Progress on the sequencing of theeuchromatic gene rich space ofchromosome 6 of Solanum lycopersicumcv. 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 TPF @ 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)

0

10

20

30

40

50

60

70

80

contig size (nts)#

cont

igs

0

1

2

3

4

5

6

7

8

9

10

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

supercontigsize (#BACs)

#s

up

erc

on

tig

s

Borders of 6E and 6H

H060A01

H315H13

H310B09H309D09

L. Telo

S. TeloH016K14H158P14

H147H20S. Telo

6SE: 0cM-10cM

6SH+6LH: 10cM-18.5cM

6LE: 18.5cM-101cM

Integrated map

► Integrated map

| 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

H060A01H315H13

H0307J13

H066I09

H021K07

Gaps between BAC supercontigs

TG115

TG482 cLEY-13-J2

H098L02

H021K07

H028D14

E005I21

M066F23

TG193

H315H13

TG215TG314

U1461

40

E014B21 M059G17

T1789

TG642

H057D21C2_At1

g1687

0

M106A20

H060A01 H182D16

H034C13

T0123T1090

cTOE-1

1-C4

SSR122

T1785

T1124

T1449

C2_At2

g2849

0

M012J12

C2_At1g18640

H169D11

TG579

E131F12

T0405

H055E14

H203N09 M099A03

H116G14

C2_At4

g2110

5T01

40

H059D21

H038F03

U144275

T0585T0798

E037L07

T1066

H084A18

M113N10

H304I22

H117B06

H246E15

T1272cLEY-15-N18 T1835

T0447

C2_At1

g2005

0

GAP

Cytogenetic map order Genetic map order BAC 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

147H2052O17107A05251G05250I21304P16134P07242H1920O1726P1823B17

(0.1)

(1.29)1.051.22

4.2713.06

0.2

2.48

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

domain chr. distance m)

gen.distance (cM)

Size (Mb)

Mb/m cM/m Mb/cM

6SE 1.8 10 4.1 2.27 5.5 0.41

6SH+6LH 8 8.5 50 6.25 1.1 5.94

6LE 29.5 82.5 26.9 0.91 2.8 0.33

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 heterochromatinbordering BACs on chromosome 6

H060A01H020O17

H091L20

H112G05

H021K07

H271L05

S. Telo

H060A01

H315H13

H310B09

H309D09

L. Telo

cen

H091L20

H003K02 10 TG178

H020O17 CT135*

H060A01 101 Ct_At1g20050

H315H13 98 TG193L. Telo

BAC cM Marker

H016K14 32

H158P14 2 T0686

H147H20 0 CT216

H107A05 3 T1188

H112G05 5.5 Mi

S. Telo

H54K13

H147H20

H176K10

S. TeloH016K14

H158P14

H147H20S. Telo

H107A05

H147H20S. Telo

single focus

Chr. 6 pericentromeric & subtelomeric BACs

H003K02

Cen

S.Telo

040F08091L20

282A22

H032D19

S.Telo

Cen

L.Telo

H309K01

H242H19S.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

TGR1+

+

--