Mapping and sequencing chromosome 6 of Solanum lycopersicum cv Mapping and sequencing chromosome 6 of Solanum lycopersicum cv. Heinz 1706 Sander Peters April 2008

Acknowledgements Erwin Datema Elio Schijlen Mark Fiers Willem Stiekema 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 Mark Fiers Sander Peters Willem Stiekema Laboratory of Genetics Dóra Szinay Hans de Jong Ludmilla Khrustaleva Song-Bin Chang Xiaobo Zhong Taco Jesse René Klein Lankhorst

Outline Strategy Sequencing and finishing Cytogenetic mapping Chr. 6 euchromatin/heterochromatin boundaries Sequencing & assembly status Chr. 6 map prototype Repeat and gene content Overall conclusions and prognosis

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

Sequencing and finishing ABI3730 (Macrogen) 1922 reads (10 x 96 x 2 + 2 BAC ends seqs) 2 kb and 6 kb libraries Shotgun clones + BAC dna Planning 454 BAC sequencing in house Finishing by primer walking on average 3 sequence gaps per BAC Shotgun clones, direct BAC sequencing Sequence gap closure Overlapping BACs (13.3 kb per extension) TOPAAS assisted (read pairs, similarity hits) Editting consensus to error rate 1:10000 (Phred 40)

Cytogenetic mapping Multi-color BAC FISH Cytogenetic position of BACs 5 fluorophores, DAEC (blue), FITC (green), Cy3 (orange), Cy3.5 (red), Cy5 (far red). Pachytene chromosomes show highly condensed DAPI stained heterochromatin and less condensed euchromatin. Cytogenetic position of BACs comparison to the linear order of anchored BACs on the genetic F2.2000 map. BAC contig scaffolding.

0-2cM 10-12 cM 40-47 cM 95-101cm a b c d e f g h i j k H016K14 H147H20 H301C21 M82G10 H144J05 M82G10 H002B19 H286N17 H023B17 H250I21 H250I21 H167M06 H040F08 H308F14 H288L09 H261A18 H309K01 H304P16 H0307J13 H024F02 H301C21 H307J13 H068M22 H215M16 H302A23 H242H19 H242H19 H194N16 H059K09 H020O17 H046G10 H309D09 H091L20 H042L06 H003K02 H026E06 H302A23 H309D09 H120H21 H066A20 H002C17 H309D09 H177K13 H042L06 H066A20 H020O17 H012O10 H052N09 H012O10 H023B17 H052N09 H304I22 H197N20 H036J15 H066I09 H261A18 H084A18 H066I09 H109C03 H246E15 H301C21 H310B09 H055E14 H117B06 H055E14 H099H05 M012J12 H116G14 H084A18 H169D11 M012J12 H304I22 H177K13 M012J12 H194N16 H034C13 H060A01 H060A01 H182D16 H315H13 H098L02 H021K07 H060A01 a b c d e f g h i j k

Cytogenetic mapping results FISHed 113 candidate BACs 84 BACs (74%) map on chr. 6 of which 54 are seeds 29 (26%) land on other chromosome 21 single focus on other chromosomes 7 multiple foci of which 3 BACs also on chr. 6 1 without signal Overall cytogenetic mapping order agrees with the linear order of anchored BACs on Tomato EXPEN2000 However, differences at distal ends, pericentromere and in 40-50 cM

Sequencing and walking progress 66 seed BACs 61 Hind, 5 Mbo 22 bidirectional, 18 unidirectional, 19 not initiated, 7 have extension overlaps >= 50% insert 92 extension BACs identified 46 Hind, 17 Eco, 29 Mbo Sequencing & assembly 45 BACs phase 3, 110 BACs finishing pipeline, 3 sequening not initiated Average overlap 13.3 kb (42 extension overlaps) 100% match 16.7 Mb redundant sequence 2.6 Mb short arm (2.4 Mb non-redundant), 14.1 Mb long arm (10.2 Mb non-redundant) 67 chr. 6 markers identified (EXPEN2000 / EXPEN1992 / Arabidopsis) confidence LOD3 LOD2 LOD<=1 No LOD Total library H M E   seed 31 1 4 19 3 7* 66 extension 7 2 33 25 15 92

Chromorome 6 integrated map 161 BACs on the physical map 28 contigs and 10 singleton BACs 3 markers mapped on multiple chromosmes (9, 11 and 12) 5 markers mapped other chromosomes (3, 5, 7 and11) Map

Gene rich space of chromosome 6 2n, 12 chromosomes, 950 Mb 75% of DNA in heterochromatin 90% non transposon genes in euchromatin of 220Mb Chr. 6 20Mb euchromatin 2.7 Mb 6SE → 4.1 Mb 17.7 Mb 6LE → 26.9 Mb Delineate euchromatin domain BAC FISH sequence composition

Euchromatin and heterochromatin bordering BACs on chromosome 6 BAC cM Marker H016K14 0 H158P14 2 T0686 H147H20 0 CT216 H107A05 3 T1188 H112G05 5.5 Mi H304P16 10 cLET-2-H1 S. Telo H54K13 H147H20 H176K10 S. Telo H016K14 H158P14 H304P16 H060A01 H315H13 H310B09 H309D09 H147H20 S.Telo Cen S. Telo H091L20 H147H20 Cen S. Telo H020O17 CT135* H060A01 101 Ct_At1g20050 H315H13 98 TG193 Cen H107A05 S. Telo H112G05 H060A01 H020O17 H091L20 L.Telo L. Telo

Pericentromeric and sub-telomeric BACs on chromosome 6 BAC cM Marker BLASTN S. Telo H003K02 Cen S.Telo H242H19 S.Telo H016K14 0 TTTAGGG Cen H242H19 12 T1063 centr. linked repeat chr. 1 A. thal. H295L11 10 T0244 Rev. transcriptase S. demissum (AAT40500) H309K01 10 cLET- CCS3 A.thal. 5-A4 (AF204876) CCS5 A.thal. (AF204878) 040F08 091L20 282A22 H032D19 L.Telo S.Telo H003K02 10 TG178 gag/pol P.sativum (AAQ82037) RNaseH M. tunc. (ABD28291) H295L11 Cen H309K01 H040F08 12 CD67 put. polyprotein Ty3 like O. sativa (AAL78097) Cen L. Telo H315H13 98 TG193 rib. Protein S4 S. lycopersicum (YP_514854) L.Telo

Conclusions Efficient and reliable extension BAC selection using STC and HICF ‘Map-as-you go’ decreases the amount of fingerprints needed and produces reliable physical mapping Annotated physical map for chr. 6 Seed BAC bias aberrant mapped markers chromosome duplications Gene poor domains in 6LE Established the borders of euchromatic/ heterochromatic domains Constructed a framework for finishing chromosome 6 12.6 Mb non redundant sequence within 155 BACs focus on Ol-1, Ol-3, Ol-5, Ty-1, Ty-2 in CBSG2 Finishing requires at least 100 BACs, funding for 60 BACs