PowerPoint Presentation

The Ph Locus and the rise of bread wheatDr Glyn Jenkins

Wheat a plant that feeds the worldCultivated area: 215,489,485 Ha (area of UK 22,933,252Ha)Production: 670,775,485 tonnesGlobal productivity: 3.1 t/HaContributes 20% of total food calories and protein in human nutritionWheat 20:20 Project aim to have achieve an average yield of 20 t/HaYield 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 10000 years ago, as part of the Neolithic RevolutionCultivation spread to the Near East 9000 years ago hexaploid bread wheat made its first appearanceThe 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

The origin and current distribution of wheat. The wheat production map was provided by Dave Hodson, CIMMYT (20). The solid line ovals in the inset indicate the putative geographic regions of origin of the cultivated forms, whereas the dotted red line indicates a southern center of domesticated emmer diversity. The approximate distributions of wild emmer and Ae. tauschii are indicated by dots, and that of wild einkorn by yellow shading (3). Numbers indicate archaeological sites where remains of domesticated cereals dating back more than 9000 years BP were found: 1, Tell Aswad; 2, Abu Hureyra; 3, Cafer Hyk; 4, Jericho; 5, Cayn; 6, Nahal Hemar; and 7, Nevali Cori [from (2)].

Also called Aegilops tauschii and Ae. squarrosaWild goat grassT. urartuAegilops searsiiAe. speltoidesKey events in the evolution of wheat

Two hybridisation and genome doubling* events

DiploidAADiploidBBxxAllotetraploidAA BBDiploidDDAllohexaploidAA BB DD*chromosome doubling may have occurred before hybridisationThe evolution of wheat - examples of spikes and grain

Shewry (2009)T. searsiiT. monococcumThe evolutionary and genome relationships between cultivated bread and durum wheats and related wild diploid grasses, showing examples of spikes and grain. Modified from Snape and Pnkov (2006), and reproduced by kind permission of Wiley-Blackwell.

Gupta et al. (2008)Bread WheatTriticum aestivum ssp. aestivum2n = 6x =42 Wheat has 3 homoeologous chromosome sets A, B and DDisomic inheritance preserves hybrid natureBehaves as a diploid at meiosis HOW?

1 2 3 4 5 6 7A

B

DTriticum tauchiiTriticum searsiiTriticum monoccoccumMeiosis 1 Diploid cell4 Haploid cellsTelophase IIProphase IDiploteneDiakinesisPachyteneZygoteneLeptoteneMetaphase IAnaphase ITelophase IProphase IIMetaphase IIAnaphase IIPaired homologues align on plateHomologues segregateSister chromatids segregateIncorrect pairing leads to unbalanced gametes and infertilityHow does wheat produce 4 haploid cells at the end of meiosis?8

Moore (2002)Pairing homoeologousInitially it was assumed that the three diploid species whose genomes had gone to make up hexaploid wheat were strongly differentiatedHow 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 relatedRiley and Chapman (1958) - discovered that homoeologous pairing is suppressed by a gene or genes on the long arm of chromosome 5B Became known as Ph1N.B. wheat contains additional Ph lociHow does Ph1 work?Sears (1976)

Ph1 in Wheat

Led by Prof Graham MooreResearch - Wheat meiosis and the Ph1 locus

http://www.jic.ac.uk/staff/graham-moore/index.htm

Ph1 is critical to maintaining genome stability in wheat Effect of Ph1Ph1+Ph1- Multivalents UnivalentsMartinez et al. (2001)Effect of Ph1

Wheat-rye hybridPh1+Ph1-Ph1 locus suppresses pairing between related chromosomes (homoeologous pairing)If Ph1 locus is deleted, pairing is induced between homoeologous chromosomesCloning - 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-Cant create segregating populations, the starting point of all previous positional cloning projects

EMS treatments dont yield mutants But X-Ray and fast neutron irradiation do -A single deletion (ph1b) of the locus = 70Mb in size

What is Ph1 ?

RiceBrachypodiumWheatDeletionsDeletionsDeletionsDefining the Ph1 locusGriffiths et al 2006Al-Kaff et al 2008

2.5 MbAl-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 locusAll defective genesLarge segment ofHeterochromatin inserted on polyploidisationHypothesis- 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 geneYousafzai and Al-kaff, 2010 Cdk2 in mammals affects histone H1 phosphorylationSo as a defective locus, does Ph1 suppress Cdk activity, hence histone H1 phosphorylation? Ph1 cdk+cyclinA compared to Cdk2+cyclinA

Protein modelingHuman Histone H1 phosphorylation sitesIs wheat histone H1 phosphorylated at Cdk2 consensus sites and is their phosphorylation altered by Ph1?_Does Ph1 affect histone H1 phosphorylation?

TPKKTPKKTPVKSPAKSPKKCdk2 phosphorylates human histone H1 atconsensus 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 Ph1copies6 Ph1copiesMetaphase I pairingBivalents

0 Ph1copiesMutivalentsReducedhomologouspairing,univalentsHomologouspairingReduced homologouspairing, univalentshomoeologouspairingHomologouspairingIncreasedCdk activityReducedCdk activityReduced homologouspairing, univalentsReduced homologous pairing, univalentsJohn DoonanMoshe Feldman1966Mutate or over-expressArabidopsis CdkgGreer et al.2012

CDKG is closely related to Cdk2 and Ph1Mutant cdkg1 shows temperature-sensitive defects in synapsis and recombination of male meiosisCdkg1 is partially asynaptic at 23oC

Zheng et al. 2014Asy1Zyp1 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 phosphorylationResult - pairing between homoeologous chromosomesSummaryDoes increased Cdk-type activity induce pairing between related chromosomes?

Detached tiller methodOkadaic acid inhibits phosphatases

Okadaic acid increases histone H1 kinase activity

Does okadaic acid induce pairing between related chromosomes?25Okadaic acid induces pairing of related chromosomes in a wheat x rye hybrid

Homoeologous pairingWheat X Rye Ph1 deletedNo okadaic acid mostly univalentsOkadaic 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 acidtreatment affect the same Cdk2 consensus site asPh1?

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 multivalentsJenkins 1983Holm, 1986,1988

DiploteneDiakinesisPachyteneZygoteneLeptoteneMetaphase IAt both these stages condensation changes occur which would be affected by histone H1 phosphorylation

42 chromosomes high stringency synapsis but some multivalents at zygotenemultivalents eliminated at pachytene21 homologous bivalentsat metaphase I Ph1+lower stringency synapsis with more multivalents at zygotenemultivalents retained at pachytenePh1-

42 chromosomes

telomereshomologoussegments What happens at the homologue recognition stage in wheat?The identical chromosomes zip up from their telomere regionsPilar Prieto et al 2004 Nat Cell Biol

Rye segment homologuestelomeres

De-condensation/elongation ofchromatinWheat

TelomeresPh1+Ph1+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 stageInterstitial segments- 15% of the wheat chromosomePilar Prieto et al 2004 Nat Cell Biol30De-condensation of chromosome segments is dependent upon their sequence similarity

Identical segmentsSegments elongated Synchronously before clustering100% pairing

Similar segmentsSegments elongated but Not Synchronously50% pairingDistinct segmentsReduced/Delayed15% pairing

Colas et al 2008 PNAS

No PairingPh1+

Some PairingPh1-PairingPh1-

Diploid-homologuesHybrid- Ph1-homoeologues In wheat-rye hybrids without Ph1 homoeologous wheat-rye chromosomes only trigger a partial conformation change Hybrid- Ph1+homoeologuesheterochromatintelomeresSynapsis in diverged (related) chromosomesHomologueswith divergentsegments

TelomeresChromosome segmentsremodelChromosome segmentsforming a circular structureColas et al., PNAS 2008Synaptic adjustmentwithout Ph1RecombinationLittle synaptic adjustmentwith Ph1

No recombination

Synaptic AdjustmentThe Ph1 effect is importantagronomically **Strategic Goal**Switch Ph1 on and off in elite wheat varieties crossed with wild species to introduce novel genes to the commercial cropWild species of wheat carry important traits for disease resistance and salt, cold and drought toleranceSummaryWheat is a global crop with a complex evolutionary history which gave it its hexaploid statusPh1 stabilises the wheat genome by controlling pairing, and effectively turns it into a diploidPh1 is related to human Cdk2 which phosphorylates histone H1 and modifies chromatin conformationPh1 could be used to introduce novel genes into commercial crops

37

ThanksDiolch

37