Validation of a procedure for BAC
end mapping on
S. lycopersicum x pennellii ILs
by:
Alessia Fiore, Giulia Falcone
ENEA, Casaccia Research Center, Rome
Steps
Choice of 12 sequenced BACs (1/chromosome)
with good end sequences on SGN
PCR primer design on BAC ends
Amplification of S. lycopersicum and S. pennellii DNA
Amplification and sequencing of 72 ILs (single pass, SP6 end)
Polymorphism detection and mapping of SP6 end
Confirmation of T7 end on selected ILs
Primer design
Parameters:
•End sequences > 500bp available on
SGN
•Amplification product @400bp
•Primer length: 18-20bp
>LE_HBa0051C14_SP6_end
CTCATCAACAGCATTGTCAAGGAGCTCTGCCAAGGCTTTAGACATCACAGAACAACA
ATGGCTATGTTAACACTCCAGTAATAGCATTTGCAATAATCCTATTACCACACAATAA
TATGGAAACGAAAAATGAATCAGCAATAGCACATACCACCAAATGCCCACTTATGG
GAAGTGGCGTTTGAGTGAAGGAACTTCGGGTGAATGTGCAAGAAACTGGAGCCTTCT
GTATAATGCCACAAATAAGATGACAACATACATATTAGATGCAAAACACATACTTGA
ATTACAGATAAAGAATTAAGCTATAAGGCGATATAGAAAGGTAGGCAAAGAAAAGA
TTGACCAACTACTCAAAACATCAGCACGCATGGTTTATGCATGGTAATGTGTTTGCTA
AGAACAATTTTGAAAAAAGGAAAAAATGGGTACTTCAGTGCATAATACTTTTAAGGG
TGGACTTGGTAGCATGCCCCTCATCAAATTTCCAGCTTTCCAGAACTGGCGAGATAG
GGGTGCTGGACATATTTGTGATGTAGAACATAGGCTTGAATCATCTATCGGAGACTG
ATCTTGATCCAAGACGGAAGTTCTACTCTGTTCAGTAGCTGTAGAATCTTGCCTCCCA
CAGCTGCTTGAGGATTTTTTATATGGCAAGGAATCAGCTTCACAGCACTCCATTTGTG
GAATTAAACTTTCAACCAATATAGGTTCCAACTTAACAGTCTCTATCTCCCCACCTCC
AAGATTGTTACTACACAAGCCAACCATATTTGT
Selected BACS
Chromosome
1
2
3
4
5
6
7
8
9
10
11
12
Bacs
LE_HBa0051C14
LE_HBa0011A02
LE_HBa0034B23
LE_HBa0006E18
LE_HBa0042B19
LE_HBa0002C17
LE_HBa0179K09
LE_HBa0005L01
LE_HBa0033H16
LE_HBa0041K23
LE_HBa0119D16
LE_HBa0021L02
Probe
T11488
TG373
T0482
T1322
cLET-8_B23
none
T0876
CT68
none
TG560
C2-At5g12200
T1211
cM
109
94
137
58
10
none
12
87
none
30
53,5
53
Example of A->G mutation
on SP6 end
LE_HBa0051C14_SP6_lyc
LE_HBa0051C14_SP6_penn
Amplification of IL DNA
SP6 end of LE_HBa0051C14:
M: 1Kb ladder, V: empty
Mapping of SP6 end
LE_HBa0006E18, SP6 end: maps to Ils 4.2, 4.3, 4.3.2
Confirmation of T7 end
Results
Chromosome
Bac
End
Positive IL
1
1
2
2
3
3
4
4
5
5
6
6
7
7
8
8
9
9
10
10
11
11
12
12
Total
LE_HBa0051C14
LE_HBa0051C14
LE_HBa0011A02
LE_HBa0011A02
LE_HBa0034B23
LE_HBa0034B23
LE_HBa0006E18
LE_HBa0006E18
LE_HBa0042B19
LE_HBa0042B19
LE_HBa0002C17
LE_HBa0002C17
LE_HBa0179K09
LE_HBa0179K09
LE_HBa0005L01
LE_HBa0005L01
LE_HBa0033H16
LE_HBa0033H16
LE_HBa0041K23
LE_HBa0041K23
LE_HBa0119D16
LE_HBa0119D16
LE_HBa0021L02
LE_HBa0021L02
Sp6
T7
Sp6
T7
Sp6
T7
Sp6
T7
Sp6
T7
Sp6
T7
Sp6
T7
Sp6
T7
Sp6
T7
Sp6
T7
Sp6
T7
Sp6
T7
No PCR product
No PCR product
2.5
2.5
3.4
3.4
4.2, 4.3, 4.3.2
4.2, 4.3, 4.3.2
5.1
5.1
6.2
6.2
7.1, 7.2
7.1, 7.2
8.3, 8.3.1
8.3, 8.3.1
9.3.1
9.3,9.3.1
10.1
No PCR product
11.2
No PCR product
12.2, 12,3.1,12.3
12.2, 12,3.1,12.3
SNPs Indels Total
Fragment
SFP
(bp)
frequency
3
11
9
2
17
17
29
9
16
6
1
2
11
11
6
5
13
0
4
3
1
2
1
4
3
1
1
0
0
1
4
1
0
4
3
15
12
3
19
18
33
12
17
7
1
2
12
15
7
5
17
357
497
516
483
509
514
430
224
402
326
425
394
439
572
440
481
379
0,84%
3,02%
2,33%
0,62%
3,73%
3,50%
7,67%
5,36%
4,23%
2,15%
0,24%
0,51%
2,73%
2,62%
1,59%
1,04%
4,49%
5
1
6
396
1,52%
5
6
184
1
0
32
6
6
216
425
461
8670
1,41%
1,30%
2,49%
Cost considerations (for 96 BACs)
Primer design, both ends
PCR amplification, gel verification
Sequencing SP6 end, seq alignment
Validation T7 end
Total
Cost €
Failure (%)
2.000
20%
3.000
12.000
5%
1.000
5%
18.000
Notes
Needs optimization