Diverse heterochromatin in Lathyrus

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1 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN: (Print) (Online) Journal homepage: Diverse heterochromatin in Lathyrus F. Ünal, A.J. Wallace & R.S. Callow To cite this article: F. Ünal, A.J. Wallace & R.S. Callow (1995) Diverse heterochromatin in Lathyrus, Caryologia, 48:1, 47-63, DOI: / To link to this article: Published online: 31 Jan Submit your article to this journal Article views: 22 View related articles Citing articles: 3 View citing articles Full Terms & Conditions of access and use can be found at Download by: [ ] Date: 28 January 2016, At: 19:11

2 CARYOLOGIA Vol. 48, n. 1: 47-63, 1995 Diverse heterochromatin in Lathyrus F. UNAL, A.J. WALLACE * and R.S. CALLOW ** Gazi Universitesi, Fen-Edebiyat Fakiiltesi, Biyoloji Boliimii, 06500, Teknikokullar, Ankara, Turkey; *Regional Molecular Genetics Laboratory, Department of Medical Genetics, St. Mary's Hospital, Hathersage Road, Manchester, M13 OJH, U.K.; and **Plant Science and Cytogenetics Group, Department of Cell and Structural Biology, Williamson Building, The University, Manchester M13 9PL, U.K. SUMMARY - Constitutive heterochromatin was observed in Lathyrus aphaca, L. cicera, L. odoratus, L. sativus and L. tingitanus. C-bands in L. odoratus and L. tingitanus are centromeric or proximal in location. In L. tingitanus, only the short arm of Chromosome 3 showed a large telomeric band; this was detected by C-banding following Q-banding but not in directly C-banded preparations. In L. odoratus, while most C-bands showed as positive Q-bands with enhanced fluorescence, small telomeric bands showed no differentiation with quinacrine. In L. tingitanus, constitutive heterochromatin showed two types of Q-band: one negative with quenched fluorescence at the centromeric regions of all the chromosomes and the other, positive with enhanced fluorescence at one or both sides of the negative bands. No other species of Lathyrus investigated showed discrete negative Q-bands. In L. aphaca and L. cicera. Q-banding patterns are characterized by large telomeric positively fluorescent bands on most members of each complement. Some chromosomes have proximal and/or intercalary bands. In L. cicera, some C-bands showed no differentiation with quinacrine staining. In L. aphaca, a large positive Q-band normally at the end of the short arm of Chromosome 6 is sometimes replaced by two small intercalary bands. Prominent telomeric positive Q-bands were also detected in L. sativus together with small proximal bands and intercalary bands. Whole short arms of Chromosomes 3 and 4 and the long arm of Chromosome 6 in L. sativus showed slightly quenched quinacrine fluorescence. The evolutionary implications of these observations are discussed. INTRODUCTION Lathyrus is a large genus (c. 300 spp.) of flowering plants with a constant basic chromosome number (x= 7); most species are diploid. Against this background, species exhibit a wide range of genome size, largely determined by the amount of moderately repetitive DNA (NARAYAN 1982). Changes in DNA amount are uniformly rather than proportionately distributed between chromosomes within genomes (NARAYAN and DuRRANT 1983).

3 48 UNAL, WALLACE and CALLOW This consistent pattern of variation in DNA amount and chromosome size provides a promising system for studies of nucleotypic responses to genomic evolution. Chiasma frequency, for example, is known to be unaffected by changes in genome size (REES and DuRRANT 1986) or in chromosome size (NARAYAN and MciNTYRE 1989). By contrast, quantitative genomic changes have influenced patterns of chromosome synapsis, numbers of synaptic sites apparently being related to the size of the moderately repetitive genomic fraction (WALLACE and CALLOW 1993a,b). Superimposed on this regular spectrum of genomic and chromosomal evolution, are patterns of variation in number of highly repetitive DNA sequences which are unrelated to genome size (NARAYAN 1982). These highly repetitive DNAs are localised within blocks of constitutive heterochromatin located at the centromeres in some species but at the telomeres in others (VERMA 1978; LAVANIA and SHARMA 1980; NARAYAN 1982; NARAYAN and DURRANT 1983; MURRAY and HAMMETT 1989). While Lathyrus species have similar DNA melting profiles and hence similar AT:GC ratios for total DNA (NARAYAN and REEs 1976), their repetitive DNAs are known to differ in sequence complexity (NARAYAN 1982; KuRIYAN and NARAYAN 1988). The possibility that heterochromatic segments may differ in base composition has been tested by comparing the Q-banded and C-banded karyotypes of five species of Lathyrus, differing in genome size and C-hand distribution. The results of this study are reported here. MATERIALS AND METHODS Five species of Lathyrus were obtained through the European Seed Exchange Scheme: L. aphaca, L. cicera, L. odoratus, L. sativus and L. tingitanus. All the material was of unknown provenance. At least twenty plants were examined from each species. Detailed karyotypes are based on a minimum of five cells from at least three individuals. Young radicles (3-5 mm) were obtained from seedlings grown on damp filter paper at 20 C. The roots were pretreated for 3 h in 0.05% colchicine and fixed in 3 : 1 ethanol:ethanoic acid. Unhanded preparations were stained in Feulgen and counterstained in lactopropionic orcein. All measurements of arm-length were based on unhanded mitotic metaphases. Staining with quinacrine dihydrochloride followed the technique of NEWTON (1985). For Giemsa C-banding, air dried preparations were treated in 5% Ba(OH)2 solution at 55 C for 10 min, washed in tap-water and submerged in 2xSSC at 55 C for 1 h, before being stained in 2% Giemsa for 10 minutes, washed, air-dried and mounted in DePex. Both Q-banded and C-banded preparations were examined from each species but only Lathyrus odoratus and L. tingitanus gave satisfactory C-banding of cells which had previously been Q-banded. Preparations were examined using a Leitz Dialux 20 microscope supplemented by an epi-illuminator with a Hg 50 lamp and a Leitz Filter block giving excitation over

4 DIVERSE HETEROCHROMATIN IN LATilYRUS nm and suppression above 430 nm. Photographs were taken with a Willd Photoautomat MPS 45 camera on Kodak AHU Microfilm, set at 25 ASA for bright, field illumination. Exposures of 2-3 min were employed for fluorescence with the film set at ASA. RESULTS Unhanded karyotypes. The five species of Lathyrus examined were all confirmed as diploid with a chromosome number of 2n=2x= 14 (Figs. 1-5). The first and seventh pair of chromosomes of each species are usually metacentric or submetacentric. Pairs 2 to 6 range between submetacentric and acrocentric. In L. aphaca (Figs. 1a-5a), Chromosomes 2,3,4 and 6 are acrocentric with arm ratios (short/long) , while Chromosomes 1, 5 and 7 are submetacentric with arm-ratios 0~ In L. cicera (Figs. 1c, 5b), Chromosomes 2,4,5 and 6 are acrocentric with arm ratios Chromosomes 1 and 3 are submetacentric, their arm ratios being 0.75 and 0.67 respectively. Chromosome 7 is perfectly metacentric. In L. sativus (Figs. 2a, 5c), Chromosomes 1 and 7 are submetacentric with arm ratios of 0.70 and 0.82 respectively. All the other chromosomes are acrocentric, their arm ratios ranging from 0.41 to Arm ratios in L. odoratus (Figs. 4c, 5d) vary from 0.42 to Chromosomes 1, 5 and 7 are submetacentric, the rest of the chromosomes being acrocentric. In L. tingitanus (Figs. 4d, 5e), arm ratios of all chromosomes vary between 0.44 and Chromosomes 1, 3, 6 and 7 are submetacentric; the rest are acrocentric. Most species have one or two nucleolar-organising chromosomes but none were detected in L. odoratus (Fig. 4c). In three species, the longest pair of chromosomes carries a secondary constriction: on the short arms in L. tingitanus (Fig. 4d) and L. cicera (Fig. 5b) but on the long arm in L. aphaca (Fig. 1a}. In contrast to these three species, L. sativus carries two secondary constrictions: one on the long arm of the third pair of chromosomes and the other on the short arm of the fifth pair (Figs. 2a, 5c). Banded karyotypes. Constitutive heterochromatin was observed in all five species examined and, on most members of each chromosome complement. i. L. aphaca and L. cicera. - The Q-banding patterns of L. aphaca and L. cicera are similar and characterized by enhanced fluorescence (Figs. 1b,d). Large telomeric bands predominate although a few small proximal bands and intercalary bands occur on some chromosomes. In both species, telomeric Q bands were observed in the short arms of all chromosome pairs except pair 6 in

5 50 UNAL, W All.ACE and CALLOW Fig Mitotic complements of Lathyrus aphaca (a,b) and L. cicera (c,d). Feulgenforcein stained preparations (a,c) are compared with Q-banded preparations (b,d). Secondary constrictions are visible on Chromosome 1 in L. aphaca (arrowheads). Bar = 10 J1m.

6 DIVERSE HETEROCHROMATIN IN LATHYRUS 51 Fig. 2. -Mitotic complement of L. sativus (a,b). Feulgen/orcein stained preparation (a) is compared with Q-banded preparation (b). Secondary constrictions are visible on Chromosome 3 (arrowheads) and Chromosome 5 (double arrowheads). Bar= 10 ~tm. L. aphaca where the telomeric band was replaced by two small intercalary bands. Both telomeric bands and intercalary bands occur in the long arm of Chromosome 5 in L. aphaca. Other intercalary bands were located in the short arm of Chromosome 4 and the long arm of Chromosome 7 (Fig. 5a). Proximal Q-bands are born on the long arms of Chromosomes 4, 6 and 7. In L. cicera (Figs. ld, 5b), proximal Q-bands were observed on the short arms of Chromosomes 3 and 6 and on the long arm of Chromosome 4. Interestingly, Q-bands appear to be absent from the longest pair of chromosomes in both L. aphaca and L. cicera. They are also absent from the smallest pair in L. cicera. Giemsa C-banding confirmed the presence of all heterochromatic regions detected under quinacrine fluorescence. Additional C-bands were detected in L. cicera: on the short arm of Chromosome 2 and on the long arms of Chromosomes 1 and 7. ii. L. sativus. - All Q-bands in L. sativus show enhanced fluorescence (Fig. 2b). Proximal Q-bands were observed in the long arm of each chromosome in L. sativus except on Chromosome 7. All the chromosomes including number 7 possess intercalary bands and/or telomeric bands (Fig. 5c). Intercalary bands occur in the long arms of Chromo-

7 52 UNAL, WALLACE and CALLOW Fig Mitotic complements of L. odoratus (a-d). Q-banded preparations (a,c) are compared with the same preparations after C-banding (b,d). One cell is incomplete (c,d). Bar= 10 jlm.

8 DIVERSE HETEROCHROMATIN IN LATHYRUS 53 Fig. 4.- Mitotic complements of L. tingitanus (a,b,d) and L. odoratus (c). Q-banded preparation (a) is compared with the same preparation after C-banding (b). c and d are Feulgen/orcein stained preparations of L. odoratus and L. tingitanus, respectivdy. Secondary constrictions are visible on Chromosome 1 in L. tingitanus (arrowheads). Bar= 10 J.1m. somes 1, 2, 3 and 5, in the short arm of Chromosome 6 and in both arms of Chromosome 7. Prominent telomeric bands were present in the long arm of Chromosomes 2, 3 and 4 and the short arm of Chromosome 5. Telomeric bands on Chromosomes 3 and 5 were in the satellites. Only Chromosome 4 had no

9 a D D b c d ~ D e c Q Bl ~ ++ Fig. 5.- Idiograms representing the basic chromosome complements of five species of Lathyrus; a: L. aphaca, b: L. cicera, c: L. sativus, d: L. odoratus and e: L. tingitanus. Segments are classified into four categories, according to their properties of C-banding (C) and Q-banding (Q). Taking euchromatin as given a standard levd of staining ( + ), C-bands show intense staining ( + +) whereas Q-bands give enhanced ( + + ), standard ( +) or quenched (-) fluorescence. The slightly quenched quinacrine fluorescence of whole arms of L. sativus (see Fig. 2b) is not represented here. One variant of Chromosome 6 (i.e. 6') was found in the heterozygous state in a single individual of L. aphaca. Bar= 12 J.l.m.

10 DIVERSE HETEROCHROMATIN IN LATHYRUS 55 intercalary band. Interestingly, the entire short arms of Chromosomes 3 and 4 and the entire long arm of Chromosome 6 all showed slightly quenched fluorescence by comparison with the remaining euchromatin. iii. L. odoratus. -The prominent C-bands in L. odoratus (Figs. 3b,d e 5d) are all proximal to the centromere. On Chromosome 2, they were either side of the centromere, whereas on Chromosome 3 there was only one proximal band in the short arm. While most show enhanced fluorescence, the small telomeric C-bands on the short arms of Chromosomes 2 and 4 were undetectable in quinacrine stained preparations (Figs. 3a,c, 5d). iv. L. tingitanus. - Unlike those of L. odoratus, the C-bands of L. tingitanus consist of two extreme types of Q-band: one positive showing enhanced fluorescence but the other negative and quenched (Figs. 4a,b, 5e). The negative Q-bands are almost all centric in location. Positive Q-bands occur on both flanks of the negative Q-bands of Chromosomes 3, 4, 6 and 7 but, only in the short arms of Chromosomes 2 and 5. In Chromosome 1, positive Q bands are present on both arms but neither is directly adjacent to the negative Q-band at the centromere. In addition to the centric band, a negative Q-band is situated in a proximal segment of the long arm of Chromosome 2. Additional positive Q-bands occur in the long arm and at the end of the short arm of Chromosome 3. Interestingly, the C-hand at the end of the short arm of Chromosome 3, observed on C-banded slides following quinacrine staining, was not detectable in directly C-banded preparations. In general, regions of quenched fluorescence stained more darkly with Giemsa than did regions of enhanced fluorescence. Interspecific comparisons. Q-banded karyotypes of the five species of Lathyrus clearly fail into two categories: those with prominent telomeric bands and those with prominent centromeric bands (Fig. 5). The telomeric group consists of three species: L. aphaca, L. cicera and L. sativus. Amongst these, the karyotypes of L. aphaca and L. cicera are remarkably similar, the principal differences being the position of the secondary constriction within Chromosome 1 and the additional telomeric Q +-band on Chromosome 5 of L. aphaca. L. sativus has fewer prominent telomeric bands than either L. aphaca or L. cicera. It also shows a diffuse pattern of quenched fluorescence along whole chromosome arms, as mentioned above (see Fig. 2b). The two species with centromeric Q-bands appear to be unrelated to those of the telomeric group. L. odoratus has the simpler karyotype, consisting mainly of centromeric or proximal Q +-bands. The centromeric Q- -bands of L. tingitanus seem unrelated to those of L. odoratus but are flanked by Q + - bands which they may have displaced.

11 56 UNAL, WALLACE and CALLOW DISCUSSION Types of chromatin. Chromatin varies in the timing of its cycle of diffusion and condensation. Expansion is associated with genic expression and results in the chromatin being unresolvable by light microscopy. Centromeres, being active during nuclear division, are therefore detectable as primary constrictions. Similarly, nucleolar organising regions (NORs) show up as secondary constrictions. They are located interstitially or distally in Lathyrus aphaca, L. cicera, L. sativus and L. tingitanus and have recently been shown to hybridize with rdna probes in L. sativus as well as in L. cassius and L. blepharicarpos (MuRRAY et al. 1992). Although secondary constrictions were not detected in L. odoratus during the present study, silver staining has exposed distal NORs on the short arms of two of the largest acrocentric chromosomes, as well as some highly proximal NORs on a range of chromosomes (MURRAY et al. 1992). Regions of constitutive heterochromatin differ from euchromatic regions in being composed predominantly of non-coding, highly repetitive DNA. They are thus permanently inactive rather than simply repressed and so, represent a distinct structural kind of chromatin and not just a peculiar state of condensation as is true of facultative heterochromatin GoHN 1988). Within chromosomes, the distribution of constitutive heterochromatin, as defined by C-banding or by fluorescence, and that of highly repeated DNA, as defined by in situ hybridization, are often, though certainly not always, coincident GoHN 1988; ScHWEIZER et al. 1990). Constitutive heterochromatin is not, however, only made up of highly repetitive sequences. Variation in base composition. Constitutive heterochromatin is enriched in highly repetitive DNA sequences which are likely to have extreme AT:GC ratios. In theory, when only 4 base pairs are sampled at random from a binomial population of equal numbers of nucleotide-pairs, the probability of getting 3AT: 1GC (or 1AT: 3GC) will be 4/16 or 25%. Should the number of base pairs increase, for example to 20, the probability of 3AT: 1GC in five repeats of 4 bp will remain at 25% but that for a non-repetitive sequence of 20 bp will decrease to about 1.5%. For 10 3 kb, 25 X 10 4 repeats of 4 bp still have a 25% likelihood of exhibiting a 3AT: 1GC base ratio, whereas the probability of such a base ratio for a single sequence is close to zero. NARAYAN and REEs (1976), working with unfractionated DNA, found eight species of Lathyrus to have similar simple melting profiles and buoyant densities with an average GC content of 39.25% ( ) and an intragenomic standard deviation (compositional heterogeneity) of 13% GC. Such

12 DIVERSE HETEROCHROMATIN IN LATHYRUS 57 results give no hint of interspecific diversity in the presence of AT- or GC-rich segments of DNA. Categories of constitutive heterochromatin. Variation in base composition of highly repetitive DNAs results in differential fluorescence of the constitutive heterochromatin when stained with quinacrine (ARRIGHI et at. 1974). Biochemical analyses have demonstrated that quinacrine fluorescence is enhanced in the presence of AT-rich DNA but quenched by GC-rich DNA (WEISBLUM and DE HASETH 1972; WEISBLUM 1973). In mammals, species exhibiting brightly fluorescing heterochromatin contain satellite DNA of low buoyant density which is therefore likely to be AT-rich Q"ALAL et al. 1974). Centromeric regions of mouse chromosomes, although AT-rich, show reduced fluorescence, apparently as a result of interspersion of GC base-pairs (WEISBLUM 1973). Evidently, AT-base-pairs must be clustered in order to give enhanced quinacrine fluorescence. A minimum of four AT-base-pairs in tandem seems to be necessary for enhancement (PACH MANN and RIGLER 1972). Both enhanced and quenched Q-bands can occur in the same species, as for example in Scilla sibirica (VosA 1973) and in Pellianeesiana (NEWTON 1985). A similar situation was described for human chromosomes by GAGNE etal. (1971). Here the Q-bands on Chromosome 3 and on the Y Chromosome show enhanced fluorescence whereas those on Chromosomes 1, 9 and 16 appear quenched. By contrast, several species of plants and mammals reveal a p9sitive relationship between GC-richness and staining with mithramycin and chro'momycin (SCHWEIZER 1976; ScHNEDL et at. 1977). In the genus Anura, three different classes of constitutive heterochromatin could be detected by comparing C- and Q-banded karyotypes: the first is Q band positive heterochromatin with a brighter fluorescence than the euchromatin (C + Q + ), the second is Q-band negative heterochromatin with a weaker fluorescence than euchromatin (C + Q-) and the third is C-hand faint Q-band negative heterochromatin with weaker fluorescence than euchromatin (C+/-Q-) (ScHMID 1980). Using the GC-specific antibiotics mithramycin (MM) and chromomycin A3 (CMA3) and AT-specific quinacrine, ScHMID (1980) concluded that the brightly fluorescing heterochromatin (C + Q +) of the Anuran chromosomes contains AT-rich sequences, whereas the weakly fluorescing heterochromatin (C + Q- and C Q-) consists mostly of GC-rich satellite DNA. Amounts of DNA in the constitutive heterochromatin of twelve Lathyrus species range from 1.89 pg/2c in L. angulatus to 8.53 pg/2c in L. sylvestris (NARAYAN 1982). Quantities of highly repetitive DNA can be deduced from Cot analyses published for the same species (NARAYAN 1982). These show a much more limited spectrum of variation from 0.32 pg/2c in L. articulatus to

13 58 UNAL, WALLACE and CALLOW 2.00 pg/2c in L. aphaca. Curiously, highly repetitive DNA ('Y) shows no significant quantitative relation to DNA (X) in the heterochromatin of this material (Y = (X); t[10]= 1.613, P>0.10). Evidently, the larger the C-bands in these species of Lathyrus, the lower their concentrations of highly repetitive DNA. In L. tingitanus, for example, while DNA in constitutive heterochromatin accounts for 7.64 pg/2c, highly repetitive DNA accounts for only 1.38 pg/2c. In this species, therefore, no more than about 18% of the DNA in the C-bands is likely to be highly repetitive. Because several species of Lathyrus show simple melting profiles and have similar base compositions (NARAYAN and REEs 1976), it may be wiser to attribute differential quinacrine fluorescence in this material to small shifts in dispersion of AT- and GC-base-pairs rather than to massive accumulations of AT- and GC-rich DNA. In the Lathyrus species examined in this study, three types of heterochromatin were detected. Heterochromatic regions exposed as C-bands show either enhanced (C + Q +) or quenched (C + Q-) quinacrine fluorescence or no differentiation with quinacrine (C +Q 0 ). While the first two categories are likely to be respectively rich and deficient in AT-clusters, the third category probably has a content of AT -clusters similar to that of the surrounding euchromatin. The only species which has both positive and negative Q-bands in its genome is L. tingitanus. KuRIYAN and NARAYAN (1988) report the presence of two large families of repetitive sequences in L. tingitanus. On the basis of Q banding, one of these may well be rich in AT-clusters and the other, deficient. Negative Q-bands were absent from the other four species examined: L. aphaca, L. cicera, L. odoratus and L. sativus. Heterochromatin showing no differentiation with quinacrine was represented by small C-bands in the long arm of Chromosome 1, in the short arm of Chromosome 2 and in one arm of Chromosome 7 in L. cicera and by small bands at the end of the short arms of Chromosomes 2 and 3 in L. odoratus. The evidence from L. tingitanus suggests that quinacrine staining may provide more sensitive detection of heterochromatin rich in AT-clusters than that given by staining with Giemsa. Amongst the larger Q-bands, regions of enhanced fluorescence consistently stain less densely with Giemsa than do those showing quenched fluorescence. Chromosomal polymorphism. C-bands have been shown to be heteromorphic in virtually all species studied in sufficient depth. In plants, there are clear differences in C-banding patterns and amounts between different populations within a species. In maize, the amount of heterochromatin varies between different strains and can be correlated not only with the total nuclear DNA content but also with geogra-

14 DIVERSE HETEROCHROMATIN IN LATHYRUS 59 phicallocation (RAYBURN et al. 1985). Southern varieties have higher nuclear DNA C-values and more heterochromatin than more northerly varieties. Marked phenotypic variation in number, size, position and intensity of fluorescence of C-bands is exhibited by Scilla sibirica (VosA 1973) and by several species of Allium (VosA 1976). Australian grasshoppers, show extensive variation in C-banding both within and between populations of the same species (SHAW et al. 1976; JoHN and KING 1983). In certain species C-bands differ in their staining intensity (WEBB et al. 1978), in their reactions with different base specific fluorochromes (JoHN et al. 1985) and in the type of satellite DNA they contain (ARNoLD and SHAW 1985). Patterns of C-banding, revealed in the present study, often contrasted markedly with those reported in the literature. Such discrepancies may bear testimony to widespread polymorphism, if not to incipient speciation or even to a need for major taxonomic revision. Observations of thick blocks of constitutive heterochromatin at the centromeric regions of all the chromosomes in L. tingitanus concord with those of VERMA (1978) and NARAYAN (1982). Intercalary bands have been detected in both arms of Chromosomes 1 in all three studies. On chromosome 2, while both previous investigators observed intercalary bands in both arms, a single intercalary band was observed only on the long arm in this study. Additional intercalary bands were observed on Chromosome 4 by VERMA (1978) and on Chromosome 5 by NARAYAN (1982); none were detected in the present study. By contrast, while neither previous investigator detected telomeric C-bands in L. tingitanus, a large C-hand was observed at the end of the short arm on Chromosome 3 in the present study. Strangely enough, this telomeric band was obvious in C-banded preparations following Q-banding but was undetectable in directly C-banded preparations. This band is the only example of its kind. In all other chromosomes and in all other species of Lathyrus investigated in this study, Giemsa C-banding revealed the same number and distribution of C bands whether employed after quinacrine staining or applied directly. Centromeric or highly proximal C-bands were detected in all chromosomes of L. odoratus, two proximal bands being in separate arms on Chromosome 2 and one, in the short arm of Chromosome 3. Small telomeric bands were observed at the end of the short arms of Chromosomes 2 and 4 but no intercalary bands were detected in L. odoratus. LAVANIA and SHARMA (1980) reported a similar distribution of centric C-bands in L. odoratus, but these authors found large telomeric bands in the short arms of Chromosomes 1, 4 and5. In L. cicera, while LAVANIA and SHARMA (1980) detected centromeric bands on all chromosomes, no centric C-bands were observed in the present study. Only small proximal bands were observed in the short arms of Chromo-

15 60 UNAL, WALLACE and CALLOW somes 3 and 6, in the long arm of Chromosome 4 and in one arm of the metacentric Chromosome 7. Intercalary bands were present in the long arm of Chromosome 1 and in the short arm of Chromosome 2 but appear to be absent from the material studied by LAVANIA and SHARMA (1980). Large telomeric bands predominate in L. cicera and, are located in the short arms of all but Chromosomes 1 and 7 in this study. LAVANIA and SHARMA (1980) observed telomeric C-bands on all chromosomes, that on Chromosome 3 being in the long arm rather than the short. In L. aphaca, centric C-bands were reported by LAVANIA and SHARMA (1980) on Chromosomes 1, 2 and 4 but were absent from the material studied here. Again, while telomeric bands were observed on the short arm in all chromosomes but numbers 1 and 7, those detected by LA VANIA and SHARMA (1980) occur on all short arms and also on a satellite in the long arm of Chromosome 3. The only chromosome with two telomeric bands, observed in the present study, was Chromosome 5. The only clear case of heteromorphism for Q-bands in Lathyrus was provided by one individual of L. aphaca. Here a large positive Q-band at the end of the short arm of one representative of Chromosome 6 is replaced by two small intercalary bands in the homologous arm. In several species of Allium, VosA (1976) defined a number of different classes of heterochromatin according to whether or not they were C-banded and whether they showed increased or decreased fluorescence or no differentiation with quinacrine or Hoechst He found that the chromosomes of Scilla sibirica showed such extensive heteromorphism on this basis that each of 20 plants studied had a unique karyotype (VosA 1973). Species relationships in Lathyrus. While changes in amounts of moderately repetitive DNA are responsible for divergence in genome size in Lathyrus, differences in content of highly repetitive DNA bear no relation to size of genome (NARAYAN 1982). By contrast, blocks of constitutive heterochromatin can provide valuable markers of chromosomal evolution. They are particularly useful for distinguishing closely related genomes such as those of goatgrass species in the genus Aegjlops (TEOH and HuTCHINSON 1983) and of subspecies of the Australian grasshopper Caledia captiva (SHAw et at. 1976). In situ hybridization (HuTCHINSON et al. 1982) and fluorescence techniques (NEWTON 1985) provide additional refinements when comparing species. The species of Lathyrus studied here display two contrasting distributions of constitutive heterochromatin: one predominantly telomeric and the other predominantly centromeric. L. aphaca and L. cicera both carry large blocks of telomeric heterochromatin which give positive staining with quinacrine dihy-

16 DIVERSE HETEROCHROMATIN IN LATHYRUS 61 drochloride. Although morphologically unrelated (SENN 1938; DAVIS 1970), these two species have the same genome size and similar amounts of repetitive and non-repetitive DNA (NARAYAN 1982). L. sativus also has prominent telomeric bands on most chromosomes but its genome is larger. Prominent telomeric bands were not detectable in the material studied by LAVANIA and SHARMA (1980). Their loss may indicate the action of transposable elements (SCHUBERT and WoBus 1985). Most species of Lathyrus display prominent centromeric C-bands (NAR AYAN 1982; LAVANIA and SHARMA 1980). The present study has shown the majority of such bands to give enhanced fluorescence with quinacrine and therefore to be enriched in AT-clusters. L. odoratus provides a clear example of this category of species. By contrast, the centric heterochromatin of L. tingitanus gives quenched quinacrine fluorescence and so appears to be deficient in AT-clusters. Proximal positive Q-bands are present on one or both sides of each central block. This distinctive pattern raises the intriguing possibility that the positive Q-bands are ancestral and have been displaced by the negative Q bands, either via transposition from a related but unknown species or by mutation followed by re-iteration possibly involving unequal crossing-over. Transposition is known to alter C-banding patterns in Allium (ScHUBERT and WoBUS 1985) and may even be responsible for major chromosomal rearrangements (NEVERS and SAEDLER 1977). Highly repetitive sequences are thought to undergo frequent cycles of expansion and contraction through unequal crossing-over (BoucHARD 1982). Further exploration of the modes of chromosomal evolution in Lathyrus must await the results of banding studies in a wider range of species. Acknowledgements. - thanks SERC (U.K.). For research studentships, FU thanks Gazi University (Turkey) and AJW REFERENCES ARNOLD M.L. and SHAW D.D., The heterochromatin of grasshoppers from the Calediacaptiva species complex. II. Cytological organisation of tandemly repeated sequences. Chromosoma, 93: ARRIGHI F.E., Hsu T.C., PATHAK S. and SAWADA H., The sex chromosomes of the Chinese hamster: constitutive heterochromatin deficient in repetitive DNA sequences. Cytogenet. Cell Genet., 13: BouCHARD R.A., Moderately repetitive DNA in evolution. Int. Rev. Cytol., 76: DAVIS P.H., Flora of Turkey. Vol. 3, Edinburgh University Press, Edinburgh. GAGNE R., TANGUAY R. and LABERGE C Differential staining patterns of heterochromatin in man. Nature New Bioi., 232: HuTCHINSON J., Mlu.ER T.E., ]AHIER}. and SHEPHERD K.W., Comparison of the chromosomes of Triticum timopheevi with related wheats using the techniques of C-banding and in situ hybridization. Theor. Appl. Genet. 64:

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