Graham Moore

To recombine, or not recombinethat is the question??

October 2014

Hexaploid (Bread) wheatTriticum aestivum

2n = 6x =42

1 2 3 4 5 6 7

A

B

D

abcd

abcd

abcd

homologues

homoeologues

To be fertile, true homologues must pair at meiosis

Diploid – Homologues distinguished from non-homologous chromosomes

Wheat - Homologues, homoeologues and non-homologous chromosomes

Meiosis

1 Diploid cell

4 Haploid cells

Telophase II

Prophase I

DiploteneDiakinesis

PachyteneZygotene

LeptoteneMetaphase

I

Anaphase I

Telophase IProphase IIMetaphase

II

Anaphase II

Homologues seen as paired via Crossovers

at metaphase I

Homologues

separateand

segregatedSister chromatids

are separated

Homologous chromosomes must recognise each other, pair correctly and recombine

Both observed that deleting chromosome 5B in wheat hybrids induced crossovers between homoeologues

What controls “polyploid” chromosome pairing in wheat??

Sir Ralph Riley UK

Dr Ernie Sears US

• Reasoned a 5B locus was the major regulator of pairing and recombination in wheat

• Termed the locus, Ph1, (Pairing homoeologous 1)• And started 50 years of intense rivalry between wheat

researchers..

Infamous Wheat genetics meetingchaired by Sir Ralph-two Ph1 speakers

XGenome A

Genome B

Genome D

Wheat 2n= 42

1 2 43 5 6 7

Rye 2n= 14

1 2 43 5 6 7

Genome AGenome B

Genome D

Rye

Wheat-rye hybrid Ph1-

Wheat- rye hybrid n =

28

No homologues

Wheat-rye hybrid Ph1+

Wheat-rye hybrid n = 28

What they saw in a wheat hybrid

Up to one crossover Up to 7 crossovers

1) Does Ph1 actually block homoeologues pairing?

Leptotene Zygotene Pachytene

homologues

Chromosome Synapsis

Basics of chromosome pairing

The power of a cell biology experiment

“ primer added”Lateral element

“Glue added”Central Element

Early meiosis

After 50 years the key antibodies to meiotic proteins are available to answer two questions

Central Element“Glue”

Lateral Element“primer”

Synaptonemal complex formed

homoeologues

In wheat-wild relatives hybrids, experimented on by Riley and Sears, there are no homologues, only homoeologues

“Pachytene”Wheat-rye hybrid Ph1-

Martin et al Nature Communications 2014

Lateral element- “primer” greenCentral element-”glue” magenta

DNA-blue

Wheat-rye hybrid Ph1+

Lateral element- “primer” greenCentral element-”Glue” magenta

DNA-blue

“P” (µm) “G” (µm) Synapsis %

Wheat-Rye - 1662.95 378.50 26%

Wheat-Rye + 1594.00 403.95 27%

Homoeologue pairing is not reduced in wheat-rye hybrid by the presence of Ph1.

Ph1 has been named incorrectly for

50 years!

Martin et al Nature Communications 2014

Meiotic progression

“Pachytene”

Genome A

Genome B

Genome D

Wheat 2n= 42

1 2 43 5 6 7

Genome AGenome B

Genome D

Rye

Wheat- rye hybrid n =

28

No homologues Ph1 can’t block the homoeologues pairing in the

hybrid

Ph1’s effect on chromosome pairing

But Ph1 can block/reduce homoeologues pairing in wheatPh1 action is to promote homologues

to pair

telomeres

homologoussegments

What happens with pairing of homologues in wheat itself??

The identical chromosomes zip up from their telomere regions

Pilar Prieto et al 2004 Nat Cell Biol

Rye

segment

homologues

telomeres

‘Elongation” ofchromatin

This conformation change now reported in C elegans

Telomeres

Ph1+ Ph1+Ph1+

Ph1-

Ph1+

In wheat- homologues can elongate asynchronously without Ph1

Interstitial segments- 15% of the wheat chromosome

Pilar Prieto et al 2004 Nat Cell BiolColas et al. 2008 PNAS 2008

XGenome A

Genome B

Genome D

Wheat 2n= 42

1 2 43 5 6 7

Rye 2n= 14

1 2 43 5 6 7

Genome AGenome B

Genome D

Rye

Wheat-rye hybrid Ph1-

Wheat- rye hybrid n =

28

Wheat-rye hybrid Ph1+

Up to one crossover Up to 7 crossovers

2) Ph1 blocks recombination between homoeologues- When?

DOUBLE HOLLIDAY JUNCTION

5’3’5’3’

Non Crossover

5’3’5’3’

5’3’5’3’

Alternative paths

DNA STRANDS CUT AT ARROWS

Crossovers

5’3’5’3’

5’3’5’3’

ONE CHROMOSOME CUT

PAIR OF HOMOLOGUES orHOMOEOLOGUES

5’3’5’3’

STRAND INVASION5’3’5’3’

DNA synthesis

EXPOSED SINGLE STRANDED 3’ END

5’3’5’3’

5’

3’5

’3’

Rad51

spo11

MLH1 complex required for resolution of Double Holliday Junctions as crossovers

# MLH1 sites at

diplotene

# sites that will crossover=

- Wheat-rye hybrid Ph1+

Metaphase I

MLH1CDK2EXO1

MLH3

leptotene

diplotene

Ph1 blocks recombination between homoeologues- When?

Robin Holliday was at JIC

MLH1- greenDAPI- blue

Wheat-rye Ph1+ - one crossover- MLH1 number expected ≈ one foci

- MLH1 number observed = 22 + 3 foci_

Wheat-rye Ph1-- 7 crossovers- MLH1 number expected ≈ 7-8 foci

- MLH1 number observed = 20 + 3 foci_

Martin et al Nature Communications 2014

Summary of data in hybrids

homoeologues

Mlh1Cdk2

• Homoeologues pair (synapse) where Ph1 is present or absent.

• Recombination machinery loads but stalls with the loading of the last complex- the MLH1 complex.

• Stalling is partially alleviated by deleting Ph1

• The MLH1 complex is composed of MLH1, MLH3, EXO1 and CDK2, and regulated by CDK2

• So what is Ph1??

Strategy for delineating the Ph1 locus

Implementation

• Screened 10 irradiated wheat populations = 15 years

• Mapping deletion breakpoints and gene content revealed by cereal synteny (our development)

Wheat mutants

• Ph1 is defined as deletion effect• Define Ph1 with overlapping deletions • Deletions scored for presence/absence of Ph1

W h e a tc h r 5 B

Ph1 region in B rachypodium (to sca le )

B rachypodium region 1 genes

B rachypodium region 2 genes

B rachypodium B A C s

W h e a tc h r 5 A

W h e a tc h r 5 D

D ele tionbreakpoints

D ele tionbreakpoints

clk1

(cl

v)

stk1

vmp1

hyp

4 (n

ew)

rdr1

zip1

(zin

c)

hsp

20-1

raf1

(ra

8)

wdb1

(wrk

y)

ps1

v

h51

l (hap

5)

at1(

AtH

ypIII

)

hsp

90

gtl1

(chic

k)

sbp1

(sel

en)

cmt1

(cc

om

)

ugg1

(udp)

hyp

6 (A

tHyp

2)

fim

2

slp1

(sub)

hyp

3 (O

sHyp

II)

bna

1236

H11

Rev a

1236

H11

Rev b

1677

L4 C

33

1417

M11

350

OsH

ypI

916G

3 R

ev

1390

E11

Uni

pep

1

fim

1

mic

1 (A

tHyp

7)

hyp

5 (m

arck

s)

R ice chr 8 genes

Ph1 region in rice chr 9 (to sca le)

R ice chr 9 genes

rlk2

hyp

scf

OsH

ypI CS379

3

CS977

raf

wdb

ps1

v

h5l

1

atat el

p

gtl

cmt

hyp

clk

scf

hsp

90

raf

wdb

h5l

gtl

cmt

pdp

gtl2

cmt2

fim

1

hyp

slp

fim

2

C84

6

CS

100

2 &

455-9

-1

CS

1010

pfk

hyp

hyp

plp

1

1 2 F 16

1 7 8 3A 2 2

1 3 9 0E 11

7 1 9 O 7

6 0 6 L2

1 0 5 1I3

1 7 7 2H 1 2

1 6 7 7L 4

5 3 2 G 11

1 6 5 D 12

1 0 5 P 17

1 6 5 3J 2

1 2 1 7B 2

9 1 6 G 3

4 3 2 L9

1 4 1 7M 11

1 5 6 8 O 9

1 6 6 4J 5

9 9 3 J2

2 8 2 J8

4 7 0 M 7

1 3 2 0C 1 4

1 4 0 5 B 1 0

3 0 4 F6

1 5 0 C 14

clk

1

rlk1

cycd

1

raf1

wdb1

mic

1

ps1

v

h5l1

at1

rlp1

AtH

yp5

gtl1

sbp1

cmt1

AtH

yp2

fim

1

slp

1

New

Hyp

cdc2

pfk

1

Marc

ks

plp

1

grp

1

KH

1

KH

4

KH

2

KH

5

KH

3

KH

6

KH

7

1 2 0 1K 2 3

hyp

2 (K

H2)

hyp

3 (K

H3)

hyp

4 (K

H4)

Sub te lom e ric insertion from chr 3A

116 2B 5

2 54 I1 0

1 8 9 8B 1 9

6 7 H 7

22 I2

563C 24

1 2 2 3B 6

9 6 2 M 8

1 2 3 6 H 11

Te lo m e reC e n tro m e re

11 9 5 H 2 2

8 4 6 A 11

hsp

90

cnb

sha

wak

lbp

fas

9 2 5 M 1 5

3 4 2F 1 0

2 1 8J 1 3

897P 24

c d c 2h o m o lo g u e s

cdc2

-4

3 0 N 2

2 8 C 1 3

3 1 8 G 1

Outside de letion region

7 7 9 O 1 2

Outside de letion region

1 5 6 7N 6

1 8 2 1A 3

1 5 9 A 2

1 4 9 7 M 6

3 1 B 11

1 2 7 5 L 15

7 0 3 L 1 6

1 2 4 P 2 2

11 4 6 J 1 3

4 3 1 E 7

1 6 2 5E 2 1

7 2 6 N 4

1 2 6 8O 8

11 8 3 B 1 0

1 4 6 2G 15

7 4 9 H 15 (A /D )

3 8 7 C 19

7 8 0 H 16

1 8 4 0B 1 7

870L14

21419

1 0 0K b

Griffiths et al Nature 2006

The gene content of the minimum region containing the Ph1 locus

Kinases

Half the genes are kinases

A cluster of 7 defective Cdk2-type genes

(Griffiths et al Nature 2006)

The gene content of the minimum region containing the Ph1 locus

Kinases

So what next??

Nearly half the genes are kinases, so does altered kinase activity induce crossovers between homoeologues?

We can increase Cdk activity/phosphorylation levels- how?

Detached tiller method

• Okadaic acid inhibits phosphatases which reduce Cdk activity

• Okadaic acid indirectly increases Cdk activity/

Phosphorylation levels

homoeologues

MLH1CDK2

Overcomestalling of MLH1 complex??

Okadaic acid treatment induces crossovers between homoeologues in a wheat x rye hybrid

Homoeologous pairing

Wheat X Rye – Ph1 deleted

No okadaic acid – mostly univalents

Okadaic acid treatment -Rod bivalents

So, okadaic acid treatment similar to deleting Ph1Emilie Knight et al., 2010

Metaphase I

Cdk2 Ph1-cdk gene

Yousafzai and Al-kaff, 2010

Ph1 cdk+cyclinA compared to Cdk2+cyclinA

Protein modelling

• MLH1 complex is regulated by CDK2• Is CDK2 activity increased by deleting Ph1?

• Increasing CDK activity is similar to deleting Ph1

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

Phosphorylation sites

(S/TP-X-Z

Azahara Martin, Ali Pendle, Alex Jones, Isabelle Colas

Greer et al., Plant Cell 2012

Wheat histone H1

Deleting Ph1 or treating with Okadaic acidincreases phosphorylation at the sameCDK2 phosphoryation site

The Ph1 locus- Summary

• In the presence of Ph1, CDK2 activity is reduced

• MLH1 complex (containing CDK2) stalls on paired homoeologues in the presence of Ph1.

• Increased CDK activity, stalling overcome, crossovers induced even in the presence of Ph1

2) blocks homoeologous recombination

1) promotes homologue pairing

• Chromatin changes with Ph1

We will make the ultimate sacrifice in the winter of……

b

* *

* * **

a

c

7 8 9 10 11 12 13 14 15 16 17 18

No. Cells

1 0 2 2 4 8 6 11 5 8 2 1

Crossover Number

JIC, US, Denmark etcWheat hybrids Ph1- (21 MLH1 sites= 7 crossovers)How to improve on this??

b

* *

* * **

a

c

7 8 9 10 11 12 13 14 15 16 17 18

No. Cells

1 0 2 2 4 8 6 11 5 8 2 1

Crossover Number

Cordoba, Feb/March (27oC!)Wheat hybrids exhibit21 MLH1 sites= over 13 crossovers

The Fly in the ointment! inthe approach

JIC grown hybrids plusCordoba fertiliser21 MLH1 sites= over 13 crossovers

May end career as a John Innes Researcher demonstratingCordoba No1 is better than John Innes No1!!

Thanks to…

Genomics- Comparative-BAC library-mutantsTracie Draeger (Foote), Michael Roberts, Lijia Qu, Terry Miller, Steve Reader, Simon Griffiths, Sebastien Allouis, Rebecca Sharp, Kath Mortimer, Emilie Knight, Nadia Al-Kaff, Vera Thole, Ruoyu Wen, Boulos Chalhoub, Shahryar Kianian, Dupont-Pioneer

ModellingFaridoon Yousafzai, Nadia Al-Kaff, David Richards, Martin Howard

Phosphoproteomics- advanced mass specAzahara Martin, Ali Pendle, Isabelle Colas, Alex Jones, Peter Shaw

Cell biology-Shahal Abbo, Luis Aragon, Fadri Martinez, Pilar Prieto, Mike Wanous, Isabelle Colas, Emma Greer, Azahara Martin, Danielle Monk, Peter Shaw

Ph1 into other species-Brachypodium/ArabidopsisRuoyu Wen, Ali Pendle,Vera Thole, Philippe Vain, John Doonan, Peter Shaw

W h e a tc h r 5 B

P h 1 reg ion in B rac h yp o d iu m (to sca le )

B rac h yp o d iu m re g io n 1 ge n es

B rac h yp o d iu m re g io n 2 ge n es

B rac h yp o d iu m B A C s

W h e a tc h r 5 A

W h e a tc h r 5 D

D ele tio nb rea kp o in ts

D ele tio nb rea kp o in ts

clk1

(cl

v)

stk1

vmp

1

hyp

4 (n

ew)

rdr1

zip

1 (z

inc)

hsp

20-1

raf1

(ra

8)

wd

b1

(wrk

y)

ps1

v

h51

l (h

ap5)

at1(

AtH

ypIII

)

hsp

90

gtl

1 (c

hic

k)

sbp

1 (s

elen

)

cmt1

(cc

om

)

ug

g1

(ud

p)

hyp

6 (A

tHyp

2)

fim

2

slp

1 (s

ub

)

hyp

3 (O

sHyp

II)

bn

a

12

36H

11 R

ev

a

12

36H

11 R

ev

b

16

77L

4 C

33

14

17M

11 3

50

Os

Hy

pI

91

6G3

Re

v

13

90E

11 U

ni

pep

1

fim

1

mic

1 (A

tHyp

7)

hyp

5 (m

arck

s)

R ice c hr 8 g e n es

P h 1 re gio n in ric e c h r 9 (to s ca le)

R ice c hr 9 g e n es

rlk2

hyp

scf

OsH

ypI CS

379

3

CS

977

raf

wd

b

ps1

v

h5l

1

atat el

p

gtl

cmt

hyp

clk

scf

hsp

90

raf

wd

b

h5l

gtl

cmt

pd

p

gtl

2

cmt2

fim

1

hyp

slp

fim

2

C84

6C

S1

002

& 4

55

-9-1

CS

10

10

pfk

hyp

hyp

plp

1

1 2 F 1 6

1 7 8 3 A 2 2

1 3 9 0 E 1 1

7 1 9 O 7

6 0 6 L 2

1 0 5 1 I 3

1 7 7 2 H 1 2

1 6 7 7 L 4

5 3 2 G 11

1 6 5 D 1 2

1 0 5 P 1 7

1 6 5 3 J 2

1 2 1 7 B 2

9 1 6 G 3

4 3 2 L 9

1 4 1 7 M 11

1 5 6 8 O 9

1 6 6 4 J 5

9 9 3 J2

2 8 2 J8

4 7 0 M 7

1 3 2 0 C 1 4

1 4 0 5 B 1 0

3 0 4 F 6

1 5 0 C 1 4

clk

1

rlk

1

cy

cd1

raf1

wd

b1

mic

1p

s1v

h5

l1

at1

rlp

1

AtH

yp

5

gtl

1sb

p1

cmt1

AtH

yp

2

fim

1

slp

1

Ne

w H

yp

cdc

2

pfk

1

Ma

rcks

plp

1

grp

1

KH

1

KH

4

KH

2

KH

5

KH

3

KH

6K

H7

1 2 0 1 K 2 3

hyp

2 (K

H2)

hyp

3 (K

H3)

hyp

4 (K

H4)

S ub te lom e ric inse rtion from ch r 3A

1 1 6 2 B 5

2 5 4 I1 0

1 8 9 8 B 1 9

6 7 H 7

2 2 I2

5 6 3 C 24

1 2 2 3 B 6

9 6 2 M 8

1 2 3 6 H 11

Te lo m e reC e n tro m e re

11 9 5 H 2 2

8 4 6 A 11

hsp

90cn

bsh

aw

aklb

p

fas

9 2 5 M 1 5

3 4 2 F 1 0

2 1 8 J 1 3

8 9 7 P 24

c d c 2h o m o lo g u e s

cdc2

-4

3 0 N 2

2 8 C 1 3

3 1 8 G 1

O u tsid e d e letio n re g io n

7 7 9 O 1 2

O u tsid e d e letio n re g io n

1 5 6 7 N 6

1 8 2 1 A 3

1 5 9 A 2

1 4 9 7 M 6

3 1 B 11

1 2 7 5 L 1 5

7 0 3 L 1 6

1 2 4 P 2 2

11 4 6 J 1 3

4 3 1 E 7

1 6 2 5 E 2 1

7 2 6 N 4

1 2 6 8 O 8

11 8 3 B 1 0

1 4 6 2 G 1 5

7 4 9 H 1 5 ( A /D )

3 8 7 C 1 9

7 8 0 H 1 6

1 8 4 0 B 1 7

8 7 0 L1 4

21419

100K b