NATURE 243: 515-516 (June 29, 1973)

DNA Differences between Flax Genotrophs

CERTAIN environmental conditions can induce genetic changes in particular varieties of plants1-5; these changes are stable and inherited over many generations. One of the best examples of this involves the flax variety Stormont Cirrus, which when supplied with certain fertilizers and grown for at least 5 weeks in a heated greenhouse can change from its original form (P1) into a large stable genotroph (L), or a small stable genotroph (S), depending on the fertilizers supplied1,6. L plants may be up to six times the weight of S plants, depending on the environment in which they are grown, with the P1 type having a weight in between those of L and S.

L and S behave as two distinct genetic types, giving equilinear inheritance when they are reciprocally crossed, and no transmission through reciprocal grafts1. L has been shown to have 16% more nuclear DNA than S as shown by Feulgen staining6. The P1 type has a DNA content intermediate between that of L and S, having 10% less DNA than L but 6% more DNA than S.

I have now characterized the nature of the DNA differences between the genotrophs, with the ultimate aim of attempting to elucidate the nature and mechanism of induction of the changes in DNA and in the phenotype. The method of extraction of DNA from the L and S genotrophs will be described elsewhere (my unpublished work). The extracted DNA was analysed by ultracentrifugation and renaturation. The DNA from both types formed two bands in neutral caesium chloride gradients (Fig. 1), The principal band had a density of 1.697 g cm‑3 and a secondary band a density of 1.688 g cm‑3. The amount of DNA contained in the satellite band was approximately 15% and was about the same in samples from both types. The densities of both the satellite and the main band increased by 0.01 g cm‑3 after heat denaturation.

The renaturation of the DNA was followed optically7 and by separation on hydroxyapatite.8 From the results (Fig. 2) it was apparent that the DNA from both L and S genotrophs contained some sequences which were highly repeated, others which were less repeated and some unique8. The highly repeated sequences make up 24% of the total DNA from L and 28.5% of the total in S (my unpublished work). The satellite DNA was included in this fraction. The difference between the proportion found in L and S could be explained as follows: if the extra DNA in L contained none of the highly repeated sequences, then the consequent dilution of such sequences in L would result in the differences observed.

The unique sequences form 45% of the total DNA in S and 40% of the total DNA in L; again this difference is accountable in terms of the dilution factor mentioned earlier. The complexity of the unique sequences, calculated against a value for E. coli of molecular weight 2.8 X 109 (ref. 9), is 2.1 X 1011; this agrees well with the value calculated from the total DNA content per nucleus10 which is 1.97 X 1011, assuming these unique sequences make up 45% of the total DNA in S and 40% of the total DNA in L.

Fig. 1 Caesium chloride equilibrium gradient run for 24 h at 44,770 r.p.m. at 25° C on a Spinco Model E analytical centrifuge. a, 2 µg of DNA from L genotroph with a principal band of density 1,697 g cm-3 and a secondary band of density 1.688 g cm-3. b, 1.5 µg of DNA from the S genotroph with a principal band of density 1.697 g cm-3 and a secondary band of density 1.688 g cm-3. Both samples had 1 µg of DNA from Streptomyces coelicolor, density 1,731 g cm-3 added as a marker. Fig. 2 Cot curves of DNA from the L genotroph (• — •) and the S genotroph (• ‑ ‑ ‑ •). The DNA was sheared by sonication to a molecular weight of 7 x 105 and incubated at 50 µg ml‑1 or 500 µg ml‑1 in 0.12 M phosphate buffer (equimolar sodium dihydrogen phosphate and disodium hydrogen phosphate) at 60° C. Single and double stranded material was separated on a hydroxyapatite column also maintained at 60° C. The degree of renaturation was plotted against the Cot value (ref. 5).

The intermediate sequences, that is neither highly repeated nor unique, make up the remainder of the DNA which is 26.5% in S and 36 in L. This intermediate fraction in L seems to be composed of two families of sequences: one renaturing at about the same rate as the intermediate sequences in S; the other renaturing more slowly. The extra DNA in L could be in this latter fraction which was not observed in S.

An increase in the concentration of any type of sequence would cause an increase in the rate of renaturation of this sequence. The extra DNA in L was found in the slowest renaturing portion of the intermediate sequences and only the unique sequences renature more slowly than this. Thus the extra sequences must have been derived by increasing some of the unique material, In that case the difference in the rates of renaturation between the additional intermediate and unique material suggests that the extra DNA is present at seventy times the multiplicity of the unique sequences. This means that the extra DNA in L would be equivalent to 0.4% of the unique sequences at a multiplicity of seventy times.

How this increase in DNA is brought about by the fertilizer treatments is still unknown. But specific portions of the genome have been shown to be differentially increased or decreased, and the difference inherited, in other systems; for example in Drosophila the number of ribosomal genes at a particular locus can be increased or decreased depending on the chromosomal constitution11. Although this effect is not caused by an external treatment, a mechanism must exist to preferentially increase and stabilize a portion of the genome and in flax something similar may occur with parts of the genome.

I thank Dr A. Durrant for P1, L and S genotrophs.

C. A. CULLIS

John Innes Institute,
Colney Lane,
Norwich NOR 70F,
Norfolk

Received February 5, 1973.

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