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BAC FISH and repeat bar-coding technology for tomato and potato

This study discusses recent advances in BAC FISH technology for tomato and potato, including improved cell spreading methods, high-resolution FISH pachytene morphology, and direct 5-color FISH centromere labeling. It also provides information on chromosome mapping and heterochromatin analysis.

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BAC FISH and repeat bar-coding technology for tomato and potato

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

  2. Recent advances FISH technology • Improved cell spreading method • High res FISH pachytene morphology • Digital improvement • Chromosome straightening • Direct 5 colour FISH centromere Cy3.5 BA76f14 FITC BA151m8 Cy3 BA180d11 DEAC BA188f1 Biotin/Cy5 BA256o1 Erik Wijnker / Boudewijn van Veen / Xiaomin Tang / Dóra Szinay

  3. 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

  4. Cot1 Cot10 Cot100 DAPI Cot100 Song-Bin Chang – Hans de Jong

  5. G a A i A A G N p a. NOR and satellite N A G D o A 0 1 2 3 4 A D r c G 1 A 1 C R R R R T / r t / t b. distal heterochromatin T S 1 A A o G A o G G G S T y 5 C G C G T T T T G 5 T T 4 a chr2 c. pericentr. heterochromatin b d. functional centromere c c. pericentr. heterochromatin chr1 d e. interstitial knobs e f f. chromomeres g. euchromatin g b. distal heterochromatin Seven chromatin classes in tomato

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

  7. Tomato telomeres 1 2 3 4 5 6 7 8 9 10 11 12 TTTAGGG TGR1 Solanum lycopersicon cv VFNT cherry - Zhong et al. 1998 E110K10, 2 cMdistal BAC chr 7Bouzayen c.s. Extended DNA fibre FISH Reveals molecular sizes of the telomere repeats euchromatin heterochromatin

  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 NL FR ES IT UK • 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 Jong 20 MB • 2. De Jong (2007) 31 MB different heterochromatin / euchromatin borders

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

  11. Heterochromatin = repetitive DNA domains? Sat + NOR • Heterochromatin • Epigenetic status of chromatin • Methylated DNAshown by Immunofluorescence • Modified Histones (e.g., H3K9, H3K27) • C-band positive • Domains of repetitive DNA sequences • Cot100 paint + BAC FISH • Repeat analysis BACs Anti-C methylated DNA - AB-FITC Marie Meznikova-Skleničková, Brno, CZ MS Ramanna, Wageningen

  12. Cot100-BAC painting 304P16 centromere • Heterochromatin border BACs • Cot100 • H304P16 border of 6S • H082G10 on the distal part of 6L heterochromatin 082G10 304P16 in Mi region 082G10 in Oi region Sequencing potentially complex due to different introgressions Dóra Szinay, Hans de Jong 1 2 3

  13. Major repeat families tomato a. TGR1 (tandem repeat) (162 bp) b. TGR2 CRT1a (8953 bp) LTR (1.8 kb) LTR (1.8 kb) 82% (1..610 bp) 82% TGR2 TGR2 (187..775 bp) c. TGR3 CRT2 (12169 bp) LTR (1.2 kb) LTR (1.2 kb) 85% (280..783 bp) (10983..11389 bp) 85% TGR3 TGR3 (1..509 bp) (1..509 bp) d. TGR4 CRT5 (10019 bp) LTR (2.2 kb) LTR (2.2 kb)

  14. Chromosome 7 1. Telomere sequences 2. Subtelomere repeat TGR-I 3. Pericentromere repeat TGR-II 4. Pericentromere repeat TGR-III 5. Centromere repeat TGR-IV 6. Ty1-copia 7. 26S rDNA Murielle Philippot and Mondher BouzayenNovember 2007

  15. Status short arm BAC FISH Extended fibre FISH of 6S telo ca. 2.7 MB TGR1: 408 ± 33kb TR: 16 ± 3kb a 107A05147H20(3 cM) (0 cM) 016K14 (32 cM) 158P14 (2 cM) 147H20 (0 cM) b FISH map 6S arm c closed gaps 304P16 158P14 – 016K14 25 BACs analyzed; 15 confirmed pericentromere heterochromatin tgr1: 408 kb d telomere repeat 16 kb a/b experiment 1; c/d experiment 2 Dóra Szinay, Chunting Lang, Song-Bin Chang, Xiaobo Zhong and Hans de Jong

  16. The 0 cM BACs enigma 114O13 166G10 054K13 112G05

  17. Long arm pooled BAC FISH More then 70 BACs analyzed; 37 confirmed randomly plotted BACs in chr 6S (Excel simulation) telomere centromere telomere In one case a gap of 23% was found Gaps can be explained by random BAC distribution

  18. New BAC positioning in gaps centromere BAC poor region telomere 120H21 307J13 H023B17 M082G10 H042L06 M012J12 H026E06 1 2 3 4 5 6 Gap-1

  19. BAC FISH around gap-1 in 6L 023B17 261A18 301C21 307J13 Gap-1 215M16 024F02 “The Dóra rainbow” 120H21 026E06 286N17 106K23 177K13 194N16 042L06 De border BACs from Gap-1 (pooled BAC FISH) are J13/C21 and H21

  20. 175H08 170D02 144J05 025B14 144J05 170D02 032D19 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.

  21. 0 158P14 4 cM (C2_At3g46780), 2 cM (T0687), 0 cM (CT216) Small inversions? 147H20 0 cM (CT216) 107A053 cM (T1188)+054K13 3 cM (T1082) 251G05 5 cM (T1198) 10 250I21 5.5 cM (Mi)+112G05 5.5 cM (Mi) 024L21 10 cM (TG436) 304P16 10 cM (cLET-2)+288L1610 cM (cLET-2) 003K0210 cM (TG178) 20 309K0110 cM (cLET-5-A4) Crossover suppression pericentromere 242H1912 cM (T1063) 30 040F08 12.5 cM (CD67) 40 082G1018.5 cM (C2_At3g56230) 023B17 25 cM (Fer) 261A18 28 cM (cLET-4-G23)+167M0628 cM (cLET-4-G23) 50 307J1332 cM (T0834) + 301C21 32 cM (T0834) 215M16 40 cM (TM43) BAC Gap Flanking BACs have markers in 42-44 cM 024F02 43 cM (TG177) 60 120H2144 cM (cLEC-15-N20) 106K2344 cM (C2_At4g10030) 286N17 45 cM (cLET-5-18) 194N16 45 cM (cLET-5-18) 026E06 47 cM (P27) 70 177K1346.5 cM (C2_At1g71950 012O10 48 cM (C2_At1g73885) 309D0950 cM (TG365)+109C03 50 cM (TG365) 66I0954 cM (CT204) 80 2C1756 cM (CT174A) 52N0957 cM (T1639) 197N20 59 cM (T1556)+310B09 59 cM (T1556) 36J1564 cM (TG292) (partly overlapping) 304I22 69 cM (T0798) 246E1569 cM (T1515) 90 55E14 73 cM (T0405) 99H0574 cM (cLET-19-J2) 169D1185 cM (cLex-2-F13) 100 021K07 97 cM (TG115) Small inversions? 182D1697.2 cM (U146140) 315H13 97.8 cM (TG193), 101 cM (TG314) RN map cM/MB cM 98L0298 cM (TG482) 60A01 101 cM (C2_At1g20050))

  22. Linkage disequilibrium tomato 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) Chromosome 6 Chromosome 9

  23. Cross-species FISH t p 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 ?

  24. Tomato – potato 6S rearrangements large 6S inversion small 6S nested inversion small 6S heterochr. Inversion? 1 2 3

  25. Consequences rearrangements • Comparison of maps • Genetic map = F2 (S. lycopersiconcv. 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

  26. Strong CO suppressed by introgression Yv TG232 Rex1 H2D1 C Tl; CD14 GP79 LC216b Moneymaker (S. lycopersicum) Vetomold S. pimpinellifolium WSL6 S. pennellii VFNT cherry S. peruvianum 83M/R S. peruvianum Motelle S. peruvianum W607 S. peruvianum Ontario-7620 S. peruvianum + S. pimpinellifolium 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 Svetlana Liharska & Pim Zabel, Wageningen University, 1998

  27. Discussions map integration tomato • 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 • 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

  28. Acknowledgements CBSG – WageningenWillem Stiekema EU-SOLRené Klein LankhorstWageningen University Centre MicrospectroscopyBoudewijn 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

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