Hereditas 13: 342-356 (1930)
Inheritance of variegation and of black flower colour in Viola tricolor L.
J. Clausen

II. INHERITANCE OF BLACK VELVETY FLOWER COLOUR

The darkest flower colour of any pansy is a dark velvety black. It is present in the so called V. tricolor maxima nigra (tricolor maxima hort. = V. Wittrockiana GAMS, a stabilized species hybrid, cf. CLAUSEN 1927 b, pp. 694-696). This large flowered type, which is used in gardens, is constant as regards flower colour.

In 1923 seeds were received from Cambridge Botanical Gardens of a Viola tricolor, which appeared to be such a black velutina, small flowered tricolor proper with n = 13 chromosomes (while V. Wittrockiana has about n = 24 and is irregular). The black flower colour was constant in the offspring from the first beginning. The darkest Viola type investigated before was the V. tricolor hortensis (CLAUSEN, 1926, Line 519, pp. 5-6) found to be recessive for three modifying and bleaching genes, which are present in the wild Viola tricolor. The pattern of tricolor hortensis is similar to the pattern of the flower shown as number four from the left, in the middle row of fig. 4.

The flower colour of Viola tricolor is determined by the cooperation of a number of bleaching and modifying genes, the darkest colours being the most recessive or hypostatic ones. Starting from tricolor hortensis (fig. 4, middle row, fourth flower from the left), the addition of one modifying gene removes the velvety colour from the two side petals (number three from the left), another gene removes the triangular velvety spot on the lower petal (number two, where only the two upper petals are velvety violet) and a third one removes velvety colour altogether, giving the wild type violet flowered Viola tricolor (the first flower from the left). This last gene is the most superior one in its effect; if it is present, the flower is non-velvety, irrespecting of the presence or absence of the two more inferior or hypostatic modifiers.

The addition of a lutea-gene in homozygous condition (LL) changes the violet flowered type to yellow (fig. 4, number one from the left in the lower row); the lower petal in this type is bright yellow and the four upper petals are bleached to yellowish white or (in older flowers) to a very light mauve tinge. A single dose (Ll) of this gene changes a violet flower to whitish or, in older flowers, to a pale violet. Finally, the yellow LL-type can be bleached to yellowish white, also on the lower petal, by the addition of a still more superior gene, (w), always present in Viola arvensis, rare in Viola tricolor proper, but often present in Viola tricolor subspec. alpestris.

In the absence of the modifiers for velvet, the L-gene cannot bleach the dark velvety colours; the flowers number two to four from the left in the lower row of fig. 4 have bright yellow lower petal, and still they show the velvety pattern, although it has been a little more sharply restricted than in the corresponding types of the row above. The types of the series in some way intergrade and the classification of them has not always been reliable, but the removal of the velvety colour from the petals follows a definite order. In some way it can be said that the three modifiers are cumulative in their effect, but they cannot well substitute one another. The absence of a reaction gene (R) changes all these colours towards different shades of reddish (rr).

Fig. 3. Diagram of the crossing V. tricolor, alba-lutea X nigra; for explanation of gene symbols, see text.

All these types of flower pattern cannot be realized if not any of a series of basic genes (A1, A2 or A3) for anthocyanine colours are present. In the absence of all these (a1a1a2a2a3a3) the colour is purely white, alba, (fig. 4, the flower in the right corner below); or it is alba-lutea (the upper flower to the right) if the lutea-gene is homozygously present. Thus the basic genes are basic for the anthocyanines only (red or violet), not for the flavones.

When these facts are borne in mind, it should be expected that the black flowered Viola tricolor nigra (fig. 4, upper flower to the left) should be extremely recessive, a Viola undressed for all the modifiers, genetically speaking exhibiting the innermost nucleus of the flower colours in Viola. From one point of view it is startling to expect a colour so dark to be very recessive to a light shade of violet; but the expectations were answered to, it proved to be extraordinarily recessive.

Fig. 4. Flowers of parents and F2 types ex V. tricolor, alba-lutea X nigra. Reading from the left, the types run as follows:

upper row: nigra, alba-lutea;

middle row: non lutea types, viz. non velutina, velutina 1, velutina 2, velutina 3 and velutina 4;

lower row: lutea types (LL), viz. lutea non velutina, velutina 1, velutina 2, velutina 3 (?), and alba (without lutea gene).

Viola tricolor alba-lutea (fig. 4, upper right flower) was selected to be crossed with nigra. This alba-lutea type was F4 ex tricolor lutea X tricolor alba (CLAUSEN 1926, p. 22, Cross IV) and a constant one. A diagram of the crossing is shown in fig. 3. The F1, V. 1217, consisted of only six plants and all these were anthocyanous and of a faintly violet flower colour with a whitish lower petal (owing to the bleaching effect of the lutea gene in single dose); the upper petals were also faintly velutina, showing that not enough modifiers were present for a total suppression of the velvet colour. The type of F1 is shown in the left flower of the upper row of fig. 5. Both of the parent types had n = 13 chromosomes, and the conjugation of the 13 pairs of chromosomes in meiosis of the hybrid was normal.

Three F1 plants were selfed and also used for backcrossing to nigra. A total of 1547 F2 plants and 317 plants of the backcrossings were grown. It was remarkable that among the 1547 F2 plants no real nigra plant appeared, the darkest type in F2 was still violet in the centre part of the flower (the right flower in the middle row of fig. 4).

On the other hand, this type was considerably darker than the tricolor hortensis (to the left for it, velutina 3) previously the darkest types analyzed.

In the backcrosses eight totally black flowered plants appeared among a total of 317 individuals. the segregated nigra type is shown in fig. 5, the bottom row to the right Apparently no difficulty was involved in distinguishing it from the type nearest to it. A comparison between fig. 5 and the parental type in fig. 4 shows how identical they look in colour. Nigra appeared in both directions of the backcrossings. Eight out of 317 is very near to one out of 32, which means that nigra in comparison with aiba-lutea is recessive for five modifying genes.

Also the alba type was rare, although by far not so rare as nigra. In F2 28 alba of 1547 individuals appeared, which is nearly one of 64 (table 4). Accordingly, nigra contains three pairs of polymeric basic genes for development of anthocyanine, a condition similar to V. tricolor hortensis, Line 519, and different from several wild growing varieties of V. tricolor, which have only two polymeric basic genes for anthocyanine.

Fig. 5. Flowers of types of the backcross tricolor, [alba-lutea X nigra] X nigra. From the left:. upper row: faintly velutina (similar to F1), velutina 1, velutina 2; lower row: velutina 3, velutina 4 and nigra. The flowers in the upper row are Ll, in the lower likely ll.

One fourth of the F2 individuals were homozygous for the lutea gene, having bright yellow lower petal (the four first flowers in the bottom row of fig. 4). Due to the bleaching effect of the LL-gene these cannot become nigra. As the expectation for nigra in F2 is as one among 1024 non-lutea and non-alba individuals, it is not so surprising that it was not found among 1084 of that category. In the backcrosses no plants can become homozygously lutea.

While one of the parent types did not occur at all in F2, one fourth of the alba individuals, should become alba-lutea. the other parent type, that is, it should occur once among each 256 plants. Only three were found, which is three below the expectation, but this type is not very vigorous in any case. In this way both of the parent types were extremely rare in F2 of this variety crossing.

The classification of the different shades of velutina was difficult in the present case, what is not to wonder about, keeping in mind how many modifying genes are in operation. Furthermore, the individuals, which have one lutea gene present (Ll-individuals), are of a lighter, more whitish shade on the lower petal than the true violet types (ll) without this gene. The types presented in figs. 4 and 5, of the F2 and the backcross respectively, must not be taken for more than approximate representations of types with one, two, three, four and five modifiers, of which the three superior ones have been analyzed in a number of earlier crossings.

The increased number of modifiers make a change in the terminology of these appropriate. In the earlier paper (CLAUSEN 1926) they were termined M1 to M3; M3 being the most superior or epistatic one. In view of the fact that the two new ones here analyzed are more hypostatic than any of the first three ones, it would appear more appropriate to begin with M1 for the most epistatic one, which by itself and in one step can bleach nigra to non-velutina violet, and continue to M5 that bleaches only the innermost part of the three lower petals (the right flower in the middle row of fig. 4); its effect cannot be seen, except when all other modifiers are absent. Beginning with nigra, the series of velutina types then will run as below:

velutina 5: m1m1m2m2m3m3m4m4m5m5

 nigra;

velutina 4: m1m1m2m2m3m3m4m4M5

 middle part of only three lower petals bleached;

velutina 3: m1m1m2m2m3m3M4

 a velutina edge left on three lower petals;

velutina 2: m1m1m2m2M3

 a velutina triangle left on the spur-bearing petal, the two side petals non-velutina;

velutina 1: m1m1M2

 only two upper petals velutina;

non-velutina: M1 

 all five petals non velvety.

TABLE 4. Segregation in the crossings alba-lutea X nigra.

segregating characters F2 F1 x nigra
observed calculated ratio observed calculated ratio
non alba
(A)
non lutea (ll + Ll) non velutina 673 |
|
|
}1084
|
|
|
      307,1 31
velutina 1 262 - - 91 |
}309
|
velutina 2 102 1141,1 193347 135
velutina 3  37 - - }83
velutina 4  10 - -  
velutina 5
(=nigra)
1,1 189 8 9,9 1
lutea (LL) non velutina 319 |
|
}435
|
|
380,7 64512      
velutina 1 70
velutina 2 30
velutina 3-4 16
alba
(a1a1a2a2a4a4)
non lutea (ll+Ll)
lutea (LL)
25
3
18,1
6,0
3072
1024
     
total 1547 1547,0 262144 317 317,0 32
non nigra 1547 1545,9 4093 309 307,1 31
nigra 1,1 3 8 9,9 1
total 1547 1547,0 4096 317 317,0 32
with spot (on style) (S) 879 933,0 3 126 131,0 1
no spot (ss) 365 311,0 1 136 131,0 1
total 1244 1244,0 4 262 262,0 2
non lutea 1109 1160,2 3
lutea 438 386,7 1
total 1547 1547,0 4
anthocyanous 1519 1522,8 63
alba 28 24,2 1
total 1547 1547,0 64

The types can be identified in this order in the upper and the middle row of fig. 4 and partly also in the lower row, as well as in fig. 5. M1 acts on all five petals, while M2 to M3 act on the three lower petals only.

Table 4 gives the total segregation; also a dominant gene for spot on style (S) was in action in most of the sowings. The proportions between the different types do not always answer very close to the expectations, but in a cross so wide, with no less than nine genes segregating, it cannot be expected that all types are equally viable. The present experiment is an example of an extremely complicated interaction of a number of genes, all determining the colour of the flowers; and although some of the genes have a superior effect, most of them to some extent modify the effect of the other ones or even substitute them, what makes it difficult to classify many of the types with any certainty. Only the classifications of anthocyanous to alba, of non-lutea to lutea, of spot to non-spot and of non-nigra to nigra are reliable in this case.

SUMMARY.

(1). A case of variegation in leaves of Viola tricolor is inherited as a non-Mendelian character, giving vegetative segregation effected by the plastids. It is transmitted by the ovules giving segregation already in F1, and it is probably also transmitted through pollen, but in this case F1 was green and in F2 among a total of 963 only three individuals were variegated.

(2). The velvety black flower colour of Viola tricolor nigra is extremely recessive as compared with wild growing, non velvety types of Viola tricolor, which have a series of five modifiers or suppressors for the black velvety colour. On the other hand, tricolor nigra contains three dominant, polymeric, basic genes for the anthocyanine colours (violet or reddish). A crossing, involving nine genes, all cooperating in determination of flower colour, is described.

This investigation is part of a series carried out by a grant of the Carlsberg Foundation, for which the author expresses his thanks to the Trustees of the Foundation.

LITERATURE CITED.

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