Progress on the sequencing of the euchromatic gene rich space of chromosome 6 of Solanum lycopersicum cv. Heinz 1706 Sander Peters Cologne Oct 2008.

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Presentation transcript:

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 ►Conclusions

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 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)

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

Integrated map ►Integrated mapmap |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)

H060A01 M082G10 H012O10 H167M06 M012J12 H261A18 H034C13 H215M16 H169D11 H194N16 H301C21 H052N09 H098L02 H060A01 M012J12 H182D16 H055E14 H309D09 H024F02 H046G10 H052N09 H060A01 H315H13 H0307J13 H066I09 H021K07 Gaps between BAC supercontigs TG115 TG482 cLEY-13-J2 H098L02 H021K07 H028D14 E005I21 M066F23 TG193 H315H13 TG215 TG314 U E014B21M059G17 T1789 TG642 H057D21 C2_At1g16870 M106A20 H060A01H182D16 H034C13 T0123 T1090 cTOE-11-C4 SSR122 T1785 T1124 T1449 C2_At2g28490 M012J12 C2_At1g18640 H169D11 TG579 E131F12 T0405 H055E14 H203N09 M099A03 H116G14 C2_At4g21105 T0140 H059D21 H038F03 U T0585 T0798 E037L07 T1066 H084A18 M113N10 H304I22 H117B06 H246E15 T1272 cLEY-15-N18 T1835 T0447 C2_At1g20050 GAP Cytogenetic map orderGenetic map orderBAC Tiling Path

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

single focus 147H20 52O17 107A05 251G05 250I21 304P16 134P07 242H19 20O17 26P18 23B17 (0.1) (1.29) Multi-color BAC FISH on pachytene

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 /  m / cM relationship Large gap on the genetic map between 73 and 93 cM 0.62 Mb/  m 1.2 Mb domainchr. distance  m) gen.distance (cM) Size (Mb) Mb/  mcM/  m Mb/cM 6SE SH+6LH LE

Gene and repeat content

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.

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

single focus Euchromatin and heterochromatin bordering BACs on chromosome 6 H060A01 H020O17 H091L20 H112G05 H021K07 H271L05 S. Telo H060A01 H315H13 H310B09 H309D09 L. Telo cen H091L20 H003K02 10 TG178 H020O17 CT135 * H060A Ct_At1g20050 H315H13 98 TG193 L. Telo BAC cM Marker H016K14 32 H158P14 2 T0686 H147H20 0 CT216 H107A05 3 T1188 H112G Mi S. Telo H54K13 H147H20 H176K10 S. Telo H016K14 H158P14 H147H20 S. Telo H107A05 H147H20 S. Telo

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