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