1/19/2016
During meiosis, chromosomes pair and recombine
Double strand breaks are repaired during pairing
PAIR OF
HOMOLOGUES or
HOMOEOLOGUES
Leptotene
Chromosome pairing occurs during the telomere
bouquet stage
Zygotene
5’
3’
5’
3’
spo11
ONE CHROMOSOME
CUT
Telomere
Bouquet
stage
EXPOSED SINGLE
STRANDED 3’ END
5’
3’
3’ 5’
3’
5’
5’
3’
Rad51
Early meiosis
Telomeres clustered as a
bouquet
Zygotene
homologues
STRAND INVASION
Pachytene
After the
Bouquet, pairing
completed
DNA synthesis
5’
DOUBLE
3’
HOLLIDAY
5’
JUNCTION 3’
Pachytene
MLH1
protein
MLH1 site
Zygotene
5’
3’
5’
3’
=
diplotene
crossover
Alternative
paths
5’
3’
5’
3’
5’
3’
5’
3’
DNA STRANDS CUT AT
ARROWS
5’
3’
5’
3’
Centromeres
clustered as 7 sites
5’
3’
5’
3’
-
Non Crossover
Crossovers
Metaphase I
Ph1 locus is the major regulator of pairing and
recombination in wheat and its hybrids
How was it first described?
Dr Ernie Sears
US
• This deletion effect of chromosome 5B was
named the Ph1 locus (Pairing homoeologous 1)
• What is it and how does it function?
Ph1+
When chromosome 5B is deleted in wheat hybrids,
homoeologous crossovers induced
Wheat-rye hybrid n = 28
1
Genome
Both deleted chromosome 5B in
wheat hybrids inducing crossovers
between homoeologues
Sir Ralph Riley
UK
Wheat-rye
hybrid
Genome
Genome
2
3
4
5
6
7
A
B
D
Rye
Wheat-rye hybrid
Ph1+
No homologues
Wheat-rye hybrid
Ph1-
Metaphase I
Up to one crossover
Up to 7 crossovers
Homoeologous crossovers at Metaphase I can used to accurately
score and map this chromosome 5B deletion effect in hybrids
The main effect of deleting chromosome 5B from
wheat is crossover failure at Metaphase I
Average number of Metaphase I chromosome
configurations per 30 meiocytes
Numbers of
Ph1+
Ph1-
Mulivalents
0
0.4
Trivalents
0
0.2
Ring bivalents
21
15
Rod bivalents
0
5
Univalents
0
1
Both crossover failure in wheat and increased
homoeologous crossover in hybrids occur when this
region is deleted
Kinases
Further deletions
Less than two chromosome arms
out of 84 engage in homoeologous
crossover without Ph1
( Al-Kaff et al Ann Bot 2008)
More than 7 chromosome arms out of
84 fail to crossover without Ph1
Only crossover failure at Metaphase I can be used to
accurately score and map this chromosome 5B deletion effect
in wheat itself
expression
CdkF-like kinase genes
Heterochromatin containing
the meiotic gene ZIP4
(Griffiths et al Nature 2006)
Homoeologous recombination,
crossover failure and a CdkF-like
kinase cluster containing a
heterochromatin-ZIP4 insertion
are linked-How??
1
1/19/2016
What happens in the wheat hybrid?
There is little homoeologue pairing at the
telomere bouquet either in the presence or
absence of Ph1
Wheat-rye hybrid n = 28
1
Genome
Genome
Genome
2
3
4
5
6
7
A
B
D
Rye
Wheat-rye hybrid
Ph1+
No homologues
Wheat-rye hybrid
Ph1-
Metaphase I
Up to one crossover
Wheat-rye hybrid Ph1Up to 7 crossovers
Most pairing starts to happen after the telomere bouquet both the
presence or absence of Ph1
Wheat-rye hybrid Ph1-
DAPI- blue
ASY1- green
ZYP1- magenta- pairing
DAPI- blue
ASY1- green
ZYP1- magenta-pairing
Pairing following the telomere bouquet
Wheat-rye hybrid Ph1+
Most pairing also happens happens after the telomere bouquet with Ph1 present
DAPI- blue
ASY1- green
ZYP1- magenta-pairing
Most pairing of homoeologues in the hybrid with or without Ph1
occurs after the telomere bouquet, with a similar level of
pairing is achieved with and without Ph1 during Pachytene
DNA-blue
Amount pairing= ”Glue” magenta
pairing
(µm)
Pairing %
Wheat-Rye -
378.50
26%
Wheat-Rye +
403.95
27%
Martin et al Nature
Communications 2014
“Pachytene”
During pairing, the recombinational machinery is loaded,
leading to 21 MLH1 sites with and without Ph1
Wheat-rye Ph17 crossovers- 7 MLH1 sites
expected
MLH1- green
DAPI- blue
Wheat-rye hybrid Ph1-
BUT most of these MLH1 sites fail to crossover whether Ph1 is present or
absent, only deleting Ph1 partially alleviates this
Wheat-rye hybrid Ph1+
Wheat-rye Ph1+
one crossover- one MLH1 site expected
Martin et al Nature
Communications 2014
2
1/19/2016
In wheat itself, delayed pairing without Ph1 leads to
incorrect pairing
Ph1+
Ph1-
Ph1-
But full pairing eventually occurs, with similar number of MLH1
sites to Ph1 present, BUT there is crossover failure
- MLH1 number observed =_48 + 4 foci
Ph1+
Telomere bouquet
16 sites on 7 chromosomes
as 8 paired sites
The delay means
homologoue pairing
to promoted by the
telomere bouquet,
so a level of
incorrect pairing
occurs
16 sites on 7 chromosomes still as 16
unpaired sites
Unpaired chromosomes
with Rad 51 sites
•
•
•
•
Ph1 promotes the early pairing of homologues in wheat,
while pairing is delayed in wheat lacking Ph1, leading to
incorrect pairing.
Despite this in wheat with or without Ph1, the chromosomes
eventually still pair to the same level.
And in wheat hybrids with or without Ph1, homoeologues
eventually still pair to the same level.
Recombination machinery also loads on these paired
homologues or homoeologues leading to similar numbers of
MLH1 sites with or without Ph1
Most MLH1 sites on the delayed pairing chromosomes or
homoeologues fail to become crossovers with and without
Ph1
• BUT deleting Ph1, partially enables more MLH1 sites to
become crossovers.
Wheat hybrids Ph121 MLH1 sites on the delayed pairing
homoeologues produce only 7 crossovers
How can this crossover failure be overcome?
b
*
* **
Glasshouse
+ fertiliser
* *
CER +
fertiliser
Now ring bivalents
c
Crossover Number
No.
Cells
- fertiliser
DAPI- blue
7
8
9
10 11 12 13 14 15 16 17 18
1
0
2
2
4
8
6
11
5
8
2
1
Our recent phosphoproteomics studies
provides an insight into the Ph1 effects
Delayed pairing in wheat without Ph1
• After premeiotic replication and double strand break (DSB) formation,
initial repair via pairing, starts with H2AX and Mre11 phosphorylation.
• Phosphorylation of H2AX, Mre11 (and SMC4-condensin) is lower without
Ph1, placing the cause of the delay during premeiotic replication.
• Ph1 maps to a CdkF region- CdkF or CAK1 regulates Cdk2/cdc28
activity- mutating CAK1 in yeast delays premeiotic replication.
MLH1 complex function on paired homoeologues
• MSH6 (part of MLH1 complex) regulates homoeologous recombination.
• Preliminary evidence suggests that Cdk2 phosphorylation sites on MSH6
show altered phosphorylation levels with and without Ph1.
Finally Crossover failure in wheat without Ph1
• The strongest expressing ZIP4 allele which promotes crossovers is
within the CdkF cluster, and deleted with Ph1
Graham Moore- My thanks to…
Up to 7 crossovers
On what the wheat hybrid is growing can partially
overcome crossover failure
a
MLH1- green
Ph1-
16 sites rarely reduce to 8Incorrect pairing occurs
Summary of data
•
- MLH1 number observed =_52 + 5 foci
Genomics- Comparative-BAC library-mutants-Tracie 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
Modelling-Faridoon Yousafzai, Nadia Al-Kaff, David
Richards, , Azahara Martin, Martin Howard, Peter Shaw
Phosphoproteomics- advanced mass spec-Azahara Martin,
Ali Pendle, Isabelle Colas, Alex Jones, Gerhard Saalbach,
Peter Shaw
Cell biology-Shahal Abbo, Luis Aragon, Fadri Martinez,
Pilar Prieto, Mike Wanous, Isabelle Colas, Emma Greer,
Azahara Martin, Danielle Monk, Lola Rey, Peter Shaw
Brachypodium/Arabidopsis- Ruoyu Wen, Ali Pendle,Vera
Thole, Philippe Vain, John Doonan, Peter Shaw
Rod bivalents
3