1. Current Sequencing Effort of Tomato Chromosome 2 - Chromosome assembly -
Finishing workshop
April. 2008
KRIBB/SNU, Korea

2. Euchromatin of chromosome 2
LG2 HK
H007F24
NOR 10 PH 13
H303I24
CEN
H163K16 20
H190N21 PH HK 30
H101G09
13 cM
H162I09
M045L06 40
H160F05
H072A04 50 60
M018P14
26Mb
268 BACs 70
M019I01
22Mb ?
230 BACs
H323A14
H204D01
H320D04
H198A03 80
H291P19
H060J03
H009K06
H164H08
H213A01 90
H134G09
H016A12
H011A02
100
M014P22
M042B19
110
H0461M08
H150M11
H210D10
120
H073P13
130
H155D20
142 cM
140
H064B17
142
H194L19
TEL
H257H21

3. 12.8 Mb <- non-redundancy :58% (22Mb)
Sequencing & assembly
153 BACs phase 3
143 Genebank submit
9 BACs in sequencing & assembly pipeline
29 contigs
- 11,182,936 bp
15 singletons
- 1,1641,963 bp
17,592,982 bp
12.8 Mb <- non-redundancy :58% (22Mb) 13 16 18 21 25 28 34
36.5 37 38
38.5 46 67
69.5 70 72
72.5 73
74.5 75 76
77.5 83
81.5
83.5 88 89
89.3 92 94 96
100
106
108/111
109
112
114
117
120
130
141
143
140
142/143
Marker-anchored seed BAC
Extended BAC
142 3

4. Chromosome assembly Contig Singleton Total number 29 15 Average BAC-
Average length
376 Kb
118Kb
Total length
11,182 kb
1,164 kb

5. The Longest Contigs Contig 45 14 1,143 Contig 27 9 554 Contig 10 699
# of Contig
BAC
Length
(kb)
Contig 45 14
1,143
Contig 27 9
554
Contig 10
699
Contig 38 6
545
Contig 19 8
630
Contig 24
503
Contig 40
595
Contig 44
497

6. How to verify BAC overlaps
Sequence comparison (>10kb, <10bp discrepancies)
Bridge BAC analysis
IL mapping
FISH

7. Hot Spot ? Case 1 11cM (27cM) (25cM) (36cM) 312,844bp
Hba0209K17(134,560bp)
Hba0320M09(136,324bp)
Hba0025A22(117,193bp)
Hba0101G09(117,515bp)
(27cM)
(25cM)
(36cM)
312,844bp
11cM

8. Cold Spot? Cold Spot? Case 2 cLEC-27-M9 Hba066C13 (46.0cM) Hba167J21
Hba323A14
T1395(72.0cM)
Hba204D01
T1625(72.5cM)
TM34(73cM)

9. Different results from marker and IL mapping
Case 3
Different results from marker and IL mapping
cLED-19-B24 (100.0 cM/chr.2)
E010H16 (84,031bp)
E012K17 (160 kb)
M095G17 (89.5kb)
26,053 bp
8,244 bp
Sle010H16 was found by marker BLAST

10. Sle010H16 has marker cLED-19-B24
Chromosome 2
Intron 1
Intron 2

11. E012K17-T7- IL Mapping ; Hinf I
Alignment Length ; 305 bp
( W / gaps )
Recognition Sequence ; GA.TC
Penn ; 305
M82 ;
Pooling IL DNA
P M
P M
P : Penn, M: M82, Chromosome 1-12
Chromosome 2 ; Fail
Chromosome 10
P M
P M 11

12. M095G17-T7- IL Mapping ; Hinf I
Alignment Length ; 454 bp
( W / gaps )
Recognition Sequence ; GA.TC
Penn ; 425
M82 ; 106, 319
Pooling IL DNA
P M
P M
P : Penn, M: M82, Chromosome 1-12
Chromosome 2 ; Fail
Chromosome 10
P M
P M 12

13. Does Sle010H16 locate on chromosome 2?
cLED-19-B24 (100.0 cM)
Chr. 2
E010H16 (84,031bp)
Chr. 10
E012K17 (160 kb)
M095G17 (89.5kb)
Chr. 10
26,053 bp
8,244 bp
Does Sle010H16 locate on chromosome 2?

14. Case 4 99.9% 77,419bp 38,184bp 15,607bp 203P08(ch. 4) 155D20(ch. 2)
C2_At4g37460
C2_At5g67370
T0634
CT59
T0769
Ch 2
137cM
Ch 4
17.6cM
Ch 4
94cM
Ch 2
129cM
Ch 2
130cM
C04Hba0203P08 (ch. 4) T0769, 94cM bp
C02Hba0155D20 (ch. 2) T0634, 130cM bp
2-K, FISH

15. Problematic clones prohibit chromosome assembly
Case 5
Problematic clones prohibit chromosome assembly
TG426
(89.3cM)
TG426
89.3
80kb
TG48
(92cM)
TG48 92
TG373
H011A02 94
TG373
(94cM)
TG147 96
TG147
(96cM)

16. C02Slm0014P22 C02Slm0065M14 TG373 (94.0cM) – contig 23
Hba0011A02 (135,832bp)
Hba0031A13 (89,105bp)
Hba0236E02 (148,233bp)
76,518bp
MboI0065M14 (97,159bp)
Hba0190P16 (95,464bp)
12,586bp
43,489bp
19,549bp
Hba0189G15 (142,305bp)
13,759bp
EcoRI0128J14 (80,800bp)
80,800bp
T0147 (96.0cM) – contig 24
MboI0014P22 (79,505bp)
Hba0189G15 (142,305bp)
61,617bp
EcoRI0128J14 (80,800bp)
21,174bp
C02Slm0014P22
C02Slm0065M14

17. Primer 1
Primer 2
Primer 1
C02SLm0014P22
C02SLm0065M14
Primer 2

18. C02SLm0014P22 should not exist in tomato genome!
M065M14-conf_2 (377bp)
Primer 2
M014P22-conf_2 (528bp)
Primer 1
Heinz
M065M14
M014P22
Heinz
M014P22
M065M14
C02SLm0014P22 should not exist in tomato genome!
TG373 (94.0cM) – contig 23
Hba0011A02 (135,832bp)
Hba0031A13 (89,105bp)
Hba0236E02 (148,233bp)
76,518bp
Hba0190P16 (95,464bp)
MboI0065M14 (97,159bp)
12,586bp
43,489bp
19,549bp
Hba0189G15 (142,305bp)
13,759bp

19. Problematic clones(?) C02SLm0108P14 X O 34,290 C02SLm0008E03 46,886
Name
Genome
BAC
Enzyme site
Size
(bp)
C02SLm0108P14 X O
34,290
C02SLm0008E03
46,886
C02HBa0075D08
66,392
C02HBa0044O16
81,404
C02SLm0014P22
79,505

20. Case 7 T0702 (76.0 cM) C locus (75.0 cM) T1492 (77.0 cM)
H044O16 (81,404bp)
H165K22 (135,795bp)
2-H
M049G16 (115,048bp)
35,695bp
M021D12 (90,430bp)
16,904bp
2-I
M073G04 (89,093bp)
40,085bp
2-I
M049G16 and H044O16 have a marker ‘cLEX-13-I15’which is mapped on chromosome 7 with high confidence.

21. Be careful! - Confusing clones
Marker
cM/chr
BAC
reason
location
rcr3
publication
?/2
C02Hba0122E16
C2_At4g18593 (98%)
Ch.12
T0266
IL(2-G)
67/2
C02Hba0059M17
C2-Atlg61620
Ch.3
HBa0059M17
C02Hba0031A21
TG25
Ch.6(IL6-2)
M049G16
IL(2-H)
75/2
C02Slm0049G16
cLEX_13_I15 (EST)
Ch.7
T0147
IL(2-J)
96/2
C02Hba0189G15
cLET-14-E21(overgo)
Ch.1
cLEC-7-L24
111.2/2
C02Hba0124N09
SSR112
Ch.9
T0634
IL(2-K)
130/2
C02Hba0155D20
T0769
Ch.4

22. Transposase From Host Genome
Case 8
Transposase From Host Genome
E.coli Tansposase 1(1.3 kb)
- C02HBa0012A12/ C02HBa0236E02/ C02HBa0177F12/ C02HBa0212C17
E.coli Transposase 2 (1.3 kb)
- C02HBa0204A09/ C02SLe0092M23/ C02HBa0138P10/ C02HBa0194N24

23. summary Difference of Genetic distance / Physical distance
Genetic marker – IL mapping
Rearranged clones
Confusing Markers
Transposase from Host genome

24. Acknowledgements PI FISH Doil Choi Dal-Hoe Koo Cheol-Goo Hur
Bioinfomatics
Jung-Eun Kim
BAC Sequencing
KRIBB
Genotech
FISH
Dal-Hoe Koo
Hae-Mi Park
BAC selection / assembly
Sung-Hwan Jo
Bo-Ra Kang
Sang-Mi Kim