2. Meiosis 1 Diploid cell 4 Haploid cells Paired homologues align on plate Homologues are separated Sister chromatids are separated Homologous chromosomes must recognise each other, pair correctly and recombine Incorrect pairing leads to unbalanced gametes and infertility

3. Hexaploid wheat Triticum aestivum 2n = 6x =42 1234567 A B D abcdabcd abcdabcd abcdabcd homologues homoeologues Ph1 locus Prevents pairing of homoeologous chromosomes and unbalanced gametes To be fertile, true homologues must pair at meiosis

4. Ph1’s effect- is important agronomically Wheat-rye hybrid Ph1+ Ph1- Ph1 locus suppresses pairing between related chromosomes (homoeologous pairing) If Ph1 locus is deleted, pairing is induced between related chromosomes **Strategic Goal** Can we from understanding Ph1 locus, switch Ph1 on and off in elite wheat varieties crossed with wild species? Wild species of wheat carry important traits for disease resistance and salt, cold and drought tolerance

5. No natural variation in Ph1 phenotype - Can’t create segregating populations, the starting point of all previous positional cloning projects EMS treatments don’t yield mutants But X-Ray and fast neutron irradiation do -A single deletion (ph1b) of the locus = 70Mb in size “Ph1 locus” arose on polyploidisation The wheat genome is very large What is Ph1 ? Cloning - the issues

6. Rice Brachypodium Wheat Deletions Defining the Ph1 locus Griffiths et al 2006 Al-Kaff et al 2008

7. Rice Wheat Cluster of Cyclin dependent kinase-like (Cdks) genes = Ph1 locus All defective genes Large segment of Heterochromatin inserted on polyploidisation Hypothesis- the defective 5B Cdk copies are suppressing the activity of the related Cdks elsewhere in the genome. But how to take the study further in wheat?

8. Ph1 Cdk-like gene shows similarity to Cdk2 Cdk2 Ph1-cdk gene Yousafzai and Al-kaff, 2010 Cdk2 in mammals affects histone H1 phosphorylation Ph1 cdk+cyclinA compared to Cdk2+cyclinA So as a defective locus, does Ph1 suppress Cdk activity, hence Histone H1 phosphorylation?? Protein modelling

9. Human Histone H1 phosphorylation sites Is wheat histone H1 phosphorylated at Cdk2 consensus sites and is their phosphorylation altered by Ph1 ??? _ Does Ph1 affect histone HI phosphorylation? TPKK TPVK SPAKSPKK Cdk2 phosphorylates human histone H1 at: Consensus motifs (S/T) –P-X-K

10. Wheat Histone H1 phosphorylated at Cdk2-type consensus (S/T) –P-X-K sites Cdk2-type phosphorylation on histone H1 is increased when Ph1 locus deleted Azahara Martinez, Ali Pendle, Alex Jones, Isabelle Colas

11. 2 Ph1 copies 6 Ph1 copies Metaphase I pairing Bivalents 0 Ph1 copies Mutivalents Reduced Homologous pairing- univalents Homologous pairing Reduced homologous Pairing-univalents Homoeologous Pairing- Homologous pairing increased Cdk activity reduced Cdk activity Reduced homologous Pairing-univalents Reduced homologous pairing-univalents John Doonan Moshe Feldman 1966 Mutate or over-express Arabidopsis Cdk Greer et al 2012

12. So key question Can we mimic the effect of deleting Ph1 by increasing histone H1 phosphorylation and hence Induce pairing between related chromosomes?? Deleting Ph1 increases Cdk activity- which increases histone H1 phosphorylation, And we have pairing between related chromosomes

13. Does increased Cdk-type activity induce pairing between related chromosomes? Detached tiller method Okadaic acid inhibits phosphatases Okadaic acid increases Histone H1 kinase activity Does Okadaic acid induce pairing between related chromosomes?

14. Okadaic acid induces pairing of related chromosomes in a wheat x rye hybrid Homoeologous pairing Wheat X Rye – Ph1 deleted No okadaic acid – mostly univalents Okadaic acid - bivalents & other chromosomes associations So, okadaic acid treatment produces a similar effect on chromosome pairing of related chromosomes as deleting Ph1 Knight et al., 2010

15. Does Okadaic acid treatment affect the same Cdk2 consensus site as Ph1??? yes The “Ph1” Cdk2-type consensus site shows increased phosphorylation with Okadaic acid treatment Increased histone H1 phosphorylation leads to more “open” /decondensed chromatin? How does this affect pairing /recombination?

16. Our detailed understanding of the genes involved in recombination has come from models: yeast, C elegans, Drosophila – Little is known about homologue recognition

17. 42 chromosomes 6 chromosomes incorrectly paired during zygotene 6 chromosomes associating incorrectly resolved at pachytene 21 correct pairs at metaphase I 42 chromosomes 14 incorrectly paired during zygotene 14 chromosomes associating incorrectly not resolved at pachytene 14 chromosomes as univalents or multivalents 14 correct pairs Ph1 effect in wheat- don’t want incorrect associations here Homologue recognition altered by Ph1 Ph1+ Ph1- Resolution of incorrect associations altered by Ph1 Holm, 1986, 1988 Homologues present At both these stages condensation changes occur which would be affected by histone H1 phosphorylation

18. Ph1 effect in wheat hybrids want incorrect associations here No increase in incorrect associations in absence of Ph1 Ph1+ Ph1- Resolution of incorrect associations altered by Ph1 Wheat-rye 28 chromosomes 30% pairing only at zygotene 1 pair at metaphase I Wheat-rye 28 chromosomes 7 pairs at metaphase I 60% pairing at pachytene Wang 1988, Wang and Holm 1988 No homologues 30% pairing Little change at pachytene 30% pairing only at zygotene <20% pairing >50% pairing 65% pairing >90% pairing Wheat-kotschyi 35 chromosomes Wheat-kotschyi 35 chromosomes increase in incorrect associations in absence of Ph1 Gillies 1987 1 pair at metaphase I 14 pairs at metaphase I

19. Effect on recognition?? Wheat Lack of Ph1 induces more incorrect pairing between homoeologues Wheat-rye Lack of Ph1 does not induce more incorrect pairing between the homoeologues in the absence of homologues?? What is happening?? Wheat-kotschyi Lack of Ph1 induces more incorrect pairing between homoeologues in the absence of homologues?? But

20. telomeres homologous segments What happens at the homologue recognition stage in wheat? The identical chromosomes zip up from their telomere regions Pilar Prieto et al 2004 Nat Cell Biol Rye segment homologues telomeres “Decondensation ‘Elongation” of chromatin This conformation change now reported in C elegans

21. Telomeres Ph1+ Ph1- Ph1+ In wheat- chromosomes remodel in both the presence and absence of Ph1 but there is asynchronous chromatin remodelling in the absence of Ph1 correlating with more incorrect associations at homologue recognition stage Interstitial segments- 15% of the wheat chromosome Pilar Prieto et al 2004 Nat Cell Biol

22. Decondensation of chromosome segments linked to presence of homologous chromosomes and affects pairing Identical segments Segments elongated Synchronously before clustering 100% pairing Similar segments Segments elongated but Not Synchronously 50% pairing Isabelle Colas et al 2008 PNAS

23. No Pairing Ph1+ Some Pairing Ph1- Pairing Ph1- Diploid- homologues hybrid- Ph1- homoeologues In wheat-rye hybrids-without Ph1 homoeologous wheat-rye chromosomes only trigger a partial conformation change hybrid- Ph1+ homoeologues heterochromatin telomeres Partial change not sufficient to induce more incorrect associations

24. Ph1+ Ph1- Resolution of incorrect associations altered by Ph1 Wheat-rye 28 chromosomes 30% pairing only at zygotene 1 pair at metaphase I Wheat-rye 28 chromosomes 7 pairs at metaphase I At pachytene Pairing increases to 60% Wang 1988, Wang and Holm 1988 30% pairing Little change at pachytene 30% pairing only at zygotene <20% pairing >50% pairing 65% pairing >90% pairing Wheat-kotschyi 35 chromosomes Wheat-kotschyi 35 chromosomes Gillies 1987 1 pair at metaphase I 14 pairs at metaphase I The processing of incorrect associations –ie associations between related chromosomes at pachytene The maintenance of incorrect associations without Ph1 correlates with significant increase in pairing (synapsis)

25. Diverged (related) chromosomes pair via pegging process Homologues with divergent segments telomeres Chromosome segments remodel Chromosome segments peg together forming a circular structure Colas et al., PNAS 2008 Synaptic adjustment Non-homologous pairing without Ph1 at pachytene/ diplotene recombination The altered chromatin with Ph1 affects Condensation/ Decondensation change at Pachytene/ Diplotene Little Synaptic adjustment with Ph1 No recombination Chromosomes resolve

26. Synaptic adjustment

27. enhances correct chromosome segregation through regulating centromere pairing Finally Metaphase I Anaphase I Meiosis I Cohesion Spindle force Chiasmata Separase Cohesion lost in arms Chromosome segregation via the centromeres

28. enhances correct chromosome segregation through regulating centromere pairing Finally Centromeres in many plants (wheat, Arabidopsis, brachypodium) pair independently from telomeres and the rest of the chromosomes Synapsis can be initiated at centromeres independently from the telomeres Ph1 increases the stringency of centromere pairing- thus the fidelity of segregation Martinez-Perez et al., 2001 Nature telomeres Centromeres

29. ( P hosphorylation (Histone) hI alters chromatin and hence homologue recognition, synaptic adjustment and chromosome segregation during meiosis Conclusions

30. 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 Ph1 into other species -Brachypodium/Arabidopsis Ruoyu Wen, Vera Thole, Philippe Vain, John Doonan, Peter Shaw Protein modelling- Faridoon Yousafzai, Nadia Al-Kaff Phosphoproteomics- advanced mass spec Azahara Martinez-Raminez, Ali Pendle, Isabelle Colas, Alex Jones, Peter Shaw Cell biology – 3-D visualisation- Luis Aragon, Fadri Martinez, Pilar Prieto, Mike Wanous, Isabelle Colas, Emma Greer, Azahara Martinez-Raminez, Peter Shaw