The Ph Locus and the rise of bread wheat Dr Glyn Jenkins

1. The Ph Locus and the rise of bread wheat Dr Glyn Jenkins

2. Wheat – a plant that feeds the world Cultivated area: 215,489,485 Ha (area of UK 22,933,252Ha) Production: 670,775,485 tonnes Global productivity: 3.1 t/Ha Contributes 20% of total food calories and protein in human nutrition Wheat 20:20 – Project aim to have achieve an average yield of 20 t/Ha Yield plateau reached - so where do we go from here? Grassini et al. (2012)

3. The origin of wheat • First cultivation of wheat (diploid and tetraploid) occurred about 10000 years ago, as part of the ‘Neolithic Revolution’ • Cultivation spread to the Near East • 9000 years ago hexaploidbread wheat made its first appearance • The main route into Europe via • Greece (8000 BP) • Balkans to the Danube (7000 BP) • Italy, France and Spain (7000 BP), • UK and Scandinavia by about 5000 BP Matsuoka (2011)

4. Key events in the evolution of wheat • Two hybridisation and genome doubling* events Aegilopssearsii Ae. speltoides T. urartu Also called Aegilopstauschiiand Ae. squarrosa Wild goat grass Diploid BB Diploid AA x Diploid DD Allotetraploid AA BB x Allohexaploid AA BB DD *chromosome doubling may have occurred before hybridisation

5. The evolution of wheat - examples of spikes and grain T. monococcum T. searsii Shewry(2009)

6. Gupta et al. (2008)

7. Bread WheatTriticumaestivumssp. aestivum2n = 6x =42 • Wheat has 3 homoeologous chromosome sets A, B and D • Disomic inheritance preserves hybrid nature • Behaves as a diploid at meiosis – HOW? 1 2 3 4 5 6 7 A B D Triticummonoccoccum Triticumsearsii Triticumtauchii

8. Prophase I Pachytene Zygotene Diplotene Diakinesis Leptotene Metaphase I Prophase II Metaphase II Telophase II Anaphase II Anaphase I Telophase I Meiosis Paired homologuesalign on plate 1 Diploid cell Homologues segregate 4 Haploid cells Sister chromatids segregate Incorrect pairing leads to unbalanced gametes and infertility How does wheat produce 4 haploid cells at the end of meiosis?

9. Moore (2002)

10. Pairing homoeologous • Initially it was assumed that the three diploid species whose genomes had gone to make up hexaploid wheat were strongly differentiated • How else could one explain the near absence of meiotic pairing in haploids of the hexaploid species? • 1952 – became clear that the corresponding chromosomes of the three different genomes are genetically very closely related • Riley and Chapman (1958) - discovered that homoeologous pairing is suppressed by a gene or genes on the long arm of chromosome 5B • Became known as Ph1 • N.B. – wheat contains additional Phloci • How does Ph1 work? Sears (1976)

11. Ph1 in Wheat • Led by Prof Graham Moore • Research - Wheat meiosis and the Ph1 locus • http://www.jic.ac.uk/staff/graham-moore/index.htm

12. Effect of Ph1 • Ph1- • Multivalents • Univalents Ph1+ Martinez et al. (2001) ∴Ph1 is critical to maintaining genome stability in wheat

13. Effect of Ph1 Wheat-rye hybrid Ph1 locus suppresses pairing between related chromosomes (homoeologous pairing) Ph1+ If Ph1 locus is deleted, pairing is induced between homoeologous chromosomes Ph1-

14. What is Ph1? Cloning - the issues • The wheat genome is very large 17 Gb. (human 3Gb, yeast 0.12Gb) • Three closely related genomes! • 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

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

16. Defining the Ph1 locus further 2.5 Mb Al-Kaff et al. (2007)

17. Cluster of 7 Cyclindependent kinase-like (Cdks) genes on the long arm of 5B All defective genes = Ph1 locus 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?

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

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

20. 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

21. Reduced homologous pairing, univalents homoeologous pairing 0 Ph1 copies Mutate or over-express Arabidopsis Cdkg Reduced homologous pairing, univalents Mutivalents Increased Cdk activity 2 Ph1 copies Homologous pairing Bivalents Homologous pairing 6 Ph1 copies Reduced homologous pairing, univalents Reduced Cdk activity Reduced homologous pairing, univalents Greer et al. 2012 Moshe Feldman 1966 John Doonan Metaphase I pairing

22. CDKG is closely related to Cdk2 and Ph1 • Mutant cdkg1shows temperature-sensitive defects in synapsis and recombination of male meiosis

23. Cdkg1 is partially asynaptic at 23oC Zheng et al. 2014 Asy1 Zyp1 DAPI

24. Summary DeletingPh1 increases Cdk activity- which increases histone H1 phosphorylation Result - pairing between homoeologous chromosomes Key question Can we mimic the effect of deleting Ph1 by increasing histone H1 phosphorylation and hence induce pairing between related chromosomes?

25. Does increased Cdk-type activity induce pairing between related chromosomes? • Okadaicacid inhibits phosphatases • Okadaic acid increases histone H1 kinase activity • Does okadaicacid induce pairing between related chromosomes? Detached tiller method

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

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

28. Zygotene Diakinesis Leptotene Metaphase I Pachytene Diplotene Ph1forms bivalents by eliminating multivalents multivalents eliminated at pachytene multivalents retained at pachytene 21 homologous bivalents at metaphase I high stringency synapsis but some multivalents at zygotene lower stringency synapsis with more multivalents at zygotene Ph1- Ph1+ 42 chromosomes 42 chromosomes At both these stages condensation changes occur which would be affected by histone H1 phosphorylation Jenkins 1983 Holm, 1986,1988

29. What happens at the homologue recognition stage in wheat? homologues De-condensation /elongation of chromatin Rye segment Wheat telomeres homologous segments telomeres The identical chromosomes zip up from their telomere regions PilarPrieto et al 2004 Nat Cell Biol

30. In wheat- chromosomes remodel in both the presence and absence of Ph1BUTthere is asynchronous chromatin remodelling in the absence of Ph1 correlating with more incorrect associations at homologue recognition stage Ph1- Interstitial segments- 15% of the wheat chromosome Telomeres Ph1+ Ph1+ Ph1+ Ph1+ PilarPrieto et al 2004 Nat Cell Biol

31. De-condensation of chromosome segments is dependent upon their sequence similarity • Identical segments Segments elongatedSynchronously before clustering 100% pairing • Similar segments Segmentselongatedbut Not Synchronously 50% pairing • Distinctsegments Reduced/Delayed 15% pairing Colas et al 2008 PNAS

32. In wheat-rye hybrids without Ph1 homoeologous wheat-rye chromosomes only trigger a partial conformation change Ph1+ Ph1- Some Pairing No Pairing Hybrid- Ph1- homoeologues Hybrid- Ph1+ homoeologues Diploid- homologues Ph1- heterochromatin telomeres Pairing

33. Synapsis in diverged (related) chromosomes Homologues with divergent segments Little synaptic adjustment with Ph1 Chromosome segments forming a circular structure Chromosome segments remodel Telomeres Synaptic adjustment without Ph1 Recombination No recombination Colas et al., PNAS 2008

34. Synaptic Adjustment

35. The Ph1 effect is important agronomically Wild species of wheat carry important traits for disease resistance and salt, cold and drought tolerance **Strategic Goal** Switch Ph1 on and off in elite wheat varieties crossed with wild species to introduce novel genes to the commercial crop

36. Summary • Wheat is a global crop with a complex evolutionary history which gave it its hexaploidstatus • Ph1stabilises the wheat genome by controlling pairing, and effectively turns it into a diploid • Ph1 is related to human Cdk2which phosphorylates histone H1 and modifies chromatin conformation • Ph1 could be used to introduce novel genes into commercial crops

37. ThanksDiolch Спасибо