PROCEEDINGS

NATIONAL ACADEMY OF SCIENCES

A CORRELATION OF RING-SHAPED CHROMOSOMES WITH
VARIEGATION IN ZEA MAYS

By BarBara McCLiIntTocK*

UNIvEeRsITy oF Missouri**

Communicated November 2, 1932

It has been concluded from cytological and genetical investigations
that ordinary rod-shaped chromosomes perpetuate themselves through
nuclear cycles by splitting into two similar halves, which pass freely to
opposite poles of a mitotic spindle figure. Through such behavior the
same genetic constitution in every nucleus of the plant is maintained.
The evidence presented here indicates that the size and genetic constitu-
tion of ring-shaped chromosomes are not the same in all the nuclei of the
plant. In one cell or a group of cells it may be larger or smaller than the
original ring chromosome contributed by one of the gametes. The method
by which a ring-shaped chromosome becomes reduced in size by loss of
a section, or even totally eliminated from the cell, has not been determined.
Nevertheless, somatic elimination of a section of a ring-shaped chromo-
some or a loss of the ring altogether should result in variegation if a genetic
marker is present in the ring. If a normal chromosome carrying a re-
cessive gene has as its homologue a ring-shaped chromosome carrying
the dominant allelomorph, sporadic loss during development of the ring
chromosome or a section of it which carries the dominant will result in
groups of cells possessing only the recessive gene.

Hight cases of ring-shaped chromosomes have been studied. In all
of these a diminution in size or loss of the ring chromosome was observed.
One case was found in untreated material. The other cases arose in the
progeny of x-rayed pollen. In five of these instances genetic markers
were present in the chromosomes concerned.

All the variegated plants described in this paper appeared in the cultures
of Dr. L. J. Stadler. The author is indebted to Dr. Stadler for the privilege
of examining these plants for the presence of ring-shaped chromosomes.

The first case to be described involved the gene B, which is associated
with sun-red plant color. The normal B-lg chromosome is easily dis-
678 BOTANY: B. MCCLINTOCK Proc. N. A. 38.

tinguishable from the others of the complement by its relative size, the
position of the insertion region and the position and size of its knob.
The regional location of the gene liguleless (/g) and the probable locations
of the genes B and virescent seedling (v4) on this chromosome are given
in figure 1.***

Pollen containing the genes Lg, B and V4 was x-rayed and then placed
upon silks of untreated plants possessing the recessive allelomorphs, Jg,
b and y%. The progeny included a seedling which was classified as ligule-
less (Ig) and probably virescent (v4). As the plant developed, stripes of
red and green tissue were noted on the stalks and leaf sheaths. The red
tissue indicated the presence of the dominant gene B; the green tissue
indicated the loss of this gene during development. Examination of the
mid-prophase of meiosis showed, in many cells from different anthers,
a large ring-shaped chromosome synapsed with the normal b-lg chromo-
some. The ring was deficient to the extent indicated by the regions be-
yond the arrows in figure 1. The breaks in the normal chromosome to
produce this ring probably occurred a short distance from the end of the

 

Le B V,
T T
FIGURE 1

short arm, thus removing the Lg gene, and through the knob on the long
arm possibly removing the V, gene. In many sporocytes, however, the
size of the ring was obviously diminished, this frequently involving a loss
of the remaining portion of the knob. Very few sporocytes showed a total
loss of the ring chromosome.

This evidence strongly suggests that the loss of the B gene, indicated
by the green regions in the stalk and leaf sheaths, is due to the absence
of that section of the ring chromosome which carries the B gene.

A second case of variegation involving the gene B was found to be
associated with a ring-shaped B-lg chromosome. In this case, /g and %
were not marked. Here, also, diminished rings were observed.

A third case of variegation involving the Pl gene associated with
purple plant color. X-rayed pollen from a plant homozygous for the
dominant gene P! was placed upon silks of a plant carrying only the re-
cessive allelomorph, pl. The variegation appeared as stripes of purple
and sun-red plant color. The locus of the Pl gene is toward the middle
of the long arm of the satellited chromosome. In the sporocytes of this
plant there were 9 normal appearing bivalents, a single satellited chromo-
some and a small ring-shaped chromosome. Anthers from different
regions of the tassel differed in the size of the rings predominating in their
VoL. 18, 1932 BOTANY: B. McCLINTOCK 679

sporocytes. In each anther there were patches of related cells with rings
much smaller than the predominating type for the anther. Moreover,
the chromosome affected is the one known to carry the gene which is
involved in the variegation.

The other two cases examined involved the brown mid-rib gene (bm)
which is associated with brown color in the veins of the leaves and leaf
sheaths. This effect is due to a brown coloration in the walls of the
cells, Pollen from Bm, plants was x-rayed and placed upon silks of bry
plants. Variegation expressed itself as patches of brown and of colorless
cell walls visible in cross-sections of the stem, or as regions with brown veins
on the stalk and leaf.

In the first of these two cases metaphase and anaphase I figures showed
10 bivalents plus a fragment chromosome, a total of 21 chromosomes.
Examination of mid-prophase of meiosis revealed the fragment as a small
ring-shaped chromosome. Furthermore, a deletion in the short arm of
the bm, chromosome comparable in extent to the most frequently ob-
served size of the ring chromosome was also present. There was one
extra insertion region to be accounted for. It is difficult to avoid the
conclusions that the insertion region was divided in the formation of the
ring and the deleted rod and that both sections of the insertion region
were capable of functioning. The ring chromosome did not synapse
with the homologous region in the normal chromosome (see McClintock,
1932). The position of the buckling in the normal rod chromosome
which compensates for the deficiency in the deleted rod chromosome
usually appeared at a little distance from the insertion region in the short
arm of the bm, chromosome. Evidence recently obtained makes it
difficult to interpret these figures with regard to homologous association
and thus to be sure of the location of the deficiency. This being so, 10
decisive statement can be made at this time regarding the origin of the
ring and the division of the insertion region.

Diminution in size as well as loss of the ring-shaped chromosome oc-
curred in this plant. Therefore, it is likely that the Bm gene was in-
cluded in the ring chromosome at the time of its formation from the
normal rod chromosome and that the diminution in size or loss of the ring
chromosome was responsible for the variegation.

The second case of variegation involving the brown mid-rib gene was
similar in some aspects to the one described above. At metaphase and
anaphase I there were 10 bivalents plus a tiny fragment chromosome, or
21 chromosomes in all. In the mid-prophase of meiosis the fragment
chromosome was seen to be a very small ring composed of not more than
several chromomeres. In synapsis of the normal bm, chromosome derived
from the female with the Bm chromosome derived from the male there
was no indication of buckling in the normal chromosome to compensate
680 BOTANY: B. MCCLINTOCK Proc. N. A. S.

for a deletion. Since the ring representing the deleted portion was so
small, it is not improbable that the deficient chromosome was slightly
stretched to compensate for the small loss. Pollen examination indicated
that the plant was approximately 50 per cent sterile. It could be con-
cluded, therefore, that a deficiency was present in one of the rod chromo-
somes. A deficiency in the Br, chromosome corresponding to the ring
fragment would account for this sterility in view of the fact that complete
absence of the ring fragment was noted in groups of sporocytes or even
large sections of the tassel. Furthermore, anaphase J was characterized
by the frequent loss of the ring fragment in those sporocytes possessing
it. The evidence, taken as a whole, suggests that in this, as in the other
Bm, case, a deletion process involving the Bm, region of the chromosome
bisected the insertion region producing a ring fragment and a deficient
rod chromosome each with a functional insertion region.

Large sections of an anther or even whole branches of the tassel were
found to lack the ring-shaped chromosome. It is probable, therefore,
that variegation in this case was more commonly associated with complete
elimination of the ring chromosome than with diminution in size.

Of a total of 9 variegated plants which were obtained from x-ray treat-
ment, 6 were examined cytologically. One of these gave such poor cyto-
logical figures that no definite conclusions could be drawn; in the small
amount of material available, no evidence of a ring chromosome was
visible. It is possible that the material examined represented a region
in which the ring had been lost. It is also possible that some other chromo-
somal condition was responsible for the variegation, but the material
was too poor to yield suggestive evidence on this point. Four of the other
five plants showed a ring-shaped chromosome which was obviously derived
from the chromosome known to be associated with the gene involved in
the variegation. The fifth case, the second Bm, — bm, variegation,
possessed a tiny ring-shaped chromosome but cytological evidence of its
origin from the Bm, chromosome was not available. The presence of
the ring chromosome in some cells and its absence in others, coupled
with the appearance of variegation, indicated its origin as being similar
to that in the other four cases.

Decrease in size of the ring-shaped chromosome through loss of con-
stituent sections was strikingly manifest in all of the 8 cases of such
chromosomes examined. In the ring with a knob marker, the decrease
was frequently associated with a loss of the knob. The extent of loss
ranged from a relatively small to a very large section of the chromosome.
Complete elimination of the ring was observed also, this being especially
frequent in those cases having the smaller rings. The evidence from the

"cases with genetic markers in the affected chromosomes suggests that these
losses are responsible for the variegation observed in the plants.
Vor. 18, 1932 BOTANY: B. McCLINTOCK 681

Two other points not directly connected with variegation are of interest
with regard to the behavior of ring-shaped chromosomes. These rings
may increase in size and in number during the course of somatic divisions.
Evidence for increase in the size of the ring was obtained not only in
measurements showing such increases in cases where a more or less ac-
curate estimate of the size of the original ring could be made, but also in
the duplication of a visible marker within the ring. The ring chromosome
in the first case described possessed a fragment of a knob. If the ring
maintained itself unchanged throughout successive nuclear divisions, only
one knob should be present regularly. In very rare cases 2 or even 4
knobs have been observed in the same ring chromosome. Here, again,
where such duplication is evident in the rings, related cells in a group
show similar conditions. Occasional accumulation of two or more rings
in the same cell was observed in isolated cells or in groups of related cells.
The several rings may or may not be of like size.

Investigations which should lead to an understanding of the mechanism
responsible for the frequent decrease and occasional increase in size of
the rings or for their loss have not as yet been conducted. Lack of uni-
formity in the splitting plane could give rise to a double sized ring with
two insertion regions or cause split halves of the ring to become inter-
locked. Subsequent movement of the two insertion regions toward
opposite poles at anaphase would cause breaks in the ring chromosomes
and thus produce changes in their size and constitution. The figures of
two interlocking rings in a somatic metaphase in Crepis given by Na-
washin? are suggestive. It is noteworthy that in the decrease or increase
in size of the rings in Zea nothing but rings have been observed to come
from rings.

* NaTIONAL RESEARCH COUNCIL FELLow in the Biological Sciences.

** Contribution from the Department of Field Crops, Missouri Agricultural Experi-
ment Station, Journal Series number 355.

#** 4 deficiency involving V4 showed this gene to be in the long arm of the B-lg
chromosome. Its assignment to a locus between the knob and the end of the chromo-
some is based only on the case described here. This plant was classified as a probable
vs. More evidence is necessary before a definite locus within the long arm can be given.

1 McClintock, B., Proc. Sixth Internat. Cong. Genet., 2, 126-128 (1932).

2 Nawashin, M., Univ. Calif. Pub. Agr. Sci., 6, 95-106 (1930).