Progress on the sequencing of the euchromatic gene rich space of chromosome 6 of Solanum lycopersicum cv. Heinz 1706 Sander Peters Sep 2007

► Selection of BAC clones ► Euchromatin and heterochromatin bordering BACs ► Sequencing & assembly status ► Map prototype ► Conclusions

Extension BAC selection by STC BAC seq. & assembly Extended consensus Seq. analysis SNaPshot & Genemapper Genoprofiler FPC Largest extending BAC Fingerprinting & mapping FISH analysis FISH Seed BAC BLASTN BAC ends Assembly BAC ends Min. overlapping BACs Alignment & scaffolding TOPAAS

Serial (bidirectional) walking from seeds seed Coverage complete Radiating out from seed BAC with extension BACs FISHED BACs used for BAC walk start ►Library depth d = 15, Av. length λ = 117 kb, L chr. 6 = 20 Mb, (L tot = 230 Mb) ►Seed BAC coverage π = 2,5 Mb, R seq = 1 / (d - 1) = 7.1% ►Average gap length ω = (L- π) / π = 7 λ = 820 kb ►Number bidirectional steps: κ 90% =~  λ = 7 κ 98% =~ 2 *  λ = 14 ( Batzoglu et al., 1999) ►Number of fingerprints based on STC 2,500 – 5,000 ►BAC associated markers and overlap sequences provide a road map for walking ►Efficiency depends on the seed BAC distribution along the chromosome ►Heterochromatin and euchromatin borders? Chr. VI M1M2M3M4M5L. TeloS. TeloCenPeri 2.7 Mb17-18 Mb

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 rDNA from S. tuberosum TR Extended DNA fibre of distal end chromosome arm 6S TGR1 + -

Sequencing and walking progress ► 64 seed BACs |61 Hind, 3 Mbo 40 initital (LOD > 3), 21 (LOD < 3), 3 new overgo |22 bidirectional, 18 unidirectional |7 have extension overlaps >= 50% insert |17 extension not initiated ► 74 extension BACs identified |40 Hind, 9 Eco, 25 Mbo ► Sequencing & assembly |9 BACs phase 3 |99 BACs ordered contigs |30 BACs in sequencing&assembly pipeline ► Average overlap 13.3 kb (42 extension overlaps) | 100% match ► 12.9 Mb |2.0 Mb short arm |10.9 Mb long arm ► 52 chr. 6 markers identified ► AGP for 8 BACs (phase 3) & TPF files have been uploaded toSGN

Mi contig on 6s Contig H250I21 H112G05 H073H07 ~148 kb ~130 kb ← centromere ~138 kb ~91 kb H019E05 ~450 kb H107A05 H054K13 ~165 kb ~160kb ~79kb E008G21 ~350kb Mi 5 cM Contig H107A05 T cM S. Telo. → T cM H147H20 CT216 0 cM H225A15 Pericentr. H225A15 H112G05 H107A05 H054K13 S. Telo H147H20 H054K13 S. Telo H176K10

Tiling path and prototype map TG178 (10 cM) CD67 (12.5 cM) T0774 ( 18cM) L.H. 6L.E.4 S. Telo S.H.1 Cen L.H.1L.H.3L.H.2S.H.2S.H.3S.E.1L.E.3L.E.1 CT216 (0) Mi (5.5) T1082 (3cM) T1188 H016K14 E008G21 H054K13H107A05 H019E05 H073H07 H250I21 H112G05 H225A15H003K02 H091L20 H282A22H147H20 M009E16 M022H01 M115P13 H024L21 TG436 (10) H295L11 H187J06 T0244 H062H07 E039G15 M098A22 E045F22 H288L16 H304P16H066P09 H095I22 cLET-2-H1 H309K01 H039P06 cLET-5-A4 H119L20 H242H19H208D24 T1063 (12) H040F08 L.H.5 T1563 M067G18 H308F14 H116O16 H302A23 H 020O17 M082G10 At3g56230 H023B17 FER ( 25 cM) H167M06 cLET-4-G23 M047K24 H261A18 H158P14 H301C21 T0834 (32 cM) H307J13 H024F02 M047M23M118M03 M201C24 H132E21 H177K13 TG240 (37.9 cM) TM43 (40 cM) E123G17 H215M16 M017O21 H057J04 H103P06 M074G21 E093P23 H120H21 cLEC-15-N20 (44 cM) C2-At5g25150 H010E05 M135H21 H103N18 H046G10 H042L06 TG472 H026E06 H118O07 H106K23 M142B18 C2-At4g10030 H194N16 cLET-5-C8 H286N17 TM4 (52) H012O10 M062L07 C2-At5g0796 E084C11 H066I09 M060G16 CT204 H023D12 H125P18 H023D12H002C17 H128E05 H052N09 H181G10 TG253 CT174 T1639 (57) TG446 L.E.3 L. Telo L.H. 7 H218K19H109C03H309D09 H159P11 H086B01 TG365 H082B05H310B09H197N20 H153G23 T1556 (59) H222J18 M074O13 H036J15 M123O13 H066D13 M127C05 TG292 H218N05H229B01 M050H06 cLET-20-H7 (67) H246E15 U M113N10 H038F03 H059D21H117B06 T0585 (71) H203N09 E131F12 H055E14 TG579 H099H05 H049I02H015I15 H046P09 cLET-19-J2 (74 cM) M099A03 H116G14 H169D11 cLEX-2-F13 (85 cM) M012J12 H034C13 U H221G10 H021K07H028D14 H217M17 H098L02 TG115 (97 cM) TG482 (98) M134B10 E129O21 M124M03 H012A08H146A12 E014B21 H060A01 M059G17 H182D16H315H13 H066A20 CT247 TG193 C2-At1g20050 (101) cTOA-4O17 T0686 (2) P27( 47 cM) H304I22 T0798 CT184 III III

Conclusions ►Efficient and reliable extension BAC selection using STC and HICF | ‘resistant’ to aberrant selection of overlapping BACs that could arise from repeats and/or chimearic inserts ►‘Map-as-you go’ decreases the amount of fingerprints needed ►TOPAAS available via |Peters et al., 2006, Plant Phys ►Expect to close 6S with 1-2 extension steps ►Combine FISH map, genetic map, and physical map ►Aim to assemble 200 BACs by the end of 2007

Taco Jesse 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 Mark Fiers Sander Peters