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Sander Peters

Progress on the sequencing of the euchromatic gene rich space of chromosome 6 of Solanum lycopersicum cv. Heinz 1706. Sander Peters. Cologne Oct 2008. Outline. Sequencing & assembly status Euchromatin and heterochromatin boundaries Integrated Map Physical gaps Gene and repeat content

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Sander Peters

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  1. Progress on the sequencing of theeuchromatic gene rich space ofchromosome 6 of Solanum lycopersicumcv. Heinz 1706 Sander Peters Cologne Oct 2008

  2. Outline • Sequencing & assembly status • Euchromatin and heterochromatin boundaries • Integrated Map • Physical gaps • Gene and repeat content • Conclusions

  3. FISH, sequencing and BAC walking progress status 155 candidate BACs 106 Hind, 34 Mbo, 15 Eco 113 BACs FISHed on Cherry VNFT and Heinz 81 BACs on VI 3 on VI and other 28 on other 1 no signal 143 BACs Physically mapped 64 seed BACs 59 Hind, 5 Mbo 79 extension BACs 39 Hind, 15 Eco, 25 Mbo Sequencing & assembly 47 BACs phase 3 96 BACs ordered contigs Average overlap 13.3 kb, 100% match AGP and TPF @ SGN 10.7 Mb 2.0 Mb short arm, 1.8 Mb pericentromere, 6.9 Mb long arm 157 markers identified (31% not mapped on chr 6)

  4. H016K14 S. Telo H158P14 H147H20 S. Telo H309D09 H310B09 H060A01 L. Telo H315H13 Borders of 6E and 6H 6SE: 0cM-10cM 6SH+6LH: 10cM-18.5cM 6LE: 18.5cM-101cM

  5. Integrated map • Integrated map • Crosslink between 157 genetic markers, 10.7 Mb of sequence and (cyto)genetic locations of 81 BACs on tomato chromosome 6 • 31% markers not mapped on EXPEN 2000 • Gaps on 6LE • 73 cM (T0405) and 93 cM (C2_At1g18640)

  6. Gaps between BAC supercontigs H301C21 M082G10 U144275 H167M06 cLEY-15-N18 TG579 T0405 T1272 T0447 H261A18 C2_At4g21105 T0798 T1066 T0585 H0307J13 T1835 T0140 H024F02 H304I22 H059D21 H055E14 M113N10 H117B06 H038F03 H116G14 E131F12 M099A03 H084A18 H246E15 E037L07 H203N09 H215M16 H194N16 H046G10 TG115 TG314 cLEY-13-J2 cTOE-11-C4 U146140 TG215 SSR122 T1090 TG482 T1449 TG642 T1785 T1789 C2_At1g16870 T1124 TG193 T0123 H021K07 H028D14 M066F23 H057D21 H060A01 H182D16 H098L02 E005I21 M106A20 H315H13 E014B21 M059G17 H034C13 H309D09 H052N09 H012O10 H052N09 H066I09 C2_At2g28490 H055E14 M012J12 M012J12 H169D11 M012J12 H060A01 H034C13 H060A01 H315H13 H182D16 H021K07 H098L02 H060A01 Cytogenetic map order Genetic map order BAC Tiling Path GAP C2_At1g20050 C2_At1g18640 H169D11

  7. Determination of gap sizes distances • Pachytene complements • measurements of physical distances on a micrometer scale • ‘global’ • ‘local’between adjacent BAC FISH positions

  8. Multi-color BAC FISH on pachytene 147H20 52O17 107A05 251G05 250I21 304P16 134P07 242H19 20O17 26P18 23B17 (0.1) (1.29) 1.05 1.22 0.2 4.27 13.06 2.48

  9. Physical gaps in integrated map • Pachytene complements • measurements of physical distances on a micrometer scale • ‘global’ and ‘local’ between adjacent BAC FISH positions • Estimation of base pair / mm / cM relationship • Large gap on the genetic map between 73 and 93 cM • 0.62 Mb/mm • 1.2 Mb

  10. Gene and repeat content

  11. Conclusions • Megabase-sized gaps which are not poorly targeted with markers and for which we cannot obtain anchored BACs (BAC-walking?) • Complement sequencing effort using NGS platforms • However, whole genome assembly (WGA) is a challenge • Sequence islands may serve as backbone for WGA • We need more anchored BACs! BAC FISH and BAC sequencing will remain indispensable. • Sequence comparison between tomato and BACs from other Solanum species combined with cross-species multicolor BAC-FISH painting may allow identification of new candidate seed BACs.

  12. Applied Bioinformatics Elio Schijlen Marjo van Staveren Jan van Haarst Marleen Henkens Roeland van Ham Bas te Lintel Hekkert Joost de Groot Thamara Hesselink Erwin Datema José van de Belt Sander Peters Willem Stiekema Laboratory of Genetics Dóra Szinay Hans de Jong Ludmilla Khrustaleva Song-Bin Chang Xiaobo Zhong René Klein Lankhorst

  13. Euchromatin and heterochromatinbordering BACs on chromosome 6 BAC cM Marker H016K14 32 H158P14 2 T0686 H147H20 0 CT216 H107A05 3 T1188 H112G05 5.5 Mi H016K14 S. Telo H158P14 H54K13 H147H20 S. Telo S. Telo H147H20 H271L05 H091L20 H003K02 10 TG178 H147H20 H176K10 cen S. Telo H021K07 H020O17 CT135* H060A01 101 Ct_At1g20050 H315H13 98 TG193 S. Telo H107A05 H112G05 H309D09 H020O17 H060A01 H310B09 H091L20 H060A01 L. Telo H315H13 L. Telo

  14. Chr. 6 pericentromeric & subtelomeric BACs - + + - TGR1 TR Extended DNA fibre of distal end chromosome arm 6S BAC cM Marker BLAST S. Telo H242H19 S.Telo S.Telo ? H016K14 32 none TTTAGGG ? Cen cen H003K02 H242H19 12 T1063 A.thal. chr. 1 centr. linked repeat H295L11 10 T0244 A. thal. chr. 4 copia like transp. H309K01 10 cLET- A.thal. conserved 5-A4 centromere sequence chr. 5 S.Telo L.Telo 282A22 H295L11 H003K02 10 TG178 A.thal. copia like transposon 091L20 Cen 040F08 H309K01 H040F08 12 CD67 A. thal. Ty3 like transposon Cen H315H13 98 TG193 L. Telo H032D19 L.Telo

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