Journ. of Genetics 33(3): 347-354 (Dec 1936)

(With One Text-figure)


1 Contribution from the Division of Cereal Crops and Diseases, Bureau of Plant Industry, U.S. Department of Agriculture, and the Farm Crops Subsection, Iowa Agricultural. Experiment Station, Ames, Iowa, co-operating. Journal Paper No, J 343, Iowa Agricultural Experiment Station, Ames, Iowa.
2 Associate Geneticist, Division of Cereal Crops and Diseases, Bureau of Plant Industry, U.S. Department of Agriculture.

It has not been possible, with the exception of a few outstanding cases (Sturtevant, 1925; Mangelsdorf & Fraps, 1931; Lindstrom & Gerhardt, 1926), to obtain reliable quantitative data on the change produced in a genetic character by varying the dosage of the major gene or genes determining the nature of that character. Data of this type are of value. A hypothesis which seeks to explain the role of a gene in ontogeny must take into account the change produced by varying the number of times it is present.

The aleurone tissue of the maize kernel offers exceptionally favourable material for the study of gene dosage and interaction. Several hundred seeds are produced on a single ear under the same environment. The aleurone is triploid in nature, since it arises from the union of a haploid sperm with two haploid polar nuclei. The seed on one ear may be of several genotypes. It is possible to vary the dosage of any allel from one to three in disomic strains, and the use of the proper primary trisome allows the dosage to range from one to five. In spite of these advantages there has been little work on the effect of gene dosage on aleurone colour because none of the previously known aleurone characters have been adapted to the obtaining of quantitative data. This paper presents a study of the effect of gene dosage on a new aleurone character in maize.

Four major genes are known to be involved in the development of aleurone colour in the maize kernel (East & Hayes, 1911; Emerson, 1918; Jenkins, 1932). These are A1, C, R, and A2 The A1 locus is in chromosome 3, the C locus in chromosome 9, the R locus in chromosome 10 and the A2 locus in chromosome 5. Aleurone colour is not formed if the recessive allel of any one of the four primary loci is homozygous, i.e. at least one dominant allel of all four loci must be present. The alternative of purple or red aleurone colour depends upon the Pr pr genes, purple being dominant to red. A ratio of 3 coloured to 1 colourless seed is obtained in F2 when one of the four primary loci is heterozygous; a 9: 7 ratio when two loci are heterozygous, etc.

In a selfed ear of Black Mexican sweet corn obtained from Dr L. F. Randolph, aleurone colour segregated into 12 self-coloured: 3 dotted: 1 colourless seeds. The Black Mexican line had been maintained for several generations by sibbing. The occurrence of colourless aleurone in a strain of corn homozygous for all the dominant allels for aleurone colour was unexpected, but more surprising was the appearance of a new dotted aleurone character. The dots or spots of colour were distributed apparently at random over the aleurone layer. They were small and fairly uniform in size (Fig. 1).

Fig. 1. Photograph of crown of maize seed showing the dotted aleurone character. The genetic constitution of this seed is a1a1a1DtDtDt and it is homozygous for the other dominant genes necessary for aleurone colour.

The colour of the dots is either purple or red depending upon the Pr pr constitution. The intensity of the colour is greatest in the central portions of the dots with a gradual fading towards the surrounding colourless tissue.

The 12: 3: 1 ratio of the original ear was correctly interpreted as the segregation of one of the four primary genes, to give 12 self-coloured: 4 non-self-coloured. (a 3: 1 ratio), and a dominant gene which interacted with the recessive primary gene to give dots of aleurone colour in three-fourths of the non-self-coloured class. The segregating primary aleurone factor proved to be A1a1. The dominant gene interacting with a1 to give the dotted character has not previously been reported. It has been designated Dt dt, the Dt allel giving dots of aleurone colour with a1, whereas dt does not.

The conclusion that the segregation of A1a1 occurred following a mutation of A1 to a1 seems unescapable, because the genetic residuum of the Black Mexican line was unchanged and out-crossing would have been detected. The Dt gene was thought, at first, to have arisen by mutation in the same gamete in which the mutation from A1 to a1 occurred, but tests of sister plants showed the Dt gene to be segregating in the Black Mexican strain, its presence not having been previously detected because the strain was homozygous for the dominant allels of genes producing aleurone colour.

Interaction of Dt with the different aleurone genes

Genotype Aleurone colour
a1CRA2DtPr Purple dots on colourless background
a1CRA2Dtpr Red dots on colourless background
A1cRA2Dt Colourless—no dots
A1CrA2Dt Colourless—no dots
A1CRa2Dt Colourless—no dots
a1CRA2dt Colourless—no dots
A1CRA2Dt Strong self-colour—no dots
a1pCRA2Dt Pale self-colour—no dots
A1a1CRA2Dt Strong self-colour—no dots
a1Pa1CRA2Dt Pale self-colour with dots
a1cRA2Dt Colourless—no dots
a1CrA2Dt Colourless—no dots
a1CRa2Dt Colourless—no dots

Dt interacts with a1 to give dots of aleurone colour only if all the remaining three primary genes have at least one dominant allel present. Dt interacts in this manner only with a1. Seeds homozygous for a2 or for c or for r, the remaining three genes in each case being present in dominant form, have colourless aleurone in the presence of Dt. The interaction of Dt with the different aleurone genes is given in Table I.

The linkage group to which the Dt gene belongs has not been established. Dt is, however, independent in its inheritance of the A1, C, R, A2 and Pr loci, genes all affecting aleurone colour, as well as of the Lg1, Su, Y and F1 loci.


There are four allels at the A1 locus. Two of these, A1 and A1b, produce deep-coloured aleurone; a1p produces pale-coloured aleurone and a1 produces colourless aleurone. Nothing is known concerning the interaction of A1 and Dt, as dots of colour, if formed, are invisible in the deep aleurone colour produced by A1. Seeds homozygous for a1p have no dots of colour in the presence of Dt, although the intensity of colour in the dots on seeds of a1Dt constitution is greater than in the pale aleurone colour and they would be visible if present. However, seeds carrying a1pand one or two a1 allels have dots of aleurone colour which are clearly evident on the pale-coloured background. Therefore it is possible to determine the effect of different dosages of a1 on the aleurone dots by varying the ratio of the a1 and a1pallels. As the aleurone is triploid the dosage of genes in it may be varied from one to three. The dosage of a1 may he varied while that of Dt is held constant, or the dosage of Dt may be varied while that of a1 is held constant.

Crosses of a1a1PDtDt x a1a1dtdt were made to determine the effect of three a1 allels as compared with one a1 allel on the aleurone dots. Two classes of seed were produced on the same ear having the following constitution:

(1) a1a1pa1pDtDtdt = pale aleurone with dots,
(2) a1a1a1DtDtdt = colourless aleurone with dots.

These two genotypes occur at random on the ear. Their environment is identical. They have the same genetic residuum except for the possibility of segregating genes linked with a1. The other aleurone factors were present in a homozygous dominant condition. The two classes, therefore, may be considered as differing only in that one of them has one a1 and the other three a1 allels.

The number of dots of aleurone colour was determined on each seed under a low-power binocular after the seed coat had been removed to increase the accuracy of counting. Counts on the seeds in both classes were made independently by two investigators. The agreement was always close. Counts were made on the kernels from four ears, approximately fifty seeds of each class being counted from each ear. No appreciable increase in the reliability of the mean number of dots per seed was obtained by counting more than fifty seeds.

The average number of dots per seed of a1a1a1 constitution was about is it three times as great as on seeds of a1a1pa1p constitution which is the same ratio as the number of a1 allels in the two classes. The data from one ear are given in Table II with a statistical analysis. The data from the three other ears also were treated statistically but the deviations from a 3: 1 ratio were not significant. The ratio of the mean numbers of dots per seed in the two classes for all four ears was 3.1: 1.0.

The effect of three a1 allels as compared with two a1 allels was studied by analysing the data obtained from the cross a1a1DtDt x a1a1pdtdt. The two classes of seed produced have the following genotypic constitution:

(1) a1a1a1pDtDtdt = pale aleurone with dots,
(2) a1a1a1DtDtdt = colourless aleurone with dots.

Data on the two classes were obtained from three ears. The ratio of the average number of dots on seeds of a1a1a1 constitution to the average number on seeds of a1a1a1p constitution was 3: 2. This is the same ratio as the dosage of the a1 allel in the two classes of seed. Data from one ear are given in Table III. The ratio of the averages for the two classes of seed for the three ears was 3.06: 20. The deviations from a 3: 2 ratio for the individual cars were not significant.

Numbers of aleurone dots on 55 seeds with three a1 genes and 55 seeds with one a1 gene from an ear
(Pedigree 2946-6 x 2944-1) of the cross a1a1pDtDt x a1a1dtdt

a1a1a1DtDtdt class   a1a1pa1pDtDtdt class
39 54 47 79   24 15 19 10
11 88 65 59   20 25 10 37
55 73 45 04   26 27 16 18
68 43 48 39   37 8 23 12
35 35 75 64   32 42 16 21
75 53 63 106   24 43 21 1
66 47 47 47   32 20 27 23
57 55 75 46   20 23 17 2
52 54 50 79   14 18 17 16
15 12 75 22   31 21 23 29
71 81 54 43   19 40 10 17
51 52 113 55   18 13 18 22
53 39 53 29   12 23 24 14
57 75 51 -   38 47 18 -
Mean = 58 dots per seed Mean = 21.8 dots per seed
t=1.6442. (108 n/F). Not significant.

The above data indicate that the effect on the dotted aleurone character of increasing the number of recessive a1 allele, as determined by the number of dots, is additive or linear.

In obtaining the data on the a1 gene the number of Dt genes was held constant. Crosses therefore were made in which the number of a1 genes remained constant while the Dt gene varied in dosage. The following cross was made reciprocally: a1a1DtDt x a1a1dtdt. When the dotted parent was the female the seeds were all a1a1a1DtDtdt. In the reciprocal cross, however, the seeds were a1a1a1Dtdtdt. These crosses are exact reciprocals and were made between lines originating from the car on. which the clotted character first appeared so that they have much the same genetic make-up. It is important to emphasize that these reciprocal crosses should be made between lines related as closely as possible because the seed produced on two different plants is to be compared. The dosages of any genes modifying the number of aleurone dots would be different in reciprocal crosses between unlike lines, as we are dealing with triploid tissue in which two-thirds of the heredity is contributed by the female parent. The mean number of dots per seed in the a1a1a1DtDtdt class was about four times that for seeds of a1a1a1Dtdtdt constitution. Data on one reciprocal cross are presented in Table IV, The data are not as extensive as might be desired but the deviation from a 4: 1 ratio of the means for the two kinds of seeds is not significant. The ratio of the means of the two seed classes for the three reciprocal crosses approximated 4: 1, the actual ratio being 4.2:1.0. There is no a priori reason for assuming theoretically that the ratio of these two means be exactly 4: 1. The data from the three ears ranged about this value so it was arbitrarily chosen. The ratio of dosages of Dt in the reciprocal crosses was 2: 1. However, the effect on the number of aleurone dots was not additive as was the case for the a2 gene. There were four tines as many dots when two Dt genes were present as there were where only one Dt gene was present.

Numbers of aleurone dots on 76 seeds with three a1 genes and 60 seeds with two a1 genes from an ear
(Pedigree 2676 x 2511-a) of the cross a1a1DtDt x a1a1pdtdt

a1a1a1DtDtdt class   a1a1a1pDtDtdt class
6 25 27 19 8 20   15 3 18 18 10
11 29 29 21 30 20   8 11 19 10 9
8 17 3 26 23 29   16 10 25 10 7
25 11 19 37 21 14   6 1 5 8 10
13 25 7 32 17 4   7 5 18 3 15
21 8 20 23 7 20   9 19 3 11 7
12 9 19 38 14 17   9 6 19 13 -
5 29 9 8 6 16   31 13 12 7 -
27 15 36 16 14 12   14 10 9 5 -
5 11 17 16 7 18   4 11 10 9 -
10 9 37 12 20 16   15 14 6 13 -
22 26 20 10 14 -   3 13 5 7 -
33 8 8 23 20 -   9 14 14 20 -
Mean = 17.6 dots per seed Mean = 11.0 dots per seed
t=0.8532. (134 D/F). Not significant.

Data on the effect of three Dt genes can be obtained from a selfed plant homozygous for the Dt gene. Obviously these data cannot be compared with those for two and one doses of Dt unless the different lines used in obtaining the three different dosages of Dt were very closely related. The only data available for three Dt genes came from seed obtained by selling a plant in a line derived from the original clotted ear. It is, therefore, related to the other lines, but some differences in genetic constitution could and undoubtedly did exist. The data should be considered with discretion. The average number of dots per seed on this selfed ear was 1850. The average numbers of dots on seeds with two and one Dt genes were 454 and 10.4, respectively. The data on the three Dt genes are, perhaps, not strictly comparable with the others, but it is possible to state, considering the data on the three classes of seeds, that the effect of increasing the dosage of the Dt gene is more nearly exponential than linear. More data will be obtained on this problem when the proper lines have been synthesized.

Numbers of aleurone dots on individual seeds from reciprocal crosses, one cross yielding seeds with
Dt genes and the other yielding seeds with one Dt gene

2951-9 x 2949-8
a1a1DtDt x a1a1dtdt
2949-8 x 2951-9
a1a1dtdt x a1a1DtDt
a1a1a1Dtdtdt seeds
55 77 55 37   8 16 30 16
29 69 19 28   4 12 15 6
45 69 32 49   7 8 7 10
20 42 35 36   8 3 8 3
17 57 56     14 11 10 -
20 52 52 -   12 17 9 -
78 16 46 -   9 1 7 -
87 27 37 -   14 10 18 -
100 20 45 -   6 12 10 -
Mean = 45.4 dots per seed Mean = 10.4 dots per seed
t = 0.7075. (60 D /F). Not significant.

If the conclusions reached on the dosage effects of a1 and Dt are valid, it should be possible to predict the outcome of previously untried combinations. A plant of a1a1pDtdt constitution was self-pollinated. The resulting seeds were classified into 102 pale coloured: 92 pale coloured with dots: 50 colourless with dots: 16 colourless. This is a close approximation to the expected ratio of 6: 6: 3: 1. It was predicted before the dots were counted that the mean number per seed in class a1Dt, colourless background with dots, would be twice that of the class a1pa1Dt, pale background with dots. The mean number of dots on the fifty seeds comprising the three a1Dt classes was 45.0, while the mean number of dots on the ninety-two seeds of the six a1pa1Dt classes was 23.0.

The dotted aleurone character is expressed only in seeds of a1CRA2Dt constitution. Dominant C, R and A2 are, therefore, as necessary for the expression of the dotted character as recessive a1 The number of dots has been shown to be dependent upon the number of closes of a1. What the effect would be if the number of doses of dominant C or R or A2 varied is a problem for the future as little information has been obtained. Certain data, however, suggest that seeds with one dominant C gene have fewer dots than seeds with two dominant C genes. This problem will, of course, be prosecuted, as it promises to open up an extensive field of investigation on the dosage effects of the several genes affecting aleurone colour.


1. A new dominant gene Dt interacts with recessive a1 in the presence of the dominant allels of the other factors concerned with aleurone colour to give coloured dots scattered over the aleurone layer. Dt is specific in its interaction with recessive a1.

2. The effect on the dotted character of varying the dosage of recessive a1 was additive. Data on three dosages of a1 were obtained.

3. The effect on the number of dots of varying the dosage of the Dt gene was non-additive.


The author is indebted to Dr A. E. Brandt and Prof. R. A. Fisher for assistance in the statistical analysis of the data. He also wishes to thank Dr L. F. Randolph for his generosity in turning the dotted character over to him for analysis.