Structure and Evolution of Wheat Centromeres and Pericentromeres

Xueyong Zhang

Institute of Crop Sciences,CAAS, Beijing

xueyongz@public.bta.net.cn

T a b le 1 S o u th e r n h y b r id iz a t io n r e s u lts o f R A P D p r o f i le b y c lo n e d g e n o m e -s p e c if ic R A P D m a r k e r s- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

G e n o m e o r s p e c ie s R A P D - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M a r k e r s A u S D E b E e S t P N s R m H I W T h . in t T h .p o n t Z 1 1 4 1 R 4 3 1- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -S t -O P N -0 1 8 1 7 _ _ _ _ _ + + + _ _ _ _ _ _ + + + + + +

S t-O P N -0 5 4 4 4 _ _ _ _ _ + + + _ _ _ _ _ _ + + + a + + a

S t -O P B -0 8 5 2 5 _ _ _ + _ + + + + _ _ + _ _ + + + + + +

S t-O P D -1 5 5 0 4 _ + b _ + b + + + + + b + + + a + b + b + a + + + + + + +

S t-O P N -0 3 7 8 0 + + b + + + + + + + + + + + + + + + + + + +

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Table 1 Southern hybridization results of RAPD profile by cloned genome-specifi RAPD markers-------------------------------------------------------------------------------------------------------------

Genome or species RAPD -------------------------------------------------------------------------------------------------- Markers Au S D Eb Ee St P Ns Rm H I W Th.int Th.pont Z1141 R431 -------------------------------------------------------------------------------------------------------------------Eb-OPF-031296 _ _ _ +++ +b _ + _ ++ _ _ _ ++ ++

Eb-OPN-01269 _ _ _ +++ +a _ +a +a +a + _ _ +++ +++

Eb-OPC-03332 + + + +++ ++ + ++ + ++a ++a + _ ++ ++

Eb-OPL-05613 _ _ _ +++ ++ + _ +b _ _ _ _ + +

Ee-OPN-03870 _ _ + + +++ _ _ _ + _ _ _ + -

Ee-OPR-05700 _ _ +b + +++ +b + _ + +b _ _ _ +

E--OPC-15291 _ +a +a +++ +++ +a +a _ _ _ _ _ +a +++

E--OPF-03373 + + + +++ +++ _ _ _ _ _ _ + + +++

------------------------------------------------------------------------------------------------------------------

"-"No hybridization signal; "+" Weak hybridization signals;

"+++" Strong hybridization signals; "a" Fragment size increased;

"b" Fragment size decreased.

P: St; B: E

EbEeExStStTh. ponticum

Th.ponticum

P: E; B: St

Centromeric and pericentromeric region may be the critical area that discriminates the St genome from the E genome in

Thinopyrum ponticum (Tall wheatgrass).

Zhang XY et al. 1996, GENOME, 39:1062-1071

Is this truth or false?

Structure and Localization of theCentromere and Kinetochore

Figure 23-38, p. 1094, Molecular Cell Biology, 3rd ed., Lodish, et al., copyright

1995, W.H. Freeman and Company, reprinted with permission.

Functions of centromeres:

Responsible for the proper segregation of chromosomes during cell division.

Sites for sister chromatids association.

Sites for microtubules of spindle to attach chromosomes.

Size:

Point centromere: 125 bp

Regional centromere: several Kb to Mb

Introduction

Structure Components of Centromeres

repetitive sequences:centromeric satellite arrays

centromere-specific retrotransposons

CENPs: CENH3, CENP-B, CENP-C, etc.

Genes

Heterochromatin proteins

Introduction

Centromeric-satellites on centromeres

They diverged rapidly, most of them are specific to only closely related species.

Serve as the core of the centomere, such as in rice, maize and Arabidopsis.

Partial of them are associated with the CENH3 proteins proved by ChIP technology, in rice, maize and Arabidopsis.

Introduction

LTR LTRgag PRO RT RNaseH INT

LTR LTRgag PRO INT RT RNaseH

Ty1/copia:

Ty3/gypsy:

Retrotransposons on centromeres

Introduction

Found in the centromere regions of all grass species studied, such as sorghum, maize, rice, barley, wheat.

Derived from Ty3/gypsy retrotransposons, frequently inserted into centromeric satellites, or into each other.

Partial of them are associated with the CENH3 proteins proved by ChIP technology, in rice, maize and Arabidopsis.

Centromeric retrotransposons (CR) family

Introduction

I. Full Sequence of Two BAC Clones Associating with Centromeres and

Isolation of Centromeric Retrotransposon

of Wheat (CRW)

Fig. Screening the BAC library with cpDNA sequence psbA gene and mtDNA sequence atp6. The pollution is less than 1%.

T. boeoticum BAC library Chen et al. 2002

17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

30ng20ng10ng5ng

30ng20ng10ng5ng

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Fig. Certification with Tsc250, A wheat centromeric repeat (Chen et al, 2003, Genetics, 164:665).

CCS1

A

B

TbBAC5 TbBAC30

RCS1

Southern hybridization of the candidate cloneswith RCS1 and CCS1

Chinese Spring probed by TbBAC5 and TbBAC30

TbBAC5 TbBAC30

1. Schematic organisation of the 90kb and 84kb contigs of BAC clones

TbBAC5 and TbBAC30

Figure 1. Schematic organisation of the 90kb and 84kb contigs of BAC clones TbBAC5 (A) and (B) TbBAC30.

Angela1 Erika1Erika2s

1 bp 10 kb 20 kb 30 kb

Wgel1p Daniela1 CRW3p Erika3s

30 kb 40 kb 50 kb 60 kb

Sukkula1 CRW1 CRW2

70 kb 80 kb 90 kb60 kb

G10

G10 C04A10

365 bp

A

30 kb 60 kb40kb 50kb

CRW8 CRW4 CRW3CRW7p WHAM1p

Angela1p

60 kb 90 kb70kb 80 kb

CRW9p CRW10p CRW11pCRW14sDerami1p Hawi1p Babara1p

1 bp 30 kb10 kb 20 kb

CRW1 CRW5 CRW2 CRW6pCRW12s CRW13s

Egug1p Athila1p

Wilma1p 365 bp

Figure 1. Schematic organisation of the 90kb and 84kb contigs of BAC clones TbBAC5 (A) and TbBAC30 (B).

B

A

B

5’LTR 3’LTRTSD TSD

PPTPBS

GAG

CCS1 CCS1

TSD TSDPPTPBS

ORF

GAG RT INT5’ LTR 3’ LTR

pBs301-like sequence RCS1 365 bp192bp CCS1

CR2-1 CR2-2 CR2-3 CR2-4 CR2-5 CR2-6 CR2-7 CR2-8

365 bp192bp CCS1

2. Schematic organisation of the autonomous and non-autonomous CRWs

Fig. Dotplot between autonomous and nonautonomous CRWs

autonomous CRWGAG RT INT5‘-LTR 3‘-LTR

3’LT

R5’

LTR

GA

G

500

1000

1500

2000

2500

3000

3500

4000

500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 6500 7000 7500

non-

auto

nom

ousC

RW

non-autonomous CRW

autonomous CRW

Fig. Phylogenic tree based on the LTR regions

3. CRWs from different genomes

II. Distributional and Functional Characterization of CRWs

1. Chromosomal localization of CRWs in Triticum species

A，B： autonomous; C-E： non-autonomous

A, B & E: CS

C: T. monoccocum

D: T. dicoccoides

2. Distribution patterns of CRWs on stretched centromeric DNA

p365-1

pINT1

2

Immunosignals generated by rice anti-CENH3 antibody

G & H: T. mono.

I & J: CS

DNA Components at Rice Centromeres

CentO

CRR

Cheng et al., 2002,Plant Cell. 14: 1691–1704

Fig. Relative fold enrichment obtained following ChIP analysis of seven wheat repetitive elements.

P1=0.0001; P2=0.0005; P3=0.0001; P4=0.0026; P5=0.0095; P6=0.0056; P7=0.0922

3. Certification of CRWs for Function of Centromeres

B M H R V Cu A u Ab S D AB AG ABD M

23.1

10.08.0

6.0

5.0

4.03.53.0

2.0

1.5

0.75

1.0

0.50

0.25

M H R V Cu Au Ab S D AB AG ABD M

1.5

0.75

1.0

0.50

0.25

0.50

0.25

23.1

10.08.0

6.05.0

4.03.53.0

2.0

1.5

0.75

1.0

1.5

0.75

1.0

0.50

0.25

A23.1

10.08.0

6.05.0

4.03.53.0

2.0

1.5

0.75

1.0

0.50

0.25

M H R V Cu Au Ab S D AB AG ABD M C

Fig. Southern hybridisation profiles generated by probing with (A) gag, (B) LTR, (C) integrase sequences of an autonomous CRW.

4. Distribution of CRWs in wheat and its relatives

T. dicoccoides Korn, ex Schweinf. 10

Arabidopsis19AD artificial amphiploid9

Millet 18Ae. tauschii Coss8

Zea mays L. 17Aegelops speltoides Tansch7

Oryza sativa L. ssp. japonica16T. monococcum L. 6

Avena sativa L. 15T. boeoticum Boiss. 5

T. aestivum L.（偃展1号）14Triticum urartu Thum. et Gandil4

T. aestivum L.（中国春）13Dasypyrum villosum (L.) Candargy3

T. araraticum Jakubz. 12Secale cereale L. 2

T. orientale Percival 11Hordeum vulgare L. 1

SpeciesNo.SpeciesNo.

Tab. Nineteen species used in Southern hybridization

A： 365bp （LTR）； B：INT （integrase）

A M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 M

Fig. Southern hybridization of CRW with Granmine species（HindIII）

B M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 M

Triticeae species Triticeae species

Ae. comosa Sibth. And Sm.24T. dicoccoides Korn, ex Schweinf.12

St95 23Artificial AD tetraploid11

Arabidopsis22Ae. tauschii Coss10

Millet21Aegelops speltoides Tansch9

Zea mays L.20Aegelops speltoides Tansch8

Oryza sativa L. ssp. japonica19T. monococcum L.7

Avena sativa L.18T. boeoticum Boiss. 6

T. aestivum L.（偃展1号）17Triticum urartu Thum. et Gandil5

T. aestivum L.（中国春）16Ae.umbellulata Zhuk. 4

T. araraticum Jakubz.15Dasypyrum villosum (L.) Candargy3

T. orientale Percival14Secale cereale L.2

T. durum13Hordeum vulgare L.1

1ng10ng100ngNo.1ng10ng100ngNo.

Tab. 24 Species used in dot blotting hybridization

A:365 bp (LTR) B:192bp (LTR)； C:INT

A B C123456789101112

131415161718192021222324

A B C

III. Other retrotransposonspresent in both the centromeric and

pericentromeric regions of the Agenome chromosomes

A,B: T. orientialC: T. araraticumD: T. aestivum

1. Chromosome localisation of sub-clone C04 in Triticum species

A：T. urartu；B：T. dicoccoides；C：T. araraticum；D：T. aestivum

２．The A genome specific dispersed retrotransposon

Fig. Distribution of A genome specific dispersed retrotransposons in wheat and its relatives.

B 23.1

10.08.0

6.05.0

4.03.53.0

2.0

1.5

0.75

1.0

0.50

0.25

M H R V Cu Au Ab S D AB AG ABD M A 23.1

10.08.0

6.05.0

4.03.53.0

2.0

1.5

0.75

1.0

0.50

0.25

M H R V Cu A u Ab S D AB AG ABD M

23.1

10.08.0

6.05.0

4.03.53.0

23.1

10.08.0

6.05.0

4.03.53.0

23.1

10.08.0

6.05.0

4.03.53.0

H R A u S D AB AG ABD M C

8.0

6.0

5.0

4.03.5

3.0

8.0

6.0

5.0

4.03.5

3.0

8.0

6.0

5.0

4.03.5

3.0

M A u S D AB AG ABD M

D

A: Wgel_TbBAC5-1 B: Erika_TbBAC5-1 C: Sukkula_TbBAC5-1 D，E: Daniela_TbBAC5-1 F: CCS1 A，B，D-F: HindIII ， C: BamHI

M H R V Cu A u Ab S D AB AG ABD M

23.1

10.08.0

6.05.0

4.03.53.0

2.0

1.5

0.75

1.0

0.50

0.25

FE 23.1

10.08.0

6.05.0

4.03.53.0

2.0

1.5

0.75

1.0

0.50

0.25

M H R Au S D AB AG ABD M

Fig. Distribution of A genome specific dispersed retrotransposons in wheat and its relatives.

A: Wgel_TbBAC5-1 B: Erika_TbBAC5-1 C: Sukkula_TbBAC5-1 D，E: Daniela_TbBAC5-1 F: CCS1 A，B，D-F: HindIII ， C: BamHI

VI. Estimation of Invasion Time of Retrotransposons Found

in TbBAC5 & TbBAC30

The eight retrotransposons in BAC5 insert

1.1-3.5 MYA

Intact CRW in TbBAC30 and TbBAC5*

TSD: Target site duplication, PBS: Primer binding site, PPT: Polypurine tractMYA: means million years ago.* Personal communication.

CRW

Length (bp)

LTR length (bp)

TSD

PBS

PPT

Insertion times

(MYA) Types

1 4305 981/981 CCTTC AGTGGTATCAG-TTTTC AAGAAGGGGAGGA 0.24 nonautonomous 2 7762 919/918 CCTAT AGTGGTATCAGA-TTTC AAGAAGGGGAGGA 0.58 autonomous 3 7861 918/918 CCTTA/G AGTGGTATCAGATTT-C AAGAAGGAGAGGA 0.75 autonomous 4 4776 982/982 ATTTG AGTGGTATCA-ATTTTC AAAAAGGGGAGGA 0.16 nonautonomous 5 4540 749/982 TAGCT AGTGGTATCAG-TTTTC AAAAAGGGGAGGA 0.86 nonautonomous 8 4769 982/983 TAGTC AGTGGTATCAG-TTTTC AAGAAGGGGAGGA 0.31 nonautonomous

2-TbBAC5* 7865 919/920 ATCC(A)G AGTGGTATCAGA-TTTC AAGAAGGGGAGGA 1.09 autonomous

Figure for wheat genome differentiation

Divergence of maize and sorghum15-20 MYA

Huang et al., 2002; Gill et al., 2004; Dvorak et al., 2004

Divergence of wheat and barley10-14 MYA

Divergence of wheat and rye7 MYA

Divergence of Triticum and Aegilops2.5-4.5 MYA Origin of tetraploid wheat

0.37-0.5 MYA

Origin of hexaploid wheat8TYA

Am and Au, 1.0MYA

Figure for wheat genome differentiation

Divergence of maize and sorghum15-20 MYA

Huang et al., 2002; Gill et al., 2004; Dvorak et al., 2004

Divergence of wheat and barley10-14 MYA

Divergence of wheat and rye7 MYA

Divergence of Triticum and Aegilops2.5-4.5 MYA Origin of tetraploid wheat

0.37-0.5 MYA

Origin of hexaploid wheat8TYA

Am and Au, 1.0MYA

Conclusions• The dominant role of the CRWs in determining the structure and

function of the centromere was confirmed by fiber-FISH and ChIP.

• The CRWs were probably amplified during the polyplodizationprocess leading to the formation of hexaploid bread wheat.

• Wgel retrotransposon is more abundant in the pericentromericregions of the A, than in those of either the B or D genome chromosomes, and this variation may contribute to the inhibitionof homoeologous chromosome pairing.

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