During prophase I, homologous chromosomes correctly recognise each other, pair and cross over Homologues seen linked by Crossovers at Metaphase I 1 Diploid cell Telophase II Diplotene Diakinesis Pachytene Zygotene Leptotene Metaphase I Anaphase I Telophase I Sister chromatids are separated during 2nd division Homologues separate and segregated 4 Haploid cells Meiosis

(Pairing homoeologous 1) controls correct pairing and recombination 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

How Ph1 was first reported? Both deleted chromosome 5B in wheat hybrids inducing crossovers between homoeologues 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

What they saw in a wheat hybrid Wheat-rye hybrid n = 28 1 2 4 3 5 6 7 Genome A Genome B Genome D Rye No homologues Wheat-rye hybrid Ph1- Wheat-rye hybrid Ph1+ Metaphase I Up to one crossover Up to 7 crossovers

Question 1. Does Ph1 actually block homoeologues pairing? After 50 years the key antibodies to meiotic proteins available to answer two questions Question 1. Does Ph1 actually block homoeologues pairing? Early meiosis Zygotene Basics of chromosome pairing Pachytene homoeologues “Glue added = amount of pairing

Ph1 has been named incorrectly for In wheat-wild relatives hybrids, experimented on by Riley and Sears, there are no homologues, only homoeologues Amount pairing= ”Glue” magenta DNA-blue Synaptonemal complex formed “Pachytene” Central Element “Glue” “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. homoeologues Wheat-rye hybrid Ph1- Wheat-rye hybrid Ph1+ Ph1 has been named incorrectly for 50 years Martin et al Nature Communications 2014

What happens in hexaploid wheat when homologues are present? Wheat- rye hybrid 1 2 4 3 5 6 7 Genome A Genome B Genome D Rye When homologues are absent, Ph1 can’t block homoeologues pairing Genome A Genome B Genome D Wheat Ph+ What happens in hexaploid wheat when homologues are present? Ph-

The pairing of 15 homologous arms reveals that with Ph1 present: 1DL chromosomes only pair at telomere bouquet stage with Ph1 present Homologous segments 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 1 2 4 3 5 6 7 Genome A Genome B Genome D Rye Wheat-rye hybrid Ph1+ Wheat-rye hybrid Ph1- Up to one crossover Up to 7 crossovers

= Ph1 blocks recombination between homoeologues- When? leptotene ONE CHROMOSOME CUT PAIR OF HOMOLOGUES or HOMOEOLOGUES 5’ 3’ STRAND INVASION DNA synthesis EXPOSED SINGLE STRANDED 3’ END Rad51 spo11 leptotene DOUBLE HOLLIDAY JUNCTION 5’ 3’ diplotene MLH1 protein MLH1 site crossover = Alternative paths Crossovers 5’ 3’ Non Crossover 5’ 3’ DNA STRANDS CUT AT ARROWS - Metaphase I

21 MLH1 sites whether Ph1 is present or absent in the hybrid Wheat-rye Ph1- 7 crossovers- 7 MLH1 sites expected MLH1- green DAPI- blue Wheat-rye Ph1+ one crossover- one MLH1 site expected Martin et al Nature Communications 2014

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 MLH1 MLH3 EXO1 MLH1 complex

How to get 21 MLH1 sites = 21 crossovers?? Wheat hybrids Ph1- 21 MLH1 sites=7 crossovers How to get 21 MLH1 sites = 21 crossovers?? It seems how well the wheat hybrid is growing…… CER + fertiliser Glasshouse + fertiliser Now ring bivalents Rod bivalents - fertiliser

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 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 Phosphorylation level Ph1+ Ph1- 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, 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, 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