Mapping and sequencing chromosome 6 of Solanum lycopersicum cv. Heinz 1706

April 2008

Sander Peters

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

Acknowledgements

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

BAC seq. & assembly

Extended consensus Seq

. a

nal

ysis

SNaPshot & Genemapper

Genoprofiler

FPC

Largest extending BAC

Fin

ge

rpri

nti

ng

& m

app

ing

FIS

H a

nal

ysi

s FISH

Seed BAC

BLASTN BAC ends

Assembly BAC ends

Min. overlapping BACs

Ali

gn

me

nt

& s

caf

fold

ing

TO

PA

A

S

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

| 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.

M82G10

H012O10

H167M06

M012J12

H261A18

H034C13

H215M16

H169D11

H194N16

H301C21

H052N09

H098L02

H060A01

M012J12

H182D16

H055E14

H309D09

H024F02

H046G10

H052N09

H060A01

H315H13

H0307J13

H066I09

H021K07

H002C17

H286N17

H309D09

H304I22

H310B09

H304I22

H040F08

H302A23

H109C03

H020O17

H177K13

H066I09

H302A23

H002B19

H309K01H308F14

H120H21

H307J13

H099H05

H012O10

H116G14

H301C21

H066A20

H197N20

H055E14 H117B06

H309D09

H066A20

H084A18

H036J15

H246E15

H042L06

H016K14

H068M22

H301C21

H147H20

H250I21H288L09

H304P16

H059K09H242H19

H003K02

H023B17

H261A18

H177K13H194N16

H084A18

H144J05

H250I21

H091L20

H060A01

H242H19

H020O17

M82G10

H023B17

H026E06

H042L06

M012J12

a b c d e f g h i j k

95-101cm

0-2cM

10-12 cM

40-47 cM

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 H M E H M E H M E  

seed 31 1 0 4 1 0 19 3 0 7* 0 0 66

extension 7 3 1 2 0 1 4 1 0 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

single focus

H060A01H020O17

H091L20

H112G05S. Telo

H060A01

H315H13

H310B09

H309D09

L. Telo

Cen

H091L20

H020O17 CT135*

H060A01 101 Ct_At1g20050

H315H13 98 TG193L.Telo

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. TeloH016K14

H158P14

H147H20S. Telo

H107A05

H147H20S. Telo

H304P16

S. Telo

Cen

Cen

Euchromatin and heterochromatinbordering BACs on chromosome 6

single focus

H003K02

Cen

S.Telo

040F08091L20

282A22

H032D19

S.Telo

Cen

L.Telo

H309K01

H242H19S.Telo

H295L11

Cen

L.Telo

H309K01 10 cLET- CCS3 A.thal. 5-A4 (AF204876) CCS5 A.thal. (AF204878) H003K02 10 TG178 gag/pol P.sativum (AAQ82037) RNaseH M. tunc. (ABD28291)

H242H19 12 T1063 centr. linked repeat chr. 1 A. thal.

H295L11 10 T0244 Rev. transcriptase S. demissum

(AAT40500)

H040F08 12 CD67 put. polyprotein Ty3 like O. sativa (AAL78097)

BAC cM Marker BLASTN

H016K14 0 TTTAGGG

S. Telo

Cen

L. Telo

H315H13 98 TG193 rib. Protein S4 S. lycopersicum (YP_514854)

Pericentromeric and sub-telomeric BACs on chromosome 6

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