Transcript Tomato Sequencing, Madison July 2006

Chr9
Antonio Granell
IBMCP-Valencia
Spain
Tomato Sequencing, Madison July 2006
Chromosome 9 has a total
of 142 markers
But...
-Most markers are in heterochromatin
-Most of them did not match any BAC
-Gap of 46cM
264 BACs from the tomato HindIII library were obtained from Cornell on July
2005. We started with a number of “seed BACs” following the recommendations of
estimated size and resent in a contig from FPC maps.
Selection of best seed BACs
(>100Kb, in a contig, with known
interest for scientific community)
6 BAC clones as
candidates for “seed
BACs”
Some did not have the
marker, others contained
the marker but were not in
a contig or did not have an
estimation of their length
10 “seed BACs” although some had
a length of <100Kb
Screening of
markers
Just 2 BACs
contained
marker
New selection of candidates as “seed
BAC”. Screening of 16 markers. 2-3
different BAC clones screened for each
marker
Sequencing
SO FAR
BACs at different
progress levels
New screening of
markers
4 seed BACs more.
Total of 14
These BACs will be used
as seed BAC, if marker is
found, despite risk
Selection of
BACs to extend
5 confirmed, 13 possible.
Several problems.
Location in Chr9 for seed
BACs done using Dani’s lines
and FISH done by Song-Bin
Chang and Steve Stack
Le_HBa0026I24
Le_HBa0148A06/SL_EcoRI0001L13
Seed BACs
Extending BACs
* Completed BACs
* BACs in progress
* Candidates Extending BACs
SL_EcoRI0130H12
Le_HBa0116C14
SL_EcoRI0019J03
Currrent status of
chromosome 9
14
Short arm
23
SL_EcoRI0004D19
Le_HBa0168F14
SL_EcoRI0103M07/ SL_EcoRI0116G11
Le_HBa0026P14
SL_EcoRI0004H20
Le_HBa0203J14
SL_EcoRI0032F16
37.5
46
48
H
SL_EcoRI0022M12
Le_HBa0300E15
SL_MobI0144H04
Seed BACs
Extending BACs
* Completed BACs
* BACs in progress
* Candidates extending BACs
H
Le_HBa0226J12
Le_HBa0099F14
Le_HBa0278J12
Le_HBa0099F14
Le_HBa0107D15
57
60
61
Le_HBa0099P03
SL_EcoRI0079M11 ! SL_EcoRI0079M11
Currrent status of
chromosome 9
Long arm
Le_HBa0008K01
Le_HBa0226D21
SL_EcoRI0049P19
107
Le_HBa0015O02
Le_HBa0165P17
Le_HBa0234F21
109
112
116
SL_MboI0017K18
Le_HBa0059I05
Le_HBa0109D11
Le_HBa0033H16
Le_HBa0183J02
Problems when extending finished BACs
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No matches found when searching in tomato BAC end database.
Too large overlaps (>20 kb, ex. SL_EcoRI0004D19 or
Le_HBa0038L16).
Some requested extending clones were contaminated with other
clones
(ex.
SL_EcoRI0004H20
contaminated
with
SL_EcoRI0004H19) or just were not in the sample sent (ex.
Le_HBa0015O02, requested twice, both times was a mix of
Le_Hba0014O02 and other clones but Le_HBa0015O02).
Estimated size
BAC inserts far from actual
size (ex.
Le_HBa0033H16)
For some clones, no restriction digest data available to check size of
the BAC although an estimation of
its length is given (ex.
Le_Hba0033H16)
Alternatives:
•Selection of new seed BACs in order to keep working waiting for
FPC contig map to be completed with new libraries.
•Go for clones from new libraries that neither have an estimation of
their length nor are present in FPC contigs. TOO RISKY!!
• MboI library and FPC
Construction of a training set for gene
prediction programa geneid
• A parameter file
constructed from
112 FL sequences
from different
Solanaceae
species (50%
tomato).
• To be refined with
the training set from
newly released 320
FL from Shibata’s
lab
Tomato Sequencing, Madison July 2006
Geneid traioned with this parameter file
applied to 6 BACs in Chr9
Tomato Sequencing, Madison July 2006
Geneid vs Geneseqer
• Geneid predicted 22
genes in the 114,526 pb
C09HBa0109D11.1 BAC
• Most of the predictions
are supported by ESTs
results as shown by
geneseqer
• Geneseqer is another
gene identification tool
based on the “spliced
alignment” of ESTs to the
genomic sequence
contained in the BAC
Tomato Sequencing, Madison July 2006
Geneid with a parameter file obtained from
100 Sol sequences has been applied to 6
BACs from Chr9
Tomato Sequencing, Madison July 2006
European Commission EU-SOL
Vicky Fernandez
Sheila Zuniga
Angela Perez
Francisco Camara
Roderic Guigó
Miguel A Botella
Antonio Granell
Tomato Sequencing, SOL Madison, July 2006