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 4:1395-1405

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)

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

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

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

90% of genes 75% heterochromatin, 25% euchromatin

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

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.

Pachytene chromosome spread Phase contrast DAPI 10 µm by hypotonic bursting

10 µm

SC 12

Improved sequencing technologies 454 & Illumina

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

≈ chromosomes Tomato DNA pseudomolecules 1 2 3 4 5 6 7 8 9 10 11 12 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

? = Head = End of short arm Ideally order and orientation of 1 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

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

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

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) 1 2 3 Pseudomolecule

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) 1 2 3

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) 1 2 3 4 1 2 3 Gaps 1-3 a b c d e f g h i

The two methods usually agree in euchromatin, Idiogram of straightened tomato pachytene chromosomes 1 2 3 4 5 6 7 8 9 10 11 12 Eu- Heterochromatin Euchromatin The two methods usually agree in euchromatin, but often disagree in pericentric heterochromatin Where 75% of the DNA is located.

Tomato DNA Pseudomolecules Nature (2012) 485:635-641 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 Nature (2012) 485:635-641 90.3Mb

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

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.

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.

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Chromosome 1 Pachytene Linkage map Pseudomolecule ? =

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.

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

Chromosome 1 Kazusa Linkage MAP Chromosome 1 Pseudomolecule

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

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.

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.

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.

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

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) 1 2 3 Gaps 1-3 Pseudomolecule

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

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

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

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

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

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

Chromosome 9

Chromosome 9

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.

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

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.

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.

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

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

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 https://solgenomics.net/organism/Solanum_lycopersicum/genome

A new estimate of genome size for tomato Scaffold total 759.9 Mb Gap total 40.1 Mb Nucleolus organizer + 25.5 Mb 825.5 Mb

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

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.

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: http://solgenomics.net

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 http://solgenomics.net

Scaffold 8 Tail – scaffold 6 Tail – scaffold 8 Head – scaffold 8 Head – scaffold 6 Scaffold 6

head tail Chromosome 1 Pseudomolecule

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

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

Gaps and Zero BACs

10 µm

A B

1.54 Mb per micrometer in euchromatin 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 2.35 μm 3.43 μm 6.26 μm 85% II 4.60 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

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

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

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

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

head tail Chromosome 1 Pseudomolecule

1 2 3 4 5 6 7 8 9 10 11 12