Hexaploid (Bread) wheat Triticum aestivum 2n = 6x = A B D abcdabcd abcdabcd abcdabcd homologues homoeologues To be fertile, true homologues must pair at meiosis Diploid – Homologues distinguished from non-homologous chromosomes Wheat - Homologues, homoeologues and non-homologous chromosomes

Meiosis 1 Diploid cell 4 Haploid cells Homologues seen as paired via Crossovers at metaphase I Homologues separate and segregated Sister chromatids are separated Homologous chromosomes must recognise each other, pair correctly and recombine

Both observed that deleting chromosome 5B in wheat hybrids induced crossovers between homoeologues What controls “polyploid” chromosome pairing in wheat?? Sir Ralph Riley UK Dr Ernie Sears US Reasoned a 5B locus was the major regulator of pairing and recombination in wheat Termed the locus, Ph1, (Pairing homoeologous 1) And started 50 years of intense rivalry between wheat researchers.. Infamous Wheat genetics meeting chaired by Sir Ralph-two Ph1 speakers

X Genome A Genome B Genome D Wheat 2n= Rye 2n= Genome A Genome B Genome D Rye Wheat-rye hybrid Ph1- Wheat- rye hybrid n = 28 No homologues Wheat-rye hybrid Ph1+ Wheat-rye hybrid n = 28 What they saw in a wheat hybrid Up to one crossoverUp to 7 crossovers

1) Does Ph1 actually block homoeologues pairing? Leptotene Zygotene Pachytene homologues Chromosome Synapsis Basics of chromosome pairing The power of a cell biology experiment Early meiosis After 50 years the key antibodies to meiotic proteins are available to answer two questions

Synaptonemal complex formed homoeologues In wheat-wild relatives hybrids, experimented on by Riley and Sears, there are no homologues, only homoeologues “Pachytene” Wheat-rye hybrid Ph1 - Martin et al Nature Communications 2014 Lateral element- “primer” green Central element-”glue” magenta DNA-blue

Wheat-rye hybrid Ph1+ Lateral element- “primer” green Central element-”Glue” magenta DNA-blue “P” (µm)“G” (µm)Synapsis % Wheat-Rye % Wheat-Rye % Homoeologue pairing is not reduced in wheat-rye hybrid by the presence of Ph1. Ph1 has been named incorrectly for 50 years! Martin et al Nature Communications 2014 Meiotic progression “Pachytene”

Genome A Genome B Genome D Wheat 2n= Genome A Genome B Genome D Rye Wheat- rye hybrid n = 28 No homologues Ph1 can’t block the homoeologues pairing in the hybrid Ph1’s effect on chromosome pairing But Ph1 can block/reduce homoeologues pairing in wheat Ph1 action is to promote homologues to pair

telomeres homologous segments What happens with pairing of homologues in wheat itself?? The identical chromosomes zip up from their telomere regions Pilar Prieto et al 2004 Nat Cell Biol Rye segment homologues telomeres ‘Elongation” of chromatin This conformation change now reported in C elegans

Telomeres Ph1+ Ph1- Ph1+ In wheat- homologues can elongate asynchronously without Ph1 Interstitial segments- 15% of the wheat chromosome Pilar Prieto et al 2004 Nat Cell Biol Colas et al PNAS 2008

X Genome A Genome B Genome D Wheat 2n= Rye 2n= Genome A Genome B Genome D Rye Wheat-rye hybrid Ph1- Wheat- rye hybrid n = 28 Wheat-rye hybrid Ph1+ Up to one crossover Up to 7 crossovers 2) Ph1 blocks recombination between homoeologues- When?

DOUBLE HOLLIDAY JUNCTION 5’ 3’ 5’ 3’ Non Crossover 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Alternative paths DNA STRANDS CUT AT ARROWS Crossovers 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ ONE CHROMOSOME CUT PAIR OF HOMOLOGUES or HOMOEOLOGUES 5’ 3’ 5’ 3’ STRAND INVASION 5’ 3’ 5’ 3’ DNA synthesis EXPOSED SINGLE STRANDED 3’ END 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Rad51 spo11 MLH1 complex required for resolution of Double Holliday Junctions as crossovers # MLH1 sites at diplotene # sites that will crossover = - Wheat-rye hybrid Ph1+ Metaphase I MLH1 CDK2 EXO1 MLH3 leptotene diplotene Ph1 blocks recombination between homoeologues- When? Robin Holliday was at JIC

MLH1- green DAPI- blue Wheat-rye Ph1+ - one crossover- MLH1 number expected ≈ one foci - MLH1 number observed = foci _ Wheat-rye Ph crossovers- MLH1 number expected ≈ 7-8 foci - MLH1 number observed = foci _ Martin et al Nature Communications 2014

Summary of data in hybrids homoeologues Mlh1 Cdk2 Homoeologues pair (synapse) where Ph1 is present or absent. Recombination machinery loads but stalls with the loading of the last complex- the MLH1 complex. Stalling is partially alleviated by deleting Ph1 The MLH1 complex is composed of MLH1, MLH3, EXO1 and CDK2, and regulated by CDK2 So what is Ph1??

Strategy for delineating the Ph1 locus Implementation Screened 10 irradiated wheat populations = 15 years Mapping deletion breakpoints and gene content revealed by cereal synteny (our development) Wheat mutants Ph1 is defined as deletion effect Define Ph1 with overlapping deletions Deletions scored for presence/absence of Ph1 Griffiths et al Nature 2006

The gene content of the minimum region containing the Ph1 locus Kinases Half the genes are kinases A cluster of 7 defective Cdk2-type genes (Griffiths et al Nature 2006)

The gene content of the minimum region containing the Ph1 locus Kinases So what next?? Nearly half the genes are kinases, so does altered kinase activity induce crossovers between homoeologues?

We can increase Cdk activity/phosphorylation levels- how? Detached tiller method Okadaic acid inhibits phosphatases which reduce Cdk activity Okadaic acid indirectly increases Cdk activity/ Phosphorylation levels homoeologues MLH1 CDK2 Overcome stalling of MLH1 complex??

Okadaic acid treatment induces crossovers between homoeologues in a wheat x rye hybrid Homoeologous pairing Wheat X Rye – Ph1 deleted No okadaic acid – mostly univalents Okadaic acid treatment -Rod bivalents So, okadaic acid treatment similar to deleting Ph1 Emilie Knight et al., 2010 Metaphase I

Cdk2 Ph1-cdk gene Yousafzai and Al-kaff, 2010 Ph1 cdk+cyclinA compared to Cdk2+cyclinA Protein modelling MLH1 complex is regulated by CDK2 Is CDK2 activity increased by deleting Ph1? Increasing CDK activity is similar to deleting Ph1

Phosphorylation sites (S/TP-X-Z Azahara Martin, Ali Pendle, Alex Jones, Isabelle Colas Greer et al., Plant Cell 2012 Wheat histone H1 Deleting Ph1 or treating with Okadaic acid increases phosphorylation at the same CDK2 phosphoryation site

The Ph1 locus- Summary In the presence of Ph1, CDK2 activity is reduced MLH1 complex (containing CDK2) stalls on paired homoeologues in the presence of Ph1. Increased CDK activity, stalling overcome, crossovers induced even in the presence of Ph1 2) blocks homoeologous recombination 1)promotes homologue pairing Chromatin changes with Ph1

We will make the ultimate sacrifice in the winter of…… JIC, US, Denmark etc Wheat hybrids Ph1- (21 MLH1 sites= 7 crossovers) How to improve on this?? Cordoba, Feb/March (27 o C!) Wheat hybrids exhibit 21 MLH1 sites= over 13 crossovers The Fly in the ointment! in the approach JIC grown hybrids plus Cordoba fertiliser 21 MLH1 sites= over 13 crossovers May end career as a John Innes Researcher demonstrating Cordoba No1 is better than John Innes No1!!

Thanks to… Genomics- Comparative-BAC library-mutants Tracie Draeger (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, Martin Howard 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, Peter Shaw Ph1 into other species-Brachypodium/Arabidopsis Ruoyu Wen, Ali Pendle,Vera Thole, Philippe Vain, John Doonan, Peter Shaw