1. BAC FISH and repeat bar-coding technology for tomato and potato San Diego, January – 2008 Hans de Jong, Dóra Szinay Laboratory of Genetics Wageningen University

2. 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

3. BAC FISH for European partners Status January 2008 Chromosome 4UK 80 Chromosome 6NL150 Chromosome 7FR 60 Chromosome 9ES 9 Chromosome 12IT - total~ 300

4. Song-Bin Chang – Hans de Jong Cot1Cot10Cot100 DAPI

5. Seven chromatin classes in tomato

6. Chromosomal anchor points 1.Telomeres 2.Centromeres 3.Borders a.heterochromatin b.euchromatin 1 2 3 3 1 2 1 1 3 RN Spread synaptonamal complexes – Steve Stack Lab

7. Tomato telomeres Extended DNA fibre FISH Reveals molecular sizes of the telomere repeats E110K10, 2 cM distal BAC chr 7 Bouzayen c.s. 123456789101112 euchromatin heterochromatin TTTAGGG TGR1 Solanum lycopersicon cv VFNT cherry - Zhong et al. 1998

8. 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

9. Chromosome arm MB estimates chr 1chr 2chr 3chr 4chr 5chr 6chr 7chr 8chr 9chr10Chr11Ch12 Short arm eu5.70.09.28.49.74.16.2 8.15.510.08.0 het29.323.513.39.125.19.624.014.917.616.011.725.6 Long arm het33.627.235.244.832.018.729.925.638.450.132.029.9 eu44.538.830.419.59.726.918.919.315.510.29.78.4 Total S+L 113.189.588.281.776.459.279.066.079.681.963.571.8 Total euchr 50.238.839.628.619.431.025.125.523.615.719.716.4 (%)44.443.444.934.1 25.4 52.431.038.629.619.131.022.8 cM ( ♂ )12410410594.583.586.588.5847983 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 estimates 1. Stack / Chang / de Jong20 MB 2. De Jong (2007) 31 MB different heterochromatin / euchromatin borders UK NLFRESIT

10. The heterochromatin borders Borders are gradual transitions of DAPI bright and DAPI weak parts!

11. 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, CZ MS Ramanna, Wageningen

13. 123 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

14. Major repeat families tomato a. TGR1 (tandem repeat) (162 bp)

15. 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

16. 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

17. The 0 cM BACs enigma cMBACs# fociPosition 32016K14Single6S 2158P14Single6S 0147H20Single6S 054K13Single6S 0114O13Repeatvarious 0176K10FewNOT 6 0166G10SingleNOT 6 0282D09SingleNOT 6 0259K16SingleNOT 6 0015K13SingleNOT 6 3107A05Single6S 114O13 054K13 112G05 166G10

18. 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

19. New BAC positioning in gaps centromere telomere 123456123456 M082G10 H023B17 H042L06 H026E06 M012J12 Gap-1 307J13 120H21 BAC poor region

20. cMmarkerBAC 32 T0834H307J13 40TM43H215M16 41.5C2_At1g03150Slm025B14 43TG177H024F02 43T0805Hba044J22 43.7T1666H170D02 43.8cTOB-6-M19Hba175H08 44cLEC-15-N2H120H21 44C2_At4g10030H106K23 44.3cLET-8-I22Hba032D19 45cLET-5-18H286N17 45cLET-5-18H194N16 46.5 C2_At1g71950 H177K13 307J13 215M16 024F02 120H21 286N17 177K13 194N16 042L06 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”

21. 032D19 175H08 144J05 170D02 025B14 cMBACChr. position 32 H307J13 40H215M16 41.5Slm025B14NOT 6 – peric. 43H024F02 43Hba044J22NOT 6 – het.chr. 43.7H170D026L + dupl chr. 43.8Hba175H08NOT 6 – peric. 44H120H21 44H106K23 44.3Hba032D19NOT 6 – peric. 45H286N17 45H194N16 46.5H177K13 30J144J056S + 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.

22. 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? Crossover suppression pericentromere BAC Gap Flanking BACs have markers in 42-44 cM RN map

23. 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)

24. Cross-species FISH ? ? p t ? 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

25. Tomato – potato 6S rearrangements large 6S inversion small 6S nested inversion small 6S heterochr. Inversion? 123

26. 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

27. Strong CO suppressed by introgression Svetlana Liharska & Pim Zabel, Wageningen University, 1998 Moneymaker(S. lycopersicum) VetomoldS. pimpinellifolium WSL6S. pennellii VFNT cherryS. peruvianum 83M/RS. peruvianum MotelleS. peruvianum W607S. peruvianum Ontario-7620S. peruvianum + S. pimpinellifolium LC216b Tl; CD14GP79 Rex1 YvTG232 H2D1C 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

28. 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 I cheesmaniae galapagense lycopersicum pimpinellifolium Clade II chmeilewskii neorickii arcanum Clade III peruvianum chilense habrochaites = hirsutum? corneliomuelleri Clade IV habrochaites = hirsutum? pennelli Clade V ochranthum juglandifolium Clade VI citiens lycopersicoides Phylogeny wild tomatos (S. Knapp) Clade I cheesmaniae galapagense lycopersicum pimpinellifolium Clade II chmeilewskii neorickii arcanum Clade III peruvianum chilense habrochaites = hirsutum? corneliomuelleri Clade IV habrochaites = hirsutum? pennelli Clade V ochranthum juglandifolium Clade VI citiens lycopersicoides

29. Acknowledgements CBSG – Wageningen Willem Stiekema EU-SOL René Klein Lankhorst Wageningen University Centre Microspectroscopy Boudewijn van Veen Genetics Lab Dóra Szinay Song-Bin Chang Xiaobo Zhong Ludmila Khrustaleva Joke van Vugt Chunting Lang Erik Wijnker Hans de Jong Maarten Koornneef WU Lab Plant Breeding Xiaomin Tang Yuling Bai Christian Bachem Theo Borm Jan de Boer Dirkjan Huigen Anja Kuipers Herman van Eck Richard Visser Piet Stam Munikote Ramanna WUR – PRI Applied Bioinformatics Elio Schijlen Marjo van Staveren Roeland van Ham Erwin Datema Sander Peters Thamara Hesselink WUR Biometrics Fred van Eeuwijk Hans Jansen Keygene Taco Jesse MPI Köln Ute Achenbach Colorado State University Steve Stack Seoul National University Tae-Jin Yang EU-SOL teams chr 4, 7, 9 and 12 (UK, FR, ES, IT) Murielle Philipot