1969, VOSA 1970 and HORN and WALDEN 1971. In addition, NATARAJAN and NATARAJAN. (1972) have shown in Rhoeo discolor that the Q-positive.
Hereditas 74: 233-238
(1973)
Identification of heterochromatic regions in the chromosomes of rye N. P. SARMA and A. T. NATARAJAN Wallenberg Laboratory, Stockholm University, Sweden
(Received February 28, 1973)
Localisation of the heterochromatic regions in the chromosomes of rye, Secale cereale L., has been achieved by (a) C-banding and (b) fluorescence banding with a bis-benzimidazole derivative. It is possible to characterise each pair of the rye chromosomes. The absence of heterochromatin at the centromeric regions and its presence in the terminal ends of the chromosomes, serve as markers and facilitate easy identification of rye chromosomes from those of wheat, in Triticale genome. Though the modern staining techniques have been effectively utilized in the identification of individual chromosomes in several mammals, their use in plant cytology has been of limited success. Quinacrine banding (Q-banding) of the heterochromatic regions of the chromosomes has been reported in some plants by CASPERSSON et al. 1969, VOSA 1970 and HORNand WALDEN1971. In addition, NATARAJAN and NATARAJAN (1972) have shown in Rhoeo discolor that the Q-positive regions are also C-positive, i.e., these chromosomal regions take up heavily Giemsa stain, following denaturation and reassociation of cytological preparations.
retical value. The results of our attempts to characterise the rye chromosomes are presented and discussed in this communication.
Materials and methods Chromosome preparations were made from root meristems which were pretreated with 0.1 % colchicine for four hours and fixed overnight in acetic alcohol (1 : 3). Feulgen squashes were made by routine methods. For banding, the fixed root tips were squashed in 45 % acetic acid directly, or after a mild hydrolysis in 0.1 N HCL for five minutes at 60" C. The slides were frozen on dry ice, cover glasses removed and transferred to absolute alcohol and then air dried.
Secale cereale L., is known to possess well defined heterochromatic regions, as revealed by the pattern of chromosome replication (LIMA DE FARIA1959). This species has been combined with the wheat genomes to produce Triticale C-banding: The preparations were treated for and many addition and substitution lines have five minutes in saturated aqueous solution of been produced involving wheat genomes to barium hydroxide and washed thoroughly in which one pair of rye chromosomes is separately running tap water (VOSAand MARCHI1972). added or substituted to the normal complement The slides were then incubated in 2 X SSC 1958; BEILIGand DRISCOLL (0.3 M NaCl and 0.03 M Na citrate) for two (RILEYand CHAPMAN 1970). Rye has also been used to study the role hours at 66" C and stained for twenty minutes of heterochromatin in the formation of chromo- with 2 % Giemsa solution (pH 6.8). some aberrations, following treatments with radiations and chemicals (NATARAJAN and AHN- Fluorescence banding: Binding of neither quinaSTROM 1970, 1972). Identification of individual crine mustard nor quinacrine dihydrochloride rye chromosomes will be of practical and theo- gave any differential fluorescence between or Hereditas 74, 1973
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within the chromosomes. The use of a bisbenzimidazole derivative (Hoechst 33258, HILWIG and GROPP1972) was successful. The air-dried preparations were stained with this dye solution (0.5 pg/ml, pH .7) for 20 minutes and differentiated by washing in water for varying intervals before viewing under Zeiss fluorescence microscope. In addition to normal rye material var. Petkus (2n = 14), irradiated material of the same was studied to explore the sensitivity of these techniques in localising the heterochromatin associated chromosome aberrations. Chromosome preparations of a hexaploid Triticale (2n=6x=42, var. 1018) were also studied to test the utility of these banding techniques in the identification of the chromosomes of the rye parent in the Triticale genome.
Results and discussion The seven pairs of rye chromosomes are presented in Fig. 1. There are three metacentric and four
submetacentric pairs. With Feulgen staining, only one pair is identifiable, i.e. the satellitebearing submetacentric one. Both C-banding and fluorescence banding with the benzimidazole derivative bring about characteristic patterns with regard to the location of heterochromatic segments which are mostly situated at the telomeric ends of the chromosomes (Fig. 2, 3). These regions correspond to the late replicating segments of rye, reported earlier (LIMA-DE-FARIA 1959; DARLINGTON and HAQUE1966; NATARAJAN and AHNSTROM1970). These heterochromatic blocks take deep Giemsa staining as well as bind heavily to the fluorochrome used in this study. (1.c.) In addition to these regions, LIMADE FARIA has reported the presence of late replicating regions near the centromeres of the rye chromosomes. However, this could neither be confirmed and HAQUE(1966) nor in our by DARLINGTON laboratory. Neither of the banding techniques used in the present investigation indicated the presence of any centromeric heterochromatin in rye chromosomes.
Fig. 1-3. Metaphase chromosomes of rye (2n = 14). - Fig. 1 . Karyotype from a Feulgen squash. - Fig. 2. C-banding showing heavily stained terminal heterochromatic regions. Arrows indicate minor bands. - Fig. 3. Fluorescence banding with Hoechst 33258, showing the brightly fluorescent terminal heterochrornatic regions. - x 1400. Hereditas 74, 1973
HETEROCHROMATIC REGIONS IN RYE CHROMOSOMES
In rye, telomeric ends of all the chromosomes do not have large blocks of heterochromatin, but every chromosome has at least one such block. In addition, some intercalary fluorescent bands can be seen. On the basis of the fluorescent patterns, five different types of chromosomes can be recognized in rye. They are: Type A - Two metacentric pairs having heterochromatic blocks at the telomeric ends of both the arms (Fig. 4 A ) . Type B - A sub-metacentric pair having a block of heterochromatin at the terminal end of the short arm and in addition, two small bands of high fluorescence at the sub-terminal region of the long arm (Fig. 4B). Type C - A sub-metacentric pair having the non-fluorescent secondary constriction in the short arm. The terminal ends of both the arms have blocks of heterochromatin (Fig. 4C). The close proximity of the heterochromatic block to the nucleolar region may indicate the possible inter-relationship between these two and GROPP1971). Type structures (NATARAJAN D - A metacentric pair with a block of heterochromatin at the terminal end of one arm and a short fluorescent band at the sub-terminal position of the other arm (Fig. 4 D ) . Type E Two sub-metacentric pairs possessing blocks of heterochromatin at the terminal ends of the short arms only (Fig. 4E). While the large blocks of terminal heterochromatin can be seen even in well condensed state of the chromosomes, the smaller bands can be seen only in early metaphase stages (Fig. 4). The smaller bands can, however, be recognized in condensed metaphases, following C-banding. A comparative analysis of the distribution of heterochromatin among the chromosomes of rye visualized by the present techniques and that of the chromomere pattern analysis of the pachytene chromosomes (LIMADE-FARIA 1952) reveals that they are in close agreement. The present study demonstrates the potentialities of these staining techniques in recognizing individual chromosomes in plants. The fluorescent regions bind Giemsa stain following denaturation and reassociation, a property very similar to most of the mammalian constitutive heterochromatin studied. However, in the plant chromosomes, it has not been possible so far to bring about banding patterns in euchromatic regions (Gbanding). This seems to be due to the different procedures adopted for chromosome preparations in plants and mammals.
235
Fig. 4 and 5. - Fig. 4. The five chromosome types of rye on the basis of fluorescence banding pattern; A: metacentric with heterochromatin on both telomeric ends; B: sub-metacentric with one large block of terminal heterochromatin in the short arm and two short intercalary bands in the long arm; C: sub-metacentric SAT-chromosome. Note the nonfluorescent secondary constriction (arrow) and the brightly fluorescent telomeric ends; D: metacentric with a large block of telomeric heterochromatin in one arm and a short intercalary band in the other arm; E: sub-metacentric with heterochromatin at the telomeric end of the short arm. Fig. 5. A: Chromosome type B; B: chromosome type C (SAT-chromosome); C: gamma ray induced dicentric involving the heterochromatic regions of the short arm of a chromosome E and an arm of chromosome A. - X 1400.
It has been established that both radiation and mitomycin C induced aberrations are prefferentially localised to the heterochromatic regions of the chromosomes in rye (NATARAJAN and AHNSTROM 1970, 1972). In such studies, it is difficult however, to decide from a Feulgen stained preparation, as to where exactly the heterochromatic block is situated in a dicentric or an acentric. Staining with this new fluorochromoe, however, enables an easy identification of Hereditas 74, I973
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Fig. 6-8. Triticafe chromosomes. - Fig. 6. C-banding. Rye chromosomes are deeply stained at the terminal ends. Some of the wheat chromosomes have intercalary bands (arrows). - Fig. 7 and 8. Fluorescent banding. Rye chromosomes can be recognized by their strong fluorescent terminal ends. Arrows show type B chromosomes of rye that are without terminal euchromatic segments in contrast to Fig. 4B and 5A. Many small intercalary bands in some wheat chromosomes are also seen. - x 1400. Hereditas 74, 1973
HETEROCHROMATIC REGIONS IN RYE CHROMOSOMES
the heterochromatic regions in an aberration (Fig. 5C). Identification of the chromosome complement of rye in the Triticale genome could also be possible with these staining techniques. Fig. 6, 7 and 8 illustrate this point. While the wheat chromosomes lack terminal blocks of heterochromatin, some of them have small blocks of centromeric heterochromatin. In addition, some pairs have small fluorescent bands dispersed along the chromosome, which may indicate the possibility of identifying some of the wheat chromosomes individually (Fig. 7, 8). Complete absence of centromeric heterochromatin and its localisation to the terminal ends of the chromosomes of rye, serve as cytological markers for identification of these from wheat chromosomes. It is also of interest to note that the rye parent involved in the Triticale, investigated in the present study, i.e., var. 1018, differs in chromosome characteristics from the variety, Petkus. The submetacentric pair with two fluorescent bands in the long arm, has a short euchromatic segment distal to them (Fig. 4B, 5A) in Petkus, whereas in the Triticale these fluorescent bands are terminal (Fig. 7, 8). This indicates that there must be variations with regard to the pattern of distribution of fluorescent bands among different varieties of rye. Analysis of rye material from diverse sources will be interesting, and such an analysis is in progress in our laboratory. Acknowledgments. - These investigations were supported by grants from the Swedish Atomic Research Council. N. P. Sarma has been a recipient of an International Atomic Energy Agency fellowship. We are grateful to Dr. Loewe, for the gift of Hoechst 33258.
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Literature cited BEILIG,L. M. and DRISCOLL, C. J. 1970. Substitution of rye chromosome 5RL for chromosome 5B of wheat and its effect o n chromosome pairing. - Genetics 65; 241-247. CASPERSSON, T., ZECH,L., MODEST,E. J., FOLEY, G. E., WAGH,U. and SIMMONSSON, E. 1969. Chemical differentiation with fluorescent alkylating agents in Vicia faba metaphase chromosomes. - Exp. Cell Res. 58: 128-140. DARLINGTON, C. D. and HAQUE,A. 1966. Organization of DNA synthesis in rye chromosomes. Chromosomes Today 1: 102-107. HILWIG,I. and GROPP,A. 1972. Staining of constitutive heterochromatin in mammalian chromosomes with a new fluorochrome. - Exp. Cell Res. 75: 122-126. HORN,J. D. and WALDEN,D. B. 1971. Fluorescent staining of euchromatin and heterochromatin in maize (Zea mays). - Can. J . Genet. Cytol. 13: 81 1-815. LIMA-DE-FARIA, A. 1952. Chromomere analysis of the chromosome complement of rye. - Chromosoma 5: 1-68. - 1959. Differential uptake of tritiated thymidine into hetero and euchromatin in Melanoplus and Secale. J. Biophys. Biochem. Cytol. 6 : 457-466. NATARAJAN, A. T. and AHNSTROM, G. 1970. The localisation of radiation induced chromosome aberrations in relation to the distribution of heterochromatin in Secale rereale. - Chromosoma 30: 2 5 G 2 5 7 . - 1972. Induced chromosomal aberrations and heterochromatin. - I n Constitutive heterochromatin in man, Symposia Medica Hoechst (in press). NATARAJAN, A. T. and GROPP, A. 1971. The meiotic behaviour of autosomal heterochromatic segments in hedgehogs. - Chromosoma 35: 143-152. N A T A R A I A N , A. T. and NATARAJAN, S. 1972. The heterochromatin of Rhoeo discolor. - Hereditas 72: 323-330. RILEY,R. and CHAPMAN, V. 1958. The production and phenotypes of wheat rye chromosome addition lines. Heredity 12: 301-315. VOSA, C. G. 1970. Heterochromatin recognition with fluorochrornes. - Chromosoma 30: 366-372. VOSA,C. G. and MARCHI, P. 1972. Quinacrine fluorescenNature New Biol. ce and Giemsa staining in plants. 237: 191-192. A. T. Natarajan Radiation Biology Institute Wallenberg Laboratory S-10405 Stockholm 50, Sweden -
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