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

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)

The origin of wheat Matsuoka (2011) First cultivation of wheat (diploid and tetraploid) occurred about years ago, as part of the ‘Neolithic Revolution’ Cultivation spread to the Near East 9000 years ago hexaploid bread 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

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

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

Gupta et al. (2008)

Bread Wheat Triticum aestivum ssp. aestivum 2n = 6x =42 – Wheat has 3 homoeologous chromosome sets A, B and D – Disomic inheritance preserves hybrid nature – Behaves as a diploid at meiosis – HOW? ABDABD Triticum tauchii Triticum searsii Triticum monoccoccum

Meiosis 1 Diploid cell 4 Haploid cells Paired homologues align on plate Homologues segregate Sister chromatids segregate Incorrect pairing leads to unbalanced gametes and infertility How does wheat produce 4 haploid cells at the end of meiosis?

Moore (2002)

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 Ph loci How does Ph1 work? Sears (1976)

Ph1 in Wheat Led by Prof Graham Moore Research - Wheat meiosis and the Ph1 locus /staff/graham- moore/index.htm

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

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

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 What is Ph1 ?

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

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

Cluster of 7 Cyclin dependent kinase-like (Cdks) genes on the long arm of 5B = 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?

Ph1 Cdk-like gene shows similarity to Cdk2 Ph1-cdk gene Yousafzai and Al-kaff, 2010 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 Protein modeling

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 H1 phosphorylation? TPKK TPVK SPAKSPKK Cdk2 phosphorylates human histone H1 at consensus motifs (S/T) –P-X-K

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

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 Cdkg Greer et al. 2012

CDKG is closely related to Cdk2 and Ph1 Mutant cdkg1 shows temperature-sensitive defects in synapsis and recombination of male meiosis

Cdkg1 is partially asynaptic at 23 o C Zheng et al Asy1 Zyp1 DAPI

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 Result - pairing between homoeologous chromosomes Summary

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?

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 and other chromosome associations Okadaic acid treatment produces a similar effect on chromosome pairing of related chromosomes as deleting Ph1 Knight et al., 2010

Does okadaic acid treatment affect the same Cdk2 consensus site as Ph1? 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? YES!

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

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 De-condensation /elongation of chromatin Wheat

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

De-condensation of chromosome segments is dependent upon their sequence similarity Identical segments Segments elongated Synchronously before clustering 100% pairing Similar segments Segments elongated but Not Synchronously 50% pairing Colas et al 2008 PNAS

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

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

Synaptic Adjustment

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

Summary Wheat is a global crop with a complex evolutionary history which gave it its hexaploid status Ph1 stabilises the wheat genome by controlling pairing, and effectively turns it into a diploid Ph1 is related to human Cdk2 which phosphorylates histone H1 and modifies chromatin conformation Ph1 could be used to introduce novel genes into commercial crops

37 Thanks Diolch Спасибо