meiosis 1 diploid cell 4 haploid cells homologues seen linked by crossovers at metaphase i...
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Meiosis
1 Diploid cell
4 Haploid cells
Telophase II
DiploteneDiakinesis
PachyteneZygoteneLeptotene Metaphase
I
Anaphase I
Telophase I
Homologues seen linked by Crossovers at
Metaphase I
Homologues separate
and segregated
Sister chromatids are separated during 2nd division
During prophase I, homologous chromosomes correctly recognise each other, pair and cross over
Bread wheat-Triticum aestivum 2n= 6x= 42
1 2 3 4 5 6 7
Genome A
Genome B
Genome D
Homoeologues
1A 1B 1D
Ph1 locus(Pairing homoeologous 1)
controls correct pairing and recombination
Homologues
1D 1D
Both deleted chromosome 5B in wheat hybrids inducing crossovers between homoeologues
How Ph1 was first reported?
Sir Ralph Riley UK
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
1 2 43 5 6 7Genome AGenome BGenome D
Rye
Wheat-rye hybrid Ph1-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
Metaphase I
Question 1. Does Ph1 actually block homoeologues pairing? Basics of chromosome
pairing
After 50 years the key antibodies to meiotic proteins available to answer two questions
“Glue added = amount of pairing
Pachytene
Early meiosisZygotene
homoeologues
Amount pairing= ”Glue” magentaDNA-blue
Martin et al Nature Communications 2014
Wheat-rye hybrid Ph1-
“Pachytene”
In wheat-wild relatives hybrids, experimented on by Riley and Sears, there are no homologues, only homoeologues
Wheat-rye hybrid Ph1+
Central Element“Glue”
Synaptonemal complex formed
homoeologues
“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 of Ph1. Ph1 has been named incorrectly
for50 years
1 2 43 5 6 7
Genome AGenome BGenome D
Rye
Wheat- rye hybrid
When homologues are absent, Ph1 can’t block homoeologues pairing
Ph+
Ph-
What happens in hexaploid wheat when
homologues are present?
Genome
AGenome BGenome D
Wheat
Centromeres also pair correctly with Ph1 Nature 2001
Homologous segments
telomeres Ph1+Ph1-
1DL chromosomes only pair at telomere bouquet stage with Ph1 present
The pairing of 15 homologous arms reveals that with Ph1 present: homologous arms pair correctly at the start of meiosis
Nature Cell Biol 2004
In the start of meiosis, telomeres clustered (bouquet stage)
So Ph1 promotes homologue pairing at the start of meiosis (rather than block homoeologue pairing)
1 2 43 5 6 7
Genome AGenome BGenome D
Rye
Wheat-rye hybrid Ph1-
Wheat- rye hybrid n = 28
Wheat-rye hybrid Ph1+
Up to one crossover Up to 7 crossovers
Question 2: When does Ph1 block recombination process between homoeologues?
DOUBLE HOLLIDAY JUNCTION
5’3’5’3’
Non Crossover
5’3’5’3’
5’3’5’3’
DNA STRANDS CUT AT ARROWS
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 protein
MLH1 site crossover =
-Metaphase I
leptotene
diplotene
Ph1 blocks recombination between homoeologues- When?
Alternative paths
Crossovers
5’3’5’3’
5’3’5’3’
MLH1- green DAPI- blue
Wheat-rye Ph1+ one crossover- one MLH1 site
expected
Wheat-rye Ph1-7 crossovers- 7 MLH1 sites expected
Martin et al Nature Communications 2014
21 MLH1 sites whether Ph1 is present or absent in the hybrid
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 EXO1 MLH
3
MLH1CDK2
MLH1 complex
Kinase
Wheat hybrids Ph1- 21 MLH1 sites=7 crossoversHow to get 21 MLH1 sites = 21 crossovers??
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
It seems how well the wheat hybrid is growing……
Glasshouse + fertiliser
- fertiliser
CER + fertiliser
Now ring bivalentsRod bivalents
Kinases
(Griffiths et al Nature 2006)
Cdk-like genes
Further deletions
The two Ph1 effects-promotion of homologue pairing in wheat and homoeologue recombination
in hybrids defined to a kinase locus
Metaphase I
Progenesis
0.00
0.02
0.04
0.06
0.08
Phosphorylation of histone H1 during meiosis
• Phosphorylation levels reduce with Ph1 present Greer et al Plant Cell 2012
• Increasing phosphorylation levels mimics effect of deleting Ph1 Knight et al Funct Intreg Genomics 2010
Consistent with the presence of a kinase locus Phosphorylation level
Ph1+ Ph1-
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 wormsPhosphorylation 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, Dupont-PioneerModelling-Faridoon Yousafzai, Nadia Al-Kaff, David Richards, , Azahara Martin, Martin Howard, Peter ShawPhosphoproteomics- advanced mass spec-Azahara Martin, Ali Pendle, Isabelle Colas, Alex Jones, Peter ShawCell biology-Shahal Abbo, Luis Aragon, Fadri Martinez, Pilar Prieto, Mike Wanous, Isabelle Colas, Emma Greer, Azahara Martin, Danielle Monk, Lola Rey, Peter ShawBrachypodium/Arabidopsis- Ruoyu Wen, Ali Pendle,Vera Thole, Philippe Vain, John Doonan, Peter Shaw