structure and evolution of wheat centromeres and ...wheat.pw.usda.gov/itmi/2008/pagxvizhang.pdf12 t....
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Structure and Evolution of Wheat Centromeres and Pericentromeres
Xueyong Zhang
Institute of Crop Sciences,CAAS, Beijing
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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