the ph locus and the rise of bread wheat dr glyn jenkins
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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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