Hexaploid wheat - Triticum aestivum 2n= 6x= 42

1. Hexaploid wheat- Triticum aestivum 2n= 6x= 42 a b c d a b c d 7D 7D a b c d Homoeologues 7D 7B 7B 7A Homoeologues pair in hybrids between wheat diploid progenitors The Ph1 locus restricts pairing to homologues. 7A 7A 1 2 3 4 5 6 7 a b c d A Similar gene orders but different content of similar repeats a b c d B a b c d D

2. Breeding Wheat hybrids In hybrids, no homologues-related chromosomes Wild species of wheat carry important traits for disease resistance, salt, cold and drought tolerance Wheat hybrid Unfortunately Ph1 locus prevents the wheat and its wild relative chromosomes from pairing /recombining - and thus from transferring of these traits Ph1+ Can we regulate this locus? Single deletion of Ph1 locus (ph1b- 70Mb in size) used in breeding Ph1- Can the process be improved? What is Ph1 doing? Can Ph1 be switched on and off?

3. Dissect pairing process centromeres Meiocyte in premeiosis telomeres Interstitial segments subterminal heterochromatin centromeres “telomere regions” Interphase configuration telomeres Meiocytes in early meiosis

4. Telomere regions- Hexaploid wheat Telomere regions of homologues pair in all the meiocytes examined in the absence of Ph1 Marked telomere regions of two homologues Ph1- Ph1- Telomere pairing of homologues in hexaploid wheat does not require Ph1

5. Summary of related issues • Kihara and Lilienfeld (1934) showed that wheat hybrids were prone to premature condensation- Implication- newly synthesized polyploids are prone to premature condensation • Premature/asynchronous condensation is a hallmark of cells that have an inadequate replication checkpoint and have begun condensation before completing replication • Overexpression of Cdc2 (checkpoint) gene activity caused premature asynchronous condensation • Implication- newly synthesized polyploids need to control Cdc2 activity- so what is Ph1 affecting?

6. Interstitial segments-Hexaploid wheat Interstitial segments- 15% of the wheat chromosome (same size as a maize chromosome, entire drosophila or Arabidopsis genome, 10 yeast genomes) Ph1- Ph1- Telomeres Ph1+ Ph1+ Ph1+ Ph1+

7. Summary of Ph1 effect- Hexaploid wheat Homologues same conformation Homologues different conformation Add Ph1 Telomere bouquet

8. Synchronisation- Hexaploid wheat Ph1- Problem, on polyploidisation homologous sites are not synchronised in their condensation Solution-Ph1 synchronises condensation of homologous sites Ph1+ One consequence of Ph1 is that pairing will occur when the chromosomes are more condensed

9. Wheat hybrids Ph1’s effect? In hybrids, no homologues-related chromosomes Heterochromatin (B chromosomes) can compensate for the absence of Ph1 in wheat hybrids- ie suppress pairing and recombination between wheat and wild relative chromosomes Heterochromatin (B chromosomes) delay S phase and cause greater compaction of chromosome regions

10. Sub-telomere regions Condensation Hexaploid wheat hybrids In hybrids, no homologues-related chromosomes Hexaploid wheat-rye hybrid premeiosis Ph1- Ph1+ meiosis meiosis Rye telomeric knobs Telomere cluster when condensation has not begun Telomere cluster (pairing) when condensation has begun

11. What is the Ph1 ? Cloning the issues • No natural variation in Ph1 phenotype- Can’t create a segregating populations, the starting point of all previous positional cloning projects- Can’t score thousands of plants for this phenotype , 5000 plants would be 150,000 metaphase spreads • Variation only occurs with dosage • 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 not present in diploids- it is specific to 5B • Heterochromatin compensates for Ph1 • Ph1 could be a multigene family or heterochromatin or both • But wheat is 5 times larger than the human genome

12. StrategyMoore, Gale, Kurata, and Flavell (1993) Nature BiotechCereal Synteny and Rice/Brachypodium to reveal gene content of wheat • Identify further deletions of the Ph1 locus (30,000 fast neutron treated wheat plants PCR screened) • Use the deletions to delimit a “small” region in wheat still defining the Ph1 locus • Reveal gene content of delimited region using synteny with small genomed cereals (140kb in rice and 180kb in Brachypodium). • Build hexaploid wheat BAC library (1,200,000 clones) • Then gets really serious…….

13. Brachypodium/Rice regions 2.5Mb Contig Ph1 5B region

14. Content of the Ph1 region a segment of subtelomeric heterochromatin inserted into this cdc2 locus Known function /Known Tissue expression profile Not Ph1 Known function/ Known Tissue expression profile Not Ph1 There is a single multigene family of cdc2 related genes

15. Ph1 arose on polyploidisation Subtelomeric repeat Chromosome 3AL Chromosome 3AL 1 2 3 Chromosome 5BL pre Ph1 cdc2 gene cluster Subtelomeric repeat 1 2 3 4 Chromosome 5BL with Ph1 1 2 3 4 5 6 7 Following polyploidisation, the some telomere regions rearrange in wheat A B D

16. Association with Ph1 activity in wheat and relatives Region contains markers consistently associated with Ph1 activity in tetraploid and hexaploid wheat and wild polyploid relatives

17. Association with Ph1 activity T. uratu AA Ae. speltoides SS T. turgidum AABB- Ph1+ Ae. tauschii DD T. timopheevi AAGG- Ph1+ T.aestivum AABBDD- Ph1+

18. Ph1 story Summary • We know the cell biological basis of Ph1 activity • We know the structure of the Ph1 “locus” • We know that there is 5B specific expression from within the locus • But we don’t yet know precisely how the locus is working • We need to be able to dissect the components of the locus using small deletions (which may or may be not technically feasible using X-Ray treatment)

19. Application? Is the future bright- is it orange? Can we improve the exploitation the introgression process? • Identification of a Ph1 mutant which does not have a large deletion • Can we use drugs (orange sponge) which affect cdc2 activity to modulate pairing in hybrids?

20. Acknowledgements Cell Biology Luis Aragon Alcaide Enrique (Fadri) Martinez-Perez Pilar Prieto Aranda Mike Wanous Thomas Haizel Physical mapping etc Tracie Foote Michael Roberts Terry Miller Steve Reader Simon Griffiths Sebastien Allouis Rebecca Sharp Kath Mortimer Isabelle Bertin Collaborators Mike Gale -Markers Peter Shaw-Cell biology RGP-Japan - Rice INRA- Evry-BACs Dupont-Sequencing Syngenta-Rush work David Baulcombe siRNA Species used Wheat (meiosis and genomics) Rice (meiosis and sequence data) Brachypodium (centromeres and sequence data) Luzula , a rush (meiosis and centromeres) Rye (meiosis)