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