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Lack of effect of B chromosomes on sister chromatid exchanges in Secale cereale
Environmental and E~-periraental Bolanr. Vol. 20, pp. 21 to 26
0098-8472/80/0101-0021 $02.00/0
Pergamon Press Lld. 1980. Primed in Great Britain
LACK OF EFFECT OF B C H R O M O S O M E S ON SISTER C H R O M A T I D E X C H A N G E S IN SECALE CEREALE FIRmRE
Institute of Applied Genetics, Free University of Berlin, Federal Republic of Germany
(Received 9 April 1979; accepted27 May 1979) FRIEBE B. Lack of effect of B chromosomes on sister chromatid exchanges in Secale cereale. ENVIRONMENTAL AND EXPERIMENTAL BOTANY 20, 21--26, 1980.--The influence of supernumerary chromosomes on the formation of SCE's has been analysed in rye containing none, two and four B chromosomes. Whereas lines with two and four B chromosomes show an increased SCE frequency per cell, depending on the higher DNA content of these forms when compared with the 14 chromosome line, neither number nor SCE frequency of B chromosomes has any effect on the SCE frequency of A chromosomes.
INTRODUCTION
ADDITION to the s t a n d a r d A chromosome c o m p l e m e n t , n u m e r o u s p l a n t a n d animal species contain a variable n u m b e r of small and often h e t e r o c h r o m a t i c B chromosomes which exhibit no chromosomal homologies to the A chromosomes. ~11'w) In some cases the fitness of B carrying individuals was affected. HS) Moreover, it has been shown that B chromosomes have a p r o n o u n c e d effect on meiotic chromosome p a i r i n g and chiasma formation and therefore could operate in regulating a species' genetic variability/9'as) Investigations on the behavior of ring chromosomes ~14'2°) and on labelling patterns of somatic chromosomes ~2a~ were the first techniques used to d e m o n s t r a t e the occurrence of sister c h r o m a t i d exchanges (SCE's); the biological significance of this p h e n o m e n o n has rem a i n e d a mystery. ~s~ By means of the recently developed fluorescent plus Giemsa ( F P G ) technique,~X2,13, a6) investigations on sister c h r o m a t i d exchanges could be accomplished more easily and with greater accuracy because of the high resolution of this method. T h e present study was initiated in order to analvse the influence of IN
B chromosomes on sister c h r o m a t i d exchanges in rye. M . & ~
AND METHODS
T h r e e lines with 2 n = 1 4 , 2 n = 1 4 + 2 B and 2n = 1 4 + 4B chromosomes of a primitive form of Secale cereale ( C R I C 1835), originating from I r a n , have been used. Differential staining of sister chromatids was accomplished according to a previously described F P G technique; ~) root tip meristems were treated in the d a r k for 2 4 h r at 25°C, which corresponds to two cell cycles, with a mixture consisting of 1 0 - 4 M 5-bromo-2'deoxyuridine, 5 x 1 0 - S M 5-fluorodeoxyuridine and 1 0 - 6 M uridine dissolved in t a p water. T h e m a t e r i a l was then transferred for 3 h r to 0.05°0 colchicine and subsequently fixed in e t h a n o l / acetic acid (3:1). After a 2 h r enzymatic processing with the stomach fluid of the snail Helix pomatis {6) or m a c e r a t i o n in 45°0 acetic acid, the meristems were squashed in 45°o acetic acid. T h e cover slips were removed by the dry--ice m e t h o d of CONGER and FAIRCHILD~4) and the p r e p a r a t i o n s passed via 96, 70, 50 and 30",, ethanol to distilled water. T h e air-dried preparations were treated for l hr with 0.01" o 91
8
8 13
8
3
5
4
6
3
4
3
2
2-
1
297
2
299
2-
1 --
Degrees of freedom
4
2-
1 1 --
16.24
61.33
Mean square
I ----
1
I
1 1
3.78
F
< 0.05
p
3.73 3.78 4.53
11.05 12.02 12.60
625 654 662
2n= 14+2B
2n= 14+4B
0
2 n = 14
Line
495
466
516
1
168
219
202
2
62
51
46
3
il
9
8
4
2
1
3
5
A chromosomes with x SCE's
0.77
0.78
0.79
i
250
119
0
116
63
1
29
14
2
5
3
3
1 m
4
0.47
0.52
B chromosomes with x SCE's
Table 2. Comparison of mean SCE frequency per chromosome (i) in rye line CRIC 1835 with dijferent numbers of B chromosomes
4822.71
4945.37
3
5
4
Sum of squares
8 12
8 10 10 15 10 10
9 10
7
7
Within lines
3
4
4
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
2 12 11 13 10 14
7
122.66
Item
1
4
4
6
Between lines
Total
3
2
1
1
5
2n=14+4B
1
4
2
1
2n=14
3
2n=14+2B
2
Line
Cells with x SCE's
Table 1. Comparison of mean SCE frequency per cell (~ )and standard deviation (s) in (ye line CRIC 1835 with different numbers of B chromosomes
7~
t~
LACK OF EFFECT OF B CHROMOSOMES
¢.¢,.,- t.,t,.,
23
(
"W \
¢ b
(b) .
#
/,
!
{ (c)
'~ '~
,lOla ,
Fro. 1. Differential staining of sister chromatids in mitotic metaphase chromosomes of { a ) C R I C 1835 121~=141 with 19 SCE's. (hi C R I C 1835 1 2 n = 1 4 + 2 B ) with 11 SCE's and ~c) C R I C 1835 1 2 n = 1 4 + 4 B t with 12 SCE's. B chromosomes are arrm~ed. Bar ~epresents 10/~m.
LACK OF EFFECT OF B CHROMOSOMES rabonuclease-A (from bovine pancreas) dissolved in 0.5 × SSC (sodium saline citrate), washed in 0.5 x SSC and then stained for 0.5 hr with the fluorochrome H33258 (1 mg H33258 dissolved in 1 ml ethanol and 0.1 ml of this solution added to 200 ml 0.5 × SSC). After rinsing in 0.5 xSSC, the slides were exposed for 1 hr in a moist chamber with 0 . 5 × S S C to u.v. light (Osram HNS, 30W ofr, 254nm) before they were incubated for 1 hr in 0.5 x SSC at 55°C. Subsequently the preparations were rinsed in 0 . 5 x S S C , then stained for 12min with 3°o Giemsa (Merck) in 0.15M S6rensen phosphate buffer adjusted to pH 6.8 and briefly washed in the same buffer. The air-dried preparations were transferred into xylol and mounted in Euparal according to requirements. With the technique described above, a sufficient number of cells could be obtained in which differential staining of sister chromatids permits the analysis of SCE's. The frequency of SCE's was determined in 100 complete rectaphase plates per line, which corresponds to 1400, 1600 and 1800 chromosomes, respectively. RF_~IJLTS A N D D I S C U S S I O N
Figure 1 shows differentially stained mitotic metaphase chromosomes of the three lines, in which numerous SCE's could be identified. The distribution pattern of cells with different amounts of SCE's is given in Table 1. Whereas the 14 chromosome line of C R I C 1835 exhibits no difference in SCE frequency per cell when compared with the previously analysed cultural variety "Karlshulder", ~71 by means of an analysis of variance a significant increase in SCE frequency per. cell could be demonstrated for forms in which two or four B chromosomes were present (F=3.78, p<0.05). No differences between lines with and without B chromosomes occurred, if only the amount of SCE's formed in the A chromosomes is considered (F=0.22, p ~ 0 . 8 ) . The distribution pattern of A and B chromosomes with different numbers of SCE's is presented in Table 2. The SCE frequency of A chromosomes is nearly identical in the three lines and the increased SCE frequency per cell in lines with 2 and 4 B chromosomes is attributable to the higher DNA content, caused by the
25
presence of B chromosomes in these forms, which approximates 5.5°~o of the total A chromosome DNA content per B chromosomeJ 1°) The influence of B chromosome SCE frequency on the amount of SCE's formed in A chromosomes was analysed, but neither of the calculated correlation coefficients was of statistical significance. h has been shown that heterochromatic chromosome regions often exhibit a lower SCE frequency than euchromatic regions, O'2'3's'19) while an increased number of SCE's could be observed in the junctions between eu- and heterochromatin. ~1'2'3~ Giemsa C-banding of B chromosomes yields one centromeric and intercalary and two distally located bands, which represent regions of constitutive heterochromatin. The distribution of SCE's within the B chromosomes was not scored, but the results obtained for the SAT chromosome of Secale cereale~7) do show that in rye, SCE formation is also reduced in heterochromatic chromosome regions, while the junctions between eu- and heterochromatin exhibit an increased SCE frequency. The data show that in the rye lines analysed, the number and SCE frequency of B chromosomes have no influence on SCE frequency of A chromosomes and that supernumeraries exhibit a DNA dependent SCE frequency similar to that of A chromosomes, which means that the control of SCE formation acts on the whole chromosome complement in a similar manner, whereby the distribution of SCE's within chromosomes could be influenced by the distribution pattern of eu- and heterochromatin.
Acknowledgements--The author wishes to thank Professor G. LINNERTfor her constant encouragement and critical reading of the manuscript and Professor W. PLARRE, who kindly provided the seeds. This research was supported by a grant from the Deutsche Forschungsgemeinschaft.
RE!TER.F.~121~.~ 1. BOSTOCK C. J. and CHRISTIE S. (1976) Analysis of
the frequency of sister chromatid exchange in different regions of chromosomes of the kangaroo rat (Dipodomys ordii). Chromosoma 5~, 275-287.
26
B. FRIEBE
2. CAam~NO A. V. and WOLVF S. (1975) Distribution of sister chromatid exchanges in the euchromatin and heterochromatin of the Indian muntjac. Chromosoma 53, 361-369. 3. CAaRANOA. V. and JOHNSTON G. R. (1977) The distribution of mitomycin C-induced sister chromatid exchanges in the euchromatin and heterochromatin of the Indian muntjac. Chromosoma 64; 97-107. 4. CONOER A. D. and FAtaCmLD L. M. (1953) A quick freeze method for making smear slides permanent. Stain Technol. ~ 281-283. 5. EvANs H. J. (1977). What are sister chromatid exchanges? Pages 315--326 m A. DE LA CHAPELLE and M. SORSA. Chromosomes today, vol. 6. Elsevier/North Holland Biomedical Press, Amsterdam. 6. FABEROg A. C. (1945) Snail stomach cytase, a new reagent for plant cytology. Stain Technol. 20~ 1-4. 7. FRIEBE B. (1978) Untersuchungen zum Schwesterchromatidenaustausch bei Secale cereale. Microsc. Acta 81~ 159-165. 8. Hsu T. C. and PATnAK S. (1976) Differential rates of sister chromatid exchanges between euchromatin and heterochromatin. Chromosoma 58, 269-273. 9. JonEs R. N. and REEs H. 11967) Genotypic control of chromosome behaviour in rye. XI. The influence of B chromosomes on meiosis. Heredity 22, 333-347. 10. JonEs R. N. and REEs H. (1968) The influence of B chromasomes upon the nuclear phenotype in rye. Chromosoma 24, 158-176. 11. JoNEs R. N. (1975) B-chromosome systems in flowering plants and animal species, b~t. Rvr.. Cytol. 40, 1-100.
12. KIM M. A. (1974) Chromatidaustausch und Heterochromatinver~inderungen menschlicher Chromosomen nach BUdR-Markierung. Nachweis mit Benzimidazolfluorochrom und Giemsafarbstoff. Humangenetik 25~ 179-188. 13. KORENBEROJ. R. and FREEDLENDERE. F. (1974) Giemsa technique for the detection of sister chromatid exchanges. Chromosoma 48, 355-360. 14. McCLINTOC~B. (1938) The production of homozygous tissues with mutant characteristics by means of the aberrant mitotic behaviour of ringshaped chromosomes. Genetics 23, 315-376. 15. M/2NTZIYOA. (1963) Effects of accessory chromosomes in diploid and tetraploid rye. Hereditas 49, 371-426. 16. PERRY P. and WOI.Fr S. (1974) New Giemsa method for the differential staining of sister chromatids. Nature (London) 251, 156-158. 17. REEs H. (1974) B-chromosome. S c i . Prog. (London) 61, 535-554. t8. REEs H. and JonEs R. N. (1977) Pages 73-88 in K. R. LEwxs and B. JOHN, eds. Genetics--Principles and perspectives, vol. 3. Chromosome genetics. Arnold, London. 19. SCnVARTZMANJ. B. and CORTES F. (1977) Sister chromatid exchanges in Allium cepa. Chromosoma 62, 119-131. 20. SCHWAaTZ D. (1953) Evidence for sister-strand crossing over in maize. Genetics 38, 251-260. 21. TAVLOaJ. H., WOODS P. S. and HuoHEs W. L. (1957) The organisation and duplication of chromosomes as revealed by autoradiographic studies using tritium-labeled thymidine. Proc. Nat. Acad. Sci. 43, 122-128.
0098-8472/80/0101-0021 $02.00/0
Pergamon Press Lld. 1980. Primed in Great Britain
LACK OF EFFECT OF B C H R O M O S O M E S ON SISTER C H R O M A T I D E X C H A N G E S IN SECALE CEREALE FIRmRE
Institute of Applied Genetics, Free University of Berlin, Federal Republic of Germany
(Received 9 April 1979; accepted27 May 1979) FRIEBE B. Lack of effect of B chromosomes on sister chromatid exchanges in Secale cereale. ENVIRONMENTAL AND EXPERIMENTAL BOTANY 20, 21--26, 1980.--The influence of supernumerary chromosomes on the formation of SCE's has been analysed in rye containing none, two and four B chromosomes. Whereas lines with two and four B chromosomes show an increased SCE frequency per cell, depending on the higher DNA content of these forms when compared with the 14 chromosome line, neither number nor SCE frequency of B chromosomes has any effect on the SCE frequency of A chromosomes.
INTRODUCTION
ADDITION to the s t a n d a r d A chromosome c o m p l e m e n t , n u m e r o u s p l a n t a n d animal species contain a variable n u m b e r of small and often h e t e r o c h r o m a t i c B chromosomes which exhibit no chromosomal homologies to the A chromosomes. ~11'w) In some cases the fitness of B carrying individuals was affected. HS) Moreover, it has been shown that B chromosomes have a p r o n o u n c e d effect on meiotic chromosome p a i r i n g and chiasma formation and therefore could operate in regulating a species' genetic variability/9'as) Investigations on the behavior of ring chromosomes ~14'2°) and on labelling patterns of somatic chromosomes ~2a~ were the first techniques used to d e m o n s t r a t e the occurrence of sister c h r o m a t i d exchanges (SCE's); the biological significance of this p h e n o m e n o n has rem a i n e d a mystery. ~s~ By means of the recently developed fluorescent plus Giemsa ( F P G ) technique,~X2,13, a6) investigations on sister c h r o m a t i d exchanges could be accomplished more easily and with greater accuracy because of the high resolution of this method. T h e present study was initiated in order to analvse the influence of IN
B chromosomes on sister c h r o m a t i d exchanges in rye. M . & ~
AND METHODS
T h r e e lines with 2 n = 1 4 , 2 n = 1 4 + 2 B and 2n = 1 4 + 4B chromosomes of a primitive form of Secale cereale ( C R I C 1835), originating from I r a n , have been used. Differential staining of sister chromatids was accomplished according to a previously described F P G technique; ~) root tip meristems were treated in the d a r k for 2 4 h r at 25°C, which corresponds to two cell cycles, with a mixture consisting of 1 0 - 4 M 5-bromo-2'deoxyuridine, 5 x 1 0 - S M 5-fluorodeoxyuridine and 1 0 - 6 M uridine dissolved in t a p water. T h e m a t e r i a l was then transferred for 3 h r to 0.05°0 colchicine and subsequently fixed in e t h a n o l / acetic acid (3:1). After a 2 h r enzymatic processing with the stomach fluid of the snail Helix pomatis {6) or m a c e r a t i o n in 45°0 acetic acid, the meristems were squashed in 45°o acetic acid. T h e cover slips were removed by the dry--ice m e t h o d of CONGER and FAIRCHILD~4) and the p r e p a r a t i o n s passed via 96, 70, 50 and 30",, ethanol to distilled water. T h e air-dried preparations were treated for l hr with 0.01" o 91
8
8 13
8
3
5
4
6
3
4
3
2
2-
1
297
2
299
2-
1 --
Degrees of freedom
4
2-
1 1 --
16.24
61.33
Mean square
I ----
1
I
1 1
3.78
F
< 0.05
p
3.73 3.78 4.53
11.05 12.02 12.60
625 654 662
2n= 14+2B
2n= 14+4B
0
2 n = 14
Line
495
466
516
1
168
219
202
2
62
51
46
3
il
9
8
4
2
1
3
5
A chromosomes with x SCE's
0.77
0.78
0.79
i
250
119
0
116
63
1
29
14
2
5
3
3
1 m
4
0.47
0.52
B chromosomes with x SCE's
Table 2. Comparison of mean SCE frequency per chromosome (i) in rye line CRIC 1835 with dijferent numbers of B chromosomes
4822.71
4945.37
3
5
4
Sum of squares
8 12
8 10 10 15 10 10
9 10
7
7
Within lines
3
4
4
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
2 12 11 13 10 14
7
122.66
Item
1
4
4
6
Between lines
Total
3
2
1
1
5
2n=14+4B
1
4
2
1
2n=14
3
2n=14+2B
2
Line
Cells with x SCE's
Table 1. Comparison of mean SCE frequency per cell (~ )and standard deviation (s) in (ye line CRIC 1835 with different numbers of B chromosomes
7~
t~
LACK OF EFFECT OF B CHROMOSOMES
¢.¢,.,- t.,t,.,
23
(
"W \
¢ b
(b) .
#
/,
!
{ (c)
'~ '~
,lOla ,
Fro. 1. Differential staining of sister chromatids in mitotic metaphase chromosomes of { a ) C R I C 1835 121~=141 with 19 SCE's. (hi C R I C 1835 1 2 n = 1 4 + 2 B ) with 11 SCE's and ~c) C R I C 1835 1 2 n = 1 4 + 4 B t with 12 SCE's. B chromosomes are arrm~ed. Bar ~epresents 10/~m.
LACK OF EFFECT OF B CHROMOSOMES rabonuclease-A (from bovine pancreas) dissolved in 0.5 × SSC (sodium saline citrate), washed in 0.5 x SSC and then stained for 0.5 hr with the fluorochrome H33258 (1 mg H33258 dissolved in 1 ml ethanol and 0.1 ml of this solution added to 200 ml 0.5 × SSC). After rinsing in 0.5 xSSC, the slides were exposed for 1 hr in a moist chamber with 0 . 5 × S S C to u.v. light (Osram HNS, 30W ofr, 254nm) before they were incubated for 1 hr in 0.5 x SSC at 55°C. Subsequently the preparations were rinsed in 0 . 5 x S S C , then stained for 12min with 3°o Giemsa (Merck) in 0.15M S6rensen phosphate buffer adjusted to pH 6.8 and briefly washed in the same buffer. The air-dried preparations were transferred into xylol and mounted in Euparal according to requirements. With the technique described above, a sufficient number of cells could be obtained in which differential staining of sister chromatids permits the analysis of SCE's. The frequency of SCE's was determined in 100 complete rectaphase plates per line, which corresponds to 1400, 1600 and 1800 chromosomes, respectively. RF_~IJLTS A N D D I S C U S S I O N
Figure 1 shows differentially stained mitotic metaphase chromosomes of the three lines, in which numerous SCE's could be identified. The distribution pattern of cells with different amounts of SCE's is given in Table 1. Whereas the 14 chromosome line of C R I C 1835 exhibits no difference in SCE frequency per cell when compared with the previously analysed cultural variety "Karlshulder", ~71 by means of an analysis of variance a significant increase in SCE frequency per. cell could be demonstrated for forms in which two or four B chromosomes were present (F=3.78, p<0.05). No differences between lines with and without B chromosomes occurred, if only the amount of SCE's formed in the A chromosomes is considered (F=0.22, p ~ 0 . 8 ) . The distribution pattern of A and B chromosomes with different numbers of SCE's is presented in Table 2. The SCE frequency of A chromosomes is nearly identical in the three lines and the increased SCE frequency per cell in lines with 2 and 4 B chromosomes is attributable to the higher DNA content, caused by the
25
presence of B chromosomes in these forms, which approximates 5.5°~o of the total A chromosome DNA content per B chromosomeJ 1°) The influence of B chromosome SCE frequency on the amount of SCE's formed in A chromosomes was analysed, but neither of the calculated correlation coefficients was of statistical significance. h has been shown that heterochromatic chromosome regions often exhibit a lower SCE frequency than euchromatic regions, O'2'3's'19) while an increased number of SCE's could be observed in the junctions between eu- and heterochromatin. ~1'2'3~ Giemsa C-banding of B chromosomes yields one centromeric and intercalary and two distally located bands, which represent regions of constitutive heterochromatin. The distribution of SCE's within the B chromosomes was not scored, but the results obtained for the SAT chromosome of Secale cereale~7) do show that in rye, SCE formation is also reduced in heterochromatic chromosome regions, while the junctions between eu- and heterochromatin exhibit an increased SCE frequency. The data show that in the rye lines analysed, the number and SCE frequency of B chromosomes have no influence on SCE frequency of A chromosomes and that supernumeraries exhibit a DNA dependent SCE frequency similar to that of A chromosomes, which means that the control of SCE formation acts on the whole chromosome complement in a similar manner, whereby the distribution of SCE's within chromosomes could be influenced by the distribution pattern of eu- and heterochromatin.
Acknowledgements--The author wishes to thank Professor G. LINNERTfor her constant encouragement and critical reading of the manuscript and Professor W. PLARRE, who kindly provided the seeds. This research was supported by a grant from the Deutsche Forschungsgemeinschaft.
RE!TER.F.~121~.~ 1. BOSTOCK C. J. and CHRISTIE S. (1976) Analysis of
the frequency of sister chromatid exchange in different regions of chromosomes of the kangaroo rat (Dipodomys ordii). Chromosoma 5~, 275-287.
26
B. FRIEBE
2. CAam~NO A. V. and WOLVF S. (1975) Distribution of sister chromatid exchanges in the euchromatin and heterochromatin of the Indian muntjac. Chromosoma 53, 361-369. 3. CAaRANOA. V. and JOHNSTON G. R. (1977) The distribution of mitomycin C-induced sister chromatid exchanges in the euchromatin and heterochromatin of the Indian muntjac. Chromosoma 64; 97-107. 4. CONOER A. D. and FAtaCmLD L. M. (1953) A quick freeze method for making smear slides permanent. Stain Technol. ~ 281-283. 5. EvANs H. J. (1977). What are sister chromatid exchanges? Pages 315--326 m A. DE LA CHAPELLE and M. SORSA. Chromosomes today, vol. 6. Elsevier/North Holland Biomedical Press, Amsterdam. 6. FABEROg A. C. (1945) Snail stomach cytase, a new reagent for plant cytology. Stain Technol. 20~ 1-4. 7. FRIEBE B. (1978) Untersuchungen zum Schwesterchromatidenaustausch bei Secale cereale. Microsc. Acta 81~ 159-165. 8. Hsu T. C. and PATnAK S. (1976) Differential rates of sister chromatid exchanges between euchromatin and heterochromatin. Chromosoma 58, 269-273. 9. JonEs R. N. and REEs H. 11967) Genotypic control of chromosome behaviour in rye. XI. The influence of B chromosomes on meiosis. Heredity 22, 333-347. 10. JonEs R. N. and REEs H. (1968) The influence of B chromasomes upon the nuclear phenotype in rye. Chromosoma 24, 158-176. 11. JoNEs R. N. (1975) B-chromosome systems in flowering plants and animal species, b~t. Rvr.. Cytol. 40, 1-100.
12. KIM M. A. (1974) Chromatidaustausch und Heterochromatinver~inderungen menschlicher Chromosomen nach BUdR-Markierung. Nachweis mit Benzimidazolfluorochrom und Giemsafarbstoff. Humangenetik 25~ 179-188. 13. KORENBEROJ. R. and FREEDLENDERE. F. (1974) Giemsa technique for the detection of sister chromatid exchanges. Chromosoma 48, 355-360. 14. McCLINTOC~B. (1938) The production of homozygous tissues with mutant characteristics by means of the aberrant mitotic behaviour of ringshaped chromosomes. Genetics 23, 315-376. 15. M/2NTZIYOA. (1963) Effects of accessory chromosomes in diploid and tetraploid rye. Hereditas 49, 371-426. 16. PERRY P. and WOI.Fr S. (1974) New Giemsa method for the differential staining of sister chromatids. Nature (London) 251, 156-158. 17. REEs H. (1974) B-chromosome. S c i . Prog. (London) 61, 535-554. t8. REEs H. and JonEs R. N. (1977) Pages 73-88 in K. R. LEwxs and B. JOHN, eds. Genetics--Principles and perspectives, vol. 3. Chromosome genetics. Arnold, London. 19. SCnVARTZMANJ. B. and CORTES F. (1977) Sister chromatid exchanges in Allium cepa. Chromosoma 62, 119-131. 20. SCHWAaTZ D. (1953) Evidence for sister-strand crossing over in maize. Genetics 38, 251-260. 21. TAVLOaJ. H., WOODS P. S. and HuoHEs W. L. (1957) The organisation and duplication of chromosomes as revealed by autoradiographic studies using tritium-labeled thymidine. Proc. Nat. Acad. Sci. 43, 122-128.