Journal of Cell Science 101, 13-24 (1992) Printed in Great Britain © The Company of Biologists Limited 1992 13 Parental genome separation in reconstructions of somatic and premeiotic metaphases of Hordeum vulgare x H. bulbosum T. SCHWARZACHER, J. S. HESLOP-HARRISON, K. ANAMTHAWAT-J6NSSON Karyobiology Group, Department of Cell Biology, JJ Centre for Plant Science Research, Norwich NR4 7UJ, UK R. A. FINCH and M. D. BENNETT Jodrell Laboratory, Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3DS, UK Summary A stable interspecific sexual plant hybrid between Hordeum vulgare cv. Tuleen 346 (barley) X H. bulbosum was shown to have seven chromosomes originating from each parent by genomic in situ hybridization. Electron microscope serial thin-section reconstructions of meta- phases and comparison with light micrograph karyo- types enabled chromosomes to be identified from their morphology. The three-dimensional positions of then- centromeres were established and analysed in the reconstructions of somatic root tip metaphases and cells at mitotic metaphase near their entry hi to meiosis. Parental genomes tended to lie in spatially separated domains in both tissues. Although varying in mor- phology, the two sets of chromosomes had similar mean sizes, so size differences did not cause the separation observed. In the EM, the centromere-associated struc- tures of the chromosomes of the more central genome, originating from H. vulgare, were larger than those of the more peripheral genome of H. bulbosum origin. Key words: nuclear organization, nuclear architecture, three-dimensional reconstruction, meiosis, Hordeum, genome separation. Introduction The spatial separation of parental genomes at meta- phase has been reported in many intergeneric and interspecific hybrids, and species, from both plants (Finch et al. 1981; Bennett, 1982; Gleba et al. 1987; Heslop-Harrison and Bennett, 1984, 1990; Linde- Laursen and Jensen, 1991) and animals (Ordartschenko and Keneklis, 1973; Zelesco and Marshall Graves, 1988; Brandriff et al. 1991). The phenomenon is important because of the consequences that spatial organization of chromosomes in the nucleus might have for mechanical chromosome behaviour, gene ex- pression (transcription) and DNA replication. Aspects of genome evolution - including speciation - may also be affected by nuclear organization. The study of the physical organization of the chromosomes within the nucleus of man-made and natural hybrids will assist the understanding of such phenomena. Wide hybrids, such as barley {Hordeum vulgare) x H. bulbosum discussed here, are important for plant breeding. The genetic base of many crops, particularly inbreeding species like barley is very narrow. New genes can be introduced by crossing with wild relatives, such as H. bulbosum, before backcrossing to produce improved varieties (see Snape et al. 1991). A second application of wide hybrids relies on uniparental chromosome elimination. New cereal crops must be uniform, which normally needs many generations of inbreeding following intercrossing of two varieties. The cross H. vulgare x H. bulbosum is used in barley breeding because, in some genotypes, the H, bulbosum chromosomes are eliminated, giving a haploid plant the chromosome number of which can be doubled with drugs such as colchicine. The doubled haploid is homozygous ("true breeding") and hence can be selected immediately for agronomic characters without variation. Understanding of stable and eliminating hybrids, and knowledge of the mechanisms of hybrid stability, may enable favourable manipulation in breed- ing programmes. We have previously described genome separation in the wide hybrids H. vulgare X Secale africanum (Finch et al. 1981) and H. chilense X S. africanum (Schwarz- acher-Robinson et al. 1987). In these intergeneric hybrids, the individual chromosomes of Hordeum origin were all smaller than those of S. africanum origin. Reconstructions of mitotic metaphase cells showed that centromeres of the two parental haploid sets tended to be spatially separate, and centromeres from one particular parent usually tended to be in the peripheral region of the metaphase plate that sur-