9 0E 11
7 1 9 O 7
6 0 6 L2
1 0 5 1I3
1 7 7 2H 1 2
1 6 7 7L 4
5 3 2 G 11
1 6 5 D 12
1 0 5 P 17
1 6 5 3J 2
1 2 1 7B 2
9 1 6 G 3
4 3 2 L9
1 4 1 7M 11
1 5 6 8 O 9
1 6 6 4J 5
9 9 3 J2
2 8 2 J8
4 7 0 M 7
1 3 2 0C 1 4
1 4 0 5 B 1 0
3 0 4 F6
1 5 0 C 14
clk
1
rlk1
cycd
1
raf1
wdb1
mic
1
ps1
v
h5l1
at1
rlp1
AtH
yp5
gtl1
sbp1
cmt1
AtH
yp2
fim
1
slp
1
New
Hyp
cdc2
pfk
1
Marc
ks
plp
1
grp
1
KH
1
KH
4
KH
2
KH
5
KH
3
KH
6
KH
7
1 2 0 1K 2 3
hyp
2 (K
H2)
hyp
3 (K
H3)
hyp
4 (K
H4)
Sub te lom e ric insertion from chr 3A
116 2B 5
2 54 I1 0
1 8 9 8B 1 9
6 7 H 7
22 I2
563C 24
1 2 2 3B 6
9 6 2 M 8
1 2 3 6 H 11
Te lo m e reC e n tro m e re
11 9 5 H 2 2
8 4 6 A 11
hsp
90
cnb
sha
wak
lbp
fas
9 2 5 M 1 5
3 4 2F 1 0
2 1 8J 1 3
897P 24
c d c 2h o m o lo g u e s
cdc2
-4
3 0 N 2
2 8 C 1 3
3 1 8 G 1
Outside de letion region
7 7 9 O 1 2
Outside de letion region
1 5 6 7N 6
1 8 2 1A 3
1 5 9 A 2
1 4 9 7 M 6
3 1 B 11
1 2 7 5 L 15
7 0 3 L 1 6
1 2 4 P 2 2
11 4 6 J 1 3
4 3 1 E 7
1 6 2 5E 2 1
7 2 6 N 4
1 2 6 8O 8
11 8 3 B 1 0
1 4 6 2G 15
7 4 9 H 15 (A /D )
3 8 7 C 19
7 8 0 H 16
1 8 4 0B 1 7
870L14
21419
1 0 0K b
Griffiths et al Nature 2006
The gene content of the minimum region containing the Ph1 locus
Kinases
Half the genes are kinases
A cluster of 7 defective Cdk2-type genes
(Griffiths et al Nature 2006)
The gene content of the minimum region containing the Ph1 locus
Kinases
So what next??
Nearly half the genes are kinases, so does altered kinase activity induce crossovers between homoeologues?
We can increase Cdk activity/phosphorylation levels- how?
Detached tiller method
• Okadaic acid inhibits phosphatases which reduce Cdk activity
• Okadaic acid indirectly increases Cdk activity/
Phosphorylation levels
homoeologues
MLH1CDK2
Overcomestalling of MLH1 complex??
Okadaic acid treatment induces crossovers between homoeologues in a wheat x rye hybrid
Homoeologous pairing
Wheat X Rye – Ph1 deleted
No okadaic acid – mostly univalents
Okadaic acid treatment -Rod bivalents
So, okadaic acid treatment similar to deleting Ph1Emilie Knight et al., 2010
Metaphase I
Cdk2 Ph1-cdk gene
Yousafzai and Al-kaff, 2010
Ph1 cdk+cyclinA compared to Cdk2+cyclinA
Protein modelling
• MLH1 complex is regulated by CDK2• Is CDK2 activity increased by deleting Ph1?
• Increasing CDK activity is similar to deleting Ph1
Progenesis
WT PH
0.00
0.02
0.04
0.06
0.08
Untreat
ed
OA_100
OA_200
0.000
0.005
0.010
0.015
0.020
0.025
rati
o p
ho
sph
o /
no
n-p
ho
sph
o
Phosphorylation sites
(S/TP-X-Z
Azahara Martin, Ali Pendle, Alex Jones, Isabelle Colas
Greer et al., Plant Cell 2012
Wheat histone H1
Deleting Ph1 or treating with Okadaic acidincreases phosphorylation at the sameCDK2 phosphoryation site
The Ph1 locus- Summary
• In the presence of Ph1, CDK2 activity is reduced
• MLH1 complex (containing CDK2) stalls on paired homoeologues in the presence of Ph1.
• Increased CDK activity, stalling overcome, crossovers induced even in the presence of Ph1
2) blocks homoeologous recombination
1) promotes homologue pairing
• Chromatin changes with Ph1
We will make the ultimate sacrifice in the winter of……
b
* *
* * **
a
c
7 8 9 10 11 12 13 14 15 16 17 18
No. Cells
1 0 2 2 4 8 6 11 5 8 2 1
Crossover Number
JIC, US, Denmark etcWheat hybrids Ph1- (21 MLH1 sites= 7 crossovers)How to improve on this??
b
* *
* * **
a
c
7 8 9 10 11 12 13 14 15 16 17 18
No. Cells
1 0 2 2 4 8 6 11 5 8 2 1
Crossover Number
Cordoba, Feb/March (27oC!)Wheat hybrids exhibit21 MLH1 sites= over 13 crossovers
The Fly in the ointment! inthe approach
JIC grown hybrids plusCordoba fertiliser21 MLH1 sites= over 13 crossovers
May end career as a John Innes Researcher demonstratingCordoba No1 is better than John Innes No1!!
Thanks to…
Genomics- Comparative-BAC library-mutantsTracie Draeger (Foote), Michael Roberts, Lijia Qu, Terry Miller, Steve Reader, Simon Griffiths, Sebastien Allouis, Rebecca Sharp, Kath Mortimer, Emilie Knight, Nadia Al-Kaff, Vera Thole, Ruoyu Wen, Boulos Chalhoub, Shahryar Kianian, Dupont-Pioneer
ModellingFaridoon Yousafzai, Nadia Al-Kaff, David Richards, Martin Howard
Phosphoproteomics- advanced mass specAzahara Martin, Ali Pendle, Isabelle Colas, Alex Jones, Peter Shaw
Cell biology-Shahal Abbo, Luis Aragon, Fadri Martinez, Pilar Prieto, Mike Wanous, Isabelle Colas, Emma Greer, Azahara Martin, Danielle Monk, Peter Shaw
Ph1 into other species-Brachypodium/ArabidopsisRuoyu Wen, Ali Pendle,Vera Thole, Philippe Vain, John Doonan, Peter Shaw
W h e a tc h r 5 B
P h 1 reg ion in B rac h yp o d iu m (to sca le )
B rac h yp o d iu m re g io n 1 ge n es
B rac h yp o d iu m re g io n 2 ge n es
B rac h yp o d iu m B A C s
W h e a tc h r 5 A
W h e a tc h r 5 D
D ele tio nb rea kp o in ts
D ele tio nb rea kp o in ts
clk1
(cl
v)
stk1
vmp
1
hyp
4 (n
ew)
rdr1
zip
1 (z
inc)
hsp
20-1
raf1
(ra
8)
wd
b1
(wrk
y)
ps1
v
h51
l (h
ap5)
at1(
AtH
ypIII
)
hsp
90
gtl
1 (c
hic
k)
sbp
1 (s
elen
)
cmt1
(cc
om
)
ug
g1
(ud
p)
hyp
6 (A
tHyp
2)
fim
2
slp
1 (s
ub
)
hyp
3 (O
sHyp
II)
bn
a
12
36H
11 R
ev
a
12
36H
11 R
ev
b
16
77L
4 C
33
14
17M
11 3
50
Os
Hy
pI
91
6G3
Re
v
13
90E
11 U
ni
pep
1
fim
1
mic
1 (A
tHyp
7)
hyp
5 (m
arck
s)
R ice c hr 8 g e n es
P h 1 re gio n in ric e c h r 9 (to s ca le)
R ice c hr 9 g e n es
rlk2
hyp
scf
OsH
ypI CS
379
3
CS
977
raf
wd
b
ps1
v
h5l
1
atat el
p
gtl
cmt
hyp
clk
scf
hsp
90
raf
wd
b
h5l
gtl
cmt
pd
p
gtl
2
cmt2
fim
1
hyp
slp
fim
2
C84
6C
S1
002
& 4
55
-9-1
CS
10
10
pfk
hyp
hyp
plp
1
1 2 F 1 6
1 7 8 3 A 2 2
1 3 9 0 E 1 1
7 1 9 O 7
6 0 6 L 2
1 0 5 1 I 3
1 7 7 2 H 1 2
1 6 7 7 L 4
5 3 2 G 11
1 6 5 D 1 2
1 0 5 P 1 7
1 6 5 3 J 2
1 2 1 7 B 2
9 1 6 G 3
4 3 2 L 9
1 4 1 7 M 11
1 5 6 8 O 9
1 6 6 4 J 5
9 9 3 J2
2 8 2 J8
4 7 0 M 7
1 3 2 0 C 1 4
1 4 0 5 B 1 0
3 0 4 F 6
1 5 0 C 1 4
clk
1
rlk
1
cy
cd1
raf1
wd
b1
mic
1p
s1v
h5
l1
at1
rlp
1
AtH
yp
5
gtl
1sb
p1
cmt1
AtH
yp
2
fim
1
slp
1
Ne
w H
yp
cdc
2
pfk
1
Ma
rcks
plp
1
grp
1
KH
1
KH
4
KH
2
KH
5
KH
3
KH
6K
H7
1 2 0 1 K 2 3
hyp
2 (K
H2)
hyp
3 (K
H3)
hyp
4 (K
H4)
S ub te lom e ric inse rtion from ch r 3A
1 1 6 2 B 5
2 5 4 I1 0
1 8 9 8 B 1 9
6 7 H 7
2 2 I2
5 6 3 C 24
1 2 2 3 B 6
9 6 2 M 8
1 2 3 6 H 11
Te lo m e reC e n tro m e re
11 9 5 H 2 2
8 4 6 A 11
hsp
90cn
bsh
aw
aklb
p
fas
9 2 5 M 1 5
3 4 2 F 1 0
2 1 8 J 1 3
8 9 7 P 24
c d c 2h o m o lo g u e s
cdc2
-4
3 0 N 2
2 8 C 1 3
3 1 8 G 1
O u tsid e d e letio n re g io n
7 7 9 O 1 2
O u tsid e d e letio n re g io n
1 5 6 7 N 6
1 8 2 1 A 3
1 5 9 A 2
1 4 9 7 M 6
3 1 B 11
1 2 7 5 L 1 5
7 0 3 L 1 6
1 2 4 P 2 2
11 4 6 J 1 3
4 3 1 E 7
1 6 2 5 E 2 1
7 2 6 N 4
1 2 6 8 O 8
11 8 3 B 1 0
1 4 6 2 G 1 5
7 4 9 H 1 5 ( A /D )
3 8 7 C 1 9
7 8 0 H 1 6
1 8 4 0 B 1 7
8 7 0 L1 4
21419
100K b
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