The Roles of Fluorescence in situ
Hybridization (FISH)
To Pachytene Chromosomes
in Sequencing the Genome of
Tomato
Stephen M. Stack1, Lindsay A. Shearer1, Suzanne M. Royer1,
Song-Bin Chang2, and Lorinda K. Anderson1
1Department of Biology, Colorado State University
2National Cheng Kung University No. 1, Tainan City, Taiwan
Tomato
(Solanum lycopersicum)
1C = 0.95 pg = ~ 917 Mb
2n = 2x = 24 chromosomes
B
New Sequencing Technologies
454
Illumina
SOLiD
Pseudomolecule
Scaffolds 1-4
1
Head
(End of short arm)
2
1
3
2
Gaps 1-3
4
3
Tail
(end of long arm)
One mapped molecular marker
or
one BAC on a Scaffold located by FISH Permits
assignment of the scaffold to a
linkage group/chromosome
and indicates order of scaffolds.
1
Head
(End of short arm)
2
1
3
2
Gaps 1-3
Pseudomolecule
4
3
Tail
(end of long arm)
One molecular marker
or
one BAC located by FISH
on a scaffold does not permit orientation of the scaffold
in regard to the head and tail of the pseudomolecule.
Orientation ?
1
Head
(End of short arm)
2
1
3
2
Gaps 1-3
4
3
Tail
(end of long arm)
Two or More Molecular Markers
or Two or More BACs Located by FISH Determine
Scaffold Orientation
a
1
Head
(End of short arm)
b
cd
1
2
e
f g
2
Gaps 1-3
3
h
3
4
i
Tail
(end of long arm)
One Molecular Marker per Scaffold?
Scaffold 1-4 order
1
Head
(End of short arm)
2
1
3
2
Gaps 1-3
4
3
Tail
(end of long arm)
BAC FISH probes can locate scaffolds
accurately on chromosome structure
regardless of crossover patterns.
Two terminal BAC FISH Markers per scaffold
permit estimates of gap sizes.
Scaffold 1-4 order
1
Head
(End of short arm)
2
1
3
2
Gaps 1-3
Pseudomolecule
4
3
Tail
(end of long arm)
1.5 Mb/µm of SC in euchromatin
8.5 Mb/µm of SC in heterochromatin
3.5 Mb/µm of SC in centromeres
Scaffold 8
Tail – scaffold 6
Tail – scaffold 8
Head – scaffold 8
Head – scaffold 6
Scaffold 6
head
______________
tail
Tail – scaffold 3 Head – scaffold 2
BAC s083L21
BAC E016I22
Gap 3-2
(centromere)
head
centromere
____________
3.5 Mb/µm X 0.7 µm = 2.4 Mb
tail
Chromosome 1 =100.2 Mb
Gaps and
Zero BACs
Tomato FISH Idiogram
-5
m SC Length
0
5
10
15
20
1
2
3
4
5
6
7
8
Chromosome Number
9
10
11
12
A
B
3.06 μm
2.35 μm
3.43 μm
6.26 μm
85%
II
4.60 μm
Dan Peterson et al. (1996)
1.54 Mb per micrometer in euchromatin
9.22 Mb per micrometer in heterochromatin
3.06 μm
85%
II
4.60 μm
2.35 μm
3.43 μm
6.26 μm
Tomato EXPEN 2000
Chromosome 1
Linkage Map
Tomato EXPEN 2000
Chromosome 1
Linkage Map
BAC E021C24
BAC s040G18
Scaffold 6
Scaffold 8
BAC s121I01
BAC s022L14
head
tail
head
centromere
tail
Chromosome 1 =100.2 Mb