BAC FISH and repeat bar-coding technology
for tomato and potato
San Diego, January – 2008Hans de Jong, Dóra Szinay
Laboratory of GeneticsWageningen University
• Improved cell spreading method• High res FISH pachytene morphology• Digital improvement • Chromosome straightening• Direct 5 colour FISH
Erik Wijnker / Boudewijn van Veen / Xiaomin Tang / Dóra Szinay
Cy3.5 BA76f14
FITC BA151m8
Cy3 BA180d11
DEAC BA188f1
Biotin/Cy5 BA256o1
centromere
Recent advances FISH technology
BAC FISH for European partners
Status January 2008
• Chromosome 4 UK 80• Chromosome 6 NL 150• Chromosome 7 FR 60• Chromosome 9 ES 9• Chromosome 12 IT -
total ~ 300
Song-Bin Chang – Hans de Jong
Cot1 Cot10 Cot100
Cot100DAPI
Seven chromatin classes in tomato
a. NOR and satelliteb. distal heterochromatin
d. functional centromere
c. pericentr. heterochromatin
g. euchromatin
e. interstitial knobs
f. chromomeres
b. distal heterochromatin
c. pericentr. heterochromatin
45S
rD
NA
5S r
DN
A
TG
R1
TGR
2TG
R3
TGR
4G
AC
A
GA
TAG
A/G
AA
Ty1/
copi
aC
ot1
Co
t10
TTTA
GG
G
a
b
c
d
e
f
g
chr1
chr2
Chromosomal anchor points
1. Telomeres2. Centromeres3. Borders
a. heterochromatin b. euchromatin
1
2
3
3
1
2
1
1
3
RN
Spread synaptonamal complexes – Steve Stack Lab
Tomato telomeres
Extended DNA fibre FISH
Reveals molecular sizes of the telomere
repeats
E110K10, 2 cMdistal BAC chr 7Bouzayen c.s.
1 2 3 4 5 6 7 8 9 10 11 12
euchromatinheterochromatinTTTAGGGTGR1
Solanum lycopersicon cv VFNT cherry - Zhong et al. 1998
Tomato centromeres• FISH with BAC H57J04 (43.5
cM)– TGR4 paints centromeres– Tomato specific (not potato)– 3 subclones also in the
centromeres
• Sequencing– Gypsode1_I retrotransposon– LTR element
(unique for tomato)– Hits on several other BACs– Complete sequence not
known yet
• ChIP for functional part of centromere planned in 2008
TGR4-274 on pachytene
TGR4-394 on metaphase I
Chromosome arm MB estimates
chr 1 chr 2 chr 3 chr 4 chr 5 chr 6 chr 7 chr 8 chr 9 chr10 Chr11 Ch12Shortarm
eu 5.7 0.0 9.2 8.4 9.7 4.1 6.2 6.2 8.1 5.5 10.0 8.0het 29.3 23.5 13.3 9.1 25.1 9.6 24.0 14.9 17.6 16.0 11.7 25.6
Longarm
het 33.6 27.2 35.2 44.8 32.0 18.7 29.9 25.6 38.4 50.1 32.0 29.9eu 44.5 38.8 30.4 19.5 9.7 26.9 18.9 19.3 15.5 10.2 9.7 8.4
Total S+L 113.1 89.5 88.2 81.7 76.4 59.2 79.0 66.0 79.6 81.9 63.5 71.8
Total euchr 50.2 38.8 39.6 28.6 19.4 31.0 25.1 25.5 23.6 15.7 19.7 16.4
(%) 44.4 43.4 44.9 34.1 25.4 52.4 31.0 38.6 29.6 19.1 31.0 22.8
cM ( ♂ ) 124 104 105 94.5 83.5 86.5 88.5 84 79 83 83 79.5
• Pachytene length measurements (Ramanna & Prakken, 1967)• DNA content estimate heterochromatin = 5.89 x euchromatin (Peterson et
al. 1995)• Total genomic DNA amount = 950 MB (Arumuganathan & Earle, 1991)
Chromosome 6 euchromatin estimates1. Stack / Chang / de Jong 20 MB
2. De Jong (2007) 31 MBdifferent heterochromatin / euchromatin borders
UK NL FR ES IT
The heterochromatin borders
Borders are gradual transitions of DAPI bright
and DAPI weak parts!
Heterochromatin = repetitive DNA domains?
• Heterochromatin– Epigenetic status of
chromatin• Methylated DNA
shown by Immunofluorescence
• Modified Histones (e.g., H3K9, H3K27)
– C-band positive
– Domains of repetitive DNA sequences
• Cot100 paint + BAC FISH
• Repeat analysis BACs
Sat + NOR
Anti-C methylated DNA - AB-FITC
Marie Meznikova-Skleničková, Brno, CZMS Ramanna, Wageningen
1 2 3
304P16
082G10
centromere
Dóra Szinay, Hans de Jong
Cot100-BAC painting
• Heterochromatin border BACs– Cot100– H304P16 border of 6S – H082G10 on the distal part of
6L heterochromatin
304P16 in Mi region
082G10 in Oi region
Sequencing potentially complex due to different introgressions
Major repeat families tomato
LTR (1.8 kb) LTR (1.8 kb)CRT1a (8953 bp)
82% (1..610 bp)
TGR2 (187..775 bp)
82%
TGR2
b. TGR2
LTR (1.2 kb) LTR (1.2 kb)CRT2 (12169 bp)
85% (280..783 bp)
TGR3 (1..509 bp)
85%
TGR3
(10983..11389 bp)
(1..509 bp)
c. TGR3
LTR (2.2 kb)CRT5 (10019 bp)LTR (2.2 kb)
d. TGR4
a. TGR1 (tandem repeat)
(162 bp)
Chromosome 7
3. Pericentromere repeat TGR-II
4. Pericentromere repeat TGR-III
5. Centromere repeat TGR-IV
6. Ty1-copia
2. Subtelomere repeat TGR-I
1. Telomere sequences
Murielle Philippot and Mondher Bouzayen
November 2007
7. 26S rDNA
Status short arm BAC FISH
ca. 2.7 MB
a
b
c
d
a/b experiment 1; c/d experiment 2
FISH map 6S arm
158P14 – 016K14
tgr1: 408 kb
telomere repeat 16 kb
pericentromere heterochromatin
Dóra Szinay, Chunting Lang, Song-Bin Chang, Xiaobo Zhong and Hans de Jong
Extended fibre FISH of 6S telo
107A05 147H20(3 cM) (0 cM)
TR: 16 ± 3kb TGR1: 408 ± 33kb
304P16 closed gaps
016K14 (32 cM)158P14 (2 cM)147H20 (0 cM)
25 BACs analyzed; 15 confirmed
The 0 cM BACs enigma
cM BACs # foci Position
32 016K14 Single 6S
2 158P14 Single 6S
0 147H20 Single 6S
0 54K13 Single 6S
0 114O13 Repeat various
0 176K10 Few NOT 6
0 166G10 Single NOT 6
0 282D09 Single NOT 6
0 259K16 Single NOT 6
0 015K13 Single NOT 6
3 107A05 Single 6S
114O13
054K13
112G05
166G10
Long arm pooled BAC FISH
randomly plotted BACs in chr 6S (Excel simulation)
centromere
telomere
Gaps can be explained by random BAC distribution
In one case a gap of 23% was found
More then 70 BACs analyzed; 37 confirmed
telomere
New BAC positioning in gaps
centromere telomere
1
2
3
4
5
6
M082G10H023B17 H042L06H026E06 M012J12
Gap-1
307J13 120H21
BAC poor region
cM marker BAC
32 T0834 H307J13
40 TM43 H215M16
41.5 C2_At1g03150 Slm025B14
43 TG177 H024F02
43 T0805 Hba044J22
43.7 T1666 H170D02
43.8 cTOB-6-M19 Hba175H08
44 cLEC-15-N2 H120H21
44 C2_At4g10030 H106K23
44.3 cLET-8-I22 Hba032D19
45 cLET-5-18 H286N17
45 cLET-5-18 H194N16
46.5 C2_At1g71950
H177K13
307J13
215M16024F02
120H21
286N17
177K13194N16042L06
026E06
261A18
023B17
106K23
BAC FISH around gap-1 in 6L
301C21
Gap-1
De border BACs from Gap-1 (pooled BAC FISH) are J13/C21 and H21
“The Dóra rainbow”
032D19
175H08
144J05
144J05
170D02
170D02
025B14
cM BAC Chr. position
32 H307J13
40 H215M16
41.5 Slm025B14 NOT 6 – peric.
43 H024F02
43 Hba044J22 NOT 6 – het.chr.
43.7 H170D02 6L + dupl chr.
43.8 Hba175H08 NOT 6 – peric.
44 H120H21
44 H106K23
44.3 Hba032D19 NOT 6 – peric.
45 H286N17
45 H194N16
46.5 H177K13
30 J144J05 6S + dupl chr.The 5 BACs in the 42 – 44 cM gap of chromosome 6 are scattered on different chromosomes. No indication for a translocation 6/9. but the chr 6 and chr 9 teams claim duplications.
251G05 5 cM (T1198)250I21 5.5 cM (Mi) + 112G05 5.5 cM (Mi)
107A05 3 cM (T1188) + 054K13 3 cM (T1082)147H20 0 cM (CT216)
158P14 4 cM (C2_At3g46780), 2 cM (T0687), 0 cM (CT216)
304P16 10 cM (cLET-2) + 288L16 10 cM (cLET-2)024L21 10 cM (TG436)
023B17 25 cM (Fer)
082G10 18.5 cM (C2_At3g56230)
215M16 40 cM (TM43)024F02 43 cM (TG177)
315H13 97.8 cM (TG193), 101 cM (TG314)
169D11 85 cM (cLex-2-F13)
261A18 28 cM (cLET-4-G23) + 167M06 28 cM (cLET-4-G23)
309D09 50 cM (TG365) + 109C03 50 cM (TG365)
021K07 97 cM (TG115)
194N16 45 cM (cLET-5-18)
197N20 59 cM (T1556) + 310B09 59 cM (T1556)
99H05 74 cM (cLET-19-J2)
304I22 69 cM (T0798)
52N09 57 cM (T1639)
36J15 64 cM (TG292)
55E14 73 cM (T0405)
60A01 101 cM (C2_At1g20050))
66I09 54 cM (CT204)
012O10 48 cM (C2_At1g73885)
307J13 32 cM (T0834) + 301C21 32 cM (T0834)
120H21 44 cM (cLEC-15-N20)106K23 44 cM (C2_At4g10030)
2C17 56 cM (CT174A)
246E15 69 cM (T1515)
98L02 98 cM (TG482)
242H19 12 cM (T1063)
003K02 10 cM (TG178)
309K01 10 cM (cLET-5-A4)
040F08 12.5 cM (CD67)
026E06 47 cM (P27)
286N17 45 cM (cLET-5-18)
177K13 46.5 cM (C2_At1g71950
182D16 97.2 cM (U146140)
0
10
20
30
40
50
60
70
80
90
100
(partly overlapping)
cMcM/MB
Small inversions?
Small inversions?
Crossover suppression
pericentromere
BAC GapFlanking BACs have
markers in 42-44 cM
RN map
Linkage disequilibrium tomato
Chromosome 6
Chromosome 9
Linkage disequilibrium along the chromosomes
Heatmap display of LD for all marker-pairs,(GGT 2.0, Ralf van Berloo)
Intensity reflects the amount of LD:
Assocations chrs 6 and 9
Courtesy:Fred van Eeuwijk (WUR)
Cross-species FISH
?
?
pt
?
p
Tomato BACs on tomato and potato, and vv
•For cross species FISH stringency of 50% to 20%plus Cot100 of tomato
•20 tomato BACs were painted on tomato and potato
•5 potato BACs op potato and tomato
Tomato – potato 6S rearrangements
large 6S inversion
small 6S nested
inversion
small 6S heterochr.Inversion?
1 2 3
Consequences rearrangements
• Comparison of maps– Genetic map = F2
(S. lycopersicon cv. VF36-Tm2a X S. pennellii LA7 16)
– Physical map: BAC libraries = tomato cv. Heinz 1706
– RN map: Cherry (VFNT?)
– FISH map: tomato cv. VFNT Cherry (LA1221)with S. peruvianum introgression
• Gaps in contigs• Problems in chromosome walking• Introgressions may have chromosomal
(micro)rearrangements
Strong CO suppressed by introgression
Svetlana Liharska & Pim Zabel, Wageningen University, 1998
Moneymaker (S. lycopersicum)
Vetomold S. pimpinellifolium
WSL6 S. pennellii
VFNT cherry S. peruvianum
83M/R S. peruvianum
Motelle S. peruvianum
W607 S. peruvianum
Ontario-7620S. peruvianum + S. pimpinellifolium
LC21
6b
Tl;
CD
14
GP79
Rex
1
Yv
TG23
2H
2D
1
C
6.6
5.5
1.9
1.0
2.0
3.3
-
3.9
44.5
44.3
25.0
45.6
44.4
42.3
-
44.8
Chromosome 6, with 8 loci
Discussions map integration tomato
• BAC selection biased: overgo false positives, picking or other errors…
• Genetic map position can be inaccurate
• Unknown repeat polymorphisms
• Possibility for gene duplications
• Cultivars /lines may have different alien introgressions
• Unknown chromosomal rearrangements
Phylogeny wild tomatos (S. Knapp)
Clade Icheesmaniaegalapagenselycopersicumpimpinellifolium
Clade IIchmeilewskiineorickiiarcanum
Clade IIIperuvianumchilensehabrochaites = hirsutum?corneliomuelleri
Clade IVhabrochaites = hirsutum?pennelli
Clade Vochranthumjuglandifolium
Clade VIcitienslycopersicoides
Phylogeny wild tomatos (S. Knapp)
Clade Icheesmaniaegalapagenselycopersicumpimpinellifolium
Clade IIchmeilewskiineorickiiarcanum
Clade IIIperuvianumchilensehabrochaites = hirsutum?corneliomuelleri
Clade IVhabrochaites = hirsutum?pennelli
Clade Vochranthumjuglandifolium
Clade VIcitienslycopersicoides
AcknowledgementsCBSG – WageningenWillem Stiekema
EU-SOLRené Klein Lankhorst
Wageningen UniversityCentre MicrospectroscopyBoudewijn van Veen
Genetics LabDóra SzinaySong-Bin ChangXiaobo ZhongLudmila KhrustalevaJoke van VugtChunting LangErik WijnkerHans de JongMaarten Koornneef
WU Lab Plant BreedingXiaomin TangYuling BaiChristian BachemTheo BormJan de BoerDirkjan HuigenAnja KuipersHerman van EckRichard VisserPiet StamMunikote Ramanna
WUR – PRIApplied BioinformaticsElio SchijlenMarjo van StaverenRoeland van HamErwin DatemaSander PetersThamara Hesselink
WUR Biometrics Fred van EeuwijkHans Jansen
KeygeneTaco Jesse
MPI KölnUte Achenbach
Colorado State UniversitySteve Stack
Seoul National UniversityTae-Jin Yang
EU-SOL teams chr 4, 7, 9 and 12(UK, FR, ES, IT)Murielle Philipot