Theoretical and Applied Genetics 40, 147--t 54 (1970) | b y Springer-Verlag t970
Y-Chromosome Hyperploidy in Rumex JERZY ZUK D e p a r t m e n t of Genetics, Polish A c a d e m y of Sciences, W a r s a w (Poland) Summary. 1. Male and female Y-chromosome hyperploid plants were obtained and morphologically and cytologically investigated. 2. Conjugation of sex chromosomes was investigated i n ~ plants with 1, 2 and 3 additional Y chromosomes. Conjugation between sex chromosomes was end-to-end. Association between Y-chromosomes was partly based on conjugation between homologous segments and partly due to heterochromatin fusion. 3. Transmission of the additional Y chromosome to the progeny was studied. I n all cases transmission of the extra Y chromosome was very low. The reasons for this phenomenon are probably nonrandom fertilization, nonrandom degeneration of megaspore, and reduced seed setting and seed fertility in plants with additional Y chromosomes. 4. The phenotypic effect of Y chromosome hyperploidy was investigated. Plants with extra Y chromosomes are shorter and die earlier. The fertility of such plants is low. Thus, although Y chromosomes are heterochromatic and nonactive (except in the premeiotic interphase of microsporogenesis) they have a marked influence on the appearance of Y-chromosome hyperploid plants.
Introduction Y chromosomes in R u m e x thyrsiflorus are heteroc h r o m a t i c a n d late-replicating, a n d t h e y have no effect on sex d e t e r m i n a t i o n (Zuk, 1963, t 969 a, 1969 b). However, t h e y are n o t genetically i n e r t as d a t a were o b t a i n e d i n d i c a t i n g t h a t t h e y are responsible for the f e r t i l i t y of male p l a n t s a n d for the sex ratio (Zuk, 1970). T h e d a t a here p r e s e n t e d concern the effect of e x t r a Y chromosomes on sex chromosome c o n j u g a t i o n a n d on the fertility a n d m o r p h o l o g y of male a n d female p l a n t s . T h e p a t t e r n of t r a n s m i s s i o n of s u p e r n u m e r a r y Y chromosomes to the p r o g e n y is also discussed.
Material and Methods I n the course of studies on polyploidy and sex determination in R. thyrsiflorus one particular female p l a n t with karyotype X X Y Y + 12 A = 16 instead of X X + + 12./1 = 14 was obtained (Fig. 1). The cross of this
female plant with a normal male p l a n t X Y Y + ! 2 A = t 5 gave m a n y male and female plants with additional Y chromosomes. Male plants X Y Y Y Y + 12 A = 17 and female plants X X Y Y + 12A = t6 were chosen as parental forms for further crosses, giving more Y-chromosome hyperploid plants with t, 2 and 3 additional Y chromosomes (Figs. 2, 3, 4). The methods of cytological analysis were the same as described previously (Zuk, 1963, t 969c).
Results I. Sex chromosome conjugation in male plants with additional Y chromosomes I n n o r m a l male p l a n t s X Y 1 Y2 + 1 2 A ---- t5 sex chromosomes form a t r i v a l e n t Y I - - X - - Y , . The homologous s e g m e n t s of X a n d Y chromosomes are v e r y short, a n d at diakinesis sex chromosomes associate e n d to e n d giving a long characteristic t r i v a l e n t
Figs. 1--4. Metaphasal plates from root tips. • 1700 Fig. t. Female plant with karyotype 16 = X X Y Y + 12 A Fig. 3. Male plant with karyotype 17 = X Y Y Y Y + t2 A t;ig. 2. Male plant with karyotype 16 = X Y Y Y + 12 A Fig. 4. Male plant with karyotype 18 = X Y Y Y Y Y + t2 A
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Figs. 5-- 8. Chromosome association at meiosis in normal male plants and in those with additional Y chromosomes. • 1700 Fig. 5. Diakinesis in normal plant with karyotype 15 = XYY Fig. 7. Diakinesis in plant with karyotype t 6 = X YY Y + + 12,4. Arrow indicates Y X Y t r i v a l e n t 12-//. Arrows indicate YXYtrivalentandunivalentformedby Y chromosome Fig. 6. MI in plant with karyotype 16 = X Y Y Y + 12A. Fig. 8. Late metaphase in PMC of plant with karyotype Arrow indicates Y X Y Y tetravalent t6 = X Y Y Y + t2 A. Note prereduction of Y Y bivalent c h r o m o s o m e , a n d this a s s o c i a t i o n s h o u l d be cons i d e r e d as a h e t e r o c h r o m a t i c fusion r a t h e r t h a n a real c o n j u g a t i o n . I n a n o t h e r t e t r a v a l e n t configur a t i o n all h o m o l o g o u s ends of Y c h r o m o s o m e s are i n v o l v e d in c o n j u g a t i o n in such a w a y t h a t t w o Y c h r o m o s o m e s ( o b v i o u s l y t w o Y1 or t w o Y~) c o n j u g a t e w i t h one end of an X c h r o m o s o m e , s c h e m a t i c a l l y
w i t h t h e X c h r o m o s o m e o c c u p y i n g t h e c e n t r a l position (Fig. 5). I n p l a n t s w i t h a d d i t i o n a l Y c h r o m o somes v a r i o u s m o d i f i c a t i o n s of sex c h r o m o s o m e conj u g a t i o n were o b s e r v e d . Conjugation in plants with an additional Y chromosome. Male p l a n t s w i t h c h r o m o s o m e c o n s t i t u t i o n XYYY + t 2 A = t 6 (Fig. 2) m a y possess e i t h e r one Y1 c h r o m o s o m e a n d t w o h o m o l o g o u s Y, c h r o m o somes or one Y2 c h r o m o s o m e a n d t w o h o m o l o g o u s Y1 chromosomes. U n f o r t u n a t e l y Y1 a n d Y, c h r o m o somes are u s u a l l y n o t m o r p h o l o g i c a l l y d i s t i n g u i s h able. I n such p l a n t s four different p a t t e r n s of sex c h r o m o s o m e c o n j u g a t i o n were o b s e r v e d : Y X Y Y (Fig. 6), Y X Y + Y (Fig. 7), X Y + Y Y and X Y + Y + Y . A s s o c i a t i o n b e t w e e n sex chromosomes was as a rule e n d - t o - e n d . Two different config u r a t i o n s of t h e t e t r a v a l e n t X Y Y Y were observed. In the chain configuration YXYY, p r e s e n t e d in Fig. 6, t w o Y c h r o m o s o m e s c o n j u g a t e w i t h two hom o l o g o u s ends of t h e X c h r o m o s o m e as in a n o r m a l t r i v a l e n t . T h e t h i r d Y c h r o m o s o m e is a t t a c h e d to t h e e n d of t h e Y c h r o m o s o m e n o t i n v o l v e d in conjug a t i o n w i t h t h e X c h r o m o s o m e . I t is k n o w n t h a t in R u m e x Y c h r o m o s o m e s are h e t e r o c h r o m a t i c a n d u n a b l e to t a k e p a r t in c o n j u g a t i o n w i t h X c h r o m o somes e x c e p t for t h e s m a l l t e r m i n a l f r a g m e n t . Thus, in t h e t e t r a v a l e n t in q u e s t i o n t h e a d d i t i o n a l Y c h r o m o s o m e is o b v i o u s l y a t t a c h e d to t h e fully h e t e r o c h r o m a t i c a n d n o n h o m o l o g o u s e n d of t h e o t h e r Y
- /Y2
Y1 - - A ~ y ,
y/Y1
or Y2 -- ~ ' \ , Y I "
The third Y chromo-
some c o n j u g a t e s w i t h t h e second a r m of t h e X chrom o s o m e in t h e n o r m a l w a y . As seen in T a b l e t, t h e m o s t f r e q u e n t associations were Y X Y + Y (triv a l e n t a n d Y c h r o m o s o m e as a u n i v a l e n t ) a n d X Y + Y Y (two b i v a l e n t s ) . I n t h e first case t h e a d d i t i o n a l Y c h r o m o s o m e does n o t affect n o r m a l sex chromosome conjugation because a normal YXY t r i v a l e n t is formed. H o w e v e r , q u i t e f r e q u e n t l y t h e e x t r a Y c h r o m o s o m e t e n d s to c o n j u g a t e w i t h its h o m o l o g u e f o r m i n g a b i v a l e n t (Y1 Y1 or Y2 Y2) a n d p r e v e n t i n g f o r m a t i o n of t h e n o r m a l sex c h r o m o s o m e t r i v a l e n t . I n such a case two sex c h r o m o s o m e biv a l e n t s were observed. T h e f o r m a t i o n of one sex c h r o m o s o m e b i v a l e n t X Y a n d two Y c h r o m o s o m e u n i v a l e n t s was also o b s e r v e d b u t this p h e n o m e n o n c a n n o t be r e a s o n a b l y e x p l a i n e d . I t is, however, possible t h a t t h e a s s o c i a t i o n b e t w e e n Y h o m o l o g u e s is less p e r s i s t e n t t h a n t h e Y - - X association, a n d t h e two Y c h r o m o s o m e s o b s e r v e d as u n i v a l e n t s are t h e result of some k i n d of d e s y n a p s i s . P r e r e d u c t i o n of
Table !. Sex chromosome conjugation in male plants of R. thyrsiflorus with one additional Y chromosome No. of plant
Karyotype
22 26 61 65
16=XYYY 16~XYYY t6=XYYY 16=XYYY Total
Pattern of sex chromosome association in diakinesis YXYY + + q+
Total
YXY + Y
XY + YY
XY + Y+ Y
12A 3 12A 6 12A 8 12A 12
42 22 18 12
40 18 11 28
6 11 14 8
91 57 51 60
29
94
97
39
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Y-Chromosome Hyperploidy in Rumex
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Table 2. Sex chromosome segregation in Anaphase I in male plants of R. thyrsiflorus with one additional Y chromosome
t49
Conjugation i n plants with 2 additional Y chromosomes. F o u r m a l e p l a n t s w i t h c h r o m o s o m e c o n s t i t u t i o n XYYYY were o b t a i n e d . I n one p l a n t t w o Y
l i k e l y t h a t this p l a n t h a d t w o h o m o l o g o u s Y1 chrom o s o m e s a n d t w o Y, chromosomes. I n this p l a n t v a r i o u s c o m b i n a t i o n s of sex c h r o m o some a s s o c i a t i o n were o b s e r v e d , as shown in T a b l e 3Most f r e q u e n t was t h e a s s o c i a t i o n X Y + Y Y + Y (Fig. 9)- This c o n f i g u r a t i o n is s i m i l a r to t h e association X + Y + Y Y o b s e r v e d f r e q u e n t l y in t h e X Y Y Y p l a n t (Table 2). S u r p r i s i n g l y , c o n f i g u r a t i o n s w i t h a n YXY t r i v a l e n t were less f r e q u e n t t h a n in p l a n t s w i t h one a d d i t i o n a l Y c h r o m o s o m e . W h e n a sex t r i v a l e n t was formed, t h e two a d d i t i o n a l Y c h r o m o s o m e s were seen as a b i v a l e n t or as t w o u n i v a l e n t s (Fig. t 0). T h e r e l a t i v e l y low f r e q u e n c y of such config u r a t i o n s m a y b e e x p l a i n e d as t h e r e s u l t of t h e presence of two h o m o l o g o u s p a i r s of Y c h r o m o s o m e s (Yx Y1 a n d Y2 Y~). I t seems t h a t in such a case h o m o l o g o u s Y c h r o m o s o m e s t e n d to c o n j u g a t e w i t h one a n o t h e r p r e v e n t i n g f o r m a t i o n of t h e sex c h r o m o some t r i v a l e n t . A few p e n t a v a l e n t s a n d t e t r a v a l e n t s w i t h one u n i v a l e n t (Fig. 11) were also o b s e r v e d . I n such c o n f i g u r a t i o n s a s s o c i a t i o n s b e t w e e n Y c h r o m o somes seem to b e t h e r e s u l t of h e t e r o c h r o m a t i c fusion, as h o m o l o g o u s p a r t s of one Y1 a n d one Y2 c h r o m o s o m e are a l r e a d y i n v o l v e d in a s s o c i a t i o n w i t h t h e X chromosome. I n one cell, o n l y u n i v a l e n t s were o b s e r v e d (Fig. 12), b u t it m i g h t h a v e b e e n m e r e l y a t e c h n i c a l artefact. S e g r e g a t i o n of sex c h r o m o s o m e s in t h i s p l a n t was n o t a n a l y z e d , b u t p r o b a b l y s e g r e g a t i o n s X Y -- Y Y Y a n d X Y Y -- Y Y were m o s t f r e q u e n t .
c h r o m o s o m e s were s h o r t a n d m e t a c e n t r i c , two o t h e r s were l o n g e r a n d s u b m e t a c e n t r i c (Fig. 3). I t is v e r y
Conjugation in plants with 3 additional Y chromosomes. I n t h e m a l e p l a n t w i t h k a r y o t y p e X Y Y Y Y Y
Pattern of sex chromosome segregation
No. of plant
Karyotype
22 26 61 65
16 = X Y Y Y + I 2 A t7 16 = X Y Y Y + 1 2 A 20
2 6
16= XYYY 16= XYYY
6 2
6 3
45
17
XY--
+ t2A + t2A
Total
YY
X-- YYY
t h e sex c h r o m o s o m e , o b s e r v e d s o m e t i m e s in M I (Fig. 8), suggests such a p o s s i b i l i t y . Sex c h r o m o s o m e s e g r e g a t i o n in A I was also a n a lyzed. As m a y b e seen from T a b l e 2, t h e m o s t freq u e n t was s e g r e g a t i o n X Y ~ Y Y . A n o t h e r possible p a t t e r n of segregation, X ~ Y Y Y , was m u c h less f r e q u e n t . T h i s fits well w i t h t h e d a t a c o n c e r n i n g c h r o m o s o m e c o n j u g a t i o n as it was to be e x p e c t e d t h a t s e g r e g a t i o n of t h e m o s t f r e q u e n t a s s o c i a t i o n s YXY+ Y and XY+ YY s h o u l d be of t h e X Y ~ Y Y t y p e . A p a r t from t h e t w o m e n t i o n e d a b o v e , no o t h e r t y p e s of sex c h r o m o s o m e segregation were found.
Table 3. Sex chromosome conjugation in male plant of R. thyrsiflorus with two additional Y chromosomes Pattern of sex chromosome association in diakinesis Karyotype
YYXYY YXYYY
17=XYYYY+I2A
4
or
YXY + YY Y X Y Y + Y or XY+ YYY
YXY+ + Y
5
1t
1o
Y
XY+ YY + Y
XY+ Y + Y+ Y
X+ Y+ Y + Y+ Y
53
9
l
Total
Figs. 9-- t 2. Chromosome association at meiosis in male plants with additional Y chromosomes. • 1700 Fig. 9. Diakinesis in plant with karyotype t 7 = X Y Y Y Y + Fig. t t. Diakinesis in plant with karyotype 17 = X Y Y Y Y + t2 A. Arrows indicate two bivalents X Y , Y Y and a Y univa12 A. Arrow indicates tetravalent formed by sex chromosolent mes Fig. 10. Diaki~esis in plant with karyotype 17 = X Y Y Y Y + Fig. 12. Diakinesis in plant with karyotype 17 = X Y Y Y Y + 12 A. Arrows indicate Y X Y trivalent and two Y univalents t2 A. Arrow indicates X and Y univalents
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Table 4. Sex chromosome conjugation in male plant of R. thyrsiflorus with three additional Y chromosomes Pattern of sex chromosome association in diakinesis Karyotype
t8=X+ YYYYY+ +t2A
YYXYY +Y
YXY +YYY
YXY + YY XY + YY +Y +YY
XY + YY +Y+Y
XY + Y+ Y YXY + Y +Y+Y +Y+Y
Total
t
1
t7
34
4
81
+ t2 A = t 8 (Fig. 4) Y1 and Y, c h r o m o s o m e s were indistinguishable. Only one such plant was obtained. Analysis of diakinesis revealed the combinations of sex chromosome associations shown in Table 4. Again, as in plants X Y Y Y and X Y Y Y Y , combinations with the X Y bivalent were more frequent t h a n combinations with the sex c h r o m o s o m e trivalent YXY. I t was observed t h a t three additional Y chromosomes m a y form a trivalent Y Y Y , or a bivalent and a univalent Y Y + Y, or only univalents Y + Y + Y. The Y c h r o m o s o m e trivalent was observed in one cell only. P e n t a v a l e n t s Y Y X Y Y were also v e r y rare and should be considered as being due to h e t e r o c h r o m a t i c fusion between Y chromosomes. Segregation of sex chromosomes was not analyzed. I n all plants with additional Y chromosomes there was no indication of loss of additional Y chromosomes in ontogenesis or during meiotic division.
2. Transmission of additional Y chromosomes to progeny Transmission of additional Y chromosomes was investigated in the p r o g e n y of two crosses: ~_XXYY+t2A = t6 • d~XYY+I2A = t5 XXYY+I2A =16 • d~XYYYY+t2A =t7 Table 6. Pollen fertility of plants with additional Y chromosomes No.
Karyotype
t 2 3 4 5 6 7 8 9
XYY+I2A XYY+t2A XYY+t2A XYYY+I2A XYYY+I2A XYYY+t2A XYYY+I2A XYYYY+t2A XYYYYY+I2A
Pollen fertility = t5 = 15 ---= 15 = 16 = 16 = 16 = 16 = 17 = t8
85% 86% 89% 78% 25% 89% 52% 90% 47%
21
3
I n b o t h crosses female plants had two additional Y chromosomes. I t seems probable t h a t in such plants two X chromosomes conjugate side to side as in a normal female plant. Two Y chromosomes m a y be a t t a c h e d (by their homologous ends) to the X - c h r o mosome bivalent or m a y form two univalents. The m o s t likely segregation of sex chromosomes is X Y and X Y , so the gametes X Y + 6 A should be the m o s t frequent. I n the first cross the male parent p r o d u c e d two types of gametes, X + 6 A = 7 and X Y + 6 A ---- 8, in equal proportion ; therefore in the p r o g e n y the k a r y o t y p e s $ X X Y + t2 A = 15 a n d ~ ) X Y Y Y + t 2 A = 16 should be the most frequent. I n the second cross, the male parent X Y Y Y Y should produce m a i n l y four types of gametes, X Y , X Y Y , Y Y Y , Y Y , r o u g h l y in equal proportions. Thus in the p r o g e n y of this cross plants with k a r y o types~ XXYYY + 12 A = 17, c~ X Y Y Y + 12 A = = 16 and c~ X Y Y Y Y + t 2 A = t 7 should be the m o s t frequent. As shown in table 5, in b o t h crosses plants with additional Y chromosomes were less frequent t h a n could have been expected from the presumed p a t t e r n of sex chromosome segregation. Most plants had n o r m a l k a r y o t y p e s or only one additional Y chromosome, which suggests t h a t transmission of extra Y chromosomes to the p r o g e n y is v e r y restricted. I t seems t h a t gametes with additional Y chromosomes m a y be eliminated in fertilization as a result of competition with normal gametes.
3. Fertility of plants with additional Y chromosomes Pollen fertility was estimated in 6 plants with !, 2 and 3 extra Y chromosomes and c o m p a r e d with t h a t of n o r m a l male plants (Table 7). All Y hyperploid and normal plants (Nos. t - - 7 in Table 6) were the p r o g e n y of the cross ~ X X Y Y + t 2 A = t6 • c~ X Y Y + t2 A = 15 described in the previous section. P l a n t No. 8 with 3 extra Y chromosomes was
Table 7. The seed fertility in crosses involving plants with extra Y chromosomes Cross
QXX--}-12A =I4xdXYYY+I2A QXX+I2A =I4• 9XX+t2A =I4• 9XXY+I2A =t5xdXYY+I2A 9XXYY+t2A=I6• Q XXYYWI2A=I6• Q XXYY+I2A=t6xc~XYYYY+I2A=t7
The number The number The percent of seeds of seeds of seed collected sown germinated = t 6 240 130 100 = 1 5 330 = 1 5 -= 1 6 13o t64
240 130 100 150 t00 130 t64
t4 23 79 24 79 42 42
= f6
9XXYY+t2A
~XYYYY+t2A
= 17 n u m b e r o f measured plants
mean height of p l a n t s in c m
15 n u m b e r o f measured plants
=
x
mean height
Karyotypes
6
24
+ t2A
2
t2
9
= 16
XXYY
1
--
9
+ 1 2 A = t7
+ 12A XXYYY
20
I0
c~
= t5
XYY
+ 12A
9
8
c~
6
94
24
125
X= X 14Q + 12 d
10
81
25
t09
+ 12A = 15Q
XXY
2
73
10
70
=
XXYY + 16Q |2A
1
45
--
--
+ 1 2 A = 17Q
XXYYY
18
79
~3
87
+ = 1 t 52CA ~
XYY
10
62
8
65
=
16(~
t
55
2
53
+
12A
XYYYY
2
2
~
=
176
+ 1 2 A = 17
+ 12A XYYYY
XYYY + i2A
= t6
XYYY
T a b l e 8. T h e height o f 9 a n d (~ p l a n t s with additional Y chromosomes
14
25
9
= t5
= 14
9
XXY
XX + 12A
~-- i 6 of p l a n t s i n c m
9XXYY+I2A x cSXYY+t2A
Cross
xc~XYYYY+I2A=I7
F r e q u e n c y of k a r y o t y p e s i n cross 9 XXYY + 12 A = 16
= 16X~XYY+t2A=I5
Frequency of karyotypes in e r o s s Q X X Y Y + t2A =
Sex
K a r y o t y p e of t h e p r o g e n y
T a b l e 5. K a r y o t y p e s i n progeny o f crosses between plants with additional Y chromosomes
t
30
--
--
+
12A
XYYYYY
1
--
c~
=
|8(~
+ 1 2 A = t8
XYYYYY
~~
~e
.r
t52
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obtained from the cross ~ X X Y Y + 12 A = t6 • X Y Y Y Y + t2 A = t7. Normal plants produced from 85 to 90% of morphologically good pollen. The pollen fertility of Y-hyperploid plants varied between 25 and 90%. It was interesting that among plants with one additional Y chromosome, one exhibited markedly reduced pollen fertility (up to 25~ while in the other two pollen fertility was more or less normal (78 and 89%). The fertility of the plant with two extra Y chromosomes was quite normal (90%), and in the plant with 3 additional Y chromosomes it was reduced to 470/0. These data indicate that in most cases additional Y chromosomes do not reduce pollen fertility in any drastic way. Fertility of plants with additional Y chromosomes was also estimated b y comparison of seed setting and seed germination in crosses involving plants with additional Y chromosomes, and in crosses between normal plants. The results are given in Table 7. Seed setting in crosses between normal plants was about t 000 seeds per cross. Seed setting in all crosses involving Y hyperploid plants was drastically reduced even to as little as 100 seeds. Seed germination varied from cross to cross but usually was much lower (t4--79%) than germination of seeds from the cross between normal plants (80~ It was surprising that in two crosses between plants with identical chromosome constitution but of different origin (crosses No. 2 and No. 3) seed germination was 23% and 79~ respectively. It resembles the variation in pollen fertility observed for male plants not differing in their karyotypes. The cause of this phenomenon remains obscure, but genetic rather than chromosomal factors should be responsible for the differences observed.
4. The phenotypic effect of Y chromosome hyperploidy Male and female plants with one additional Y chromosome are indistinguishable in their morphology from normal plants. The presence of two or more additional Y chromosomes usually influences strongly the morphological features of plants. Leaves of such individuals, especially when young, are curled, pale green and their shape different than that typically observed in normal seedlings. Moreover, the number of leaves and flower stems is markedly reduced and such plants are shorter than normal individuals. Table 8 presents the results of measurements of the progeny obtained from two crosses between plants with additional Y chromosomes. It is seen that the height of the plant is inversely proportional to the number of additional Y chromosomes. Even plants with one additional Y chromosome, whose leaf morphology is not changed, are shorter than normal plants. Also, the viability of Y chromosome hyperploid plants is lower than that of normal plants. R. thyrsiflorus is a perennial species and normal diploid individuals remain vigorous for several years;
the longevity of Y hyperploid plants was markedly reduced in comparison to that of normal plants. Discussion
In mammals including man (Hayward, 1962; Utakoji, 1966; Mittwoch, 1967; Solari, 1969), and in dioecious plants (Jacobsen, 1957; Westergaard, t958; Smith, 1964), the most common association between X and Y chromosomes during meiosis is the end-to-end configuration. It has been demonstrated in various organisms that the end-to-end association of the X -- Y pair is the result of chiasma formation between minute homologous regions (Ohno, Kaplan and Kinosita, t959; Utakoji, 1966; Solari, t969). Data presented in this paper show that in Rumex thyrsiflorus, both in normal and Y-chromosome hyperploid plants, associations between sex chromosomes are usually end to end. Autoradiographical data and cytological observation (Zuk, t969 a) showed that both Y chromosomes are entirely late-replicating (heterochromatic) and the X chromosome is entirely euchromatic. Thus if the association between sex chromosomes Y X Y in Rumex is based on synapsis between homologous segments of X and Y chromosomes, these homologous segments in Y chromosomes are very short, below the resolving power of autoradiography. Thus it is possible that Y chromosomes in R. thyrsiflorus are composed of large differential heterochromatic segments, and very small euchromatic ones homologous to the X chromosome. In Rumex, as well as forming chiasmata between the distal homologous ends of X and Y chromosomes, Y~ and Y2 chromosomes can produce associations b y heterochromatic fusion. If a sex trivalent is formed in an X Y Y Y + 12 A = t6 plant, association between the chromosomes I/-1, X and Y2 is based on the homology between Y1, Y2 and X chromosomes. Association of an additional Y1 or Y, chromosome with the trivalent is based on heterochromatic fusion because homologous segments of Y and X chromosomes are already involved in association. It seems that association b y heterochromatic fusion can take place mainly between partly homologous }71 + I71 or Y2 -~- Y2 chromosomes, because in normal Y X Y + + 12 A = t 5 plants it has never been observed. This is not surprising, because in the opposite case the regular behaviour of the sex chromosome mechanism would be highly disturbed. Association between the nonhomologous Y and the heterochromatic S chromosome has already been reported in Rumex (Zuk, 1969b) and in other organisms (Riley and Law, 1965). Recently Maguire (1969) has shown that distribution of large heterochromatic knobs in premeiotic interphase nuclei of maize is not random. Two large heterochromatic K t0 regions were found to be localized very significantly nearer to each other than random expectations predicted. Thus it seems that the tendency of heterochromatin to clump during inter-
Vol. 4o. No. 4
Y-Chromosome Hyperploidy in Rumex
phase, especially during premeiotic interphase, m a y be the reason for nonhomologous heterochromatic association in meiosis. Analysis of anaphase cells in plants with one additional Y chromosome revealed t h a t segregation of sex chromosomes was nonrandom. S e g r e g a t i o n X Y - - Y Y was about 3 times more frequent than segregation XY Y Y , and not one case of segregation Y --XYY was observed. Nonrandom segregation of sex chromosomes in X Y Y Y + 12 A = t6 plants is partly the result of the pattern of sex chromosome association in meiosis. Associations Y X Y + Y and X Y + Y Y were very frequent. The most frequent segregation resulting from such association should be X Y -- Y Y . Non-random segregation of sex chromosomes has also been shown in X Y Y males of Drosophila b y Grell (i969). In Rumex, transmission of extra Y chromosomes to the progeny was greatly reduced. This was true for both female and male gametes. Low transmission of extra chromosomes of the trisomics have been reported in m a n y plants (Burnham, t962). This is also true for heterochromatic Y sex chromosomes in R. thyrsiflorus. W h a t mechanism m a y be responsible for low transmission of additional Y chromosomes to the progeny. In male plants with extra Y chromosomes no case of loss of the extra Y chromosome during ontogenetic development or during meiotic division was observed. All Y chromosomes were transmitted to the gametes. Pollen fertility of male plants with additional Y chromosomes is usually quite good, so t h a t selective degeneration of pollen grains with additional Y chromosomes is almost excluded. Therefore the most probable explanation is nonrandom fertilization. The more Y chromosomes are present in pollen grains the poorer their chance to fertilize. This m a y be connected with the deleterious effect of extra Y chromosomes on germination and growth of pollen tubes. Meiosis in female plants was not investigated but it is probable, t h a t if the basal megaspore contains th.e extra Y chromosome it fails to produce the embryo sac. Consequently the embryo sac m a y be produced b y another megaspore with the normal haploid complement. A similar possibility was postulated b y McClintock and Hill (i931) to explain the failure of the extra chromosome to be transmitted to 50% of the progeny in trisomics of maize. In Rumex, even in normal plants, transmission of Y Y + 6 A gametes is much lower than of X + 6 A, and the resulting female plants are much more numerous in the population (Zuk, 1963). I t m a y be speculated t h a t in both cases the mechanism of low transmission of Y chromosomes is the same. Y-chromosome hyperploid individuals have been obtained in Drosophila (Dronamraju, 1965; Mittwoch, 1967; Lewis and John, t 968; Grell, 1959, t969),
153
in humans (Chu, 1964; Jacobs, Brunton and Melville, t965), in mouse (Cattanach, 1961; Russell and Chu, t 9 6 t ; Mittwoch, 1967) and in Melandrium (Westergaard, t958). In man, mouse and Melandrium, individuals with an extra Y chromosome were male, because in these organisms the Y chromosome is strongly male-determining. In Drosophila the Y chromosomes carry no male-determining genes but the addition of an extra Y chromosome affects fertility. The fertility of X Y Y males is only about 0,5--3.0~o. Similarly, b y comparing females with 0, 1, 2 Y chromosomes Grell (t969) has found t h a t the fertility varied inversely to the hyperploidy. In man, X Y Y males are mentally subnormal and behave aggressively, and X X Y and X X Y Y have Klinefelter's syndrome. X X Y mice are morphologically indistinguishable from normal males, but they are sterile. I t is possible, as postulated b y Mittwoch (1967), t h a t the apparent lack of phenotypic abnormalities in mice with an abnormal sex chromosome is connected with the fact t h a t the chromosomes in mouse contain a large proportion of heterochromatin, so t h a t modifications of the k a r y o t y p e m a y be less damaging than those in man. The same is also true for Rumex: b o t h the Y chromosomes are heterochromatic. Male and female plants with one extra Y chromosome are almost normal in appearance. They are only about 20 cm lower. Morphological pollen fertility of male plants with one extra Y chromosome ranges between 50--90%. Seed setting and germination of female plants with one additional Y chromosome is 30%. Two or three extra Y chromosomes have a much more pronounced effect. Female plants are about 30 cm and male plants about 30 cm lower than normal ones. The leaves are often curled and pale. Seed setting and seed germination are much lower than in normal plants. The female plant with three extra Y chromosomes is completely sterile. Thus additional Y chromosomes in Rumex have some effect on the phenotype and fertility of plants. This effect is much less pronounced t h a n in other organisms because Y chromosomes are heterochromatic and have no male-determining genes, and probably because in plants the shift in chromosomal balance has less drastic effects than in organisms with a much higher level of organization like m a m m a l s including man. Acknowledgement. I wish to thank Professor W. Gajewski and Dr. Z. Swietlifiska for much help in the course of this investigation, and for reading and commenting on the manuscript.
Zusammenfassung Die Arbeit berichtet von Untersuchungen tiber die Wirkung tiberz/ihliger Y-Chromosomen auf die Paarung der Geschlechtschromosomen und die Fertilit/it und Morphologie m/innlicher und weiblicher Pflanzen von Rumex thyrsiflorus.
J. ~uk: Y-Chromosome Hyperploidy in Rumex
t54
Die G e s c h l e c h t s c h r o m o s o m e n bei d e n u n t e r s u c h t e n c~ P f l a n z e n m i t 1, 2 u n d 3 iiberz~ihligen Y-Chromosom e n zeigen e n d to e n d - P a a r u n g . Die Assoziation der Y - C h r o m o s o m e n b a s i e r t teils auf einer P a a r u n g homologer Segmente, teils auf einer H e t e r o c h r o m a t i n fusion, I n allen u n t e r s u c h t e n F~llen war eine 0 b e r t r a g u n g des tiberz~hligen Y-Chromosoms auf die N a c h k o m m e n s c h a f t sehr gering. Die Griinde hierftir sind m6glicherweise pr~iferentielle B e f r u c h t u n g , pr~iferentielle D e g e n e r a t i o n der Megaspore sowie geringer S a m e n a n s a t z u n d geringe Fertilit~it der P f l a n z e n m i t iiberz~ihligen Y-Chromosomen. Auch der ph/inot y p i s c h e Effekt einer H y p e r p l o i d i e y o n Y-Chromosom e n w u r d e u n t e r s u c h t . P f l a n z e n m i t iiberzAhligen Y - C h r o m o s o m e n sind kiirzer u n d s t e r b e n friiher ab. Ihre F e r t i l i t i i t ist gering. Obwohl die Y - C h r o m o s o m e n h e t e r o c h r o m a t i s c h u n d i n a k t i v sind (aul3er i n der pr~meiotischen I n t e r p h a s e der Mikrosporogenese), h a b e n sie doch e i n e n d e u t l i c h e n EinfluB auf den H a b i t u s v o n P f l a n z e n m i t tiberz~ihligen Y-Chromosomen.
References
t. Burnham, C. R. : Discussion in cytogenetics. Minneapolis: Burgess Publishing Co. t962. -- 2. Cattanach, B. M.: X X Y mice. Gen. Research 2, t 5 6 - - t 5 8 (t96t). -3. Chi-Chang, Ch., Grant, W. F. : Trisomic transmission in Lotus pedunculatus. Can. J. Genetics and Cytology X, No. 3, 648--653 (1968). -- 4. Chu, E. H. Y.: Abnormalities of sex chromosomes. Mammalian cytogenetics and related problems in radiobiology, Proc. of a Symposium held in Brasil t962. Oxford: Pergamon Press t964. -5. Dronamraju, K. R. : The function of the Y-chromosome in man, animals and plants. Advances in Genet. t3, 227--310 (t965). -- 6. Grell, R. F.: The D u b i n i n effect and Y chromosomo. Genetics 44, 911--922 (t959). -7. Grell, R. F. : Sterility, lethality and segregation ratios in X Y Y males of Drosophila melanogaster. Genetics, Suppl. 6t, No. 2, Part 2, 23 (1969). -- 8. Hayward, M. D.:
Received December 8, 1969 Communicated b y H. Stubbe
The structure and function of the sex chromosomes in Man. Cytologia 27, 30--42 (t962). -- 9. Jacobs, P . A . , Brunton, M., Melville, M.M.: Aggressive behaviour, mental sub-normality and the X Y Y male. Nature 2o8, t 35 t -- 1352 (1965). -- t 0. Jacobsen, P. : The sex chromosomes in Hurnulus. Hereditas 43, 357--370 (t957). -t t. Lewis, K. R., John, B. : The chromosomal basis of sex determination. Intern. Review of Cytology 23, 277--379 (t968). -- 12. Maguire, M. P.: Apparent loose pairing of homologous K t 0 heterochromatic regions in early premeiotic, late premeiotic and tapetal interphase nuclei. Genetics, Suppl. 61, No. 2, Part 2, 38 (1969). -- 13. McClintock, B., Hill, H. E. : The cytological identification of the chromosome associated with the R-9 linkage group in Zea mays. Genetics 16, t75--190 (1931). -- t4. Mittwoch, U. : Sex chromosomes. New York-London: Academic Press t967. -- 15. Ohno, S., Kaplan, W . D . , Kinosita, R. : On the end-to-end association of the X and Y chromosomes of Mus rausculus. Exptl. Cell. Res. 18, 282--290 (t959). -- 16. Riley, R., Law, C. N.: Genetic variation in chromosome pairing. Advances in Genet. t 3, 57--1t4 (1965). -- 17. Russell, L. B., Chu, E. H. Y.: An X X Y male in the mouse. Proc. Natl. Acad. Sci. U.S. 47, 571--575 (196t). -- t8. Smith, B . W . : The evolving karyotype of Rumex hastatulus. Evolution i8, 9 3 - - t 0 4 (1964). -- t9. Solari, A. J. : Changes in the sex chromosomes during meiotic prophase in mouse spermatocytes. Genetics, Suppl. 61, No. 1, 1t3--120 (1969). -- 20. Utakoji, T. : On the homology between the X and the Y c'lromosomes of the Chinese hamster. Chromosoma (Berl.) t8, 449--452 (t966). -- 21. Westergaard, M.: The mechanism of sex determinations in dioecious floweringeplants. Advances in Genet. 9, 217--28t (1958). -22. Zuk, J. : An investigation on polyploidy and sex determination within the Genus Rumex. Acta Soc. Botan. Polon. X X X I I , 5--67 (1963). -- 23. 9ink, J.: Autoradiographic studies in Rumex with special reference to sex chromosomes. 2nd Oxford Chromosome Conf. (1969a). -24. guk, J. : Analysis of Y chromosome heterochromatin in Rumex thyrsiflorus. Chromosoma (Berl.) 27, 338-- 353 (t969b). -- 25. ~uk, J . : T h e additional heterochromatic chromosome and its influence on sex chromosome pairing in Rumex. Heredity 24, 69--74 (1969c). -- 26. ~uk, J.: Function of Y chromosomes in Rumex thyrsiflorus. Theor. Appl. Gen. 4o, 124-- 129 (t 970).
Dr. Jerzy ~uk Department of Genetics Polish Academy of Sciences Rakowiecka 36 Warsaw (Poland)
Y-Chromosome Hyperploidy in Rumex JERZY ZUK D e p a r t m e n t of Genetics, Polish A c a d e m y of Sciences, W a r s a w (Poland) Summary. 1. Male and female Y-chromosome hyperploid plants were obtained and morphologically and cytologically investigated. 2. Conjugation of sex chromosomes was investigated i n ~ plants with 1, 2 and 3 additional Y chromosomes. Conjugation between sex chromosomes was end-to-end. Association between Y-chromosomes was partly based on conjugation between homologous segments and partly due to heterochromatin fusion. 3. Transmission of the additional Y chromosome to the progeny was studied. I n all cases transmission of the extra Y chromosome was very low. The reasons for this phenomenon are probably nonrandom fertilization, nonrandom degeneration of megaspore, and reduced seed setting and seed fertility in plants with additional Y chromosomes. 4. The phenotypic effect of Y chromosome hyperploidy was investigated. Plants with extra Y chromosomes are shorter and die earlier. The fertility of such plants is low. Thus, although Y chromosomes are heterochromatic and nonactive (except in the premeiotic interphase of microsporogenesis) they have a marked influence on the appearance of Y-chromosome hyperploid plants.
Introduction Y chromosomes in R u m e x thyrsiflorus are heteroc h r o m a t i c a n d late-replicating, a n d t h e y have no effect on sex d e t e r m i n a t i o n (Zuk, 1963, t 969 a, 1969 b). However, t h e y are n o t genetically i n e r t as d a t a were o b t a i n e d i n d i c a t i n g t h a t t h e y are responsible for the f e r t i l i t y of male p l a n t s a n d for the sex ratio (Zuk, 1970). T h e d a t a here p r e s e n t e d concern the effect of e x t r a Y chromosomes on sex chromosome c o n j u g a t i o n a n d on the fertility a n d m o r p h o l o g y of male a n d female p l a n t s . T h e p a t t e r n of t r a n s m i s s i o n of s u p e r n u m e r a r y Y chromosomes to the p r o g e n y is also discussed.
Material and Methods I n the course of studies on polyploidy and sex determination in R. thyrsiflorus one particular female p l a n t with karyotype X X Y Y + 12 A = 16 instead of X X + + 12./1 = 14 was obtained (Fig. 1). The cross of this
female plant with a normal male p l a n t X Y Y + ! 2 A = t 5 gave m a n y male and female plants with additional Y chromosomes. Male plants X Y Y Y Y + 12 A = 17 and female plants X X Y Y + 12A = t6 were chosen as parental forms for further crosses, giving more Y-chromosome hyperploid plants with t, 2 and 3 additional Y chromosomes (Figs. 2, 3, 4). The methods of cytological analysis were the same as described previously (Zuk, 1963, t 969c).
Results I. Sex chromosome conjugation in male plants with additional Y chromosomes I n n o r m a l male p l a n t s X Y 1 Y2 + 1 2 A ---- t5 sex chromosomes form a t r i v a l e n t Y I - - X - - Y , . The homologous s e g m e n t s of X a n d Y chromosomes are v e r y short, a n d at diakinesis sex chromosomes associate e n d to e n d giving a long characteristic t r i v a l e n t
Figs. 1--4. Metaphasal plates from root tips. • 1700 Fig. t. Female plant with karyotype 16 = X X Y Y + 12 A Fig. 3. Male plant with karyotype 17 = X Y Y Y Y + t2 A t;ig. 2. Male plant with karyotype 16 = X Y Y Y + 12 A Fig. 4. Male plant with karyotype 18 = X Y Y Y Y Y + t2 A
t48
J. ~uk
:
Theoret. AppL Genetics
Figs. 5-- 8. Chromosome association at meiosis in normal male plants and in those with additional Y chromosomes. • 1700 Fig. 5. Diakinesis in normal plant with karyotype 15 = XYY Fig. 7. Diakinesis in plant with karyotype t 6 = X YY Y + + 12,4. Arrow indicates Y X Y t r i v a l e n t 12-//. Arrows indicate YXYtrivalentandunivalentformedby Y chromosome Fig. 6. MI in plant with karyotype 16 = X Y Y Y + 12A. Fig. 8. Late metaphase in PMC of plant with karyotype Arrow indicates Y X Y Y tetravalent t6 = X Y Y Y + t2 A. Note prereduction of Y Y bivalent c h r o m o s o m e , a n d this a s s o c i a t i o n s h o u l d be cons i d e r e d as a h e t e r o c h r o m a t i c fusion r a t h e r t h a n a real c o n j u g a t i o n . I n a n o t h e r t e t r a v a l e n t configur a t i o n all h o m o l o g o u s ends of Y c h r o m o s o m e s are i n v o l v e d in c o n j u g a t i o n in such a w a y t h a t t w o Y c h r o m o s o m e s ( o b v i o u s l y t w o Y1 or t w o Y~) c o n j u g a t e w i t h one end of an X c h r o m o s o m e , s c h e m a t i c a l l y
w i t h t h e X c h r o m o s o m e o c c u p y i n g t h e c e n t r a l position (Fig. 5). I n p l a n t s w i t h a d d i t i o n a l Y c h r o m o somes v a r i o u s m o d i f i c a t i o n s of sex c h r o m o s o m e conj u g a t i o n were o b s e r v e d . Conjugation in plants with an additional Y chromosome. Male p l a n t s w i t h c h r o m o s o m e c o n s t i t u t i o n XYYY + t 2 A = t 6 (Fig. 2) m a y possess e i t h e r one Y1 c h r o m o s o m e a n d t w o h o m o l o g o u s Y, c h r o m o somes or one Y2 c h r o m o s o m e a n d t w o h o m o l o g o u s Y1 chromosomes. U n f o r t u n a t e l y Y1 a n d Y, c h r o m o somes are u s u a l l y n o t m o r p h o l o g i c a l l y d i s t i n g u i s h able. I n such p l a n t s four different p a t t e r n s of sex c h r o m o s o m e c o n j u g a t i o n were o b s e r v e d : Y X Y Y (Fig. 6), Y X Y + Y (Fig. 7), X Y + Y Y and X Y + Y + Y . A s s o c i a t i o n b e t w e e n sex chromosomes was as a rule e n d - t o - e n d . Two different config u r a t i o n s of t h e t e t r a v a l e n t X Y Y Y were observed. In the chain configuration YXYY, p r e s e n t e d in Fig. 6, t w o Y c h r o m o s o m e s c o n j u g a t e w i t h two hom o l o g o u s ends of t h e X c h r o m o s o m e as in a n o r m a l t r i v a l e n t . T h e t h i r d Y c h r o m o s o m e is a t t a c h e d to t h e e n d of t h e Y c h r o m o s o m e n o t i n v o l v e d in conjug a t i o n w i t h t h e X c h r o m o s o m e . I t is k n o w n t h a t in R u m e x Y c h r o m o s o m e s are h e t e r o c h r o m a t i c a n d u n a b l e to t a k e p a r t in c o n j u g a t i o n w i t h X c h r o m o somes e x c e p t for t h e s m a l l t e r m i n a l f r a g m e n t . Thus, in t h e t e t r a v a l e n t in q u e s t i o n t h e a d d i t i o n a l Y c h r o m o s o m e is o b v i o u s l y a t t a c h e d to t h e fully h e t e r o c h r o m a t i c a n d n o n h o m o l o g o u s e n d of t h e o t h e r Y
- /Y2
Y1 - - A ~ y ,
y/Y1
or Y2 -- ~ ' \ , Y I "
The third Y chromo-
some c o n j u g a t e s w i t h t h e second a r m of t h e X chrom o s o m e in t h e n o r m a l w a y . As seen in T a b l e t, t h e m o s t f r e q u e n t associations were Y X Y + Y (triv a l e n t a n d Y c h r o m o s o m e as a u n i v a l e n t ) a n d X Y + Y Y (two b i v a l e n t s ) . I n t h e first case t h e a d d i t i o n a l Y c h r o m o s o m e does n o t affect n o r m a l sex chromosome conjugation because a normal YXY t r i v a l e n t is formed. H o w e v e r , q u i t e f r e q u e n t l y t h e e x t r a Y c h r o m o s o m e t e n d s to c o n j u g a t e w i t h its h o m o l o g u e f o r m i n g a b i v a l e n t (Y1 Y1 or Y2 Y2) a n d p r e v e n t i n g f o r m a t i o n of t h e n o r m a l sex c h r o m o s o m e t r i v a l e n t . I n such a case two sex c h r o m o s o m e biv a l e n t s were observed. T h e f o r m a t i o n of one sex c h r o m o s o m e b i v a l e n t X Y a n d two Y c h r o m o s o m e u n i v a l e n t s was also o b s e r v e d b u t this p h e n o m e n o n c a n n o t be r e a s o n a b l y e x p l a i n e d . I t is, however, possible t h a t t h e a s s o c i a t i o n b e t w e e n Y h o m o l o g u e s is less p e r s i s t e n t t h a n t h e Y - - X association, a n d t h e two Y c h r o m o s o m e s o b s e r v e d as u n i v a l e n t s are t h e result of some k i n d of d e s y n a p s i s . P r e r e d u c t i o n of
Table !. Sex chromosome conjugation in male plants of R. thyrsiflorus with one additional Y chromosome No. of plant
Karyotype
22 26 61 65
16=XYYY 16~XYYY t6=XYYY 16=XYYY Total
Pattern of sex chromosome association in diakinesis YXYY + + q+
Total
YXY + Y
XY + YY
XY + Y+ Y
12A 3 12A 6 12A 8 12A 12
42 22 18 12
40 18 11 28
6 11 14 8
91 57 51 60
29
94
97
39
259
Y-Chromosome Hyperploidy in Rumex
Vol. 4 o. No. 4
Table 2. Sex chromosome segregation in Anaphase I in male plants of R. thyrsiflorus with one additional Y chromosome
t49
Conjugation i n plants with 2 additional Y chromosomes. F o u r m a l e p l a n t s w i t h c h r o m o s o m e c o n s t i t u t i o n XYYYY were o b t a i n e d . I n one p l a n t t w o Y
l i k e l y t h a t this p l a n t h a d t w o h o m o l o g o u s Y1 chrom o s o m e s a n d t w o Y, chromosomes. I n this p l a n t v a r i o u s c o m b i n a t i o n s of sex c h r o m o some a s s o c i a t i o n were o b s e r v e d , as shown in T a b l e 3Most f r e q u e n t was t h e a s s o c i a t i o n X Y + Y Y + Y (Fig. 9)- This c o n f i g u r a t i o n is s i m i l a r to t h e association X + Y + Y Y o b s e r v e d f r e q u e n t l y in t h e X Y Y Y p l a n t (Table 2). S u r p r i s i n g l y , c o n f i g u r a t i o n s w i t h a n YXY t r i v a l e n t were less f r e q u e n t t h a n in p l a n t s w i t h one a d d i t i o n a l Y c h r o m o s o m e . W h e n a sex t r i v a l e n t was formed, t h e two a d d i t i o n a l Y c h r o m o s o m e s were seen as a b i v a l e n t or as t w o u n i v a l e n t s (Fig. t 0). T h e r e l a t i v e l y low f r e q u e n c y of such config u r a t i o n s m a y b e e x p l a i n e d as t h e r e s u l t of t h e presence of two h o m o l o g o u s p a i r s of Y c h r o m o s o m e s (Yx Y1 a n d Y2 Y~). I t seems t h a t in such a case h o m o l o g o u s Y c h r o m o s o m e s t e n d to c o n j u g a t e w i t h one a n o t h e r p r e v e n t i n g f o r m a t i o n of t h e sex c h r o m o some t r i v a l e n t . A few p e n t a v a l e n t s a n d t e t r a v a l e n t s w i t h one u n i v a l e n t (Fig. 11) were also o b s e r v e d . I n such c o n f i g u r a t i o n s a s s o c i a t i o n s b e t w e e n Y c h r o m o somes seem to b e t h e r e s u l t of h e t e r o c h r o m a t i c fusion, as h o m o l o g o u s p a r t s of one Y1 a n d one Y2 c h r o m o s o m e are a l r e a d y i n v o l v e d in a s s o c i a t i o n w i t h t h e X chromosome. I n one cell, o n l y u n i v a l e n t s were o b s e r v e d (Fig. 12), b u t it m i g h t h a v e b e e n m e r e l y a t e c h n i c a l artefact. S e g r e g a t i o n of sex c h r o m o s o m e s in t h i s p l a n t was n o t a n a l y z e d , b u t p r o b a b l y s e g r e g a t i o n s X Y -- Y Y Y a n d X Y Y -- Y Y were m o s t f r e q u e n t .
c h r o m o s o m e s were s h o r t a n d m e t a c e n t r i c , two o t h e r s were l o n g e r a n d s u b m e t a c e n t r i c (Fig. 3). I t is v e r y
Conjugation in plants with 3 additional Y chromosomes. I n t h e m a l e p l a n t w i t h k a r y o t y p e X Y Y Y Y Y
Pattern of sex chromosome segregation
No. of plant
Karyotype
22 26 61 65
16 = X Y Y Y + I 2 A t7 16 = X Y Y Y + 1 2 A 20
2 6
16= XYYY 16= XYYY
6 2
6 3
45
17
XY--
+ t2A + t2A
Total
YY
X-- YYY
t h e sex c h r o m o s o m e , o b s e r v e d s o m e t i m e s in M I (Fig. 8), suggests such a p o s s i b i l i t y . Sex c h r o m o s o m e s e g r e g a t i o n in A I was also a n a lyzed. As m a y b e seen from T a b l e 2, t h e m o s t freq u e n t was s e g r e g a t i o n X Y ~ Y Y . A n o t h e r possible p a t t e r n of segregation, X ~ Y Y Y , was m u c h less f r e q u e n t . T h i s fits well w i t h t h e d a t a c o n c e r n i n g c h r o m o s o m e c o n j u g a t i o n as it was to be e x p e c t e d t h a t s e g r e g a t i o n of t h e m o s t f r e q u e n t a s s o c i a t i o n s YXY+ Y and XY+ YY s h o u l d be of t h e X Y ~ Y Y t y p e . A p a r t from t h e t w o m e n t i o n e d a b o v e , no o t h e r t y p e s of sex c h r o m o s o m e segregation were found.
Table 3. Sex chromosome conjugation in male plant of R. thyrsiflorus with two additional Y chromosomes Pattern of sex chromosome association in diakinesis Karyotype
YYXYY YXYYY
17=XYYYY+I2A
4
or
YXY + YY Y X Y Y + Y or XY+ YYY
YXY+ + Y
5
1t
1o
Y
XY+ YY + Y
XY+ Y + Y+ Y
X+ Y+ Y + Y+ Y
53
9
l
Total
Figs. 9-- t 2. Chromosome association at meiosis in male plants with additional Y chromosomes. • 1700 Fig. 9. Diakinesis in plant with karyotype t 7 = X Y Y Y Y + Fig. t t. Diakinesis in plant with karyotype 17 = X Y Y Y Y + t2 A. Arrows indicate two bivalents X Y , Y Y and a Y univa12 A. Arrow indicates tetravalent formed by sex chromosolent mes Fig. 10. Diaki~esis in plant with karyotype 17 = X Y Y Y Y + Fig. 12. Diakinesis in plant with karyotype 17 = X Y Y Y Y + 12 A. Arrows indicate Y X Y trivalent and two Y univalents t2 A. Arrow indicates X and Y univalents
t 50
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Table 4. Sex chromosome conjugation in male plant of R. thyrsiflorus with three additional Y chromosomes Pattern of sex chromosome association in diakinesis Karyotype
t8=X+ YYYYY+ +t2A
YYXYY +Y
YXY +YYY
YXY + YY XY + YY +Y +YY
XY + YY +Y+Y
XY + Y+ Y YXY + Y +Y+Y +Y+Y
Total
t
1
t7
34
4
81
+ t2 A = t 8 (Fig. 4) Y1 and Y, c h r o m o s o m e s were indistinguishable. Only one such plant was obtained. Analysis of diakinesis revealed the combinations of sex chromosome associations shown in Table 4. Again, as in plants X Y Y Y and X Y Y Y Y , combinations with the X Y bivalent were more frequent t h a n combinations with the sex c h r o m o s o m e trivalent YXY. I t was observed t h a t three additional Y chromosomes m a y form a trivalent Y Y Y , or a bivalent and a univalent Y Y + Y, or only univalents Y + Y + Y. The Y c h r o m o s o m e trivalent was observed in one cell only. P e n t a v a l e n t s Y Y X Y Y were also v e r y rare and should be considered as being due to h e t e r o c h r o m a t i c fusion between Y chromosomes. Segregation of sex chromosomes was not analyzed. I n all plants with additional Y chromosomes there was no indication of loss of additional Y chromosomes in ontogenesis or during meiotic division.
2. Transmission of additional Y chromosomes to progeny Transmission of additional Y chromosomes was investigated in the p r o g e n y of two crosses: ~_XXYY+t2A = t6 • d~XYY+I2A = t5 XXYY+I2A =16 • d~XYYYY+t2A =t7 Table 6. Pollen fertility of plants with additional Y chromosomes No.
Karyotype
t 2 3 4 5 6 7 8 9
XYY+I2A XYY+t2A XYY+t2A XYYY+I2A XYYY+I2A XYYY+t2A XYYY+I2A XYYYY+t2A XYYYYY+I2A
Pollen fertility = t5 = 15 ---= 15 = 16 = 16 = 16 = 16 = 17 = t8
85% 86% 89% 78% 25% 89% 52% 90% 47%
21
3
I n b o t h crosses female plants had two additional Y chromosomes. I t seems probable t h a t in such plants two X chromosomes conjugate side to side as in a normal female plant. Two Y chromosomes m a y be a t t a c h e d (by their homologous ends) to the X - c h r o mosome bivalent or m a y form two univalents. The m o s t likely segregation of sex chromosomes is X Y and X Y , so the gametes X Y + 6 A should be the m o s t frequent. I n the first cross the male parent p r o d u c e d two types of gametes, X + 6 A = 7 and X Y + 6 A ---- 8, in equal proportion ; therefore in the p r o g e n y the k a r y o t y p e s $ X X Y + t2 A = 15 a n d ~ ) X Y Y Y + t 2 A = 16 should be the most frequent. I n the second cross, the male parent X Y Y Y Y should produce m a i n l y four types of gametes, X Y , X Y Y , Y Y Y , Y Y , r o u g h l y in equal proportions. Thus in the p r o g e n y of this cross plants with k a r y o types~ XXYYY + 12 A = 17, c~ X Y Y Y + 12 A = = 16 and c~ X Y Y Y Y + t 2 A = t 7 should be the m o s t frequent. As shown in table 5, in b o t h crosses plants with additional Y chromosomes were less frequent t h a n could have been expected from the presumed p a t t e r n of sex chromosome segregation. Most plants had n o r m a l k a r y o t y p e s or only one additional Y chromosome, which suggests t h a t transmission of extra Y chromosomes to the p r o g e n y is v e r y restricted. I t seems t h a t gametes with additional Y chromosomes m a y be eliminated in fertilization as a result of competition with normal gametes.
3. Fertility of plants with additional Y chromosomes Pollen fertility was estimated in 6 plants with !, 2 and 3 extra Y chromosomes and c o m p a r e d with t h a t of n o r m a l male plants (Table 7). All Y hyperploid and normal plants (Nos. t - - 7 in Table 6) were the p r o g e n y of the cross ~ X X Y Y + t 2 A = t6 • c~ X Y Y + t2 A = 15 described in the previous section. P l a n t No. 8 with 3 extra Y chromosomes was
Table 7. The seed fertility in crosses involving plants with extra Y chromosomes Cross
QXX--}-12A =I4xdXYYY+I2A QXX+I2A =I4• 9XX+t2A =I4• 9XXY+I2A =t5xdXYY+I2A 9XXYY+t2A=I6• Q XXYYWI2A=I6• Q XXYY+I2A=t6xc~XYYYY+I2A=t7
The number The number The percent of seeds of seeds of seed collected sown germinated = t 6 240 130 100 = 1 5 330 = 1 5 -= 1 6 13o t64
240 130 100 150 t00 130 t64
t4 23 79 24 79 42 42
= f6
9XXYY+t2A
~XYYYY+t2A
= 17 n u m b e r o f measured plants
mean height of p l a n t s in c m
15 n u m b e r o f measured plants
=
x
mean height
Karyotypes
6
24
+ t2A
2
t2
9
= 16
XXYY
1
--
9
+ 1 2 A = t7
+ 12A XXYYY
20
I0
c~
= t5
XYY
+ 12A
9
8
c~
6
94
24
125
X= X 14Q + 12 d
10
81
25
t09
+ 12A = 15Q
XXY
2
73
10
70
=
XXYY + 16Q |2A
1
45
--
--
+ 1 2 A = 17Q
XXYYY
18
79
~3
87
+ = 1 t 52CA ~
XYY
10
62
8
65
=
16(~
t
55
2
53
+
12A
XYYYY
2
2
~
=
176
+ 1 2 A = 17
+ 12A XYYYY
XYYY + i2A
= t6
XYYY
T a b l e 8. T h e height o f 9 a n d (~ p l a n t s with additional Y chromosomes
14
25
9
= t5
= 14
9
XXY
XX + 12A
~-- i 6 of p l a n t s i n c m
9XXYY+I2A x cSXYY+t2A
Cross
xc~XYYYY+I2A=I7
F r e q u e n c y of k a r y o t y p e s i n cross 9 XXYY + 12 A = 16
= 16X~XYY+t2A=I5
Frequency of karyotypes in e r o s s Q X X Y Y + t2A =
Sex
K a r y o t y p e of t h e p r o g e n y
T a b l e 5. K a r y o t y p e s i n progeny o f crosses between plants with additional Y chromosomes
t
30
--
--
+
12A
XYYYYY
1
--
c~
=
|8(~
+ 1 2 A = t8
XYYYYY
~~
~e
.r
t52
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obtained from the cross ~ X X Y Y + 12 A = t6 • X Y Y Y Y + t2 A = t7. Normal plants produced from 85 to 90% of morphologically good pollen. The pollen fertility of Y-hyperploid plants varied between 25 and 90%. It was interesting that among plants with one additional Y chromosome, one exhibited markedly reduced pollen fertility (up to 25~ while in the other two pollen fertility was more or less normal (78 and 89%). The fertility of the plant with two extra Y chromosomes was quite normal (90%), and in the plant with 3 additional Y chromosomes it was reduced to 470/0. These data indicate that in most cases additional Y chromosomes do not reduce pollen fertility in any drastic way. Fertility of plants with additional Y chromosomes was also estimated b y comparison of seed setting and seed germination in crosses involving plants with additional Y chromosomes, and in crosses between normal plants. The results are given in Table 7. Seed setting in crosses between normal plants was about t 000 seeds per cross. Seed setting in all crosses involving Y hyperploid plants was drastically reduced even to as little as 100 seeds. Seed germination varied from cross to cross but usually was much lower (t4--79%) than germination of seeds from the cross between normal plants (80~ It was surprising that in two crosses between plants with identical chromosome constitution but of different origin (crosses No. 2 and No. 3) seed germination was 23% and 79~ respectively. It resembles the variation in pollen fertility observed for male plants not differing in their karyotypes. The cause of this phenomenon remains obscure, but genetic rather than chromosomal factors should be responsible for the differences observed.
4. The phenotypic effect of Y chromosome hyperploidy Male and female plants with one additional Y chromosome are indistinguishable in their morphology from normal plants. The presence of two or more additional Y chromosomes usually influences strongly the morphological features of plants. Leaves of such individuals, especially when young, are curled, pale green and their shape different than that typically observed in normal seedlings. Moreover, the number of leaves and flower stems is markedly reduced and such plants are shorter than normal individuals. Table 8 presents the results of measurements of the progeny obtained from two crosses between plants with additional Y chromosomes. It is seen that the height of the plant is inversely proportional to the number of additional Y chromosomes. Even plants with one additional Y chromosome, whose leaf morphology is not changed, are shorter than normal plants. Also, the viability of Y chromosome hyperploid plants is lower than that of normal plants. R. thyrsiflorus is a perennial species and normal diploid individuals remain vigorous for several years;
the longevity of Y hyperploid plants was markedly reduced in comparison to that of normal plants. Discussion
In mammals including man (Hayward, 1962; Utakoji, 1966; Mittwoch, 1967; Solari, 1969), and in dioecious plants (Jacobsen, 1957; Westergaard, t958; Smith, 1964), the most common association between X and Y chromosomes during meiosis is the end-to-end configuration. It has been demonstrated in various organisms that the end-to-end association of the X -- Y pair is the result of chiasma formation between minute homologous regions (Ohno, Kaplan and Kinosita, t959; Utakoji, 1966; Solari, t969). Data presented in this paper show that in Rumex thyrsiflorus, both in normal and Y-chromosome hyperploid plants, associations between sex chromosomes are usually end to end. Autoradiographical data and cytological observation (Zuk, t969 a) showed that both Y chromosomes are entirely late-replicating (heterochromatic) and the X chromosome is entirely euchromatic. Thus if the association between sex chromosomes Y X Y in Rumex is based on synapsis between homologous segments of X and Y chromosomes, these homologous segments in Y chromosomes are very short, below the resolving power of autoradiography. Thus it is possible that Y chromosomes in R. thyrsiflorus are composed of large differential heterochromatic segments, and very small euchromatic ones homologous to the X chromosome. In Rumex, as well as forming chiasmata between the distal homologous ends of X and Y chromosomes, Y~ and Y2 chromosomes can produce associations b y heterochromatic fusion. If a sex trivalent is formed in an X Y Y Y + 12 A = t6 plant, association between the chromosomes I/-1, X and Y2 is based on the homology between Y1, Y2 and X chromosomes. Association of an additional Y1 or Y, chromosome with the trivalent is based on heterochromatic fusion because homologous segments of Y and X chromosomes are already involved in association. It seems that association b y heterochromatic fusion can take place mainly between partly homologous }71 + I71 or Y2 -~- Y2 chromosomes, because in normal Y X Y + + 12 A = t 5 plants it has never been observed. This is not surprising, because in the opposite case the regular behaviour of the sex chromosome mechanism would be highly disturbed. Association between the nonhomologous Y and the heterochromatic S chromosome has already been reported in Rumex (Zuk, 1969b) and in other organisms (Riley and Law, 1965). Recently Maguire (1969) has shown that distribution of large heterochromatic knobs in premeiotic interphase nuclei of maize is not random. Two large heterochromatic K t0 regions were found to be localized very significantly nearer to each other than random expectations predicted. Thus it seems that the tendency of heterochromatin to clump during inter-
Vol. 4o. No. 4
Y-Chromosome Hyperploidy in Rumex
phase, especially during premeiotic interphase, m a y be the reason for nonhomologous heterochromatic association in meiosis. Analysis of anaphase cells in plants with one additional Y chromosome revealed t h a t segregation of sex chromosomes was nonrandom. S e g r e g a t i o n X Y - - Y Y was about 3 times more frequent than segregation XY Y Y , and not one case of segregation Y --XYY was observed. Nonrandom segregation of sex chromosomes in X Y Y Y + 12 A = t6 plants is partly the result of the pattern of sex chromosome association in meiosis. Associations Y X Y + Y and X Y + Y Y were very frequent. The most frequent segregation resulting from such association should be X Y -- Y Y . Non-random segregation of sex chromosomes has also been shown in X Y Y males of Drosophila b y Grell (i969). In Rumex, transmission of extra Y chromosomes to the progeny was greatly reduced. This was true for both female and male gametes. Low transmission of extra chromosomes of the trisomics have been reported in m a n y plants (Burnham, t962). This is also true for heterochromatic Y sex chromosomes in R. thyrsiflorus. W h a t mechanism m a y be responsible for low transmission of additional Y chromosomes to the progeny. In male plants with extra Y chromosomes no case of loss of the extra Y chromosome during ontogenetic development or during meiotic division was observed. All Y chromosomes were transmitted to the gametes. Pollen fertility of male plants with additional Y chromosomes is usually quite good, so t h a t selective degeneration of pollen grains with additional Y chromosomes is almost excluded. Therefore the most probable explanation is nonrandom fertilization. The more Y chromosomes are present in pollen grains the poorer their chance to fertilize. This m a y be connected with the deleterious effect of extra Y chromosomes on germination and growth of pollen tubes. Meiosis in female plants was not investigated but it is probable, t h a t if the basal megaspore contains th.e extra Y chromosome it fails to produce the embryo sac. Consequently the embryo sac m a y be produced b y another megaspore with the normal haploid complement. A similar possibility was postulated b y McClintock and Hill (i931) to explain the failure of the extra chromosome to be transmitted to 50% of the progeny in trisomics of maize. In Rumex, even in normal plants, transmission of Y Y + 6 A gametes is much lower than of X + 6 A, and the resulting female plants are much more numerous in the population (Zuk, 1963). I t m a y be speculated t h a t in both cases the mechanism of low transmission of Y chromosomes is the same. Y-chromosome hyperploid individuals have been obtained in Drosophila (Dronamraju, 1965; Mittwoch, 1967; Lewis and John, t 968; Grell, 1959, t969),
153
in humans (Chu, 1964; Jacobs, Brunton and Melville, t965), in mouse (Cattanach, 1961; Russell and Chu, t 9 6 t ; Mittwoch, 1967) and in Melandrium (Westergaard, t958). In man, mouse and Melandrium, individuals with an extra Y chromosome were male, because in these organisms the Y chromosome is strongly male-determining. In Drosophila the Y chromosomes carry no male-determining genes but the addition of an extra Y chromosome affects fertility. The fertility of X Y Y males is only about 0,5--3.0~o. Similarly, b y comparing females with 0, 1, 2 Y chromosomes Grell (t969) has found t h a t the fertility varied inversely to the hyperploidy. In man, X Y Y males are mentally subnormal and behave aggressively, and X X Y and X X Y Y have Klinefelter's syndrome. X X Y mice are morphologically indistinguishable from normal males, but they are sterile. I t is possible, as postulated b y Mittwoch (1967), t h a t the apparent lack of phenotypic abnormalities in mice with an abnormal sex chromosome is connected with the fact t h a t the chromosomes in mouse contain a large proportion of heterochromatin, so t h a t modifications of the k a r y o t y p e m a y be less damaging than those in man. The same is also true for Rumex: b o t h the Y chromosomes are heterochromatic. Male and female plants with one extra Y chromosome are almost normal in appearance. They are only about 20 cm lower. Morphological pollen fertility of male plants with one extra Y chromosome ranges between 50--90%. Seed setting and germination of female plants with one additional Y chromosome is 30%. Two or three extra Y chromosomes have a much more pronounced effect. Female plants are about 30 cm and male plants about 30 cm lower than normal ones. The leaves are often curled and pale. Seed setting and seed germination are much lower than in normal plants. The female plant with three extra Y chromosomes is completely sterile. Thus additional Y chromosomes in Rumex have some effect on the phenotype and fertility of plants. This effect is much less pronounced t h a n in other organisms because Y chromosomes are heterochromatic and have no male-determining genes, and probably because in plants the shift in chromosomal balance has less drastic effects than in organisms with a much higher level of organization like m a m m a l s including man. Acknowledgement. I wish to thank Professor W. Gajewski and Dr. Z. Swietlifiska for much help in the course of this investigation, and for reading and commenting on the manuscript.
Zusammenfassung Die Arbeit berichtet von Untersuchungen tiber die Wirkung tiberz/ihliger Y-Chromosomen auf die Paarung der Geschlechtschromosomen und die Fertilit/it und Morphologie m/innlicher und weiblicher Pflanzen von Rumex thyrsiflorus.
J. ~uk: Y-Chromosome Hyperploidy in Rumex
t54
Die G e s c h l e c h t s c h r o m o s o m e n bei d e n u n t e r s u c h t e n c~ P f l a n z e n m i t 1, 2 u n d 3 iiberz~ihligen Y-Chromosom e n zeigen e n d to e n d - P a a r u n g . Die Assoziation der Y - C h r o m o s o m e n b a s i e r t teils auf einer P a a r u n g homologer Segmente, teils auf einer H e t e r o c h r o m a t i n fusion, I n allen u n t e r s u c h t e n F~llen war eine 0 b e r t r a g u n g des tiberz~hligen Y-Chromosoms auf die N a c h k o m m e n s c h a f t sehr gering. Die Griinde hierftir sind m6glicherweise pr~iferentielle B e f r u c h t u n g , pr~iferentielle D e g e n e r a t i o n der Megaspore sowie geringer S a m e n a n s a t z u n d geringe Fertilit~it der P f l a n z e n m i t iiberz~ihligen Y-Chromosomen. Auch der ph/inot y p i s c h e Effekt einer H y p e r p l o i d i e y o n Y-Chromosom e n w u r d e u n t e r s u c h t . P f l a n z e n m i t iiberzAhligen Y - C h r o m o s o m e n sind kiirzer u n d s t e r b e n friiher ab. Ihre F e r t i l i t i i t ist gering. Obwohl die Y - C h r o m o s o m e n h e t e r o c h r o m a t i s c h u n d i n a k t i v sind (aul3er i n der pr~meiotischen I n t e r p h a s e der Mikrosporogenese), h a b e n sie doch e i n e n d e u t l i c h e n EinfluB auf den H a b i t u s v o n P f l a n z e n m i t tiberz~ihligen Y-Chromosomen.
References
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Received December 8, 1969 Communicated b y H. Stubbe
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Dr. Jerzy ~uk Department of Genetics Polish Academy of Sciences Rakowiecka 36 Warsaw (Poland)
