Giemsa C-banded karyotypes of Hordeum secalinum, H. capense and their interspecific hybrids with H. vulgare

C-banded karyotypes of and their hybrids with H. The European H. secalinum (2n=4x=28) and the South African H. capense (2n=4x=28) had similar karyotypes with ten pairs of metacentrics, three of submetacentrics, and one of SAT-chromosomes. The C-handed karyotypes of H. secalinum from northern Europe were characterized by banding patterns with few hands, those of H. secalinum from Spain and H. capense by handing patterns with more bands. The bands were mostly small or very small and had no preferential disposition. Intraplant handing pattern polymorph- ism was observed in North European H. secalinum, in agreement with an outbreeding reproductive system. No banding pattern polymorphism was present within plants of H. secalinum from Spain and H. capense, suggesting self-pollination. In both species banding pattern polymorphism was prevalent among plants. To-gether with other evidence the fairly similar basic C-banded karyotypes of the two species indicate a rather close relationship.

Giemsa C-banding of chromosomes revealing constitutive heterochromatin is a valuable tool in cytogenetic studies in plants. In a biosystematic investigation of the genus Hordeum (BOTHMER and JACOBSEN 1979a) it was used successfully to investigate species relationships and to elucidate the chromosomal composition of interspecific hybrids (LINDE-LAURSEN et al. 1980, 1986LINDE-LAURSEN 1981;BOTHMER 1984a, b, 1986a, b). The present paper reports the C-banded karyotypes of H . secalinum Schreb. from Europe, of H . capense Thunb. from southern Africa (cf. BOTHMER and JACOBSEN 1979b), and of their interspecific hybrids with H . vulgare L. (cf. BOTHMER et al. 1983(cf. BOTHMER et al. , 1985.

Material and methods
The plant material used is given in Tables 1 and 2. The material of the two species was derived from seeds collected in nature. The interspecific hybrids were produced as described in BOTHMER et al. (1983). Voucher specimens of the plant material are kept at the Botanical Museum, Copenhagen (C). The plants were grown and their C-banded idiograms constructed as reported in LINDE-LAURSEN et al. (1980). A C-band is here defined as large if it covers more than ten per cent of a chromosome arm. The mean length of the chromosomes and the ratio longestlshortest chromosome of each plant measured are given in Table 1. The number of chromosomes with nucleolus-forming capacity was determined through staining of interphases with AgNO, according to LINDE-LAURSEN (1984).

H, secalinum Schreb.
This species, which has a European and North African distribution, is generally reported to be a tetraploid (2n=4x=28). Di-and triploids have been reported from Portugal, but these reports are doubt- Apart from the missing chromosome of H 231-25, the complements of all three plants were similar with ten pairs of metacentrics, three of subrnetacentrics, and one of satellite (SAT) chromosomes (Fig. Za, b, c), with satellite lengths about two-thirds of the supporting short arms. The presence of two SAT-chromosomes matched observations of AgN0,-stained interphases with a maximum of two nucleoli (Table 1; Fig. Id). The ratio longest/shortest chromosome was nearly the same in all three plants (Table 1). In comparison to the C-banded karyotype of the plant from Spain, H 296-33 ( Fig.  2c), the karyotypes of the two North European plants were characterized by a lower level of banding ( Fig. 2a, b), also reflected in a lower content of constitutive heterochromatin (Table 1). The Cbanding patterns of all plants had from zero to eight bands per chromosome with no preferential disposition ( Fig. la, 2a,   bands. The number of conspicuous and large bands in a karyotype corresponded to the number and size of the chromocentres of interphases (Fig. lc).
The C-banding patterns of the homologous chromosomes of H 296-33 were homomorphic, whereas those ofthe North European plants H 192-27 and H 231-25 showed a certain level of banding pattern polymorphism (Fig. 2a, b, c). However, the polymorphism did not interfere with a safe identification of homologues except among the four k unbanded chromosomes in H 192-27 (Fig. 2a). Banding pattern polymorphism was prevalent among the three karyotypes, preventing identification of homologues beyond the level reached by considering chromosome morphology, i.e., SAT-chromo-somes (Fig. 2a, b, c). The banding patterns gave no indication of homoeology among chromosome pairs.

H. secalinum x H. vulgare
The plant was derived from the cross of a sister plant of H 296-33 (cf.  1983,1985). We studied the chromosomal composition in ten C-banded metaphases with 2n=18-23 (Tables 2, 3  complement. In only three of the cells, two with 2n=20 and one with 2n=21, was the quality of the metaphases sufficient for a safe identification of all the remaining chromosomes. The C-banding pattern of each of these matched a similar C-banding pattern of H 296-33, thus giving no indication of intrapopulational banding-pattern polymorphism. However, a specific H . secalinurn chromosome (marked with asterisks in Fig. 2c) was missing in the three cells. Further, it was absent from a cell with 2n=22 and one with 2n=23.
In cells with more than 20 chromosomes and thus with more than one homologue of one or more H. secalinum chromosomes it could be ascertained that the multiplication had affected different H. secah u m chromosomes. No cell contained more than two chromosomes with clearly visible nucleolar constrictions (Table 2). These were always H. vulgare chromosomes 6 and 7. However, the observation of a faint secondary constriction in a third chromo-organizer of the H. secalinum SAT-chromosome was incomplete (Fig. le) (cf. LINDE-LAURSEN 1984). In most cells investigated, the genomes were arranged concentrically with the H . vulgare genome closest to the metaphase centre (cf. Fig. lb Fig. 3a). The complement of plant H 334 had ten pairs of metacentrics, three of submetacentrics, and one of SAT-chromosomes, with satellites about two-thirds the length of the supporting short arms (Fig. 4a). Compared to this, H 335 had an extra pair of metacentrics instead of one pair of submetacentrics (Fig. 4b). However, in another cell of H 335 the latter chromosome pair was classified as submetacentric. (The pair in question is demarcated by triangles in Fig. 4b). The presence in both plants of two chromosomes with visible nucleolar constrictions was supported by observations of a maximum of two nucleoli in AgN0,-stained interphases (Table 1 ; Fig. 3d). The ratio longest/shortest chromosome deviated somewhat between the two plants, probably because of squashing (Table 1).
The C-banded karyotypes of the two plants were fairly similar (Fig. 4a, b) as was also reflected in the fairly similar contents of constitutive heterochromatin (Table 1). The banding patterns had from three to eleven bands per chromosome, with no preferential disposition (Fig. 3a, 4a, b). The bands varied in size from very small to large. The SAT-chromosome pair of H 335 only had two large bands. The rather low number of conspicuous bands corresponded to the number of conspicuous chromocentres in interphases (Fig. 3c).
In both plants the chromosome pairs were identified by banding patterns and chromosome morphology. However, banding pattern polymorphism prevented a safe identification of homologues between plants beyond the level reached by the use of chromosome morphology, i.e., the SAT-chromosomes and the smallest pair of submetacentrics. Comparisons of banding patterns gave no indication of homoeology among chromosome pairs. some later ideniified as the H . secalinurn SAT-H. capense H. vulgare chromosome (cf. Fig. 2c) and the observation in AgN0,-stained interphases of three nucleoli (   1981). Bars = 10 pm. MER et al. 1983). The chromosomal composition was studied in three C-banded metaphases with 2n=19+t, 21 and 22 (Tables 2, 3; Fig. 3b). In the three cells C-banding patterns identified seven chromosomes with H . vulgare chromosomes 1-7. The remainder of the chromosomes could be identified with their homologues in H 334 (cf. Most metaphases observed contained three SATchromosomes (Table 2). In these, the nucleolar constriction of H . vulgare chromosome 6 was the one most frequently observed and most clearly expressed. The observation of three SAT-chromosomes agrees with the observation of up to three standard nucleoli, one always smaller than the other two, in many interphases (Fig. 3e). However, one cell in addition had a micronucleolus.

LINDE-LAURSEN
In the few cells examined the chromosomes of the H. vulgure genome were situated closer to the metaphase centre than the H. capense chromosomes (cf. Fig. 3b).
The variation in C-banding patterns between the plants from northern Europe and Spain was larger than normally observed within a cytotype of a Hordeum taxon (VOSA 1976; LINDE-LAURSEN 1981; LINDE- BOTHMER 1984a, 1986a). Another differentiation of the plants from northern Europe and that from Spain was the difference in banding pattern polymorphism. Both plants from northern Europe showed intraplant banding pattern polymorphism in agreement with an outbreeding reproductive system (BOTHMER and JACOBSEN 1979b), whereas no banding pattern polymorphism was observed in the material from Spain, suggesting self-pollination of plants of the population. The hypothesis is supported by a high seed-setting upon isolation of the plants. Further, a rather pronounced differentiation in morphology (BOTHMER and JACOBSEN, 1979b) and biochemical characters (JBRGENSEN unpubl.) has been observed between materials from the two areas. No banding pattern polymorphism was observed within the two H. capense plants; this supports the theory that the species is mainly self-pollinating (BOTHMER and JACOBSEN 1979b). The high level of banding pattern polymorphism among plants of different populations of the two species agrees with observations in all other Hordeum species previously examined (VOSA 1976; LINDE- LAURSEN et al. 1980LAURSEN et al. ,1986LINDE-LAURSEN 1981 ;LINDE-LAURSEN and BOTHMER 1984a).
VOSA ( 1986;JORGENSEN 1986, and unpubl.).  (RAJHATHY and SYMKO 1974;SUBRAHMANYAM 1980), whereas no haploids of H . tetraploidum have been obtained as yet from the latter combination (BOTHMER et al. 1983(BOTHMER et al. ,1985. The difference between the two groups of hybrids with respect to the preservation of the H . vulgare genome suggests inequalities in the interaction of parental genomes. The mechanism may be similar to that observed in H . marinum x H . vulgareand H. vulgare x H. bulbosum hybrids by FINCH (1983) and in a H . vulgare x Psathyrostachys fragilis hybrid by LINDE-LAUREN and BOTHMER (1984b). The arrangement of the parental genomes in the present hybrids with the H . vulgare genome closest to the metaphase centre agrees with previous observations of a concentric arrangement of genomes in other interspecific and intergeneric hybrids (see BENNEIT 1984;LINDE-LAURSEN and JENSEN 1984;LINDIYLAURSEN et al. 1986;LINDE-LAURSEN and BOTHMER 1986a). As in these hybrids, the concentric arrangement was accompanied by a more or less pronounced suppression of the activity of the nucleolar organizers of the outside genome.