Скачать презентацию Mapping and sequencing chromosome 6 of Solanum lycopersicum Скачать презентацию Mapping and sequencing chromosome 6 of Solanum lycopersicum

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

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

Outline ► Strategy ► Sequencing and finishing ► Cytogenetic mapping ► Chr. 6 euchromatin/heterochromatin 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

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

Sequencing and finishing ► ABI 3730 (Macrogen) | 1922 reads (10 x 96 x Sequencing and finishing ► ABI 3730 (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 | 5 fluorophores, DAEC (blue), FITC (green), Cy Cytogenetic mapping ► Multi-color BAC FISH | 5 fluorophores, DAEC (blue), FITC (green), Cy 3 (orange), Cy 3. 5 (red), Cy 5 (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 F 2. 2000 map. | BAC contig scaffolding.

H 144 J 05 H 301 C 21 M 82 G 10 H 016 H 144 J 05 H 301 C 21 M 82 G 10 H 016 K 14 H 147 H 20 0 -2 c. M H 002 B 19 H 286 N 17 H 023 B 17 H 250 I 21 H 167 M 06 H 261 A 18 H 0307 J 13 H 024 F 02 H 250 I 21 H 288 L 09 H 304 P 16 H 308 F 14 H 309 K 01 H 040 F 08 H 301 C 21 H 307 J 13 H 215 M 16 H 242 H 19 H 302 A 23 H 020 O 17 H 194 N 16 H 046 G 10 H 068 M 22 H 242 H 19 H 059 K 09 10 -12 c. M H 309 D 09 H 091 L 20 H 003 K 02 H 042 L 06 H 026 E 06 H 309 D 09 H 302 A 23 H 120 H 21 H 066 A 20 H 309 D 09 H 002 C 17 H 042 L 06 H 177 K 13 H 066 A 20 40 -47 c. M H 020 O 17 H 052 N 09 H 012 O 10 H 052 N 09 H 304 I 22 H 036 J 15 H 084 A 18 H 246 E 15 H 055 E 14 H 034 C 13 a b H 109 C 03 c H 301 C 21 H 310 B 09 H 117 B 06 H 055 E 14 H 099 H 05 H 116 G 14 H 084 A 18 H 304 I 22 H 060 A 01 H 182 D 16 H 098 L 02 H 060 A 01 H 261 A 18 H 066 I 09 M 012 J 12 H 177 K 13 H 194 N 16 95 -101 cm H 315 H 13 H 021 K 07 d H 023 B 17 H 197 N 20 H 066 I 09 M 012 J 12 H 169 D 11 H 012 O 10 e f g h i j k

Cytogenetic mapping results ► FISHed 113 candidate BACs | 84 BACs (74%) map on 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 EXPEN 2000 ► However, differences at distal ends, pericentromere and in 40 -50 c. M

Sequencing and walking progress ► 66 seed BACs | 61 Hind, 5 Mbo | 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 (EXPEN 2000 / EXPEN 1992 / Arabidopsis) confidence LOD 3 LOD<=1 LOD 2 Total No LOD library H M E seed 31 1 0 4 1 0 19 3 0 7 extension 7 3 1 2 0 1 4 1 0 * 0 0 66 33 25 15 92

Chromorome 6 integrated map ► 161 BACs on the physical map ► 28 contigs 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 and 11) ► Map

Gene rich space of chromosome 6 ► 2 n, 12 chromosomes, 950 Mb ► Gene rich space of chromosome 6 ► 2 n, 12 chromosomes, 950 Mb ► 75% of DNA in heterochromatin ► 90% non transposon genes in euchromatin of 220 Mb ► Chr. 6 20 Mb euchromatin | 2. 7 Mb 6 SE → 4. 1 Mb | 17. 7 Mb 6 LE → 26. 9 Mb ► Delineate euchromatin domain | BAC FISH | sequence composition

Euchromatin and heterochromatin bordering BACs on chromosome 6 BAC c. M Marker single focus Euchromatin and heterochromatin bordering BACs on chromosome 6 BAC c. M Marker single focus S. Telo. H 016 K 14 0 H 158 P 14 H 54 K 13 H 147 H 20 H 304 P 16 H 158 P 14 2 T 0686 S. Telo H 147 H 20 Cen H 147 H 20 0 CT 216 H 107 A 05 3 T 1188 Cen H 176 K 10 S. Telo H 112 G 05 5. 5 Mi H 147 H 20 H 304 P 16 10 c. LET-2 -H 1 H 091 L 20 H 112 G 05 S. Telo H 107 A 05 Cen H 020 O 17 CT 135 * H 060 A 01 H 020 O 17 H 309 D 09 H 310 B 09 H 091 L 20 H 060 A 01 101 Ct_At 1 g 20050 L. Telo H 315 H 13 98 TG 193 H 315 H 13 L. Telo

Pericentromeric and sub-telomeric BACs on chromosome 6 single focus S. Telo BAC c. M Pericentromeric and sub-telomeric BACs on chromosome 6 single focus S. Telo BAC c. M Marker BLASTN H 016 K 14 0 TTTAGGG Cen S. Telo H 242 H 19 Cen H 242 H 19 12 T 1063 centr. linked repeat chr. 1 A. thal. H 003 K 02 H 295 L 11 10 T 0244 Rev. transcriptase S. demissum (AAT 40500) L. Telo H 309 K 01 10 c. LET- CCS 3 A. thal. 5 -A 4 (AF 204876) CCS 5 A. thal. (AF 204878) 003 K 02 10 TG 178 gag/pol P. sativum H (AAQ 82037) RNase. H M. tunc. (ABD 28291) H 040 F 08 12 CD 67 put. polyprotein Ty 3 like O. sativa (AAL 78097) H 315 H 13 98 TG 193 rib. Protein S 4 S. lycopersicum (YP_514854) L. Telo S. Telo 282 A 22 Cen H 295 L 11 091 L 20 040 F 08 H 309 K 01 Cen L. Telo H 032 D 19

Conclusions ► Efficient and reliable extension BAC selection using STC and HICF ► ‘Map-as-you 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 6 LE ► 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 CBSG 2 ► Finishing requires at least 100 BACs, funding for 60 BACs