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The Role of Fluorescence in situ hybridization (FISH)
in Sequencing the Tomato Genome
Short arm
Centromere
Long arm India
17 16 10 13 1124 26 26 19 11 20 27
Japan
Spain
Italy
euchromatinheterochromatinAT-rich satellite DNA
Mb1 10 11 129
USA C
hin
a
UK
Chin
a
USA Kore
a France
62 3 4 7
The N
eth
erl
ands
5 8
Maize 1C = 2634 Mb
Tomato 1C = 916 Mb
Arabidopsis 1C = 157 Mb
77% of the DNA is in heterochromatin 23% of DNA is in euchromatin
77% of the DNA is in heterochromatin 23% of the DNA is in euchromatin
Thus,
0.23 x 916 Mb = 211 Mb
77% of the DNA is in heterochromatin 23% of the DNA is in euchromatin
Thus,
0.23 x 916 Mb = 211 Mb
Arabidopsis 1C = 157 Mb
HindIII library
15 tomato genome equivalents129,000 clones
Averaging 117 kb
EXPEN 2000 MolecularLinkage Map of Tomato Chromosome 10(SGN)
Anchor (seed) BAC LE_HBa0234C10 Probe TG285
Anchor (seed) BAC LE_HBa0234C10 Probe TG285
aatgcctaggcatgaatcttggccatc
Anchor (seed) BAC LE_HBa0234C10 Probe TG285
aatgcctaggcatgaatcttggccatcgctacgcttagaa
Seed BAC LE_HBa0234C10 Probe TG285
Seed BAC LE_HBa0234C10 Probe TG285
Seed BAC LE_HBa0234C10 Probe TG285
Seed BAC
Contig
LE_HBa0234C10 Probe TG285
Fluorescence in situ Hybridization(FISH)
ChinaFrance
The NetherlandsKoreaJapanUSA
Fluorescence in situ Hybridization(FISH)
ChinaFrance
The NetherlandsKoreaJapanUSA
Nick translation
biotin- digoxygenin-
dinitrophenol-labeled nucleotides
FISH Probes
BAC DNA
Hybridization mixture50-100-fold excess of
unlabeled tomato Cot 100 DNA
Chromosomal in situ Suppression(CISS) Hybridization
Functions of FISH in Sequencing the Tomato Genome
1. To determine the locations of anchor BACs2. To define eu-heterochromatin borders3. To determine distances in Mb4. To locate problem BACs
Green LabelSpread % Distance
126-06 86.23126-28 81.92126-29 82.28126-30 84.27126-32 85.77126-33 85.2126-34 83.9126-37 87.63126-38 87.2
Mean 84.93Std. Dev. 2.02
BAC 116C14, Slide 126, Chromosome 9, Short (p) Arm
%
%
Pachytene Chromosome 9
Pachytene chromosome 9
Functions of FISH in Sequencing the Tomato Genome
1. To determine the locations of anchor BACs2. To define eu-heterochromatin borders3. To determine distances in Mb4. To locate problem BACs
Functions of FISH in Sequencing the Tomato Genome
1. To determine the locations of anchor BACs2. To define eu-heterochromatin borders3. To determine distances in Mb4. To locate problem BACs
Functions of FISH in Sequencing the Tomato Genome
1. To determine the locations of anchor BACs2. To define eu-heterochromatin borders3. To determine distances in Mb4. To locate problem BACs
177 BACs FISHED
RESULTS SO FAR
177 BACs FISHED
126 BACS (74%) successfully localized.
RESULTS SO FAR
177 BACs FISHED
51 failed to localize because they either gave no FISH signal or there was more than one signal in spite of CISS hybridization.
126 BACS (74%) successfully localized.
RESULTS SO FAR
Of 126 BACs localized22 (17.5%) FISHed to wrong chromosomes
Of 126 BACs localized22 (17.5%) FISHed to wrong chromosomes
Of these, 11 have been checked by sequencing:7 were overgo false positives1 was due to a picking error 1 was due to a typographical error 2 were due to mapping errors
Of 126 BACs localized22 (17.5%) FISHed to wrong chromosomes
Of these 11 have been checked by sequencing:7 were overgo false positives1 were due to a picking error 1 were due to a typographical error 2 were due to mapping errors
Suggesting ≤ 3% mapping errors on the EXPEN 2000 linkage map
FUTURE ACTIVITIES
an increasing emphasis on defining the size of gaps in sequencing
1) within BACs,2) between contigs,3) between contigs and euchromatin-
heterochromatin borders
Senior Personnel UndergraduatesLorrie Anderson Madeline FujishiroSong-Bin Chang Lauren LarsenSuzanne Royer Dylan Westfall Lindsay Shearer Jessica WuSteve Stack
The Role of BAC Fluorescence in situ hybridization (FISH)
in Sequencing the Tomato Genome