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
• 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
Matsuoka (2011)
Key events in the
evolution of wheat
• Two hybridisation and
genome doubling*
events
Diploid x
AA
T. urartu
Also called
Aegilops
tauschii and
Ae. squarrosa
Wild goat
grass
Diploid
BB
Allotetraploid x Diploid
AA BB
DD
Allohexaploid
AA BB DD
*chromosome doubling may have occurred before hybridisation
Aegilops searsii
Ae. speltoides
The evolution of wheat - examples of spikes and grain
T. monococcum
Shewry (2009)
T. searsii
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?
1
Triticum monoccoccum A
Triticum searsii
B
Triticum tauchii D
2
3
4
5
6
7
Meiosis
Paired homologues
align on plate
1 Diploid cell
Prophase I
Zygotene
Pachytene
Diplotene
Leptotene
Diakinesis
Metaphase I
Anaphase I
Homologues
segregate
Telophase II
4 Haploid cells
Telophase I
Anaphase II
Metaphase II
Prophase II
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
http://www.jic.ac.uk
/staff/grahammoore/index.htm
Effect of
Ph1
Ph1• Multivalents
• Univalents
Ph1+
Martinez et al. (2001)
∴ Ph1 is critical to maintaining genome stability in wheat
Effect of Ph1
Wheat-rye hybrid
Ph1 locus suppresses pairing
between related chromosomes
(homoeologous pairing)
If Ph1 locus is deleted, pairing is
induced between homoeologous
chromosomes
Ph1+
Ph1-
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
Defining the
Ph1 locus
Deletions
Rice
Deletions
Griffiths et al 2006
Al-Kaff et al 2008
Deletions
Wheat
Brachypodium
Defining the Ph1 locus further
2.5 Mb
Al-Kaff et al. (2007)
Cluster of 7 Cyclin dependent 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?
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
Does Ph1 affect histone H1 phosphorylation?
Human Histone H1 phosphorylation sites
Cdk2 phosphorylates human histone H1 at
consensus motifs (S/T) –P-X-K
H11_HUMAN
H1T_HUMAN
H15_HUMAN
H12_HUMAN
H13_HUMAN
H14_HUMAN
Consensus
(1)
(1)
(1)
(1)
(1)
(1)
(1)
H11_HUMAN
H1T_HUMAN
H15_HUMAN
H12_HUMAN
H13_HUMAN
H14_HUMAN
Consensus
(79)
(80)
(79)
(76)
(77)
(76)
(81)
H11_HUMAN
H1T_HUMAN
H15_HUMAN
H12_HUMAN
H13_HUMAN
H14_HUMAN
Consensus
(154)
(151)
(157)
(156)
(157)
(156)
(161)
_
1
80
MSETVPPAPAAS--AAPEKPLAGKKAKKPAKAAAASKKKPAGPSVSELIVQAASSSKERGGVSLAALKKALAAAGYDVEK
MSETVPAASASAGVAAMEKLPTKKRGRKPAGLISAS-RKVPNLSVSKLITEALSVSQERVGMSLVALKKALAAAGYDVEK
MSETAPAETATP--APVEKSPAKKKATKKAAGAGAAKRKATGPPVSELITKAVAASKERNGLSLAALKKALAAGGYDVEK
MSETAPAAPAAA--PPAEKAPVKKKAAKKAGGTP---RKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEK
MSETAPLAPTIP--APAEKTPVKKKAKKAGATAGK--RKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEK
MSETAPAAPAAP--APAEKTPVKKKARKSAGAAK---RKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEK
MSETAPAAPAAP APAEKTPVKKKAKK
AGAAGAS RKASGPPVSELITKAVAASKERSGVSLAALKKALAAAGYDVEK
TPVK
81
160
NNSRIKLGIKSLVSKGTLVQTKGTGASGSFKLNKKASSVETKPGASKVATKT--KATGASKKLKKATGASK---KSVKTP
NNSRIKLSLKSLVNKGILVQTRGTGASGSFKLSKKVIPKSTRSKAKKSVSAKTKKLVLSR-----DSKSPK----TAKTN
NNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAT--PKKAKKAAGAKKAVKKTP
NNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKVKKAGGTKPKKPVGAAKKPKKAAGGATPKKSAKKTP
NNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKKAASGEGKPKAKKAGAAKPRKPAGAAKKPKKVAGAATPKKSIKKTP
NNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAKAKKPAGAAKKPKKATGAATPKKSAKKTP
TPKK
TPKK
NNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKKAASGEAKPKAKKAGAAK KKPAGAAKKPKKATGAATPKKSAKKTP
161
231
KKAKKPAATRKSSKNP---KKPKTVK-PKKVAKSPAKAKAVKPKAAKARVTKPKTAKPKKAAPKKK----KRAKKPRATTPKTVRS--GRKAKGAK-GKQQQKSPVKARASK-----SKLTQHHEVNVRKATSKK-----KKAKKPAAAGVKKVAK-SPKKAKAAAKPKKATKSPAKPKAVKPKAAKPKAAKPKAAKPKAAKAKKAAAKKK
KKAKKPAAATVTKKVAKSPKKAKVAK-PKKAAKSAAKAVKP-------KAAKPKVVKPKKAAPKKK----KKVKKPATAAGTKKVAKSAKKVKTPQ-PKKAAKSPAKAKAPKPKAAKPKSGKPKVTKAKKAAPKKK----KKAKKPAAAAGAKKAK-SPKKAKAAK-PKKAPKSPAKAKAVKPKAAKPKTAKPKAAKPKKAAAKKK----KKAKKPAAAA TKK A SPKK
SPKKAKAAK PKKAAKSPAKAKAVKPKAAKPKAAKPK
AKPKKAAPKKK
SPAK
Is wheat histone H1 phosphorylated at Cdk2 consensus sites and is their
phosphorylation altered by Ph1?
Wheat histone H1
phosphorylated at Cdk2type consensus
(S/T) –P-X-K sites
Progenesis
0.08
0.06
0.04
0.02
PH
W
T
0.00
Cdk2-type phosphorylation on histone H1 is
increased when Ph1 locus deleted
Azahara Martinez, Ali Pendle, Alex Jones, Isabelle Colas
Reduced homologous
pairing, univalents
homoeologous
pairing
Mutate or over-express
Arabidopsis Cdkg
Reduced homologous
pairing, univalents
0 Ph1
copies
Mutivalents
Increased
Cdk activity
2 Ph1
copies
Bivalents
Homologous
pairing
Homologous
pairing
6 Ph1
copies
Reduced
Cdk activity
Reduced
homologous pairing,
univalents
John Doonan
Greer et al.
2012
Reduced
homologous
pairing,
univalents
Moshe Feldman
1966
Metaphase I pairing
• 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
Asy1
Zyp1
DAPI
Summary
Deleting Ph1 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?
Does increased Cdk-type activity induce
pairing between related chromosomes?
•Okadaic acid inhibits
phosphatases
•Okadaic acid increases histone
H1 kinase activity
•Does okadaic acid induce
pairing between related
chromosomes?
Detached tiller method
Okadaic acid induces pairing of related chromosomes in a
wheat x rye hybrid
No okadaic acid – mostly
univalents
Okadaic acid treatment
produces a similar effect
on chromosome pairing of
related chromosomes as
deleting Ph1
Knight et al., 2010
Okadaic acid - bivalents
and other chromosome
associations
Wheat X Rye – Ph1 deleted
Homoeologous pairing
Does okadaic acid
treatment affect the same
Cdk2 consensus site as
Ph1? YES!
Progenesis
0.08
0.06
0.04
0.02
0.025
0.020
0.015
0.010
0.005
U
O
A
_1
00
0.000
nt
re
at
ed
ratio phospho / non-phospho
W
T
PH
0.00
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?
Ph1 forms bivalents by eliminating multivalents
Pachytene
Zygotene
Diplotene
Diakinesis
Leptotene
Metaphase I
Ph1+
42 chromosomes
Ph142 chromosomes
Jenkins 1983
Holm, 1986,1988
high stringency
synapsis but some
multivalents at
zygotene
multivalents
eliminated at
pachytene
21 homologous
bivalents
at metaphase I
multivalents
lower stringency
retained at
synapsis with more
pachytene
multivalents at
zygotene
At both these stages condensation changes occur which would
be affected by histone H1 phosphorylation
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
Pilar Prieto et al 2004 Nat Cell Biol
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
Ph1-
Interstitial
segments- 15%
of the wheat
chromosome
Telomeres
Ph1+
Ph1+
Pilar Prieto et al 2004 Nat Cell Biol
Ph1+
Ph1+
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
In wheat-rye hybrids without Ph1 homoeologous wheat-rye
chromosomes only trigger a partial conformation change
Diploidhomologues
Hybrid- Ph1homoeologues
Ph1-
Hybrid- Ph1+
homoeologues
Ph1+
Ph1-
heterochromatin
telomeres
Pairing
Some Pairing
No Pairing
Synapsis in diverged (related) chromosomes
Homologues
with divergent
segments
Chromosome segments
remodel
Little synaptic
adjustment
with Ph1
Telomeres
Chromosome segments
forming a circular
structure
Synaptic adjustment
without Ph1
Recombination
Colas et al., PNAS 2008
No recombination
Synaptic Adjustment
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
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
Thanks
Diolch
Спасибо
37