During prophase I, homologous chromosomes correctly
recognise each other, pair and cross over
Homologues seen linked by
Crossovers at Metaphase I
1 Diploid cell
Zygotene
Leptotene
Telophase
II
4 Haploid cells
Pachytene
Diplotene
Diakinesis
Metaphase I
Sister chromatids
are separated during 2nd division
Meiosis
Anaphase I
Telophase I
Homologues
separate
and segregated
Bread wheat-Triticum aestivum 2n= 6x= 42
1
2
3
4
5
6
7
Genome A
Genome B
Genome D
Homologues
1D 1D
Homoeologues
1A 1B 1D
Ph1 locus
(Pairing homoeologous 1)
controls correct pairing and
recombination
How Ph1 was first reported?
Sir Ralph Riley
UK
Both deleted chromosome 5B in wheat
hybrids inducing crossovers between
homoeologues
Dr Ernie Sears
US
• The major regulator of pairing and recombination in wheat on 5B
• Termed the locus, Ph1, (Pairing homoeologous 1), assuming it
blocked homoeologous pairing, therefore recombination
What they saw in a wheat hybrid
Wheat-rye hybrid n = 28
Genome A
Genome B
Genome D
Rye
Wheat-rye hybrid
1
2
Ph1+
3
4
5
No homologues
6
7
Wheat-rye hybrid
Ph1-
Metaphase I
Up to one crossover
Up to 7 crossovers
After 50 years the key antibodies to meiotic
proteins available to answer two questions
Question 1. Does Ph1 actually block homoeologues
pairing?
Early meiosis
Basics of chromosome pairing
Zygotene
homoeologues
Pachytene
In wheat-wild relatives hybrids, experimented on by Riley and
Sears, there are no homologues, only homoeologues
Synaptonemal
complex formed
homoeologues
DNA-blue
Amount pairing= ”Glue” magenta
“Pachytene”
“Glue” or pairing
(µm)
Pairing or Synapsis
%
Wheat-Rye -
378.50
26%
Wheat-Rye +
403.95
27%
Homoeologue pairing is not reduced in wheat-rye
hybrid
by the
presence
Ph1.
Wheat-rye
hybrid
Ph1-ofWheat-rye
hybrid Ph1+
Ph1 has been named incorrectly for
50 years
Martin et al Nature Communications 2014
Wheat- rye hybrid
1
Genome
Genome
Genome
Rye
2
3
4
A
B
D
Wheat
Ph+
Genome
A
Genome
B
Genome
D
Ph-
5
6
7
When homologues are absent,
Ph1 can’t block homoeologues
pairing
What happens in
hexaploid wheat when
homologues are present?
Homologous segments
1DL chromosomes only pair at telomere
bouquet stage with Ph1 present
Nature Cell
Biol 2004
Ph1+
Ph1- telomeres
In the start of meiosis, telomeres clustered (bouquet stage)
The pairing of 15 homologous arms reveals that with Ph1 present:
homologous arms pair correctly at the start of meiosis
Centromeres also pair correctly with Ph1 Nature 2001
So Ph1 promotes homologue pairing at the start of meiosis
(rather than block homoeologue pairing)
Question 2: When does Ph1 block recombination
process between homoeologues?
Wheat- rye hybrid n = 28
Genome
Genome
Genome
Rye
Wheat-rye hybrid
A
B
D
Ph1+
Up to one crossover
1
2
3
4
5
6
7
Wheat-rye hybrid
Up to 7 crossovers
Ph1-
Ph1 blocks
recombination between
homoeologues- When?
PAIR OF
HOMOLOGUES or
HOMOEOLOGUES
leptotene
5’
3’
5’
3’
spo11
ONE CHROMOSOME
CUT
EXPOSED SINGLE
STRANDED 3’ END
5’
3’
5’
3’ 5’
3’
5’
3’
Rad51
STRAND INVASION
5’
3’
5’
3’
DNA synthesis
5’
DOUBLE
3’
HOLLIDAY 5’3’
JUNCTION
MLH1 protein
MLH1 site
=
crossover
diplotene
Alternative
paths
5’
3’
5’
3’
5’
3’
5’
3’
DNA STRANDS CUT AT
ARROWS
5’
3’
5’
3’
-
5’
3’
5’
3’
Crossovers
Metaphase I
Non Crossover
21 MLH1 sites whether Ph1 is present or absent in
the hybrid
Wheat-rye Ph17 crossovers- 7 MLH1 sites
expected
MLH1- green
Wheat-rye Ph1+
one crossover- one MLH1 site expected
Martin et al Nature
Communications 2014
DAPI- blue
Summary of data in hybrids
• Homoeologues pair whether Ph1 is present or absent.
• Recombination machinery loads with or without Ph1, but
stalls with the loading of the last protein- MLH1.
• Stalling is partially alleviated by deleting Ph1.
• Ph1 affects the MLH1 complex and whether crossovers
occur or not
Kinase
CDK2
EXO1
MLH1
MLH3
MLH1 complex
Wheat hybrids Ph121 MLH1 sites=7 crossovers
How to get 21 MLH1 sites = 21 crossovers??
It seems how well the wheat hybrid is growing……
a
b
*
* **
*
CER +
fertiliser
Glasshouse
+ fertiliser
*
c
Crossover Number
Rod bivalents
- fertiliser
No.
Cells
7
8
9
10
11
12
13
14
15
16
17
18
1
0
2
2
4
8
6
11
5
8
2
1
Now ring bivalents
The two Ph1 effects-promotion of homologue pairing in
wheat and homoeologue recombination in hybrids defined to
a kinase locus
Further deletions
Cdk-like genes
Kinases
(Griffiths et al Nature 2006)
Consistent with the presence of a kinase locus
Phosphorylation level
0.06
0.04
0.02
W
• Increasing phosphorylation
levels mimics effect of deleting
Ph1 Knight et al Funct Intreg Genomics 2010
Ph1H
Ph1+
T
0.00
P
• Phosphorylation levels reduce
with Ph1 present Greer et al Plant Cell 2012
Progenesis
0.08
Phosphorylation of histone H1
during meiosis
Metaphase I
Wheat-rye hybrid treated with
Okadaic acid-Rod bivalents
We have shown that:
•
•
•
•
Ph1 maps to a kinase locus region –Nature 2006
Ph1 reduces meiotic phosphorylation levels-Plant Cell 2012
Ph1 promotes correct pairing of centromeres-Nature 2001
Ph1 promotes correct pairing of chromosome arms-Nature Cell Biol 2004
Ph1 regulates the conversion of MLH1 sites to crossovers- Nature
Communs 2014
•
Increased phosphorylation levels mimic the effect of deleting Ph1 •
Funct Intreg Genomics 2010- Plant Cell 2012
•
At increased phosphorylation levels, the ratio of MLH1 sites to
crossovers is closer to 1:1 in healthy plants
A C elegans study (PLoS Genetics 2014) concluded:
reduced meiotic phosphorylation levels promote:
correct pairing of centromeres
correct pairing of chromosome arms
the conversion of MLH1 sites to crossovers
At increased phosphorylation levels, the ratio of MLH1 sites
to crossovers is closer to 1:1 in healthier worms
Phosphorylation may be a universal regulator of correct
meiotic prophase chromosome dynamics
•
•
•
•
Graham Moore- My thanks to…
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, DupontPioneer
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, 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