MUTABLE LOCI IN. MAIZE

BarparA McCrintock

During the past year, the study of insta-
bility of action of the genetic factors at
various loci in maize chromosomes was
continued. This study, extending over a
six-year period, has shown that many
different genetic factors may exhibit such
instability. By appropriate. methods, it is
possible to detect the instability of expres-
sion of the factors at a particular locus
shortly after the event at the locus leading
to this behavior has occurred. Different
origins of instability at any one locus may
thus be discerned. Comparisons of such
‘cases have shown that the genetically active
components at any one locus may vary in
type and in range of expression, and that
differences may exist in their mode of con-
trol. From this evidence interpretations
have been derived regarding the responsi-
ble mechanisms. These have been devel-
oped in an article now in press (Cold
Spring Harbor Symposium on Quantita-
tive Biology, vol. 16, 1951) and therefore

need not be stated here. This report will
be limited to brief reviews of newly ac-
quired information concerning the origin
and behavior of mutable conditions at vari-
ous loci.

Two new cases of instability have ap-
peared involving the bronze locus in the
short arm of chromosome 9 (Bz, dark red
or purple color in plant and aleurone; dz,
recessive allele, plant and aleurone color
a bronze shade). Each appeared in a single
tested gamete of a plant that carried Ds
and Ac, and they are much alike in be-
havior. Both are Ac-controlled; and they
appear to have originated by transposition
of Ds to the bronze locus, for Ds-type
breaks in the chromosome occur at this
locus, as well as mutations from dz to the
apparently full Bz phenotypic expression.
Their behavior resembles in many respects
that of c”*, described in previous reports.
They have been designated 5z”7* and
bam,
DEPARTMENT OF GENETICS

The Wx locus has been concerned in sev-
eral other studies. The factors at this locus
are associated with production of amylose
starch in the pollen and in the endosperm
of the kernel. When only the recessive,
wx, 1s present, the starch consists of amylo-
pectin, and this stains reddish brown with
iodine—potassium iodide solutions. The
normal dominant, Wx, in single, double,
or triple doses, produces amylose starch,
iN quantities amounting to approximately
22, 25, or 28 per cent, respectively, of the
total starch content of the endosperms.
This starch, in each of these doses of the
dominant, stains a dark blue with I-KI
solutions.

In Year Book No. 48 (1948-1949) a series
of alleles produced by mutations of the Ac-
controlled wx"* locus was described.
Many of these alleles may be distinguished
by the physical appearance of the starch
in the endosperm, but a more accurate
criterion is the intensity of the blue stain
when a solution of I and KI is applied.
The intensity of the staining reaction is an
indication of the amount of amylose
starch produced by a particular allele; and
a progressive increase in intensity of stain
is exhibited by kernels carrying single to
triple doses of the particular allele. (One
and two doses are obtained by combina-
tions with the recessive, wx.) The alleles
arising from mutations at wx”? produce
amounts of amylose ranging from only a
fraction of 1 per cent of that produced by
the normal allele, Wx, to approximately
the same amount as Wx. Mutations at
wx" occur only when Ac is present in the
nucleus, and the alleles so produced may
again mutate if Ac is present. When Ac is
absent, the alleles appear to be stable. A
number of different germinal mutations of
wx" have occurred in plants carrying
wx” and one Ac factor. Those carried in
gametes in which Ac was not present
served as the source of stable strains of

175

particular alleles. The action of these se-
lected alleles in single, double, and triple
doses was examined visually by means of
the iodine-solution test. Chemical analyses
to determine the amylose content produced
by some of the alleles in single to triple
doses were conducted by Dr. B. Brimhall
and Dr. G. F. Sprague, of Iowa State Col-
lege. Table 1 reproduces the data supplied
by these investigators.

 

 

 

 

TABLE 1
PERCENTAGE AMYLOSE STARCH
PRODUCED BY ALLELE IN SINGLE
ALLELE TO TRIPLE DOSES
DESIGNATION
Single Double Triple
dose dose dose
4744A-3.......... 2.4 4.0 5.8
5220A........... 3.5 4.6 7.3
4744E-1.......... 3.5 4.6 7.2
4744E-2.......... 3.7 5.8 .
2d sample...... wees 5.3 wee
4744G-2.......... 4.3 6.5 *
$013D........... 4.6 5.0 9.3
4742B-2.......... 6.5 7.3 13.2
ATIF 0... ee. 16.8 22.2 25.0
4744C.. 2. .......f 18.5 20.0 25.5
S750B........... 25.0 27.0 28.0
5016A........... 23.0 27.0 *

 

 

 

 

*Sample having triple dose of allele not available at
time of analysis.

Selection of the alleles whose action is
shown in table 1 was not random; they
included some of the lower and some of
the higher ones. Other alleles with still
lower activity, as estimated by the iodine
staining reaction, have been isolated. Some
others that may fall between isolates 4742B-2
and 4721F of the table were not available
in graded doses to be included in the
analyses. The samples analyzed, together
with the others mentioned, suggest that
mutations at wx" may give rise to a
series of alleles distinguished by small dif-
ferences in the amount of amylose starch
176

they produce. Thus, a relatively large
number of quantitative alleles may be
anticipated,

Several other cases of independently
originating instability at the Wx locus have
been investigated. One of them, desig-
nated wx”"*, was detected by differences in
starch constitution in regions of an individ-
ual kernel on an ear coming from the cross
of a female plant carrying a previously
normally behaving Wx locus in each chro-
mosome 9, by a male plant homozygous
for the stable recessive, wx. The modifica-
tion producing this instability occurred at
the Wx locus in one of the chromosomes
g of the female parent. Studies of the
gametes produced by the plant arising
from this kernel, and tests of its progeny,
revealed the type of instability expression.
It appears that wx” is autonomous; no
separate activator factor is required for the
occurrence of mutations. The types of mu-
tation differ markedly from those produced
at wx™"*, No alleles showing intermediate
expressions of the action of the factors at
this locus have been isolated, and only very
rarely does a kernel exhibit a region that
indicates mutation to intermediate action.
The mutations are nearly always from re-
cessive to apparent full-dominant expres-
sion. The dominant so produced may mu-
tate again to the full recessive.

Two additional mutable conditions at
the Wx locus have recently been found.
Both arose from modifications at a pre-
viously normally behaving Wx locus. Each
was first recognized in an individual ker-
nel, which had regions showing visible
changes in appearance of the starch as well
as the associated less intense blue staining
with iodine solutions. A plant was ob-
tained from each kernel, and the study was
continued. Neither of these cases appears
to require a separate activator factor, but
the studies are too incomplete to permit

CARNEGIE INSTITUTION OF WASHINGTON

adequate description of behavior. In one
of them, however, the rate of production
of germinal mutations is high. Selections
were made of some of these, each showing
a particular rate of reaction, and the plants
arising from them were crossed to tester
stocks this past summer to determine the
subsequent stability of the selected mutants.

Variegation in starch composition re-
sembling instability of action of factors at
the Wx locus commonly occurs in kernels
that have Ac located a few crossover units
to the left of this locus. Areas showing
various grades of intensity of blue stain
with iodine solutions may appear in ker-
nels that have received from their female
parent a chromosome g carrying the stable
recessive wx, and no Ac, and from their
male parent a chromosome 9 carrying Ac
at this location and also a normal Wx. At-
tempts are now being made to determine
whether these altered phenotypic expres-
sions arise from modifications at the Wx
locus and as a consequence of the spatial
relation between Ac and Wx.

Unstable behavior of the factors located
at a number of other positions in the chro-
mosome complement has also been ex-
amined. Because the number of such cases
is continually expanding, it has not been
possible to examine each with the required
degree of thoroughness. For many of them,
only a cursory examination was attempted,
with a view to gaining some information,
even if limited, about the nature of insta-
bility in each case. The presence of these
mutable conditions is detected by variega-
tion in a plant or endosperm character.
Ten of those examined are associated with
production of pigment in the aleurone
layer of the kernel, or in both kernel and
plant tissues. The loci of six of them are
known. Three are at the A: locus in chro-
mosome 3, one is at the J locus in chromo-
some 9, and two are at the R locus in chro-
DEPARTMENT OF GENETICS

mosome 10. One involves a previously un-
identified locus whose factors resemble in
action those of the R locus. One suggests
alterations at Pr (purple anthocyanin pig-
ment; pr, recessive allele, red pigment;
located in chromosome 5). Its mutations
are from the recessive, which results in red
pigmentation, to various grades of inten-
sity of the dominant expression, which is
purple pigmentation. Tests for the loca-
tions of the remaining two have not been
completed, but the mutation types do not
suggest that the factors at any of the previ-
ously known loci are involved.

Study of instability of expression of fac-
tors associated with anthocyanin pigmenta-
tion in the plant tissues only has been con-
fined to determining the patterns of this
expression; no attempt has been made to
determine the loci involved. One such case
suggests alterations at the B (booster of
plant color) locus in chromosome 2; an-
other suggests alterations at the P/ (purple
plant color) locus in chromosome 6; and
a third suggests instability at the R locus
in chromosome 10 that alters the antho-
cyanin pigmentation in the plant tissue
only.

In another case, the factors at the locus
involved are associated with pigmentation
of the pericarp layer of the kernel. Al-
though this mutable condition has received
only cursory examination, its action will be
mentioned because of an interesting rela-
tion between mutations in the pericarp and
intensity of pigment formation in the cells
of the aleurone layer underlying the mu-
tant areas. The pericarp tissue forms the
outer layer of the kernel. It is maternal in
origin, whereas the underlying aleurone
layer is derived from the endosperm. In
this case, the pericarp is colorless until
a mutation occurs. The mutations result
in pigment formation in the pericarp. The
type of pigmentation produced by muta-

177

tion resembles that associated with the
cherry allele at the R locus. Some diffusible
substance is produced as a consequence of
these mutations, and this substance is used
by the cells of the underlying aleurone to
intensify their own pigmentation. This is
evident because the aleurone cells located
under the regions of colored pericarp are
more intensely pigmented than those un-
derlying the pericarp areas in which no
mutations have occurred. If the pericarp -
layer is peeled off, its pattern of mutations
is still revealed in the aleurone by regions
of more intense pigmentation,

The alteration at the J locus is of some
interest because of the nature of the action
of I, which had been analyzed earlier. J
acts to produce an inhibitor of pigment
formation in the aleurone layer when its
allele C (aleurone color) is also present.
Kernels homozygous for I, however, are
completely colorless. In kernels with J I C
constitutions, aleurone color does not de-
velop. In 7 C C constitutions a faint blush
of color may appear at the base of some of
the kernels. The dosage action of the C
allele has been described in previous re-
ports; the higher the dose, the more intense
the pigmentation. The expression of both
I and C is quantitative. I appears to be
concerned with the production of some sub-
stance or substances that compete for re-
active sites with the substance or substances
associated with C action. As the dose com-
binations given indicate, I appears to be
more effective than C in this respect. If
more C factors were present, however, the
increment might result in the development
of considerable amounts of pigment in the
aleurone layer. This possibility was ex-
amined by using a chromosome 9 with a
duplication of the short arm, each mem-
ber of which carried a C factor. When
two such chromosomes were contributed
by the female parent and a single J factor
178

by the male parent, the dose ratio in the
endosperm was four C factors to one /
factor. The aleurone layer in such kernels
was uniformly pigmented. The intensity
of this pigmentation was slightly less than
that produced by a single dose of C (C cc
constitutions). The alteration at the J locus
effected a change that lowered the capacity
of J to inhibit C activity.

The above-mentioned alteration occurred
at a previously normally behaving I
locus. It was detected in an aberrant
kernel on an ear resulting from the cross
of a female plant homozygous for C by a
male plant carrying I. Much pigmentation
appeared in the aleurone layer of this ker-
nel. Variegation was exhibited by small
areas that were either lighter or darker in
color. The plant arising from this kernel
was self-pollinated, crossed reciprocally
with plants carrying C, and also crossed to
plants carrying c. Examination of the re-
sulting ears indicated the nature of the
action attributable to the alteration at the /
locus. Kernels homozygous for this modi-
fied I were completely colorless, as were
those carrying this J and also c. The re-
duced capacity for functioning was ex-
hibited in combinations with C. A single
dose of modified J with a double dose of C
produced kernels that were darkly pig-
mented. Those having a double dose of /
and a single dose of C were more lightly
pigmented. In some of the kernels with
I C C constitutions, variegation was ex-
hibited; regions that were colorless or more
deeply colored than the surrounding areas
were present.

During the year, much effort was fo-
cused on examination of alterations affect-
ing the action of factors at the 4: locus
in chromosome 3 (Au, aleurone and plant
anthocyanin color; a1, recessive allele, col-
orless aleurone and altered plant color).
_ Three independent cases of inception of
instability at this locus have been isolated,

CARNEGIE INSTITUTION OF WASHINGTON

and they will be described shortly. Atten-
tion will first be given, however, to sum-
marizing the results of efforts to produce
mutations of the known stable recessive
allele, ai.

Experiments aimed at the production of
mutations of ai were described in Year
Book No. 49 (1949-1950). During the past
year, these experiments were considerably
expanded. Because the methods employed
have been described, only a summary of
the additional results need be included
here. Thirteen plants homozygous for a
and having the desired constitutions with
respect to chromosome g (one carrying a
long terminal deficiency of the short arm,
the other having a duplication of this arm
in the reverse order) were used in crosses
to plants homozygous for a: and having
normal chromosome constitutions. No Dt
(Dotted) factor was present in any of these
plants. As was mentioned in the previous
report, the purpose of the experiment was
to determine whether or not mutations to
A, would occur in the absence of Dz and,
if so, whether these mutations would be
of the type produced by Dz. At the pres-
ent writing, the kernels on 315 ears derived
from these crosses have been individually
examined; and on 86 of the ears one, or
occasionally two or several, kernels show-
ing one or more spots of the 4, phenotype
were found. Among the 93,078 kernels
examined, 117 had such mutant areas; and
all these areas were small, resembling those
produced by the De factor. In 93 of them,
only one A: spot was present. The other
24 were distributed as follows: g with
two A; dots, 4 with three, 6 with four,
I with five, 2 with seven, 1 with seventeen,
and 1 with eighty-four. Except in the one
kernel having eighty-four spots, the Ai
spots were confined to one area of the ker-
nel. This would be expected if the event
leading to mutation depended upon an
earlier event that evolved a Dr-like factor.
DEPARTMENT OF GENETICS

Mutations at a:, of the Dz type, would ap-
pear only after such an event had occurred,
and only in that area of the kernel derived
from the cell in which it had taken place.
In the kernel having eighty-four of them,
the A: dots were distributed throughout
the aleurone layer. In the plant grown
from this kernel no mutations to the Ai
phenotype appeared; and no such muta-
tions occurred when this plant was crossed
to and by plants homozygous for a. The
alteration responsible for the mutations to
the 4: phenotype in the kernel was proba-
bly present in only one of the two sperms
that functioned in the origin of this ker-
nel—the one that entered the endosperm
nucleus.

A control experiment, accompanying the
one described above, was made by inter-
crossing plants homozygous for a: but hav-
ing normal chromosome constitutions.
Only one kernel among a total of 21,464
examined had a single small spot of the
A, phenotype.

The origins of the three independent in-
ceptions of instability at the locus of A,
mentioned above, may be reviewed briefly.
The first case, designated a.”"*, was detected
in a single aberrant kernel on an ear result-
ing from a cross in which the male parent
was homozygous for ai and the female
parent heterozygous (41/a1). The aleurone
layer of this kernel was variegated; both
colored and colorless areas were present.
The types of variegation and color expres-
sion suggested that mutations were occur-
ring from the recessive to higher alleles of
Ax. The plant derived from this kernel
was likewise variegated for anthocyanin
pigmentation. Tests of its progeny con-
firmed the presence of an unstable a: locus
and showed that this instability had arisen
in one of the chromosomes 3 contributed
by the female parent. This ai%* is au-
tonomous, in that no separate activator
factor is required for expression of insta-

179

bility. A number of different types of mu-
tation occur. They affect the type and
amount of pigment that will be formed in
the aleurone and plant tissues, and the
time and place of its formation.

The second inception of instability was
detected in much the same way as was
a:""*, It was present in a single aberrant
kernel on an ear from a cross in which
the male plant had been homozygous for
a, and the female plant heterozygous
(A:/a:). In this kernel the variegation was
expressed by dots of the 4: phenotype. The
plant derived from it also showed variega-
tion for anthocyanin pigmentation. Sub-
sequent tests indicated that this mutable
condition, designated ai”, arose in one of
the chromosomes 3 contributed by the fe-
male parent.

The third mutable condition, a.” *, was
detected by variegation for anthocyanin
color in the aleurone layer of some kernels
on the self-pollinated ear of an individual
plant and on the ears coming from recipro-
cal crosses with plants carrying a. Both
parents of this plant were homozygous for
Ax, and no instability affecting its behavior
had been detected in these parents or their
antecedents. Six sister plants were also
self-pollinated and crossed reciprocally with
plants homozygous for ai, and no instabil-
ity of phenotypic expression of A: was
detected.

Both a”? and a:”°° arose in plants that
carried Ds and Ac. Preliminary evidence
suggests that the mutations are controlled
in each case by an activator. This may
prove to be Ac. Until the possible rela-
tion with Ac has been clarified, a descrip-
tion of the mutational types given by these
two mutable loci will be postponed.

Some other loci carrying factors asso-
ciated with the development of kernel
characters have exhibited mutability.
Changes at one such locus affected the
production of yellow pigment in the endo-
180

sperm cells. They were first detected in
some kernels on an ear of a plant that
was homozygous for y (Y, yellow pigment
formation; y, recessive allele, no yellow pig-
ment formed). In these kernels, areas
were present showing various grades of
intensity of yellow pigment. Study of this
case was confined to determining that the
mutable condition was heritable. Insta-
bility of the action of factors at other loci
has resulted in altered development of
either the aleurone layer or the under-
lying endosperm cells. These types have
proved to be heritable, but no attempt has
been made to study their behavior in
detail.

A number of cases of instability have
appeared that were originally revealed by
somatic changes affecting chlorophyll de-
velopment. Except in three cases that ap-
peared early in the study, no attempt has
been made to investigate in detail the
nature of the mutation processes con-
cerned. The study was confined to deter-
mining whether or not the mutable con-
dition was heritable.

The above description of different muta-
ble loci and their origins has been given in
order to indicate the range of genetic ex-
pression of this kind that can occur in a
single organism and the large number of
known loci as well as previously unknown
loci that may be concerned. On the basis
of the types of mutation and their modes
of control, it has been possible to subdivide
the mutable conditions into classes. It is
now known that mutable conditions be-
longing to different classes may arise at any
one locus. No theory of gene action or
mutation that has been seriously consid-
ered by the majority of geneticists is ade-
quate to account for these various types of
expression of the factors at a locus and
their modes of control. It has been neces-
sary, therefore, as was pointed out early in
this report, to reconsider former concepts

CARNEGIE INSTITUTION OF WASHINGTON

and to develop others, where required, in
order to incorporate the new evidence.
All the types of mutation described in
this and previous reports have been herita-
ble. Some cases have been found, how-
ever, in which the phenotypic expression of
instability did not prove to be heritable.
Several of them have been given some at-
tention. The general pattern of such insta-
bility may be illustrated by the following
case. A plant homozygous for the recessive
factors ¢ and wx in chromosome g and pr
in chromosome 5 was crossed by one carry-
ing the dominant alleles. All the kernels
on the resulting ear showed bizarre types
of variegation. Every kernel had several,
and some many, regions showing unex-
pected phenotypes. Some regions showed
the ¢ phenotype in the aleurone layer.
The underlying cells might be all Wx,
some Wx and others wx, or all wx. Other
regions obviously were produced because
dicentric chromatid formations, involving
the chromosome g carrying the dominant
factors, had occurred in the ancestor cell
that produced the region. Chromosome 5
also was involved in chromosome aberra-
tions, for many areas of the pr phenotype
appeared. Of considerable significance was
the appearance. of twin areas, in large
numbers. These are adjacent regions of
similar size, which suggest origin from sis-
ter cells. Several classes of such twin areas
appeared: (1) one area of colorless alev-
rone, the other having a deeper color than
that of the surrounding aleurone cells;
(2) one area showing the red (pr) pheno-
type and the other a deeper purple (Pr)
than the surrounding aleurone cells; and
(3) two regions showing reciprocal quanti-
tative differences in color intensity—that
is, one lighter and one darker than the
surrounding areas. That some if not all
of this variegation resulted from chromo-
some aberrations was suggested by the con-
comitant losses of C and Wx in some
DEPARTMENT OF GENETICS

areas, indicating deletion, and the occur-
rence of breakage-fusion-bridge cycles in
others, indicating dicentric chromatid for-
mations. Plants were obtained from some
of these kernels and crosses were made to
test for recurrence of the expression in the
following generation. None appeared on
the many test ears examined, and the ex-
periment was discontinued at this stage.

181

A second case, very similar to the one
just described, was also examined. It like-
wise proved to be nonheritable, in that the
abnormal phenotypes did not reappear in
the following generations. No evidence
has appeared to suggest what factors are
responsible for these nonheritable types of
variegation.