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Reversion frequency of waxy pollen type in normal and hypoploid maize plants
352
R E V E R S I O N F R E Q U E N C Y OF W A X Y AND H Y P O P L O I D MAIZE PLANTS
MUTATI()N RESEARCH
P O L L E N T Y P E 1N NORMAL
A. B I A N C H I AND C. T O M A S S I N I
Istituto di Genetica, Universit~ di Milano, Milano, Istituto di Genetica Vegetale, Universit~ Cattolica, Piacenza and Istituto di Allevamento Vegetale, Bologna* (Italy) ( R e c e i v e d M a r c h 29th, 1965)
SUMMARY
Maize pollen grains of constitution Y g I S h B z W x were used to fertilize a stock possessing the corresponding recessive genetic markers y g C sh bz w x of the short arm of chromosome 9. Appropriate screening of the ensuing seedlings, together with the results of appropriate testcrossing and scoring of the pollen, made it possible to isolate hypoploid plants in which the chromosome 9 carrying the dominant markers had lost at least the W x gene. This gene is known to affect the chemical nature of the starch, both in endosperm and in pollen grains. The pollen produced b y such plants was only influenced by the w x gene of the undamaged chromosome. The reversion rate of this gene--on the basis of the appearance of pollen grains giving the W x chemical reaction (with iodine solution)-was measured and compared with the reversion rate of normally diploid w x w x plants. On the basis of results with yeast, the expectation was that the normal chromosome condition should have produced a higher reversion rate than the genotype in which the hypoploid condition associated with the deficiency of the genetically marked region provided no opportunity for pairing and crossing-over, which was supposed to be responsible for that high reversion rate. In maize the experimental data failed to confirm a similar behaviour. This finding suggests t h a t the w x alleles investigated are likely to be due to base substitution. In such a case unequal crossing-over, causing base losses and insertions in the DNA and, therefore, restoration of a sequence compatible with normal functioning, appears unlikely. Mutagenesis in these cases appears to rely only on mutational events s e n s u stricto and associated with a suppressor system. In maize such a requirement seems to be satisfactorily met with the controlling elements.
INTRODUCTION
During the past thirty years great efforts have been made to understand the mechanism of origin and the nature of mutational events in m a n y organisms, especially on the basis of the artifical induction of mutations. Much less knowledge has * P r e s e n t a d d r e s s of t h e a u t h o r A. BIANCHI.
Mutation Research 2 (1965) 352-365
.\. BIAN('HI ANI) (.-I'~)M.\SSINI
D
C
1~'
A
Fig. I. The results o{ backcrossing ye~lo~-green p l a n t s of the cross yg C sh bz ~,x . irradiated Y g I Sh B z W x b y Yg C sh be' w.r. l:rom left: A. E a r showing segregation of all the endosperm markers, and, consequently, d e m o n s t r a t i n g t h a t the breakage point was b e t w e e n Yg and 1 ; notice also, as c o n f i r m a t o r y d a t u m , the almost n o r m a l fertility. B. E a r in which the I factor is absent: the b r e a k has been induced between l and Sh. The see}] setting is reduced. C. Ii1 this ear no donlin a n t character is detectable, the fertility is very poor. The breakage m u s t have been induced beyond W*, so as to cause the loss of ah the genetic m a r k e r s of c h r o m o s o m e 9. l). E a r in which only t h e W x m a r k e r has been lost, all other e n d o s p e r m d o m i n a n t t r a i t s being recognizable. The interstitial deficiency c a n n o t have been large, in fact the fertility is not greatly reduced.
E R R A T U M : This figure should be readj?om right to left.
Mutation Research 2 (1965) 352-365
R E V E R S I O N F R E Q U E N C Y OF w a x y POLLEN T Y P E IN MAIZE
353
accumulated on the intimate phenomeI~a responsible for the occl~rrence of spontaneous mutation. However, the present information on the structure of DNA, and the data obtained from the studies of artificially induced and spontaneously occurring mutations seem to suggest that the so-called point mutations can be grouped into two main classes: base substitutions and base deletions or insertions. In Saccharomyces cerevisiae MAGNI et al.7-1 o present convincing evidence that, at least for mutants supposed to be of the latter class, an important fraction of the spontaneously occurring mutations are associated with (unequal) crossing-over. MAGNI9 also points out that similar behaviour is not restricted to this species, provided that special caution is adopted in collecting and analysing the data. However, no special studies have been performed to demonstrate a more general occurrence of this phenomenon, with the exception of some experiments which are being carried out as a consequence of the MAGNI finding. Of course, it is generally accepted that the Bar mutants in Drosophila are a product of unequal crossing-over. Moreover, in maize, the occurrence of unequal crossing-over at the R region in chromosome IO was explained b y STADLER et al. ~°-~ as a source of a large fraction of mutants. However, although the important role in spontaneous mutagenesis of the "oblique synapsis" was accepted, no a t t e m p t was made to compare the mutation rate caused b y this process with the mutation rate in situations in which regular pairing between homologous chromosomes was not possible. On the other hand, L A U G H N A N ~, in the fine structure analysis of the alpha derivatives from the A L p complexes in maize, finds that these mutants are at least as frequent among the progeny of deficiency heterozygotes--where obviously there was no opportunity for exchanges between the homologous p a r t n e r s - - a s they are from normally diploids. This work has been undertaken in order to compare the reversion frequency of maize plants, in which normal synapsis and crossing-over occur, with that of individuals in which these processes are entirely missing, and, consequently, to confirm the undifferentiated behaviour of mutagenesis in the two chromosome situations, such as found b y LAUGHNAN~, o r to recognize also for maize the "meiotic effect" on spontaneous mutations as found b y MAGNI9 in Saccharomyces, at least for a class of mutants. MATERIAL AND METHODS
Spontaneous mutation is usually a rare event; consequently special devices and/or appropriate material are needed to obtain data from which to draw dependable conclusions, with feasible work and in reasonable time. In maize these requirements are uniquely satisfied with the waxy trait which, for a long time, has been known to affect the chemical nature of the pollen starch. With appropriate iodine solution the waxy pollen grain stains brown; the W x type stains dark blue. NELSON is suggested appropriate details for exploiting these properties in studies of the genetic fine structure and of the intracistron recombination. With minor modifications, this technique was used b y BIANCHI AND CONTIN1 in the study of artificial mutagenesis. The same fixing and staining procedures have been adopted for the present work. The waxy allele employed was kindly provided several years ago b y Dr. B. Mutation Research 2 (1965) 352-365
354
A. B I A N C H I A N D C. T O M A S S I N I
McCLINTOCK, and has been maintained by us during this period by self-pollinating the plants of the successive generations. The stock possesses, besides the complementary colour factors on the different chromosomes such as A 1, A 2, R in homozygous conditions, the following markers of chromosomes 9 : Yg2 C Shl bz wx. This strain has been used for estimating the reversion rate of the wx allele to l~'x in the mature pollen grain population in the normally diploid condition. For the study of the reversion in conditions that lacked homologous pairing and crossing-over phenomena, hypoploid counterpart has been obtained as follows: the McClintock stock was fertilized with pollen of a stock obtained from the Maize Genetics Cooperative, Urbana, Ill. (courtesy of Dr. E. PATTERSON).The constitution of thisstockwasA 1, A 2, R, Yg~I Shl Bz W x . Before pollination the pollen was X-irradiated with 10oo-2ooo R. The kernels produced were planted in the field and the seedlings scored for chlorophyll-deficient characters. The great majority of them were normally green and were eliminated: the very few yellow-green seedlings, carefully protected from any possible damage, were grown to maturity. Some of the yellowgreen plants were unable to extrude anthers and/or silk, and they were discarded too. The remaining individuals were pollinated by the McClintock stock, and their tassel, at the very beginning of anthesis, was fixed for the study of the pollen. Another wx source was kindly provided b y Dr. O. E. NELSON in the form of an unstable allele of this locus, known as wx -~t-lA. As mentioned above, the technical procedure for fixing, staining and scoring of the pollen was essentially the same as that of NELSON. However, for special purposes, single anthers were scored too. With the aid of teasing needles, the anther content was poured into a drop of the standard staining solution. During the procedure the pollen sample was carefully protected from air with a cover-glass of appropriate size on microscope slides. For special comparisons the compound type wxg°/wx c, obtained by crossing the strains bearing the two pseudoalleles wx 9° and wxC (again received from Dr. NELSON), was also studied with single anther analysis. RESULTS
As mentioned above, the critical material was to be found in some plants of the cross yg C sh bz wx × Y g I Sh B z Wx. Following X-irradiation, the loss of the I Sh Bz markers is very frequently accompanied by the loss of the W x locus 2. It was therefore considered that, once screening for yg seedlings had been made, the loss of Y g could be used as a first appropriate index of loss likely to involve W x too. In fact, in a sample of 57 yellow-green plants so obtained, the results of the baekcross to the multiple recessive yg C sh bz wx demonstrated that 2o were deficient for only Yg, 4 for Y g and I, 2o for all the markers, i for only W x (Fig. I), and 2 were almost completely sterile. Of course, the partial sterility of these plants generally increases, going from the first group to the last. Moreover, out of 57 tassels collected from yellowgreen plants, 37 showed practically only wx pollen grains associated with at least 5o% sterility, whereas 18 presented also W x grains (about 50%), and 2 W x grains in values of lO-12% (these two groups did not have very severe pollen sterility). Obviously, only the plants which turned out to be deficient for W x were considered and used in the successive pollen analysis. ~Iutation Research 2 (I965) 352-365
R E V E R S I O N F R E Q U E N C Y OF w a x y P O L L E N T Y P E I N M A I Z E TABLE
355
I
REVERSION RATE AT THE W,~ LOCUS OF HYPOPLOID AND NORMAL TASSELS FROM PLANTS GROWN IN TWO DIFFERENT YEARS
Year and chromosome type
Tassel No.
1963; h y p o p l o i d
lO92- 3 lO64-1 lO69-2 lO69-1 lO64- 4
Number of W x pollen grains
Frequency of W x pollen grains ( × Io -6)
572669 112616 733 192 3865oi 1o34oo 19o8378
28 I 68 7° o I67
4.9 o.9 9.3 18.1 o.o 8.7
14.o - 2 2 . 9 o.o - 3.5 7.5 - l O . 2
56-1
618o21 695767 1540347 2256349 2o3o517 3288372 308646o 13515833
13 21 229 374 168 128 48 981
2. I 3.0 14.9 16.6 8. 3 3.9 1. 5 7.2
I. i 1.9 13.o 14.9 7.1 3 .2 I.I 6.8
- 3.6 - 4.6 -16.9 -18.3 - 9.6 - 4 .6 - 2.1 - 7.7
13o9 1291 1298 1282 129o 1283 1291
448860 73OLO 4o663 ° 74o8o 914850 98955 ° 1117350 402433 °
29 o 12 15 6 7 19 88
6.4 o.o 2.9 20.2 o.6 0.7 1. 7 2.2
4.3 o.o 1.5 1.3 0.2 0-3 i.o 1. 7
- 9.3 - 5.0 - 5.1 -31.7 - 1. 4 - 1.4 - 2.6 - 2. 7
1267-1 1267-2 1267-3 1267- 4
1967200 34388o 3° o o 6 8 0 66921o 598o97 o
14 4 57 7 82
0. 7 1.2 1.9 i.o 1. 4
0. 4 0. 3 1.4 0. 4 i.I
- 1.2 - 3.0 - 2.4 - 2.1 - 1. 7
Total and average 1963 ; n o r m a l
56- 7 56-11 56-16 56-2 56-3 56- 4 Total and average 1964; h y p o p l o i d
Total and average 1964; n o r m a l
Total and average
Estimated number of pollen grains
Fiducial limits for P o.o 5 level* ( × Io -5) 3 .2 - 7 .1 o . o 2 - 4.9
7.2 - I 1.8
* C a l c u l a t e d a c c o r d i n g t o STEVENS 24.
The pollen preparations of such plants, moreover, not only demonstrated the lack of the Wx-bearing chromosome segment but also, as expected, the presence of at least 50% of empty pollen grains. The results of these analyses are summarized in Table I. This shows that the reversion rate is highly variable, and that this variability may derive from different sources. The table immediately suggests that the year is an important factor, producing a 4-5 fold effect in the hypoploid as well as in the normal chromosome condition on the overall reversion rate. However, if instead of the overall frequency the median value (which of course has the advantage of being less influenced by the higher rates) is considered, the discrepancy between the two-year data is statistically less significant (in the case of the hypoploid genotype) or scarcely significant (for the diploid series). On the other hand, within data for both years, there is no indication that the normal condition is associated with a higher rate of reversion; actually in both years this rate is rather lower than in the hypoploid*. * E v e r y e f f o r t h a s b e e n m a d e t o a v o i d c o n t a m i n a t i o n b y c o n d u c t i n g e x p e r i m e n t s in p r o p e r l y i s o l a t e d f i e l d s ; b u t i t is p o s s i b l e , e v e n if u n l i k e l y , t h a t t h e h i g h e r r a t e of W x g r a i n s in 1963 is d u e t o s o m e w i n d - b o r n e p o l l e n f r o m r a r e a n d d i s t a n t fields.
Mutation Research 2 (1965) 3 5 2 - 3 6 5
356
A. BIANCHI AND C. TOMASSINI Another
plants,
important
which,
s o u r c e of v a r i a t i o n
although
detectable
for both
appears
to be that
genotypes,
of t h e i n d i v i d u a l
is u n q u e s t i o n a b l y
more
e v i d e n t i n t h e h y p o p l o i d c l a s s , B u t t h i s h e t e r o g e n e i t y m a y b e t h e c o n s e q u e n c e of t h e heterogeneity
which can be detected
orderly and separately
a t d i f f e r e n t l e v e l s , w h e n t h e p o l l e n a n a l y s i s is
carried out on the different branches
of t h e t a s s e l , o n t h e
d i f f e r e n t r e g i o n s of t h e b r a n c h e s , d o w n t o t h e d i f f e r e n t s i n g l e a n t h e r s . F o r t h e s a k e of brevity,
the detailed data obtained
examples
may be extracted
i n t h i s k i n d of a n a l y s i s a r e o m i t t e d ,
and presented,
but a few
s i n c e t h e y m a y b e of u s e in d i s c u s s i n g
later the general results. Table II reproduces
an example
of t h i s k i n d of d a t a
for the normal
diploid
TABLE II INCIDENCE
OF
~TX P O L L E N
GRAINS IN DIFFERENT
P O S I T I O N S O F A T A S S E L O F T H E N O R M A L ¢~)X fI;x
GENOTYPE
Branch
Position in the branch
rom bottom to top I.
Total I[
Total iII
Total IV
Total V
Total Grand total
Estimated Number number of of W x stainable pollen pollen grains grains
Rate of Wx pollen grains ( X IO-~)
Fiducial limits for the Wx rate of pollen grains ( × xo -5)
I 2 3 4 5 1- 5
3905 ° 53600 23300 3tSOO 34433 181883
o o o o I r
o.o o.o o.o o.o 2.1 o. 5
I 2 3 4 5 1-5
42866 53633 4455 ° 35667 179o0 I94616
o o I I 2 4
o.o o.o 2.2 2.8 I 1.2 2.I
o.o 8.6 o.o - 6.9 o . o 5 - I 2. 5 o.I -15.6 1.3 -4o.3 o.6 - 5-3
I 2 3 4 1- 4
4435 ° 8 3 ioo 43333 6255 ° 233333
o o i o I
o.o o.o 2. 3 o.o o. 4
o.o - 8,3 o.o - 4.4 o.o5-I2.8 o.o - 5-9 O.Ol- 2.4
i 2 3 4 5 i- 5
839o0 72000 54700 34833 3o3oo 275733
14 4 21 18 i 58
I6. 7 5-5 38. 4 51.7 3.3 21 .o
9. I 1.5 23.8 30.6 o.i 16.o
i 2 3 4 5 6 7 8 9 io i-io
83233 84566 748o0 763 °0 50900 85300 4895 ° 5035 ° 38833 6155 ° 654782
18 4 33 5 27 2 5 36 28 7 165
21.6 4.7 44 -1 6.5 53.0 2.3 lO.2 71.4 72.1 i 1.4 25.2
12.8 -34.2 1. 3 - i 2 . 1 3o.4 -62.0 2.1 -15.3 33.0 -73 .0 0.3 - 8.5 3-3 -23.8 5 ° .i - 9 9 . I 48.O-lO4.3 4 .6 - 2 3 ' 4 21.5 -29.4
154o347
229
14.9
x4.o -16.o
Mutation Research 2 (I965) 352-365
o.o o.o o.o o.o o. 7 o.o
- 9.4 - 6.9 -I5.8 -II.7 -16.2 - 3.1
-28.0 -I4.2 -56.9 -81.7 -18. 4 -27.2
R E V E R S I O N F R E Q U E N C Y OF TABLE
357
w a x y POLLEN T Y P E IN MAIZE
In
INCIDENCE OF
WX POLLEN GRAINS IN DIFFERENT POSITIONS OF A TASSEL OF TIlE HYPOPLOID WX
GENOTYPE
Branch
Position in the branch
Estimated number of stainable pollen grains
I 2 3 4 5 I-5
31267 2o167 15o33 13767 133oo 93534
o I I 2 I 5
o.o 4-9 6.6 14. 5 7.5 5.3
o.oo.Io.21. 7 o.21.7-
11.8 27.6 37.o 52.4 41.9
I 2 3 4 1- 4
2655 ° 15o5o 2o45o 24833 86883
o o 3 I 4
o.o o.o 14.7 4.o 4 .6
o.oo.o3.oo.I1.2-
13. 9 24. 5 42.9 27.2 I 1.8
i 2 3 I- 3
14467 I4567 1o6oo 39634
n 6 o 17
76.o 41.2 o.o 42.9
37.9-136.o I 5 . 1 - 89.6 o . o - 34.8 2 5 . o - 68.7
i 2 3 1-3
424 °o 25574 17400 85374
9 17 i 27
21.2 66. 5 5.7 31.6
9 - 7 - 4o.3 38.7-1o6.4 o . i - 32.o 2 o . 9 - 46.o
i 2 3 4 1-4
16900 lO567 16567 15500 59534
o i 1i 2 14
o.o 9.4 66. 4 12.9 23.5
o . o - 21.8 2 . 4 - 52.7 33.1-118.8 1.5- 46.6 1 2 . 8 - 39.4
364959
67
18. 3
1 4 . 2 - 23. 3
from bottom to top I
Total II
Total III
Total IV
Total V
Total Grand total
Number o/Wx pollen grains
Rate o / W x pollen grains ( × Io s)
Fiducial limits for the W x rate of pollen grains ( × io-S)
12.5
condition, and Table n I an example from a hypoploid tassel. The heterogeneity, often highly significant, in the reversion rate at the wx locus in both genotypes can be easily demonstrated with the Z* test (as has been done). This holds true for the different branches as well as for the various positions within the same branch. Anyway, in the tables, in order to make comparison more immediate, the fiducial limits (calculated according to STEVENSZ4 at the 0.05 P level) are reported. The fact that the distribution of the W x grains is often of non-Poisson type, would suggest the use of other statistical approaches. Better estimates, as those based on median values mentioned above, require other sets of data, not always obtainable from our material. However, for asymmetric distributions as those of the W x frequency classes, the use of STEVENS tables should not lead to serious bias. The mentioned heterogeneity is detectable also at the anther level. In other words, if the single anthers are scored for W x grains and the frequencies of the different classes (possessing o,I,2 . . . . n W x grains) are obtained, the distributions of Table IV can be prepared. Inspection of such a table suggests that these distributions often show an excess of some classes; a sort of clustering of the W x grains is often evident. The Mutation Research 2 (1965) 3 5 2 - 3 6 5
358 TABLE
A. BIANCHI AND C. TOMASSINI
IV
FREQUENCY
DISTRIBUTION
OF THE
H/'X P O L L E N
GRAINS
IN SINGLE
ANTHER
SMEARS
Entire Anthers showing Wx poUen grains in Genotype tassel (T) number of or single o --~ 2 3 4 5 6 7 >/8 branch (B)
Z2 value
Degrees V"2~ of -j~'eedom ~,'2ti±i (n)
1"
T B T B T B T B T B T B
5362.1 491.2 1512.o 80.0 1828.3 443.0 II5I.O 292.0 1413. 7 200.0 1287. 4 89.0
lO67 161 1169 IiO 899 149 899 176 973 143 832 87
v e r y small v e r y small v e r y small 0.98 v e r y snlall very small v e r y small very small very small 0.003 v e r y small 0.4o
lOO6 15 ° 1129 1°5 887 147 894 175 338 31 296 14
43 8 l 5 i i 8 i 1 I o i 35 5 t o o o 5 1 o o o o io I 1 r o o i i o i o o 5 i o o o o i 1 o o o o 283 194 90 45 13 6 52 31 16 7 4 i 224 139 lO7 37 18 9 22 20 20 7 2 2
o o o o o o o o i o i o
3 o o o o o o o 4 2 2 o
normal normal normal normal hypoploid hypoploid hypoploid hypoploid c o m p o u n d wxg°/wx c c o m p o u n d wxg°/wx ¢ c o m p o u n d wx'°/wx c compoundwx'°/wx ¢
57.4 13. 4 6.6 --2.2 18.o 12. 5 5.6 5.3 9.0 3.0 9.9 0.2
Fig. 2. S m e a r of a single a n t h e r c o n t e n t s h o w i n g t h r e e d a r k - s t a i n i n g grains (Wx type) g r o u p e d in t h e c e n t e r as c o n t r a s t e d to t h e b r o w n - s t a i n i n g g r a i n s of t h e wx t y p e . O n e of t h e d a r k - s t a i n i n g g r a i n s is clearly s m a l l e r t h a n t h e s t a n d a r d t y p e (see text). Fig. 3- Pollen g r a i n s in a n a n t h e r of a p l a n t w x M - 1 A w x M-1A s t a i n e d w i t h a p p r o p r i a t e iodine s o l u t i o n : t h e n o n - r a n d o m d i s t r i b u t i o n of t h e g r a i n s s t a i n i n g differentially, is evident.
s t a t i s t i c a l t e s t (Z 2 f o r P o i s s o n d i s t r i b u t i o n ) c l e a r l y c o n f i r m s t h e d e v i a t i o n o f t h e d i s t r i bution from normality. In a few cases single anther smears showed two or more Wx p o l l e n g r a i n s s t i l l g r o u p e d t o g e t h e r ( F i g . 2). T h e d e v i a t i o n b u t i o n is o f t e n f o u n d , a l s o w h e n o n l y t h e d a t a g a t h e r e d single branches
are considered
from the Poissonian distrifrom the earlier flowers of
(in the table the distribution
indicated
with B). The
heterogeneity underlying this behaviour can be so great as to be unmasked, at times, by the intracistron recombination, which, however, occurs in the compound wxg°/wx c at a rate much higher (lower part of Table IV) than that of the hemi- and homozygous type.
Mutation Research 2 (1965) 352-365
R E V E R S I O N F R E Q U E N C Y OF 'TABLE
V
INCIDENCE OF W x POLLEN GRAINS IN ANTHERS OF
wxM-1Awx M-1A PLANTS Degrees of freedom
P
32.58
2
307.46
2
0.96
2
~ 0.60
2.91 3.78 1.96 2.84
15.19
2
995
3 .08
571.71
3
335 ° 23 ° 0 23oo 795 °
lO 4 94 89 287
3.1o 4.o9 3.87 3.61
4.4 °
2
~o.I 5
I 2 3 i- 3
230o 225 o 340o 795 °
52 58 50 16o
2.26 2.58 1.47 2.Ol
9.42
2
~ o.oi
I 2 3 1- 3
325 ° 32oo 2550 90o0
42 53 71 166
1.29 1.66 2.78 1.84
18.54
2
I 2 3 1- 3
26oo 29oo 290o 8400
86 76 78 24o
3.31 2.62 2.69 2.86
2.77
2
~ o.25
333o0
853
2.56
66.o9
3
Plant
Flower
Anther
Total Number number of of W x pollen pollen grains grains
I
I
i 2 3 1- 3
19oo 36oo 365 o 915o
33 41 io 84
1.73 1.13 o.27 o.92
I 2 3 1- 3 i 2 3 1- 3
215o 285o 295 o 795 ° 3000 3° o o 175° 775 °
32o 153 77 55 ° 58 53 38 149
14.88 5.36 2.61 6.92 1.93 1.76 2.17 1.92
I 2 3 1- 3
2400 235o 27oo 745 o
7° 89 53 212
323 ° o
I 2 3 1- 3
II
III
IV
Between flowers
]1
359
w a x y P O L L E N T Y P E IN MAIZE
I
II
III
IV
Between flowers
°/o incidence of the W x type
X~ value
This being the case, an analogous analysis, b y single anthers, has been performed on the w x m - l a stock, known to produce reversion at an unusually high rate. Data from such agenotype were thought to be of interest in discussing andinterpreting the data on the material, normal and hypoploid, which was the main object of this work. Table V demonstrates clearly the significant variation of the incidence of W x pollen grains in the different anthers. Fig. 3 gives a pictorial representation of this behaviour. Mutation Research 2 (1965) 3 5 2 - 3 6 5
360
A. BIANCHIAND C. TOMASSIN1
DISCUSSION
The results reported deal, of course, with the scoring of fixed pollen grains, whose properties cannot be properly analysed further with appropriate genetic tests, in order to demonstrate the inheritable nature of the variation observed. However, NELSON17 demonstrated that the rate of blue-staining elements estimated by pollen analysis is similar to Wx frequency estimated by conventional genetic techniques, at least in compound genotypes. On the basis of this result, taking into account the fact that the phenotypic characters exhibited by the pollen grains correspond strictly to known genetic differences, and that the comparison is made between plants possessing similar genetic background growing in the same environmental conditions, it seems more than reasonably justified to discuss the rate of blue-staining pollen grains in terms of W x frequency. As mentioned in INTRODUCTION,in yeast the exchange-associated spontaneous mutations, which MAGNI9 calls "meiotic effect", have been assumed to occur for a class of mutants that are thought to be due to base losses or insertions in the DNA. For such mutants, according to this interpretation, unequal crossing-over leads to restoration of a sequence compatible with normal function. However, the same author points out that such is not the case for mutation due to base substitution. Some preliminary data suggest, in fact, that mutations thought to belong to this class, do not revert with meiosis at a rate higher than in mitosis, where no crossing-over phenomena are involved. Mutants considered as belonging to such a class, on the other hand, appear to revert mainly for mutations occurring in suppressors. The results presented in this paper indicate that the phenomena of pairing and crossing-over at the w x locus, with the allele investigated, do not play a significant role in varying the reversion rate at this locus, because this rate is not lowered in a hypoploid condition where the participation of the homologue is impossible. From this point of view, this conclusion is therefore not at variance with that reached by LAUGHNAN6 in the analysis of the A 1 locus*, although in his case forward mutations were scored, and the frequency of this event is about IO fold higher than that observed for reversion at the w x locus. In this respect, it is also important to consider the fact that the reversion rate of the yeast auxotrophs to prototrophy is, on the contrary, at least IOO fold lower than that of the w a x y mutant. However, the assumption that crossing-over may be responsible for the origin of mutational events in maize cannot be completely ruled out. In fact the evidence obtained by SCHWARTZ19, that sister-strand crossing-over occurs in maize, might well account for a similarity in the behaviour of the two different chromosome conditions, provided, obviously, that sister-strand crossing-over is postulated to occur at a sufficiently high rate, at least in the hypoploid type. This may not be unlikely, taking into account some not infrequent chromosome behaviour of maize, such as nonhomologous pairing and autosynthesis. But the crossing-over hypothesis meets some other difficulties with our data. The strikingly high heterogeneity (mainly due, as suggested by the single anther analysis, to the clustering of the W x grains, and to the * Rather extensive data now available on the frequency of alpha occurrences from Ab hemizygous plants (heterozygous for the a - X 1 and a - X 3 deficiencies) as compared with those from A0 normal diploid plants, again confirm that the yield of alpha cases from the former genotype is higher than in the normal condition (LAUGHNAN,personal communication). Mutation
Research
2 (1965) 352-365
REVERSION FREQUENCY OF w a x y POLLEN TYPE IN MAIZE
361
excess of zero-class; Table IV) of the reversion rate encountered in the different pollen mother cells tissues within the same tassel is much better explained on the assumption t h a t the W x grains observed are derived not only b y mutation occurring along with crossing-over, but also b y duplication of the genetic material which has undergone m u t a t i o n earlier. T h a t a similar interpretation holds good for such an unstable allele as ~:x M lA is certainly true. The data repclted fGr this allele (Table V and Fig. 3), as u-ell as its behavi6ur in endosFerm tissue, ~here patches of blue-staining cells are inteimingled with the underlying mass staining brown, are ~easGnably suggestive of this interpretation. However, extension of this interpretation to the reversion of normally stable alleles cannot be only a m a t t e r of generalisation. This, rather, rests mainly on the significant heterogeneity which characterizes t h e rate of reversion in different tassel sectors, and which becomes prominent with some clusters of W x grains in the single anther smears. If the reversions are, to a significant degree, not necessarily associated with crossing-over, but with mutational events s e n s u stricto, a difference between the genotype in which normal crossing-over occurs (the diploid plants) and that where it is excluded, is not to be expected. If this is the case, the w a x y mutants could be considered as corresponding to the base substitution group for which mutagenesis appears t o be controlled b y suppressor factors. The existence in maize of the controlling elements, which show some parallels with the gene control system of microorganisms is, appears here to be the condition sufficient and adequate to explain the data of the reversion at the w x locus. Mutations occurring either at the operator or the regulator unit, not necessarily restricted at the crossing-over time, m a y well provide the basis for genetical events which account for the changes in the activity of the w a x y alleles which, in this framework, could be considered structural genes. The specific response of the w a x y locus to a dual system of gene control (the d i s s o c i a t i o n - a c t i v a t o r system) has been demonstrated b y McCLINTOCI~13 since 1948; it is interesting to note t h a t even if in that case the reversion rate was exceptionally high under the influence of such a system, in some of our cases revertant grains of distinctly smaller size (Fig. 4) have been observed similar to those observed by McCLINTOCK, who attributed the finding to chromosomal disturbances also induced b y the d i s s o c i a t i o n element. Finally, the failure to obtain indication of higher reversion in the normally diploid condition as compared with the hypoploid genotype is in agreement with the finding of SPRAGUEet al.*a and of PALENZONAAND SCOSSIROLIis who found that doubled maize monoploids, which would be expected to be completely homozygous since they originated from a single monoploid gamete, showed significant variability, interpreted as resulting from some type of mutational type hardly to be attributed to u n e q u a l crossing-over phenomena. APPENDIX
As indicated in the text of this paper, the main purpose of this work was t h a t of comparing the reversion rate of a normally diploid condition with the reversion of the hypoploid type obtained following X-irradiation and screening of appropriate genotypes. On the basis of the yeast results the expectation was t h a t the normal condition should have produced a higher reversion rate than the genotype in which no opportunity was offered for normal pairing and crossing-over which were supposed M u t a t i o n Research 2 (1965) 352-365
362
A. BIANCHI AND C. T()MASSINI
to be responsible for that higher reversion rate. In maize the experiments failed to confirm a similar behaviour. In fact the general average for the normal genotype turned out to be slightly lower than that of the hypoploid one. On the other hand, the difference, in the opposite sense to the expected one, could have been larger, had two exceptional tassels ot hypoploid plants been included in the data reported in Table I. The exceptional specimens have been excluded because of their extremely high heterogeneity in the reversion rate (Table VI). This heterogeneity, because of its size (several pollen samples exhibit .~°/;~-~°//o o f the Wx type)* is likely to be due
,,
[:ig. 4" E a r o b t a i n e d i r o m t h e s a m e b a c k c r o s s i n d i c a t e d in Fig. 1 b e a r i n g a b o u t half kernels w h i c h e x h i b i t t h e t y p i c a l v a r i e g a t i o n of b r e a k a g e - f u s i o n - b r i d g e cycle s t a r t i n g d i s t a l l y to I m a r k e r . R i g h t , a kernel s h o w i n g s u c h a p a t t e r n a t a h i g h e r m a g n i f i c a t i o n . T h e o t h e r half of t h e k e r n e l s s h o w t h e e x p e c t e d recessive c h a r a c t e r s in t h e n o r m a l p a t t e r n ( t h e y are easily recognized b y t h e i r s h r u n k e n a p p e a r a n c e a n d u n i f o r m l y b r o n z e colour). T h e v e r y d a r k coloured k e r n e l a t t h e t o p left is a k e r n e l in w h i c h t h e b r e a k a g e - f u s i o n - b r i d g e cycle s t a r t e d b e t w e e n I a n d t h e c e n t r o m e r e , following t h e c o m p l e t e loss of t h e Inhibitor factor, * Single a n t h e r s m e a r s r e v e a l e d t h a t in a s a m p l e of 96, b e s i d e s 68 a n t h e r s w i t h no W x grains, t h e r e were single cases of t h e r e m a i n i n g 28 w i t h t h e following n u m b e r s of W x pollen g r a i n s : 2 - 7 8-I4-23-24-3o-37-42-56-62-78-78-87 92-115-117-118-127-143-146-i64-166-171-189-263. Mutation Research 2 (1965) 352-365
REVERSION FREQUENCY TABLE
363
O F WaA;y P O L L E N T Y P E I N M A I Z E
VI
x Y g l Sh
FREQUENCY" OF W x POLLEN IN EXCEPTIONAL TASSELS FROM THE c R o s s y g C s h b z w x /~g W x WHOSE DOMINANT GAMETE WAS X - I R R A D I A T E D
Tassel 13o 3
Branch i
Total 2
Total
3
Total number of pollen grains
N u m b e r o/ W x pollen grains
Rate of W x grains ( × Io -s)
I
21850
2
9.1
2 3
17200 31500
i 3
5.8 9.5
2045 ° 1485o I o 5 85 °
27 io 43
132"° 67. 3 4°.6
4 5 I-5 i
16800
o
2
1745 °
3
3 4 5 6 I-6
20200 1723° 17500 21350 I i o 53 °
IOI 56 13 o 173
o.o 17-2 5oo.o 3 2 5 .0 74-3 o.o 156.5
I
23633
2 3 4 5 I-5
33 1 6 6 329 oo 3445 ° 3285 ° 156999
23 ° 585 479 288 742 2324
1763.8 1445.9 836.o 2858"7 157 o . °
I
I
41000
o
o.o
2
I 2 3 I-3
19000 15400 164 °° 5° 8oo
I o o I
52.6 o.o o.o 2.o
I 2 I-2
21150 13600 3475 °
4 o 4
18.9 o.o I 1.5
2122400
o
o.o
2765 ° 2865 ° 3255 ° 329 °0 17050 31 16o 225 °° 19246o
23 14 24 4 4° 39 15 159
83.2 48.9 73.7 12.2 234"6 I25-2 66"7 82.6
Total
I292
Position in the branch
Total 3 Total
4
I
5
I 2 3 4 5 6 7 1- 7
Total
973.2
to some special phenomena besides the mutation event sensu stricto invoked above. Those were possibly breakage-fusion-bridge cycles initiated distally to Wx, and which were conducive to the loss of this dominant factor especially in the later development of the tassel, namely the lateral branches 5. The latter, in fact (the lower numbered cases in Table VI), present no W x grains or very few, especially in the late developing sectors. Such grains are, on the contrary, strikingly numerous in the tassel sectors which trace back to parts likely to be formed during early developmental stages, when the broken chromosome arms bearing the dominant markers had not Mutation Research 2 (1965) 3 5 2 - 3 6 : 5
364
a . t~IANCHI AN]_) C. T()MASSIN1
yet been lost following the cycle of events (the branch 3 ill tassel I3o3, and 5 in tassct 1292; moreover in both tassels the central sectors of m a n y branches also show, as expected according to our interpretation involving the developmental process, higher Wx-pollen rates). This interpretation is also supported by the fact that in the two tassels a very strong correlation was present between the frequency of Wx pollen grains of the early flower and that of the late one on the same spikelet. Factual evidence of the occurrence of such a kind of cycle has been given by an ear (Fig. 4) in which most of the typical variegation produced by the cycle is visible in m a n y kernels. Most of these kernels actually show the phenotype expected to originate from a distal breakage point. Moreover, few kernels from the late developing part of the ear show phenotypes indicative of the loss of more proximal dominant factors of the genetically marked region. McCLINTOCKn,12 produced evidence that in the sporophyte tissue of maize only the breakage-fusion-bridge cycle of chromosome type can occur. This required in her type of experiments that both egg and sperm nuclei contributed a chromosome with a freshly broken end. Since our plants derive from zygotes in which only the sperm nuclei m a y have been broken by the irradiation treatment, the cases just described can be interpreted either admitting that in the sporophyte also a chromatid type of cycle occurs, as a result of a breakage induced in the sperm nuclei, or that two broken ends of different chromosomes undergo rejoining and start a cycle of chromosome type. ACKNOWLEDGEMENTS
This work has been in part financially supported b y C.N.R., Rome (with a fellowship to Dr. C. TOMASSINI) and b y I.A.E.A., Vienna (Research Contract 64 US). The laboratory work has been carried out mainly in the Institute of Genetics of Milan University, the field work mainly at Piacenza (Plant Genetics Institute of the Catholic University), whereas the calculation work as well as the preparation of the manuscript has been performed in Bologna (Plant Breeding Institute). The critical reading of the manuscript b y Dr. G. E. MAGNI, Parma, and Dr. O. E. NELSON, Purdue University, Lafayette, Ind., is also gratefully acknowledged.
REFERENCES
I BIANCttI, A. AND M. CONTIN, R a d i o g e n e t i c a l e x p e r i m e n t s w i t h h i g h e r resolving p o w e r in maize. Atti Assoc. Genet. ltal. Pavia, 8 (1963) 257-267. 2 EMMERLING, M. H., A n a n a l y s i s of i n t r a g e n i c a n d e x t r a g e n i c m u t a t i o n s of p l a n t color c o m p o n e n t of t h e R r gene c o m p l e x in Zea mays. Cold Spring Harbor Syrup. Quant. Biol., 23 (1958) 393-407 • 3 FABERGt~,A. C., T h e a n a l y s i s of i n d u c e d c h r o m o s o m e a b e r r a t i o n s b y m a i z e e n d o s p e r m p h e n o t y p e s . Z. Vererbungslehre, 87 (1956) 392-420. 4 HAWTHORNE, D. C. AND 1R. H. MORTIMER, S u p e r - s u p p r e s s o r s in y e a s t s . Genetics, 48 (I963) 617-62o. 5 KIESSELBACH, T. A., T h e s t r u c t u r e a n d r e p r o d u c t i o n of corn. Nebraska Univ. Agr. Expt. Sta. Res. Bull., 161 (1949) 1-96. 6 LAUGHNAN, J. t{., A n e v a l u a t i o n of m u l t i p l e crossover a n d s i d e c h a i n h y p o t h e s e s b a s e d on an a n a l y s i s of a l p h a d e r i v a t i v e s f r o m t h e A b - P c o m p l e x e s in maize. Genetics, 46 (1961) 1347-1372. 7 MAGNI, G. E., M u t a t i o n r a t e s d u r i n g t h e m e i o t i c process in y e a s t . I n F. H. SOBELS, Repair from Genetic Radiation Damage, P e r g a m o n Press, Oxford, 1963, p. 71-85 . Mutation Research 2 (1965) 352-365
REVERSION FREQUENCY OF w a x y POLLEN TYPE IN MAIZE
365
8 MAGNI, G. E., T h e origin of s p o n t a n e o u s m u t a t i o n d u r i n g meiosis. Proc. Natl. Acad. Sci. U . S . 5 ° (1963) 975-98o. 9 MAGNI, G. E., S t u d i sull'origine della m u t a z i o n e s p o n t a n e a . Atti Assoc. Genet. Ital. Pavia, IO (I965) 3-26. lO MAGNI, G. E. AND R. C. VON BORSTEL, D i f f e r e n t r a t e s of s p o n t a n e o u s m u t a t i o n d u r i n g m i t o s i s a n d meiosis in y e a s t . Genetics, 47 (1962) lO97-11o8. i i McCLINTOCK, B., T h e s t a b i l i t y of b r o k e n e n d s of c h r o m o s o m e in Zea mays. Genetics, 26 (1941) 234-282. 12 McCLINTOCK, B., T h e f u s i o n of b r o k e n e n d s of c h r o m o s o m e following n u c l e a r division. Proc. Natl. Acad. Sci. U.S., 28 (I942) 458-463 . 13 McCLINTOCK, B., M u t a b l e loci in maize. Carnegie Inst. Wash. Publ., 47 (I948) I 5 5 - I 6 9 . 14 McCLINTOCK, B., Controlling e l e m e n t s a n d t h e gene. Cold Spring Harbor Syrup. O,uant. Biol., i6 (1956) 13-47. 13 McCLINTOCK, B., S o m e parallels b e t w e e n gene c o n t r o l s y s t e m s in m a i z e a n d in bacteria. Am. Naturalist, 95 (I96I) 265-277. i6 NELSON, O. E., I n t r a c i s t r o n r e c o m b i n a t i o n of t h e W x / w x locus. Maize Genet. Coop. News Letter, 33 (1959) lO3-1o7. [7 NELSON, O. E., T h e waxy locus in maize. I. I n t r a l o c u s r e c o m b i n a t i o n f r e q u e n c y e s t i m a t e s b y pollen a n d b y c o n v e n t i o n a l analysis. Genetics, 47 (1962) 737-742 . [8 PALENZONA, D. L. AND S. SCOSSIROLI, Ricerche sulla m u t a b i l i t g poligenica i n d o t t a nei mais. Atti Assoc. Genet. Ital. Pavia, 7 (1962) 257-265. 19 Sc~I'aTaR:rz, D., E v i d e n c e for s i s t e r - s t r a n d c r o s s i n g - o v e r in maize. Genetics, 38 (1953) 251-26o. 20 STADLER, L. J. AND M. 0 . NUFFER, P r o b l e m s of gene s t r u c t u r e . II. S e p a r a t i o n of R r e l e m e n t s (S) a n d (P) b y u n e q u a l crossing-over. Science, 117 (1953) 471-472. 21 STADLER, L. J. AND M. H. EMMERLING, P r o b l e m s of gene s t r u c t u r e . I I I . R e l a t i o n s h i p of u n e q u a l c r o s s i n g - o v e r to t h e i n d e p e n d e n c e of R r e l e m e n t s (P) a n d (S). Genetics, 41 (1954) I 2 4 - I 3 7 . 22 STABLER, L. J. AND M. I-{. EMMERLING, iRelation of u n e q u a l c r o s s i n g - o v e r to t h e i n d e p e n d e n c e of R r e l e m e n t s (P) a n d (S). Genetics, 41 (1956) 124-137. 2 3 SPRAGUE, G. F., W. A. RUSSELL AND L. H. PENNY, M u t a t i o n affecting q u a n t i t a t i v e t r a i t s in selfed p r o g e n y of d o u b l e d m o n o p l o i d m a i z e stocks. Genetics, 45 (196o) 855-865. 24 STEVENS, W. L., A c c u r a c y of m u t a t i o n rate. J. Genet., 43 (1942) 3Ol-3O7.
21lutation'~Research 2 (1965) 352-365
R E V E R S I O N F R E Q U E N C Y OF W A X Y AND H Y P O P L O I D MAIZE PLANTS
MUTATI()N RESEARCH
P O L L E N T Y P E 1N NORMAL
A. B I A N C H I AND C. T O M A S S I N I
Istituto di Genetica, Universit~ di Milano, Milano, Istituto di Genetica Vegetale, Universit~ Cattolica, Piacenza and Istituto di Allevamento Vegetale, Bologna* (Italy) ( R e c e i v e d M a r c h 29th, 1965)
SUMMARY
Maize pollen grains of constitution Y g I S h B z W x were used to fertilize a stock possessing the corresponding recessive genetic markers y g C sh bz w x of the short arm of chromosome 9. Appropriate screening of the ensuing seedlings, together with the results of appropriate testcrossing and scoring of the pollen, made it possible to isolate hypoploid plants in which the chromosome 9 carrying the dominant markers had lost at least the W x gene. This gene is known to affect the chemical nature of the starch, both in endosperm and in pollen grains. The pollen produced b y such plants was only influenced by the w x gene of the undamaged chromosome. The reversion rate of this gene--on the basis of the appearance of pollen grains giving the W x chemical reaction (with iodine solution)-was measured and compared with the reversion rate of normally diploid w x w x plants. On the basis of results with yeast, the expectation was that the normal chromosome condition should have produced a higher reversion rate than the genotype in which the hypoploid condition associated with the deficiency of the genetically marked region provided no opportunity for pairing and crossing-over, which was supposed to be responsible for that high reversion rate. In maize the experimental data failed to confirm a similar behaviour. This finding suggests t h a t the w x alleles investigated are likely to be due to base substitution. In such a case unequal crossing-over, causing base losses and insertions in the DNA and, therefore, restoration of a sequence compatible with normal functioning, appears unlikely. Mutagenesis in these cases appears to rely only on mutational events s e n s u stricto and associated with a suppressor system. In maize such a requirement seems to be satisfactorily met with the controlling elements.
INTRODUCTION
During the past thirty years great efforts have been made to understand the mechanism of origin and the nature of mutational events in m a n y organisms, especially on the basis of the artifical induction of mutations. Much less knowledge has * P r e s e n t a d d r e s s of t h e a u t h o r A. BIANCHI.
Mutation Research 2 (1965) 352-365
.\. BIAN('HI ANI) (.-I'~)M.\SSINI
D
C
1~'
A
Fig. I. The results o{ backcrossing ye~lo~-green p l a n t s of the cross yg C sh bz ~,x . irradiated Y g I Sh B z W x b y Yg C sh be' w.r. l:rom left: A. E a r showing segregation of all the endosperm markers, and, consequently, d e m o n s t r a t i n g t h a t the breakage point was b e t w e e n Yg and 1 ; notice also, as c o n f i r m a t o r y d a t u m , the almost n o r m a l fertility. B. E a r in which the I factor is absent: the b r e a k has been induced between l and Sh. The see}] setting is reduced. C. Ii1 this ear no donlin a n t character is detectable, the fertility is very poor. The breakage m u s t have been induced beyond W*, so as to cause the loss of ah the genetic m a r k e r s of c h r o m o s o m e 9. l). E a r in which only t h e W x m a r k e r has been lost, all other e n d o s p e r m d o m i n a n t t r a i t s being recognizable. The interstitial deficiency c a n n o t have been large, in fact the fertility is not greatly reduced.
E R R A T U M : This figure should be readj?om right to left.
Mutation Research 2 (1965) 352-365
R E V E R S I O N F R E Q U E N C Y OF w a x y POLLEN T Y P E IN MAIZE
353
accumulated on the intimate phenomeI~a responsible for the occl~rrence of spontaneous mutation. However, the present information on the structure of DNA, and the data obtained from the studies of artificially induced and spontaneously occurring mutations seem to suggest that the so-called point mutations can be grouped into two main classes: base substitutions and base deletions or insertions. In Saccharomyces cerevisiae MAGNI et al.7-1 o present convincing evidence that, at least for mutants supposed to be of the latter class, an important fraction of the spontaneously occurring mutations are associated with (unequal) crossing-over. MAGNI9 also points out that similar behaviour is not restricted to this species, provided that special caution is adopted in collecting and analysing the data. However, no special studies have been performed to demonstrate a more general occurrence of this phenomenon, with the exception of some experiments which are being carried out as a consequence of the MAGNI finding. Of course, it is generally accepted that the Bar mutants in Drosophila are a product of unequal crossing-over. Moreover, in maize, the occurrence of unequal crossing-over at the R region in chromosome IO was explained b y STADLER et al. ~°-~ as a source of a large fraction of mutants. However, although the important role in spontaneous mutagenesis of the "oblique synapsis" was accepted, no a t t e m p t was made to compare the mutation rate caused b y this process with the mutation rate in situations in which regular pairing between homologous chromosomes was not possible. On the other hand, L A U G H N A N ~, in the fine structure analysis of the alpha derivatives from the A L p complexes in maize, finds that these mutants are at least as frequent among the progeny of deficiency heterozygotes--where obviously there was no opportunity for exchanges between the homologous p a r t n e r s - - a s they are from normally diploids. This work has been undertaken in order to compare the reversion frequency of maize plants, in which normal synapsis and crossing-over occur, with that of individuals in which these processes are entirely missing, and, consequently, to confirm the undifferentiated behaviour of mutagenesis in the two chromosome situations, such as found b y LAUGHNAN~, o r to recognize also for maize the "meiotic effect" on spontaneous mutations as found b y MAGNI9 in Saccharomyces, at least for a class of mutants. MATERIAL AND METHODS
Spontaneous mutation is usually a rare event; consequently special devices and/or appropriate material are needed to obtain data from which to draw dependable conclusions, with feasible work and in reasonable time. In maize these requirements are uniquely satisfied with the waxy trait which, for a long time, has been known to affect the chemical nature of the pollen starch. With appropriate iodine solution the waxy pollen grain stains brown; the W x type stains dark blue. NELSON is suggested appropriate details for exploiting these properties in studies of the genetic fine structure and of the intracistron recombination. With minor modifications, this technique was used b y BIANCHI AND CONTIN1 in the study of artificial mutagenesis. The same fixing and staining procedures have been adopted for the present work. The waxy allele employed was kindly provided several years ago b y Dr. B. Mutation Research 2 (1965) 352-365
354
A. B I A N C H I A N D C. T O M A S S I N I
McCLINTOCK, and has been maintained by us during this period by self-pollinating the plants of the successive generations. The stock possesses, besides the complementary colour factors on the different chromosomes such as A 1, A 2, R in homozygous conditions, the following markers of chromosomes 9 : Yg2 C Shl bz wx. This strain has been used for estimating the reversion rate of the wx allele to l~'x in the mature pollen grain population in the normally diploid condition. For the study of the reversion in conditions that lacked homologous pairing and crossing-over phenomena, hypoploid counterpart has been obtained as follows: the McClintock stock was fertilized with pollen of a stock obtained from the Maize Genetics Cooperative, Urbana, Ill. (courtesy of Dr. E. PATTERSON).The constitution of thisstockwasA 1, A 2, R, Yg~I Shl Bz W x . Before pollination the pollen was X-irradiated with 10oo-2ooo R. The kernels produced were planted in the field and the seedlings scored for chlorophyll-deficient characters. The great majority of them were normally green and were eliminated: the very few yellow-green seedlings, carefully protected from any possible damage, were grown to maturity. Some of the yellowgreen plants were unable to extrude anthers and/or silk, and they were discarded too. The remaining individuals were pollinated by the McClintock stock, and their tassel, at the very beginning of anthesis, was fixed for the study of the pollen. Another wx source was kindly provided b y Dr. O. E. NELSON in the form of an unstable allele of this locus, known as wx -~t-lA. As mentioned above, the technical procedure for fixing, staining and scoring of the pollen was essentially the same as that of NELSON. However, for special purposes, single anthers were scored too. With the aid of teasing needles, the anther content was poured into a drop of the standard staining solution. During the procedure the pollen sample was carefully protected from air with a cover-glass of appropriate size on microscope slides. For special comparisons the compound type wxg°/wx c, obtained by crossing the strains bearing the two pseudoalleles wx 9° and wxC (again received from Dr. NELSON), was also studied with single anther analysis. RESULTS
As mentioned above, the critical material was to be found in some plants of the cross yg C sh bz wx × Y g I Sh B z Wx. Following X-irradiation, the loss of the I Sh Bz markers is very frequently accompanied by the loss of the W x locus 2. It was therefore considered that, once screening for yg seedlings had been made, the loss of Y g could be used as a first appropriate index of loss likely to involve W x too. In fact, in a sample of 57 yellow-green plants so obtained, the results of the baekcross to the multiple recessive yg C sh bz wx demonstrated that 2o were deficient for only Yg, 4 for Y g and I, 2o for all the markers, i for only W x (Fig. I), and 2 were almost completely sterile. Of course, the partial sterility of these plants generally increases, going from the first group to the last. Moreover, out of 57 tassels collected from yellowgreen plants, 37 showed practically only wx pollen grains associated with at least 5o% sterility, whereas 18 presented also W x grains (about 50%), and 2 W x grains in values of lO-12% (these two groups did not have very severe pollen sterility). Obviously, only the plants which turned out to be deficient for W x were considered and used in the successive pollen analysis. ~Iutation Research 2 (I965) 352-365
R E V E R S I O N F R E Q U E N C Y OF w a x y P O L L E N T Y P E I N M A I Z E TABLE
355
I
REVERSION RATE AT THE W,~ LOCUS OF HYPOPLOID AND NORMAL TASSELS FROM PLANTS GROWN IN TWO DIFFERENT YEARS
Year and chromosome type
Tassel No.
1963; h y p o p l o i d
lO92- 3 lO64-1 lO69-2 lO69-1 lO64- 4
Number of W x pollen grains
Frequency of W x pollen grains ( × Io -6)
572669 112616 733 192 3865oi 1o34oo 19o8378
28 I 68 7° o I67
4.9 o.9 9.3 18.1 o.o 8.7
14.o - 2 2 . 9 o.o - 3.5 7.5 - l O . 2
56-1
618o21 695767 1540347 2256349 2o3o517 3288372 308646o 13515833
13 21 229 374 168 128 48 981
2. I 3.0 14.9 16.6 8. 3 3.9 1. 5 7.2
I. i 1.9 13.o 14.9 7.1 3 .2 I.I 6.8
- 3.6 - 4.6 -16.9 -18.3 - 9.6 - 4 .6 - 2.1 - 7.7
13o9 1291 1298 1282 129o 1283 1291
448860 73OLO 4o663 ° 74o8o 914850 98955 ° 1117350 402433 °
29 o 12 15 6 7 19 88
6.4 o.o 2.9 20.2 o.6 0.7 1. 7 2.2
4.3 o.o 1.5 1.3 0.2 0-3 i.o 1. 7
- 9.3 - 5.0 - 5.1 -31.7 - 1. 4 - 1.4 - 2.6 - 2. 7
1267-1 1267-2 1267-3 1267- 4
1967200 34388o 3° o o 6 8 0 66921o 598o97 o
14 4 57 7 82
0. 7 1.2 1.9 i.o 1. 4
0. 4 0. 3 1.4 0. 4 i.I
- 1.2 - 3.0 - 2.4 - 2.1 - 1. 7
Total and average 1963 ; n o r m a l
56- 7 56-11 56-16 56-2 56-3 56- 4 Total and average 1964; h y p o p l o i d
Total and average 1964; n o r m a l
Total and average
Estimated number of pollen grains
Fiducial limits for P o.o 5 level* ( × Io -5) 3 .2 - 7 .1 o . o 2 - 4.9
7.2 - I 1.8
* C a l c u l a t e d a c c o r d i n g t o STEVENS 24.
The pollen preparations of such plants, moreover, not only demonstrated the lack of the Wx-bearing chromosome segment but also, as expected, the presence of at least 50% of empty pollen grains. The results of these analyses are summarized in Table I. This shows that the reversion rate is highly variable, and that this variability may derive from different sources. The table immediately suggests that the year is an important factor, producing a 4-5 fold effect in the hypoploid as well as in the normal chromosome condition on the overall reversion rate. However, if instead of the overall frequency the median value (which of course has the advantage of being less influenced by the higher rates) is considered, the discrepancy between the two-year data is statistically less significant (in the case of the hypoploid genotype) or scarcely significant (for the diploid series). On the other hand, within data for both years, there is no indication that the normal condition is associated with a higher rate of reversion; actually in both years this rate is rather lower than in the hypoploid*. * E v e r y e f f o r t h a s b e e n m a d e t o a v o i d c o n t a m i n a t i o n b y c o n d u c t i n g e x p e r i m e n t s in p r o p e r l y i s o l a t e d f i e l d s ; b u t i t is p o s s i b l e , e v e n if u n l i k e l y , t h a t t h e h i g h e r r a t e of W x g r a i n s in 1963 is d u e t o s o m e w i n d - b o r n e p o l l e n f r o m r a r e a n d d i s t a n t fields.
Mutation Research 2 (1965) 3 5 2 - 3 6 5
356
A. BIANCHI AND C. TOMASSINI Another
plants,
important
which,
s o u r c e of v a r i a t i o n
although
detectable
for both
appears
to be that
genotypes,
of t h e i n d i v i d u a l
is u n q u e s t i o n a b l y
more
e v i d e n t i n t h e h y p o p l o i d c l a s s , B u t t h i s h e t e r o g e n e i t y m a y b e t h e c o n s e q u e n c e of t h e heterogeneity
which can be detected
orderly and separately
a t d i f f e r e n t l e v e l s , w h e n t h e p o l l e n a n a l y s i s is
carried out on the different branches
of t h e t a s s e l , o n t h e
d i f f e r e n t r e g i o n s of t h e b r a n c h e s , d o w n t o t h e d i f f e r e n t s i n g l e a n t h e r s . F o r t h e s a k e of brevity,
the detailed data obtained
examples
may be extracted
i n t h i s k i n d of a n a l y s i s a r e o m i t t e d ,
and presented,
but a few
s i n c e t h e y m a y b e of u s e in d i s c u s s i n g
later the general results. Table II reproduces
an example
of t h i s k i n d of d a t a
for the normal
diploid
TABLE II INCIDENCE
OF
~TX P O L L E N
GRAINS IN DIFFERENT
P O S I T I O N S O F A T A S S E L O F T H E N O R M A L ¢~)X fI;x
GENOTYPE
Branch
Position in the branch
rom bottom to top I.
Total I[
Total iII
Total IV
Total V
Total Grand total
Estimated Number number of of W x stainable pollen pollen grains grains
Rate of Wx pollen grains ( X IO-~)
Fiducial limits for the Wx rate of pollen grains ( × xo -5)
I 2 3 4 5 1- 5
3905 ° 53600 23300 3tSOO 34433 181883
o o o o I r
o.o o.o o.o o.o 2.1 o. 5
I 2 3 4 5 1-5
42866 53633 4455 ° 35667 179o0 I94616
o o I I 2 4
o.o o.o 2.2 2.8 I 1.2 2.I
o.o 8.6 o.o - 6.9 o . o 5 - I 2. 5 o.I -15.6 1.3 -4o.3 o.6 - 5-3
I 2 3 4 1- 4
4435 ° 8 3 ioo 43333 6255 ° 233333
o o i o I
o.o o.o 2. 3 o.o o. 4
o.o - 8,3 o.o - 4.4 o.o5-I2.8 o.o - 5-9 O.Ol- 2.4
i 2 3 4 5 i- 5
839o0 72000 54700 34833 3o3oo 275733
14 4 21 18 i 58
I6. 7 5-5 38. 4 51.7 3.3 21 .o
9. I 1.5 23.8 30.6 o.i 16.o
i 2 3 4 5 6 7 8 9 io i-io
83233 84566 748o0 763 °0 50900 85300 4895 ° 5035 ° 38833 6155 ° 654782
18 4 33 5 27 2 5 36 28 7 165
21.6 4.7 44 -1 6.5 53.0 2.3 lO.2 71.4 72.1 i 1.4 25.2
12.8 -34.2 1. 3 - i 2 . 1 3o.4 -62.0 2.1 -15.3 33.0 -73 .0 0.3 - 8.5 3-3 -23.8 5 ° .i - 9 9 . I 48.O-lO4.3 4 .6 - 2 3 ' 4 21.5 -29.4
154o347
229
14.9
x4.o -16.o
Mutation Research 2 (I965) 352-365
o.o o.o o.o o.o o. 7 o.o
- 9.4 - 6.9 -I5.8 -II.7 -16.2 - 3.1
-28.0 -I4.2 -56.9 -81.7 -18. 4 -27.2
R E V E R S I O N F R E Q U E N C Y OF TABLE
357
w a x y POLLEN T Y P E IN MAIZE
In
INCIDENCE OF
WX POLLEN GRAINS IN DIFFERENT POSITIONS OF A TASSEL OF TIlE HYPOPLOID WX
GENOTYPE
Branch
Position in the branch
Estimated number of stainable pollen grains
I 2 3 4 5 I-5
31267 2o167 15o33 13767 133oo 93534
o I I 2 I 5
o.o 4-9 6.6 14. 5 7.5 5.3
o.oo.Io.21. 7 o.21.7-
11.8 27.6 37.o 52.4 41.9
I 2 3 4 1- 4
2655 ° 15o5o 2o45o 24833 86883
o o 3 I 4
o.o o.o 14.7 4.o 4 .6
o.oo.o3.oo.I1.2-
13. 9 24. 5 42.9 27.2 I 1.8
i 2 3 I- 3
14467 I4567 1o6oo 39634
n 6 o 17
76.o 41.2 o.o 42.9
37.9-136.o I 5 . 1 - 89.6 o . o - 34.8 2 5 . o - 68.7
i 2 3 1-3
424 °o 25574 17400 85374
9 17 i 27
21.2 66. 5 5.7 31.6
9 - 7 - 4o.3 38.7-1o6.4 o . i - 32.o 2 o . 9 - 46.o
i 2 3 4 1-4
16900 lO567 16567 15500 59534
o i 1i 2 14
o.o 9.4 66. 4 12.9 23.5
o . o - 21.8 2 . 4 - 52.7 33.1-118.8 1.5- 46.6 1 2 . 8 - 39.4
364959
67
18. 3
1 4 . 2 - 23. 3
from bottom to top I
Total II
Total III
Total IV
Total V
Total Grand total
Number o/Wx pollen grains
Rate o / W x pollen grains ( × Io s)
Fiducial limits for the W x rate of pollen grains ( × io-S)
12.5
condition, and Table n I an example from a hypoploid tassel. The heterogeneity, often highly significant, in the reversion rate at the wx locus in both genotypes can be easily demonstrated with the Z* test (as has been done). This holds true for the different branches as well as for the various positions within the same branch. Anyway, in the tables, in order to make comparison more immediate, the fiducial limits (calculated according to STEVENSZ4 at the 0.05 P level) are reported. The fact that the distribution of the W x grains is often of non-Poisson type, would suggest the use of other statistical approaches. Better estimates, as those based on median values mentioned above, require other sets of data, not always obtainable from our material. However, for asymmetric distributions as those of the W x frequency classes, the use of STEVENS tables should not lead to serious bias. The mentioned heterogeneity is detectable also at the anther level. In other words, if the single anthers are scored for W x grains and the frequencies of the different classes (possessing o,I,2 . . . . n W x grains) are obtained, the distributions of Table IV can be prepared. Inspection of such a table suggests that these distributions often show an excess of some classes; a sort of clustering of the W x grains is often evident. The Mutation Research 2 (1965) 3 5 2 - 3 6 5
358 TABLE
A. BIANCHI AND C. TOMASSINI
IV
FREQUENCY
DISTRIBUTION
OF THE
H/'X P O L L E N
GRAINS
IN SINGLE
ANTHER
SMEARS
Entire Anthers showing Wx poUen grains in Genotype tassel (T) number of or single o --~ 2 3 4 5 6 7 >/8 branch (B)
Z2 value
Degrees V"2~ of -j~'eedom ~,'2ti±i (n)
1"
T B T B T B T B T B T B
5362.1 491.2 1512.o 80.0 1828.3 443.0 II5I.O 292.0 1413. 7 200.0 1287. 4 89.0
lO67 161 1169 IiO 899 149 899 176 973 143 832 87
v e r y small v e r y small v e r y small 0.98 v e r y snlall very small v e r y small very small very small 0.003 v e r y small 0.4o
lOO6 15 ° 1129 1°5 887 147 894 175 338 31 296 14
43 8 l 5 i i 8 i 1 I o i 35 5 t o o o 5 1 o o o o io I 1 r o o i i o i o o 5 i o o o o i 1 o o o o 283 194 90 45 13 6 52 31 16 7 4 i 224 139 lO7 37 18 9 22 20 20 7 2 2
o o o o o o o o i o i o
3 o o o o o o o 4 2 2 o
normal normal normal normal hypoploid hypoploid hypoploid hypoploid c o m p o u n d wxg°/wx c c o m p o u n d wxg°/wx ¢ c o m p o u n d wx'°/wx c compoundwx'°/wx ¢
57.4 13. 4 6.6 --2.2 18.o 12. 5 5.6 5.3 9.0 3.0 9.9 0.2
Fig. 2. S m e a r of a single a n t h e r c o n t e n t s h o w i n g t h r e e d a r k - s t a i n i n g grains (Wx type) g r o u p e d in t h e c e n t e r as c o n t r a s t e d to t h e b r o w n - s t a i n i n g g r a i n s of t h e wx t y p e . O n e of t h e d a r k - s t a i n i n g g r a i n s is clearly s m a l l e r t h a n t h e s t a n d a r d t y p e (see text). Fig. 3- Pollen g r a i n s in a n a n t h e r of a p l a n t w x M - 1 A w x M-1A s t a i n e d w i t h a p p r o p r i a t e iodine s o l u t i o n : t h e n o n - r a n d o m d i s t r i b u t i o n of t h e g r a i n s s t a i n i n g differentially, is evident.
s t a t i s t i c a l t e s t (Z 2 f o r P o i s s o n d i s t r i b u t i o n ) c l e a r l y c o n f i r m s t h e d e v i a t i o n o f t h e d i s t r i bution from normality. In a few cases single anther smears showed two or more Wx p o l l e n g r a i n s s t i l l g r o u p e d t o g e t h e r ( F i g . 2). T h e d e v i a t i o n b u t i o n is o f t e n f o u n d , a l s o w h e n o n l y t h e d a t a g a t h e r e d single branches
are considered
from the Poissonian distrifrom the earlier flowers of
(in the table the distribution
indicated
with B). The
heterogeneity underlying this behaviour can be so great as to be unmasked, at times, by the intracistron recombination, which, however, occurs in the compound wxg°/wx c at a rate much higher (lower part of Table IV) than that of the hemi- and homozygous type.
Mutation Research 2 (1965) 352-365
R E V E R S I O N F R E Q U E N C Y OF 'TABLE
V
INCIDENCE OF W x POLLEN GRAINS IN ANTHERS OF
wxM-1Awx M-1A PLANTS Degrees of freedom
P
32.58
2
307.46
2
0.96
2
~ 0.60
2.91 3.78 1.96 2.84
15.19
2
995
3 .08
571.71
3
335 ° 23 ° 0 23oo 795 °
lO 4 94 89 287
3.1o 4.o9 3.87 3.61
4.4 °
2
~o.I 5
I 2 3 i- 3
230o 225 o 340o 795 °
52 58 50 16o
2.26 2.58 1.47 2.Ol
9.42
2
~ o.oi
I 2 3 1- 3
325 ° 32oo 2550 90o0
42 53 71 166
1.29 1.66 2.78 1.84
18.54
2
I 2 3 1- 3
26oo 29oo 290o 8400
86 76 78 24o
3.31 2.62 2.69 2.86
2.77
2
~ o.25
333o0
853
2.56
66.o9
3
Plant
Flower
Anther
Total Number number of of W x pollen pollen grains grains
I
I
i 2 3 1- 3
19oo 36oo 365 o 915o
33 41 io 84
1.73 1.13 o.27 o.92
I 2 3 1- 3 i 2 3 1- 3
215o 285o 295 o 795 ° 3000 3° o o 175° 775 °
32o 153 77 55 ° 58 53 38 149
14.88 5.36 2.61 6.92 1.93 1.76 2.17 1.92
I 2 3 1- 3
2400 235o 27oo 745 o
7° 89 53 212
323 ° o
I 2 3 1- 3
II
III
IV
Between flowers
]1
359
w a x y P O L L E N T Y P E IN MAIZE
I
II
III
IV
Between flowers
°/o incidence of the W x type
X~ value
This being the case, an analogous analysis, b y single anthers, has been performed on the w x m - l a stock, known to produce reversion at an unusually high rate. Data from such agenotype were thought to be of interest in discussing andinterpreting the data on the material, normal and hypoploid, which was the main object of this work. Table V demonstrates clearly the significant variation of the incidence of W x pollen grains in the different anthers. Fig. 3 gives a pictorial representation of this behaviour. Mutation Research 2 (1965) 3 5 2 - 3 6 5
360
A. BIANCHIAND C. TOMASSIN1
DISCUSSION
The results reported deal, of course, with the scoring of fixed pollen grains, whose properties cannot be properly analysed further with appropriate genetic tests, in order to demonstrate the inheritable nature of the variation observed. However, NELSON17 demonstrated that the rate of blue-staining elements estimated by pollen analysis is similar to Wx frequency estimated by conventional genetic techniques, at least in compound genotypes. On the basis of this result, taking into account the fact that the phenotypic characters exhibited by the pollen grains correspond strictly to known genetic differences, and that the comparison is made between plants possessing similar genetic background growing in the same environmental conditions, it seems more than reasonably justified to discuss the rate of blue-staining pollen grains in terms of W x frequency. As mentioned in INTRODUCTION,in yeast the exchange-associated spontaneous mutations, which MAGNI9 calls "meiotic effect", have been assumed to occur for a class of mutants that are thought to be due to base losses or insertions in the DNA. For such mutants, according to this interpretation, unequal crossing-over leads to restoration of a sequence compatible with normal function. However, the same author points out that such is not the case for mutation due to base substitution. Some preliminary data suggest, in fact, that mutations thought to belong to this class, do not revert with meiosis at a rate higher than in mitosis, where no crossing-over phenomena are involved. Mutants considered as belonging to such a class, on the other hand, appear to revert mainly for mutations occurring in suppressors. The results presented in this paper indicate that the phenomena of pairing and crossing-over at the w x locus, with the allele investigated, do not play a significant role in varying the reversion rate at this locus, because this rate is not lowered in a hypoploid condition where the participation of the homologue is impossible. From this point of view, this conclusion is therefore not at variance with that reached by LAUGHNAN6 in the analysis of the A 1 locus*, although in his case forward mutations were scored, and the frequency of this event is about IO fold higher than that observed for reversion at the w x locus. In this respect, it is also important to consider the fact that the reversion rate of the yeast auxotrophs to prototrophy is, on the contrary, at least IOO fold lower than that of the w a x y mutant. However, the assumption that crossing-over may be responsible for the origin of mutational events in maize cannot be completely ruled out. In fact the evidence obtained by SCHWARTZ19, that sister-strand crossing-over occurs in maize, might well account for a similarity in the behaviour of the two different chromosome conditions, provided, obviously, that sister-strand crossing-over is postulated to occur at a sufficiently high rate, at least in the hypoploid type. This may not be unlikely, taking into account some not infrequent chromosome behaviour of maize, such as nonhomologous pairing and autosynthesis. But the crossing-over hypothesis meets some other difficulties with our data. The strikingly high heterogeneity (mainly due, as suggested by the single anther analysis, to the clustering of the W x grains, and to the * Rather extensive data now available on the frequency of alpha occurrences from Ab hemizygous plants (heterozygous for the a - X 1 and a - X 3 deficiencies) as compared with those from A0 normal diploid plants, again confirm that the yield of alpha cases from the former genotype is higher than in the normal condition (LAUGHNAN,personal communication). Mutation
Research
2 (1965) 352-365
REVERSION FREQUENCY OF w a x y POLLEN TYPE IN MAIZE
361
excess of zero-class; Table IV) of the reversion rate encountered in the different pollen mother cells tissues within the same tassel is much better explained on the assumption t h a t the W x grains observed are derived not only b y mutation occurring along with crossing-over, but also b y duplication of the genetic material which has undergone m u t a t i o n earlier. T h a t a similar interpretation holds good for such an unstable allele as ~:x M lA is certainly true. The data repclted fGr this allele (Table V and Fig. 3), as u-ell as its behavi6ur in endosFerm tissue, ~here patches of blue-staining cells are inteimingled with the underlying mass staining brown, are ~easGnably suggestive of this interpretation. However, extension of this interpretation to the reversion of normally stable alleles cannot be only a m a t t e r of generalisation. This, rather, rests mainly on the significant heterogeneity which characterizes t h e rate of reversion in different tassel sectors, and which becomes prominent with some clusters of W x grains in the single anther smears. If the reversions are, to a significant degree, not necessarily associated with crossing-over, but with mutational events s e n s u stricto, a difference between the genotype in which normal crossing-over occurs (the diploid plants) and that where it is excluded, is not to be expected. If this is the case, the w a x y mutants could be considered as corresponding to the base substitution group for which mutagenesis appears t o be controlled b y suppressor factors. The existence in maize of the controlling elements, which show some parallels with the gene control system of microorganisms is, appears here to be the condition sufficient and adequate to explain the data of the reversion at the w x locus. Mutations occurring either at the operator or the regulator unit, not necessarily restricted at the crossing-over time, m a y well provide the basis for genetical events which account for the changes in the activity of the w a x y alleles which, in this framework, could be considered structural genes. The specific response of the w a x y locus to a dual system of gene control (the d i s s o c i a t i o n - a c t i v a t o r system) has been demonstrated b y McCLINTOCI~13 since 1948; it is interesting to note t h a t even if in that case the reversion rate was exceptionally high under the influence of such a system, in some of our cases revertant grains of distinctly smaller size (Fig. 4) have been observed similar to those observed by McCLINTOCK, who attributed the finding to chromosomal disturbances also induced b y the d i s s o c i a t i o n element. Finally, the failure to obtain indication of higher reversion in the normally diploid condition as compared with the hypoploid genotype is in agreement with the finding of SPRAGUEet al.*a and of PALENZONAAND SCOSSIROLIis who found that doubled maize monoploids, which would be expected to be completely homozygous since they originated from a single monoploid gamete, showed significant variability, interpreted as resulting from some type of mutational type hardly to be attributed to u n e q u a l crossing-over phenomena. APPENDIX
As indicated in the text of this paper, the main purpose of this work was t h a t of comparing the reversion rate of a normally diploid condition with the reversion of the hypoploid type obtained following X-irradiation and screening of appropriate genotypes. On the basis of the yeast results the expectation was t h a t the normal condition should have produced a higher reversion rate than the genotype in which no opportunity was offered for normal pairing and crossing-over which were supposed M u t a t i o n Research 2 (1965) 352-365
362
A. BIANCHI AND C. T()MASSINI
to be responsible for that higher reversion rate. In maize the experiments failed to confirm a similar behaviour. In fact the general average for the normal genotype turned out to be slightly lower than that of the hypoploid one. On the other hand, the difference, in the opposite sense to the expected one, could have been larger, had two exceptional tassels ot hypoploid plants been included in the data reported in Table I. The exceptional specimens have been excluded because of their extremely high heterogeneity in the reversion rate (Table VI). This heterogeneity, because of its size (several pollen samples exhibit .~°/;~-~°//o o f the Wx type)* is likely to be due
,,
[:ig. 4" E a r o b t a i n e d i r o m t h e s a m e b a c k c r o s s i n d i c a t e d in Fig. 1 b e a r i n g a b o u t half kernels w h i c h e x h i b i t t h e t y p i c a l v a r i e g a t i o n of b r e a k a g e - f u s i o n - b r i d g e cycle s t a r t i n g d i s t a l l y to I m a r k e r . R i g h t , a kernel s h o w i n g s u c h a p a t t e r n a t a h i g h e r m a g n i f i c a t i o n . T h e o t h e r half of t h e k e r n e l s s h o w t h e e x p e c t e d recessive c h a r a c t e r s in t h e n o r m a l p a t t e r n ( t h e y are easily recognized b y t h e i r s h r u n k e n a p p e a r a n c e a n d u n i f o r m l y b r o n z e colour). T h e v e r y d a r k coloured k e r n e l a t t h e t o p left is a k e r n e l in w h i c h t h e b r e a k a g e - f u s i o n - b r i d g e cycle s t a r t e d b e t w e e n I a n d t h e c e n t r o m e r e , following t h e c o m p l e t e loss of t h e Inhibitor factor, * Single a n t h e r s m e a r s r e v e a l e d t h a t in a s a m p l e of 96, b e s i d e s 68 a n t h e r s w i t h no W x grains, t h e r e were single cases of t h e r e m a i n i n g 28 w i t h t h e following n u m b e r s of W x pollen g r a i n s : 2 - 7 8-I4-23-24-3o-37-42-56-62-78-78-87 92-115-117-118-127-143-146-i64-166-171-189-263. Mutation Research 2 (1965) 352-365
REVERSION FREQUENCY TABLE
363
O F WaA;y P O L L E N T Y P E I N M A I Z E
VI
x Y g l Sh
FREQUENCY" OF W x POLLEN IN EXCEPTIONAL TASSELS FROM THE c R o s s y g C s h b z w x /~g W x WHOSE DOMINANT GAMETE WAS X - I R R A D I A T E D
Tassel 13o 3
Branch i
Total 2
Total
3
Total number of pollen grains
N u m b e r o/ W x pollen grains
Rate of W x grains ( × Io -s)
I
21850
2
9.1
2 3
17200 31500
i 3
5.8 9.5
2045 ° 1485o I o 5 85 °
27 io 43
132"° 67. 3 4°.6
4 5 I-5 i
16800
o
2
1745 °
3
3 4 5 6 I-6
20200 1723° 17500 21350 I i o 53 °
IOI 56 13 o 173
o.o 17-2 5oo.o 3 2 5 .0 74-3 o.o 156.5
I
23633
2 3 4 5 I-5
33 1 6 6 329 oo 3445 ° 3285 ° 156999
23 ° 585 479 288 742 2324
1763.8 1445.9 836.o 2858"7 157 o . °
I
I
41000
o
o.o
2
I 2 3 I-3
19000 15400 164 °° 5° 8oo
I o o I
52.6 o.o o.o 2.o
I 2 I-2
21150 13600 3475 °
4 o 4
18.9 o.o I 1.5
2122400
o
o.o
2765 ° 2865 ° 3255 ° 329 °0 17050 31 16o 225 °° 19246o
23 14 24 4 4° 39 15 159
83.2 48.9 73.7 12.2 234"6 I25-2 66"7 82.6
Total
I292
Position in the branch
Total 3 Total
4
I
5
I 2 3 4 5 6 7 1- 7
Total
973.2
to some special phenomena besides the mutation event sensu stricto invoked above. Those were possibly breakage-fusion-bridge cycles initiated distally to Wx, and which were conducive to the loss of this dominant factor especially in the later development of the tassel, namely the lateral branches 5. The latter, in fact (the lower numbered cases in Table VI), present no W x grains or very few, especially in the late developing sectors. Such grains are, on the contrary, strikingly numerous in the tassel sectors which trace back to parts likely to be formed during early developmental stages, when the broken chromosome arms bearing the dominant markers had not Mutation Research 2 (1965) 3 5 2 - 3 6 : 5
364
a . t~IANCHI AN]_) C. T()MASSIN1
yet been lost following the cycle of events (the branch 3 ill tassel I3o3, and 5 in tassct 1292; moreover in both tassels the central sectors of m a n y branches also show, as expected according to our interpretation involving the developmental process, higher Wx-pollen rates). This interpretation is also supported by the fact that in the two tassels a very strong correlation was present between the frequency of Wx pollen grains of the early flower and that of the late one on the same spikelet. Factual evidence of the occurrence of such a kind of cycle has been given by an ear (Fig. 4) in which most of the typical variegation produced by the cycle is visible in m a n y kernels. Most of these kernels actually show the phenotype expected to originate from a distal breakage point. Moreover, few kernels from the late developing part of the ear show phenotypes indicative of the loss of more proximal dominant factors of the genetically marked region. McCLINTOCKn,12 produced evidence that in the sporophyte tissue of maize only the breakage-fusion-bridge cycle of chromosome type can occur. This required in her type of experiments that both egg and sperm nuclei contributed a chromosome with a freshly broken end. Since our plants derive from zygotes in which only the sperm nuclei m a y have been broken by the irradiation treatment, the cases just described can be interpreted either admitting that in the sporophyte also a chromatid type of cycle occurs, as a result of a breakage induced in the sperm nuclei, or that two broken ends of different chromosomes undergo rejoining and start a cycle of chromosome type. ACKNOWLEDGEMENTS
This work has been in part financially supported b y C.N.R., Rome (with a fellowship to Dr. C. TOMASSINI) and b y I.A.E.A., Vienna (Research Contract 64 US). The laboratory work has been carried out mainly in the Institute of Genetics of Milan University, the field work mainly at Piacenza (Plant Genetics Institute of the Catholic University), whereas the calculation work as well as the preparation of the manuscript has been performed in Bologna (Plant Breeding Institute). The critical reading of the manuscript b y Dr. G. E. MAGNI, Parma, and Dr. O. E. NELSON, Purdue University, Lafayette, Ind., is also gratefully acknowledged.
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365
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21lutation'~Research 2 (1965) 352-365