1. Using fluorescence in situ hybridization (FISH) to improve assembly of the tomato genome sequence
Lindsay A. Shearer1, Lorinda K. Anderson1, Hans de Jong2, Jose Luis Goicoechea3, Bruce A. Roe4, Axin Hua4, James J. Giovannoni5, Stephen M. Stack1
Fluorescence In Situ Hybridization and Optical Mapping to Correct Scaffold Arrangement in the Tomato Genome G3 (2014) volume 4:

3. When the tomato sequencing project was being planned 13 years ago, the small Arabidopsis thaliana genome (1C = 157 Mb) had just been sequenced using overlapping DNA inserts in bacterial artificial chromosomes (BACs)

4. 6X as much work Tomato 1C = ~916 Mb Arabidopsis 1C = 157 Mb X 6
(2n = 2x = 10)
X 6
Tomato 1C = ~916 Mb
Solanum lycopersicum
6X as much work

5. 75% heterochromatin, 25% euchromatin
Repeat-rich, gene poor
75% heterochromatin, 25% euchromatin

6. 75% heterochromatin, 25% euchromatin
Repeat-poor, gene rich
75% heterochromatin, 25% euchromatin

7. 90% of genes
75% heterochromatin, 25% euchromatin

8. Arabidopsis 1C = 157 Mb
Tomato 1C = 916 Mb
X 0.25
229 Mb

9. To guide sequencing to euchromatin by
Fluorescence in situ
Hybridization (FISH)
of DNA in
Bacterial Artificial Chromosomes (BACs) from tomato BAC libraries to
tomato pachytene
chromosomes
that were spread by hypotonic bursting.

10. Pachytene chromosome spread
Phase contrast
DAPI
10 µm
by hypotonic bursting

11. 10 µm

14. SC 12

15. Improved sequencing
technologies
454
&
Illumina

16. DNA pseudomolecule representing
one chromosome
Sequenced scaffolds 1-4 1 2 3 4
Head
(end of short arm)
Tail
(end of long arm)
Gaps 1-2, 2-3, 3-4

17. ≈ chromosomes Tomato DNA pseudomolecules 1 2 3 4 5 6 7 8 9 10 11 12
49.9 Mb
46.0 Mb
53.4 Mb
65.5 Mb
63.0 Mb
64.8 Mb
64.1 Mb
65.0 Mb
65.3 Mb
64.8 Mb
67.7 Mb
Tomato DNA pseudomolecules
≈ chromosomes
90.3Mb

18. ? = Head = End of short arm Ideally order and orientation of1
Head = End
of short arm ? =
Ideally order and
orientation of
scaffolds in the DNA
pseudomolecule
for chromosome 1
Corresponding
straightened
pachytene
chromosome 1
Tail = End
of long arm

19. Kazusa Linkage MAP for tomato Chromosome 1 Pseudomolecule
Mapped DNA
sequences were
identified in
scaffolds.
Chromosome 1
Kazusa Linkage MAP for tomato
Chromosome 1
Pseudomolecule

20. Fluorescence in situ
Hybridization (FISH) of
scaffold sequence DNA in
Bacterial Artificial
Chromosomes (BACs) to
tomato pachytene
chromosomes.

21. Pseudomolecule or location of one BAC in a scaffold by FISH
Identification of one mapped molecular marker in a scaffold or
location of one BAC in a scaffold by FISH
permits assignment of the scaffold to a
linkage group/chromosome/pseudomolecule
and indicates the order of scaffolds, but their not orientation. 1 2 3 4
Head
(End of short arm)
Tail
(end of long arm)
Pseudomolecule

22. 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 2 3 4
Head
(End of short arm)
Tail
(end of long arm)

23. Two or More mapped molecular markers on a scaffold
or two or more BACs located by FISH on a scaffold
determine scaffold head/tail orientation
Head
(End of short arm)
Tail
(end of long arm)
Gaps 1-3
a b c d e f g h i

24. The two methods usually agree in euchromatin,
Idiogram of straightened tomato pachytene chromosomes
Eu-
Heterochromatin
Euchromatin
The two methods usually agree in euchromatin,
but often disagree in pericentric heterochromatin
Where 75% of the DNA is located.

25. Tomato DNA Pseudomolecules Nature (2012) 485:635-641
49.9 Mb
46.0 Mb
53.4 Mb
65.5 Mb
63.0 Mb
64.8 Mb
64.1 Mb
65.0 Mb
65.3 Mb
64.8 Mb
67.7 Mb
Tomato DNA Pseudomolecules
Nature (2012) 485:
90.3Mb

26. FISH results should be given
preference for ordering and
orienting scaffolds in tomato
pseudomolecules for the
following reasons:

27. 1) The linkage map method only
works if there is mapped
sequence in a scaffold. This
may or may not be the case for
small scaffolds and scaffolds in
heterochromatin, but known
BACs are always available in
scaffolds for use in FISH.

28. 2) FISH is a more direct method.
The linkage map method only works if there is
mapped sequence in a scaffold.
2) FISH is a more direct method.

29. 12

30. Chromosome 1
Pachytene Linkage map Pseudomolecule ? =

31. 3) FISH is accurate in euchromatin and in heterochromatin.
The linkage map method only works
if there is mapped sequence in a scaffold.
2) FISH is a more direct method.
3) FISH is accurate in euchromatin
and in heterochromatin.

32. 75% of genomic DNA is in pericentric heterochromatin
where crossing over is suppressed.

33. Chromosome 1
Kazusa Linkage MAP
Chromosome 1
Pseudomolecule

34. BAC/FISH can locate scaffolds accurately in euchromatin and heterochromatin without reference to crossing over. 12

35. 4) Optical sequencing agrees with FISH.
The linkage map method only works if there
is mapped sequence in a scaffold.
2) FISH is a more direct method.
3) FISH is accurate in euchromatin and in
heterochromatin.
4) Optical sequencing agrees
with FISH.
Optical sequencing is an independent
physical method for ordering and
orienting scaffolds in both euchromatin
and in heterochromatin.

36. While optical mapping only
covered 38 scaffolds (= 32% of the sequenced tomato genome), optical mapping results were invariably compatible with FISH results, while they were incompatible with the linkage map for 16 out of 38 scaffolds covered.

37. 5) FISH permits estimates of gaps sizes between scaffolds,
The linkage map method only works if there
is mapped sequence in a scaffold.
2) FISH is a more direct method.
3) FISH is accurate in euchromatin and in
heterochromatin.
4) Optical sequencing agrees with FISH.
5) FISH permits estimates of
gaps sizes between scaffolds,
while the linkage map does
not.

38. Density of DNA (Mb/µm SC)
For this we first determined the amount of DNA/µm for tomato pachytene chromosomes (= SCs) in:
Density of DNA (Mb/µm SC)
Chromatin type
1.5
euchromatin
8.5
heterochromatin
3.5
centromere

39. Pseudomolecule Then we localized a BAC at each end of every scaffold
and measured gap lengths in micrometers.
Scaffold 1-4 order 1 2 3 4
Head
(End of short arm)
Tail
(end of long arm)
Gaps 1-3
Pseudomolecule

40. BAC s083L21
Tail – scaffold 3
BAC E016I22
Head – scaffold 2
Gap 3-2
(centromere)
Chromosome 1

41. Gap 3-2 = 0.7 µm of centromeric chromatin
X 3.5 Mb/µm centromeric chromatin
2.45 Mb in chromosome 1 gap 3-2

42. Gap sizes were measured between all 91 scaffolds on all 12 chromosomes
Tail – scaffold 6
Head – scaffold 6
Tail – scaffold 8
Head – scaffold 8
Scaffold 6

43. Chromosome 1 Pseudomolecule http://solgenomics.net head centromere
tail
centromere
Chromosome 1 =100.2 Mb
Chromosome 1
Pseudomolecule
____________

44. 6) FISH can localize chromosome 0 BACs
The linkage map method only works if
there is mapped sequence in a scaffold.
2) FISH is a more direct method.
3) FISH is accurate in euchromatin and in
heterochromatin.
4) Optical sequencing agrees with FISH.
5) FISH permits estimates of gaps sizes.
6) FISH can localize
chromosome 0 BACs

45. Chromosome 0 BACs are
sequenced BACs with no linkage
mapped sequences, and their
sequences do not fit into any
scaffold.

46. Chromosome 9

47. Chromosome 9

49. 7) Finally, FISH relates DNA pseudomolecules directly to
The linkage map method only works if there is mapped
sequence in a scaffold.
2) FISH is a more direct method.
3) FISH is accurate in euchromatin and in heterochromatin.
4) Optical sequencing agrees with FISH.
5) FISH permits estimates of gaps sizes.
6) FISH can localize chromosome 0 BACs
7) Finally, FISH relates DNA
pseudomolecules directly to
chromosome structure, unlike
the linkage map.

51. Using FISH, we have ordered and oriented all 91
scaffolds and determined gap sizes for the 12 tomato pseudomolecules

52. Discrepancies are shown in red between ordering and orienting scaffolds using the linkage map versus FISH. They vary from chromosome to chromosome, but they are mostly in pericentric heterochromatin where a lack of crossing over makes the linkage map inaccurate.

53. Based on FISH, scaffolds that were ordered and oriented using the linkage map can be in the wrong order and/or orientation (inverted), and one case, two scaffolds overlapped.

54. Chromosome/pseudomolecule 8 as an example
of linkage map-based versus FISH-based order and
orientation of scaffolds and gap sizes

55. In summary, FISH rearranged 45 of
the 91 scaffolds. This amounts to
rearranging 34% of the total tomato
genome.

56. See the Sol Genomics website
Because of its advantages, the order and orientation of scaffolds determined by FISH is now used in the newest build (SL2.50) of the tomato genome.
See the Sol Genomics website

57. A new estimate of genome
size for tomato
Scaffold total Mb
Gap total Mb
Nucleolus organizer Mb
825.5 Mb

58. FISH can be used to correct
linkage maps, especially in
heterochromatin.

59. And finally, these results suggest similar errors exist in other sequencing projects that ordered and oriented scaffolds using linkage maps, and these errors can be corrected using BAC/FISH and/or optical mapping.

61. Sol Genomics website: http://solgenomics.net
Chromosome/pseudomolecule 8 as an example
of linkage map-based versus FISH-based order and
orientation of scaffolds and gap sizes
Sol Genomics website:

62. Because of the advantages of FISH
FISH is a more direct method.
FISH is accurate in both eu- and
heterochromatin.
3) Optical sequencing agrees with FISH.
4) FISH permits estimates of gap sizes.
5) FISH can localize chromosome 0 BACs.
Sol Genomics website

63. Scaffold 8
Tail – scaffold 6
Tail – scaffold 8
Head – scaffold 8
Head – scaffold 6
Scaffold 6

64. head
tail
Chromosome 1
Pseudomolecule

65. Tail – scaffold 3
BAC s083L21
Head – scaffold 2
BAC E016I22
Gap 3-2
(centromere)

66. 3.5 Mb/µm X 0.7 µm = 2.4 Mb head centromere ____________ tail
Chromosome 1 =100.2 Mb
____________
3.5 Mb/µm X 0.7 µm = 2.4 Mb

67. Gaps and
Zero BACs

70. 10 µm

71. A B

73. 1.54 Mb per micrometer in euchromatin
3.06 μm μm μm 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

75. 3.06 μm μm μm μm
85% II
4.60 μm

76. Tomato EXPEN 2000
Chromosome 1
Linkage Map

77. Tomato EXPEN 2000
Chromosome 1
Linkage Map

79. BAC E021C24
BAC s040G18
Scaffold 6

80. Scaffold 8
BAC s121I01
BAC s022L14

82. head
tail

83. head
centromere
tail
Chromosome 1 =100.2 Mb

85. (Solanum lycopersicum)
Tomato
(Solanum lycopersicum)
1C = 0.95 pg = ~ 917 Mb
2n = 2x = 24 chromosomes

87. One Molecular Marker per Scaffold?
Scaffold 1-4 order 1 2 3 4
Head
(End of short arm)
Tail
(end of long arm)
Gaps 1-3

88. BAC FISH probes can locate scaffolds accurately on chromosome structure regardless of crossover patterns.

89. 1.5 Mb/µm of SC in euchromatin
8.5 Mb/µm of SC in heterochromatin
3.5 Mb/µm of SC in centromeres

90. head
tail
Chromosome 1
Pseudomolecule