Despite the economic and scientific importance of tomato (Lycopersicon esculentum), to our knowledge a
Cot curve has not been prepared for this plant, possibly due to difficulties in isolating large quantities of
polyphenol-free nuclear DNA from some plants (e.g., see Katterman and Shattuck 1983; Couch and Fritz 1990).
We have developed a method for isolating milligram quantities of tomato nuclear DNA free of polyphenolic
contamination (see accompanying TGCR paper, this issue). Using DNA isolated by this method, we performed a
Cot analysis of the tomato genome. Briefly, tomato (Cal-Ace) nuclear DNA was sheared to a mean fragment
length of 375 bp using a Vertis homogenizer (see Davidson et al. 1973). The DNA was dialyzed into TE buffer (10
mM Tris, 1 mM EDTA, pH 7.0), run through a Chelex column to remove metal ions (Britten et al. 1974), ethanol
precipitated, and dissolved in 0.03, 0.12, or 0.48 M sodium phosphate buffer. DNA samples in phosphate buffer
were sealed in siliconized glass ampoules or siliconized glass microcapillary tubes and stored at -70°C.
Hydroxyapatite (HAP) was prepared according to Bernardi (1971), A 2 mm high layer of glass beads was placed
in a BioRad water jacketed column (cat. # 737-6131) fitted with a BioRad flow adapter (cat. # 738-0015). HAP was
added to a height of 1 cm (an approximate volume of 1 cm3). The column was maintained at 60°C using a Lauda
model B1 circulating water bath. A fresh HAP column was prepared for each Cot sample (see Britten et al. 1974).
The tube from which eluant exited the HAP column was directly connected to a flow-cell cuvette in a Hewlett
Packard (HP) 8453 spectrophotometer connected to a HP Vectra VL Series 3 computer. To determine a Cot point,
an ampoule/capillary tube was thawed and placed in boiling water for 5-10 minutes to denature the DNA. The
sample was then immediately placed in a water bath set at a temperature that was 20-25°C below the melting
temperature for tomato DNA in the same buffer as the sample. Each sample was incubated to a desired Cot value
as described by Britten et al. (1974). At the end of the incubation period, any sample in 0.48 M or 0.12 M buffer
was immediately diluted in a 50 fold excess of 0.03 M sodium phosphate buffer (60°C) and loaded onto a HAP
column. Sample in 0.03 M sodium phosphate buffer were applied directly to a HAP column without dilution.
Absorbance readings at 260 nm (A260) of the eluant were taken every three seconds and displayed in graphic
form on a computer screen. Single-stranded DNA was eluted by adding 0.12 M sodium phosphate buffer. Once
elution of the single-stranded DNA was complete, double-stranded DNA was eluted by adding 0.48 M phosphate
buffer. Elution of single-stranded DNA and double-stranded DNA was visualized on the computer screen as two
separate peaks in A260. The single-stranded DNA peak was collected in one polypropylene tube, and the double-
stranded DNA peak was collected in another tube. The volumes of both the single-stranded fraction and the
double-stranded fraction were determined. 0.9 ml was taken from each tube and mixed with 0.1 ml of aqueous 10
N potassium hydroxide to denature the DNA. A solution consisting of 9 parts 0,12 M sodium phosphate buffer and
1 part 10 N KOH was used to blank the spectrophotometer. The A260 value (adjusted for light scatter at 320 nm)
of the single-stranded DNA/KOH mixture was determined. The spectrophotometer was then blanked with a
solution composed of 9 parts 0.48 M sodium phosphate buffer and 1 part 10 N KOH, and the A260 value
(adjusted for light scatter at 320 nm) of the denatured "double-stranded" DNA/KOH mixture was determined. For a
particular Cot value, the percentage of single-stranded DNA (% SS DNA) was calculated as follows:
(Vss x Ass) x 100
(Vss x Ass) + (Vds x Ads) = % SS DNA
where Vss = total volume of single-strand fraction, Vds = total volume of double-strand fraction, Ass = A260 for the
KOH-denatured single-strand fraction, and Ads = A260 for the KOH-denatured double-strand fraction.
The logarithms of Cot values were plotted against corresponding percentages of single-stranded DNA. A
least squares analysis of the Cot data was performed, and a best-fit Cot curve was generated using the computer
program of Pearson et al. (1977) (Figure 1). Approximately 12% of the tomato genome was reassociated at the
lowest Cot point (i.e., Cot = 0.000001). This DNA is most likely composed of fold-back sequences (see Britten et
al. 1974). Additionally, 11% of the tomato genome did not reassociate even at the highest Cot value (i.e., Cot
20233). This DNA presumably is degraded and would never reassociate. The curve itself consists of two main
components. The first component consists of repetitive sequences and
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