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THE EFFECT OF STRUCTURAL HETEROZYGOSITY ON THE
DEGREE OF PREFERENTIAL PAIRING I N
ALLOTETRAPLOIDS O F ZEAl
DONALD L. SHAVER
Department of Botany, Indiana Uniuersity, Bloomington, Indiana and
Department of Biology, Brookhaven National Laboratory, Upton, N e w York2
Received November 30, 1962
INCE somewhere between 30 and 50 percent of the extant angiosperms are
polyploids it is clear that the ploidy pathway to new plant forms is of vital
importance in evolution, and therefore potentially so to the plant breeder. Nearly
all existing polyploids, however, have been successful in becoming functionally
diploid, and during meiosis display none of the tetrasomic complications well
known to the ploidy experimentalist. Few, if any, of the “raw” allopolyploids
produced by man are perfectly diploidized per se, and modern experience is so
short that we probably have not yet witnessed the creation of a new allopolyploid, and then noted the pathway such a new form might take to attain stable
disomy. Since a stable allotetraploid has a new increment of built-in heterozygosity through constant, linear heterozygosity (epistases, or hetero-allelic
gene interaction) as well as of conventional paraheterozygosity (intra-allelic
gene interaction), the means by which plants may attain this state are naturally
of great theoretical importance and practical interest.
STEBBINS(1947) in his comprehensive review work stresses the importance
of loss of homology through mutation or through cryptic structural deletions
(SAX1933) as the prime means by which raw allotetraploids become functionally
diploid. FISHER
(1935) has shown that in such a new allotetraploid, the more
mutable of the pairs of duplicate factors would be lost, but according to CLAUSEN
and CAMERON
(1950), the process would be very slow. At any rate, the usual
view is that diploidization proceeds by way of a gradual loss of homology through
small changes until the partially tetrasomic condition is eliminated in favor of
functional disomy. STEBBINS(1950). CLAUSEN(1941), and others definitely
disfavor the concept that large chromosome rearrangements participate in the
process. The finding that the natural allotetraploid Nicotiana tabacum has
fewer duplicate factors than its newly synthesized counterpart ( CLAUSEN
1941)
supports STEBBINS’
idea. Since tetraploids in general are released from the stringent sexual cycle selection against even slight losses of chromatin imposed upon
diploids. it does not seem possible at present to choose between the two alternatives
1 A portion of the research was carried out at Brookhaven National Laboratory under the
auspices of the U. S. Atomic Energy Commission.
2 Present address.
Genetics 48: 515-524 4pni 1903
516
DONALD L. SHAVER
favored by STEBBINS,
and it seems likely that the two processes work together in
a complementary, rather than in a mutually exclusive fashion.
Recent work by DOYLE
(1960) on preferential pairing in structurally hybrid
autotriploids and autotetraploids of maize due to a cytologically defined large
inversion indicates that macro-rearrangements exert a profound influence upon
gene segregation. In the 4n duplex A,Zn 3a/A,Zn 3a/al/a,, he obtained a testcross
result of 89.2 percent A,, compared to the control value of 79.17 percent. The
present work will test the possibility that macro-rearrangements such as maize
Inversion 3a might participate in the process of diploidizing the allotetraploid
hybrid 4n maize x 4n teosinte, by increasing the degree of preferential pairing
(SHAVER
1962a) found in this hybrid.
MATERIAL A N D METHODS
A strain of 4n maize, homozygous for Inversion 3a, and for the gene markers
a, and lg*, which are within the inverted region, was employed in making hybrids with a clonal derivative of the original collection of 4n perennial teosinte
from Mexico made in 1921. The position of the maize inversion (RHOADESand
DEMPSEY
1953) in chromosome 3 and the approximate locations of the genes
a, and Lgz in the inverted region are shown in Figure 1. Genetic testcross results
were obtained by crossing this interspecific hybrid with recessive (nulliplex) 4n
maize tester stock homozygous for the loci a, Lg, A , C R' P'. Testcross progeny
kernels were first classified for a, on the basis of aleurone color, and then germinated for classification of liguleless-2. Standard corrections were applied for non-
.4
--
-101
--'%
.95-NORMAL CHROM. 3
FIGURE1.-Representation
approximate.
INVERTED CHROM. 3
of breakpoints for maize Inversion 3a. Gene locations are
P A I R I N G I N ALLOTETRAPLOIDS
517
viable seeds. Several lots of seed were germinated both in the sand bench and
out-of-doors. Cytological studies were made using the acetocarmine technique.
All quantitations were from whole cell analysis. All scorable cells were studied
in a preparation to ensure randomness. Controls were established as shown in
Figure 2, to include duplex 4n maize with no inversion, duplex maize with
duplex Inversion 3a, and the duplex 4n interspecific hybrid with no inversion
in chromosome 3.
EXPERIMENTAL RESULTS
Genetic results: The testcross results given in Tables 1 and 2 show that the
presence of the inversion in pure maize markedly altered the frequency of recessive gametes for a, from 17.8 percent to 12.4 percent, and for Zgefrom 17.4 percent to 12.6 percent. These results correspond satisfactorily to those of DOYLE
(1960) who found that the (duplex) presence of Inversion 3a reduced the frequency of al gametes from 20.8 percent to 12.6 percent. The proportion of crossover type gametes was even more markedly reduced by the presence of the
inversion from 16.9 percent (two classes combined) to only 5.0 percent. The
physical basis of preferential segregation may be deduced from Figure 3. As
shown, with random bivalent pairing, five-sixths of the testcross progeny will
be plus type. With random disjunction of quadrivalents, ignoring double reduc-
DUPLEX AUTO TETRAPLOID
MAIZE
DUPLEX
HYBRID 4 n
INTERSPECIFIC
MAIZE x4n TEASINTE
5
HETEROZYGOUS
MAIZE
STRUCTURALLY HETEROZYGOUS DUPLEX
INTERSPECIFIC HYBRID 4 n MAIZE x 4 n TEASINE
FIGURE
2.-Diagrammatic representation of the four types of heterozygotes studied. The
straight line represents maize chromatin, the wavy line represents teosinte chromatin, and the
boxed-in regions represent the relatively inverted region in maize.
518
DONALD L. SHAVER
TABLE 1
Testcross results from four types of duplexes
k9
B'
a1 k,
Total
114
8.6%
122
9.2%
1326
,420
41
2.6%
39
2.4%
159
10.0%
1589
,7411
2953
86.1%
49
1.4%
25 7
7.5%
170
5.0%
3429
,483
3407
96.3%
11
0.3%
25
0.7%
96
2.7%
3540
.833
A , Lg,
A,
98 1
74.0%
109
8.2%
Structural!y heterozygous
duplex 4n maize
1350
85.0%
Duplex hybrid of 4n
maizex4.n teosinte
Structurally heterozygous
duplex hybrid of 4n
maizeX4n teosinte
_______
Duplex 4n maize
~-
a, Lg2
* Beta values are coefficients of tetraploid linkage calculated from the function given by SHAVER(1960, 1963). A value
of unity indicates coniplete linkage, a value of zero indicates no linkage.
-;- This value ir imdouhtedly somewhat biased because uf bridge-related loses of ~narkers( D O Y L E 19152, personal comniuniration 1
TABLE 2
Single-gene testcross dula from four types of duplexes
Perrent
a,
S value'
a,
0.0
'gr
S value'
18,
17.4
0.0
Percent
Duplex 4n maize
17.8
Structurally heterozygous duplex
4n maize
12.45
,301
12.58
,277
Duplex hybrid of 4n
maize~4.n
teosinte
12.4
.301
6.4
,633
3.4
,806
3.0
.827
Structurally heterozygous duplex
hybrid of 4n m a i z e ~ 4 . n
teosinte
* S values m e <oelficients of preferential segregation according to SH.hvm il962a). A value of zero indicates a lack o f
preferential srgiegation, and B value of unity indicates completely preferential segregation.
tion, the testcross results are identical. Because there is no reason (SHAVER
196213)
to presume preferential disjunction from quadrivalents. it is assumed that in
this material a n increase in preferential segregation stems directly from a n increase in the frequency of preferential pairing, i.e., a n increase in the frequency
of preferential bivalents. T h e crossover type gametes, though infrequent, are
expected from the cytological data of DOYLE(1960), who found evidence of a n
appreciable amount of heterosynaptic crossing over in the loop from his study
of bridge frequency. However, the Beta statistic, designed by SHAVER(1963) to
give a measure of linkage in tetrasomics which would be essentially free of the
swamping effects of preferential segregation, indicates a reduction in crossover
potential between relatively inverted regions, presumably even when the four
homologues associate as a quadrivalent (Table 1 ) . However, because of bridgerelated losses of dominant markers, crossing-over data m a y be biased somewhat.
519
PAIRING I N ALLOTETRAPLOIDS
PREFERENTIAL PAIRING
M I
14:
14:
GAMETES
A I
___,
ALL OF TEST CROSS
PROGENY
PLUS TYPE
NON-PREFERENTIAL PAIRING
( I)
AS ABOVE. OR
4
@@
THREE POSSIBLE
ORIENTATIONS:
ALTERNATE
ADJACENT-I
ADJACENT-2
THREE- FOURTHS OF TEST
PROGENY PLUS TYPE
.--*
CROSS
FIVE-SIXTHS OF TEST CROSS
PROGENY PLUS TYPE
FIGURE
3.-Physical basis for the effects of preferential and nonpreferential pairing on
testcross progeny ratios. Double reduction and numerical nondisjunction are ignored for the
sake of clarity.
This apparent reduction in crossing-over potential agrees with a similar observation based upon cytological data by DOYLE (1960).
The testcross results for the two types of interspecific duplexes (Tables 1 and 2)
show that the presence of the inversion exerts an important effect on the degree
of preferential segregation. Thus as seen in Table 2 the proportion of algametes
was 12.4 percent without the inversion, and only 3.4 percent when the inversion
was present. Likewise, the frequency of Zg, gametes was reduced from 6.4 percent
to only 3.0 percent. Coefficients of preferential segregation were altered from .301
to .806 and from .633 to .827 respectively. The proportion of crossover gametes
was more markedly reduced, from 8.9 percent to only 1.O percent.
Cytological observations: Cytological studies of MI, AI and AI1 in the two types
of interspecific hybrid, given in Tables 3 and 4, show that the number of chromosome associations at diakinesis was larger in the structural heterozygote than in
the noninverted interspecific version (control), 17.19 us. 16.84 ( t = 2.800, P =
0.02).
The tabulated bridge frequency in the structural heterozygote was slightly
less than that of the control. None of the bridges scored appeared to be typical
inversion bridges as illustrated by DOYLE
(1960) in 4n maize. Rather, all appeared to be misdividing, bridge-formingunivalents, as shown in Figure 4. Thus
it appears that no evidence of inversion-related bridge formation was found in
over 474 dividing cells.
520
DONALD L. S H A V E R
TABLE 3
Meiotic anaphase bridge frequency in normal and structurally heterozygous
4 n hybrids of maize and perennial teosinte
Anaphase IT
Anaphase 1
No hndge
Normal plants
7922
7924 1st collection
7924. 2nd collection
7925 1st collection
7925 2nd collection
62
50
70
103
6
I
29 1
Structurally heterozygous plants
7913 1st collection
7913 2nd collection
7914
7920 1st collection
7920 2nd collection
7920 3rd collection
7920 4th collection
Bridge
No bridge
1
3
31
0
46
10
5
2
0
0
_
_
_
6 = 2.06%
1 = 1.09%
92
153
0
0
3
37
48
51
94
12
45
25
312
-
Bridge
0
0
0
6 = 1.92%
156
0
TABLE 4
Number of chromosome associations present at diakinesis in normal and structurally
heterozygous 4n hybrids of maize arid perennial teosinte
Normal plants
7922
7924 1st collection
7924 2nd collection
7925 1st collection
7925 2nd collection
Number
of cells
No. chraqoscme
82
53
35
67
16.97 +- .I7
15.77 4 .21
16.49 k .23
17.04. i .I6
17.98 ?- .25
41)
277
Structurally heterozygous plants
7913 1st collection
7913 2nd collection
7914
7920 1st collection
7920 2nd collection
32
66
42
92
31
263
as6c€latIons
average 16.84 k .09
16.65 i .23
17.15 -C . I 4
17.17 & .I8
17.70 k .I4
16.35 t .28
average 17.19 -+ .08
Uniformity of data: In obtaining testcross data, usually several ears from each
of several different plants composed any given sample of gametes. Chi-square
values were obtained for each sample component. Among 22 chi-squares testing
the homogeneity of separate ear ratios pollinated from the same plant, only one
521
P A I R I N G I N ALLOTETRAPLOIDS
FIGURE
4.-Photomicrogrnph
maize
x
of n hritlgc-forming univiilmt at i\l
111
thv intt.npt.cific hyhrid
perennial t m i n t c . 2000 X .
value indicated a significant deviation at the five percent level. This indicates
that the data were derived by means of satisfacto? techniques. However. among
eleven chi-squares testing the homogeneity of data among the progenies of different plants of the same genotypes, three significant deviations were noted, one
at the five percent level and two at the one percent level of confidence. This lack
of uniformity of testcross data from different plants of identical genotypes found
here corresponds to similar results obtained by SHAVER(1960, 1962a), DOYLE
il960) and WELCH(1962). However, there appear to be no data as yet which
form a reasonable basis from which to choose between the several intriguing
possible explanations of this phenomenon, which now seems to be real.
DISCUSSION
The failure to find cytological bridges: The fact that no inversion bridges were
found in the allotetraploid inversion heterozygote is somewhat discordant with
the testcross data showing a frequency of crossover gametes of 1.0 percent. It
seems best at present to consider that this figure represents the resolution limit of
the experiment. and that these ‘Lcrossover”gametes are not real. Reasons for the
low level of resolution in this experiment (by diploid standards) may relate to
misclassification of progeny especially for Ig, due to dosage effects in tetraploids.
or due to induced mutation hecause of the presence of teosinte and maize chromatin in the same genome ( MANCELWORP
1958). If. however, one percent is the
limit of accuracy, this is still small when compared with the very large effect of
the type of structural hybridity studied here on preferential segregation.
Elfects on chromosome stabiliry of a macro-inuersion in tetmploids: In autotetraploid maize, CATCHESIDE
(1 956) calculated that in functional female gametes, the proportion in which a chromosome in any homologous set was deficient
or in excess was about 3.8 percent. In his study of 4n maize duplex for Inversion
321, DOYLE(1960) found that 11.8 percent of AI cells had single bridges. 0.7 per-
5 22
DONALD L. S H A V E R
cent had double bridges, 1.5 percent had two bridges, and 5 percent of AI1 cells
had a single bridge. Since gametes of tetraploids tolerate excesses or deficiencies
of whole chromosomes, it is reasonable to expect that partially duplicate o r deficient chromosomes would be transmitted. For the purposes of rough calculation,
we may assume that each bridge results in two duplicate o r deficient chromosomes. Hence the phenomenon of bridge formation studied by DOYLE
should as
an over-all result produce about 16 percent of gametes with duplicate or deficient
chromosome 3. This seems more than sufficient to swamp any possible reduction
in CATCHESIDC'S
figure of 3.82 percent through a reduction in quadrivalent frequency. Even though it is as yet impossible to say whether any bridge-altered
chromosomes may be of special advantage in some future situation in the autotetraploid, it is nevertheless certain that Inversion 3a does not contribute to
chromosomal stability per se.
In the allotetraploid studied here, the situation is quite different. Since interspscific crossing over is abolished in the relatively inverted region, this structnral hybridity per se does not immediately bring with it the attending disadvantage of duplicate-deficient chromosome production. If we make numerical
nondisjunction proportional to quadrivalent frequency, one can estimate numerical nondisjunction to result in 1.2 percent of female gametes unbalanced for
chromosome 3. Because quadrivalent frequency for chromosome 3 is reduced by
the presence of Inversion 3a in the structurally hybrid allotetraploid, one can
calculate that numerical nondisjunction for chromosome 3 would be reduced to
only 0.5 percent. Although there are more possible advantages and disadvantages
that could be considered, the parameters do not seem as yet to be at hand to make
further speculation meaningful. A more obvious point is that since preferential
pairing is still not perfect, some sexual derivatives may be expected to be triplex
or simplex for the inversion. One should now expect bridge-related complications,
unless the decrease in crossover potential in the loop noted by DOYLE
(1960) in
maize operates in this case to below a threshold level. However, such simplex
and triplex individuals would be rare, and if successful ecological competition
depended otherwise upon balanced hybridity for the inverted segment, these
would already be functionally inviable.
Evolutionary considerations: Both cytological and genetic data indicate that
chromosome 3 would be more faithfully transmitted to progeny by the structurally hybrid allotetraploid than by the normal allotetraploid because of a reduction in quadrivalent frequency Hence this type of macro-rearrangement would
be a positive rather than a negative force in diploidization. It is possible then to
extrapolate that ordinary divergence in chromosome structure between related
species may yroceed through mutation or cryptic structural changes up to a
threshold level, which appears to be surprisingly low. Beyond this threshold level,
allotetraploids if produced in nature, may retain the specific integrity of large
blocks of chromatin because of the abolition of interspecific crossing over due to
the heterozygous presence of large inversions. DOYLE
(1960) noted that structural
hybridity reduced the crossing-over potential in the rearranged region. I n the
present study, this reduction appears to have been reinforced by the addition of
P A I R I N G I N ALLOTETRAPLOIDS
523
interspecific nonhomology with the net result of essentially no effective pairing
between relatively inverted segments. It is important to note that if one can
generalize from the results reported here, direct cytological observations of allotetraploids do Got allow a decision as to the presence or absence of inversions.
In the present case, structural hybridity would have been undetectable in the
absence of prior knowledge of its existence.
That the threshold phenomenon may be general in this allotetraploid may be
inferred from a previous study by the author (SHAVER
1962a). From genetic data,
he concluded that perennial teosinte has a large inversion in short arm chromosome 9. Hence any hybrid between 4n maize and perennial teosinte would be
duplex for this inversion. However, he has been unable to demonstrate bridge
formation due to this putative inversion in a total now of over 1000 AI cells.
SUMMARY
By mating appropriate stocks of 4n maize with 4n perennial teosinte, two
types of allotetraploids were produced, one carrying Inversion 3a as a structurally heterozygous region, and the other without structural hybridity. Similarly,
structurally heterozygous and non-heterozygous autotetraploids of pure maize
were produced. The genetic effect of the defined chromosome rearrangement on
preferential segregation was measured from testcross ratios of two linked gene
markers in the loop. I n the allotetraploid, the average segregation ratio for the
contained markers was altered from 10.6: 1 to 31.2: 1 by the insertion of structural hybridity. In the autotetraploid the average testcross ratio was altered from
5.7: 1 to 8.0: 1. Calculations based upon bridge frequency indicate that since there
is a high production of duplicate and deficient chromosome types in the structurally hybrid autotetraploid, this macro rearrangement would not be a stabilizing influence, in spite of its notable effect of increasing the degree of preferential
segregation. In the allotetraploid, however, inversion-bridge formation appeared
to be absent, presumably because of a threshold effect. It is concluded that in
nature, this type of structural hybridity would be a positive influence in diploidizing this allotetraploid by greatly increasing preferential pairing, and by preserving large blocks of chromatin inviolate against interspecific crossing over.
ACKNOWLEDGMENTS
The author is indebted to G. G. DOYLE
for the 4n maize inversion stock used
in making the hybrids studied here. He is indebted to L. F. RANDOLPH for the
clone of perennial teosinte. For critical readings of the manuscript and many
STEBBINSand G. G.
useful suggestions thereto he is indebted to G. LEDYARD
DOYLE.
524
DONALD L. SHAVER
LITERATURE CITED
CATCHESIDE,
D. G., 1956 Double reduction and numerical nondisjunction in tetraploid maize.
Heredity 10: 205-218.
CLAUSEN,
R. E., 1941 Polyploidy in Nicotiana. Am. Naturalist 75: 291-306.
CLAUSEN,
R. E., and D. R. CAMERON,
1950 Inheritance in Nicotiunu tubacum. Duplicate factors
for chlorophyll production. Genetics 35: 4-10.
DOYLE,G. G., 1960 Preferential pairing in structural heterozygotes of Zeu mays. Ph.D. thesis.
University of Illinois, Urbana, Illinois.
FISHER,R. A., 1935 The sheltering of lethals. Am. Naturalist 69: 446-455.
MANGELSDORF,
P. C., 1958 The mutagenic effect of hybridizing maize and teosinte. Cold Spring
Harbor Symp. Quant. Biol. 23 : 409-421.
RHOADES,
M. M., and ELLENDEMPSEY,1953 Cytogenetic studies of deficient-duplicate chromosomes derived from inversion heterozygotes in maize. Am. J. Botany 40: 405-421.
SAX,K., 1933 Species hybrids in Platanus and Campsis. J. Arnold Arboretum 14: 274-278.
SHAVER,
D. L., 1960 Cytogenetic studies of tetraploid hybrids of Euchluenu perennis and Zea
mays. Ph.D. thesis. University of Illinois, Urbana, Illinois.
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Botany 49 : 348-354.
1962b A study of meiosis in perennial teosinte, in tetraploid maize and in their tetraploid
hybrid. Caryologia 15: 43-57.
1963 Linkage i n autotetraploids and allotetraploids of Zea. (In preparation.)
G. L., 1947 Types of polyploids: their classification and significance. Advan. Genet.
STEBBINS,
1: 403429.
1950 Variation and Evolution in Plants. pp. 298-379. Columbia University Press, New York.
WELCH,J. E., 1962 Linkage in autotetraploid maize. Genetics 47:367-396.