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 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. squarrosaWild goat grass

T. urartuAegilops searsiiAe. speltoides

Key events in the evolution of wheat

• Two hybridisation and genome doubling* events

DiploidAA

DiploidBB

x

xAllotetraploidAA BB

DiploidDD

AllohexaploidAA BB DD

*chromosome doubling may have occurred before hybridisation

The evolution of wheat - examples of spikes and grain

Shewry (2009)

T. searsiiT. monococcum

Gupta et al. (2008)

Bread WheatTriticum 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?

1 2 3 4 5 6 7

A

B

DTriticum tauchii

Triticum searsii

Triticum monoccoccum

Meiosis 1 Diploid cell

4 Haploid cellsTelophase II

Prophase IDiplotene

DiakinesisPachyteneZygotene

Leptotene

Metaphase I

Anaphase I

Telophase IProphase IIMetaphase IIAnaphase II

Paired homologues align on plate

Homologues segregate

Sister chromatids segregate

Incorrect pairing leads to unbalanced gametes and infertilityHow 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

http://www.jic.ac.uk/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 Ph1Wheat-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

DeletionsDeletionsDeletions

Defining the Ph1 locus

Griffiths et al 2006Al-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 ofHeterochromatin 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 phosphorylationSo 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? 1 80 H11_HUMAN (1) MSETVPPAPAAS--AAPEKPLAGKKAKKPAKAAAASKKKPAGPSVSELIVQAASSSKERGGVSLAALKKALAAAGYDVEK H1T_HUMAN (1) MSETVPAASASAGVAAMEKLPTKKRGRKPAGLISAS-RKVPNLSVSKLITEALSVSQERVGMSLVALKKALAAAGYDVEK H15_HUMAN (1) MSETAPAETATP--APVEKSPAKKKATKKAAGAGAAKRKATGPPVSELITKAVAASKERNGLSLAALKKALAAGGYDVEK H12_HUMAN (1) MSETAPAAPAAA--PPAEKAPVKKKAAKKAGGTP---RKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEK H13_HUMAN (1) MSETAPLAPTIP--APAEKTPVKKKAKKAGATAGK--RKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEK H14_HUMAN (1) MSETAPAAPAAP--APAEKTPVKKKARKSAGAAK---RKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEK Consensus (1) MSETAPAAPAAP APAEKTPVKKKAKK AGAAGAS RKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEK 81 160 H11_HUMAN (79) NNSRIKLGIKSLVSKGTLVQTKGTGASGSFKLNKKASSVETKPGASKVATKT--KATGASKKLKKATGASK---KSVKTP H1T_HUMAN (80) NNSRIKLSLKSLVNKGILVQTRGTGASGSFKLSKKVIPKSTRSKAKKSVSAKTKKLVLSR-----DSKSPK----TAKTN H15_HUMAN (79) NNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAT--PKKAKKAAGAKKAVKKTP H12_HUMAN (76) NNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKVKKAGGTKPKKPVGAAKKPKKAAGGATPKKSAKKTP H13_HUMAN (77) NNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKKAASGEGKPKAKKAGAAKPRKPAGAAKKPKKVAGAATPKKSIKKTP H14_HUMAN (76) NNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAAKKPKKATGAATPKKSAKKTP Consensus (81) NNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAK KKPAGAAKKPKKATGAATPKKSAKKTP 161 231 H11_HUMAN (154) KKAKKPAATRKSSKNP---KKPKTVK-PKKVAKSPAKAKAVKPKAAKARVTKPKTAKPKKAAPKKK----- H1T_HUMAN (151) KRAKKPRATTPKTVRS--GRKAKGAK-GKQQQKSPVKARASK-----SKLTQHHEVNVRKATSKK------ H15_HUMAN (157) KKAKKPAAAGVKKVAK-SPKKAKAAAKPKKATKSPAKPKAVKPKAAKPKAAKPKAAKPKAAKAKKAAAKKK H12_HUMAN (156) KKAKKPAAATVTKKVAKSPKKAKVAK-PKKAAKSAAKAVKP-------KAAKPKVVKPKKAAPKKK----- H13_HUMAN (157) KKVKKPATAAGTKKVAKSAKKVKTPQ-PKKAAKSPAKAKAPKPKAAKPKSGKPKVTKAKKAAPKKK----- H14_HUMAN (156) KKAKKPAAAAGAKKAK-SPKKAKAAK-PKKAPKSPAKAKAVKPKAAKPKTAKPKAAKPKKAAAKKK----- Consensus (161) KKAKKPAAAA TKK A SPKKAKAAK PKKAAKSPAKAKAVKPKAAKPKAAKPK AKPKKAAPKKK

TPKKTPKK

TPVK

SPAKSPKK

Cdk2 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

Progenesis

WT PH

0.00

0.02

0.04

0.06

0.08

Azahara Martinez, Ali Pendle, Alex Jones, Isabelle Colas

2 Ph1copies

6 Ph1copies

Metaphase I pairing

Bivalents

0 Ph1copies

Mutivalents

Reducedhomologouspairing,univalents

Homologouspairing

Reduced homologouspairing, univalentshomoeologouspairing

Homologouspairing

IncreasedCdk activity

ReducedCdk activity

Reduced homologouspairing, univalents

Reduced homologous pairing, univalents

John Doonan Moshe Feldman1966

Mutate or over-expressArabidopsis 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 23oC

Zheng et al. 2014

Asy1Zyp1 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 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 acidtreatment affect the same Cdk2 consensus site asPh1?

The “Ph1” Cdk2-type consensus site shows increased phosphorylation with okadaic acid treatment

Progenesis

WT PH

0.00

0.02

0.04

0.06

0.08

Untreat

ed

OA_100

OA_200

0.000

0.005

0.010

0.015

0.020

0.025

rati

o p

ho

sph

o /

no

n-p

ho

sph

o

Increased histone H1 phosphorylation leads to more “open” /decondensed chromatin? How does this affect pairing /recombination?

YES!

Ph1 forms bivalents by eliminating multivalents

Jenkins 1983Holm, 1986,1988

DiploteneDiakinesis

PachyteneZygotene

LeptoteneMetaphase I

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 bivalentsat metaphase I

Ph1+

lower stringency synapsis with more multivalents at zygotene

multivalents retained at pachytene

Ph1-

42 chromosomes

telomeres

homologoussegments

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+

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

• Distinct segments

Reduced/Delayed

15% 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) chromosomesHomologueswith divergentsegments

Telomeres

Chromosome segmentsremodel

Chromosome segmentsforming a circular

structure

Colas et al., PNAS 2008

Synaptic adjustmentwithout Ph1

Recombination

Little synaptic adjustmentwith Ph1

No recombination

Synaptic Adjustment

The 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 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

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ThanksDiolchСпасибо