the role of fluorescence in situ hybridization (fish) in sequencing the tomato genome

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