Ann Kenton

Characterization of the Nicotiana tabacum L. genome by molecular cytogenetics

Nicotiana tabacum (2n=48) is a natural amphidiploid with component genomes S and T. We used non-radioactive in situ hybridization to provide physical chromosome markers for N. tabacum, and to determine the extant species most similar to the S and T genomes. Chromosomes of the S genome hybridized strongly to biotinylated total DNA from N. sylvestris, and showed the same physical localization of a tandemly repeated DNA sequence, HRS 60.1, confirming the close relationship between the S genome and N. sylvesfris. Results of dot blot and in situ hybridizations of N. tabacum DNA to biotinylated total genomic DNA from N. tomentosiformis and N. otophora suggested that the T genome may derive from an introgressive hybrid between these two species. Moreover, a comparison of nucleolus-organizing chromosomes revealed that the nucleolus organizer region (NOR) most strongly expressed in N. tabacum had a very similar counterpart in N. otophora. Three different N. tabacum genotypes each had up to 9 homozygous translocations between chromosomes of the S and T genomes. Such translocations, which were either unilateral or reciprocal, demonstrate that intergenomic transfer of DNA has occurred in the amphidiploid, possibly accounting for some results of previous genetic and molecular analyses. Molecular cytogenetics of N. tabacum has identified new chromosome markers, providing a basis for physical gene mapping and showing that the amphidiploid genome has diverged structurally from its ancestral components.

Genomic in situ hybridization reveals the allopolyploid nature of Milium montianum (Gramineae)

Molecular techniques that “paint” chromosomes offer exciting new opportunities for testing genome relationships.Milium montianum (2n=22) is a grass whose distinctive bimodal karyotype comprises 8 large (L-) and 14 smaller (S-) chromosomes. The proposal thatM. montianum is an allotetraploid, with diploidMilium vernale (2n=8) as the L-chromosome genome donor, has been impossible to confirm by classical means. To test this hypothesis, biotinylated total genomic DNA of diploidM. vernale (2n=8) was hybridized in situ to root tip chromosomes ofM. montianum. TheM. vernale probe hybridized preferentially to all L-chromosomes, but not to the S-chromosomes. These results (i) confirm the allopolyploid nature ofM. montianum, (ii) strongly support the theory that the L-chromosomes ofM. montianum were donated byM. vernale, or a closely related genotype and (iii) show that subsequently the L-chromosomes have largely retained their genomic integrity in the new allopolyploid backgroud. Clearly, genomic in situ hybridization (GISH) is a potentially powerful tool for studying genome evolution and biosystematics. It will often be useful for investigating the origins of wild and cultivated polyploid plant species, especially where conventional methods have failed, for studying introgression, and for understanding the mechanism(s) of origin of bimodal karyotypes.

Chromosomal location of endogenous geminivirus-related DNA sequences inNicotiana tabacum L.

TheN. tabacum (tobacco) nuclear genome carries approximately 25 multiple direct repeats of a geminivirus-related DNA (GRD) sequence that probably arose by illegitimate recombination, following geminivrus infection, duringNicotiana evolution. Each GRD repeat carries sequences similar to the geminiviralAL1 gene of the tomato golden mosaic virus (TGMV), encoding a protein required for viral DNA replication, plus thecis-essential replication origin. Using a cloned 14-kb GRD repeat sequence as a probe for fluorescencein situ hybridization (FISH), we identified a unique tobacco chromosome carrying GRD. Translocations between chromosomes of the tobacco S and T genomes were used as physical markers by sequentially hybridizing chromosomes with labelled GRD and total genomic DNA fromN. sylvestris (equivalent to the S genome). The 25S, 18S and 5.8S ribosomal gene clusters were detected in double-labelling experiments for use as additional markers to identify the chromosomal location of GRD. GRD occupies one site on a homologous pair of small submetacentrics from the T genome characterized by a lack of either translocated segments from the S genome or ribosomal genes. GRD provides an additional marker for the small chromosomes of the T genome and a useful phylogenetic tool.

Heterochromatin accumulation, disposition and diversity in Gibasis karwinskyana (Commelinaceae)

C-banding differences within Gibasis karwinskyana (Roem & Schult.) Rohw. were reassessed using dual fluorochrome staining. Pronounced differences in C-band pattern between two subspecies with identical basic karyotypes were due to different chromosomal locations of AT-rich and GC-rich heterochromatin. The AT-rich component had an equilocal distribution in the karyotype and has evidently been accumulated at telomeres, as shown by its prevalence in supernumerary segments and B chromosomes. The GC-rich component also varied in amount, but was limited to nucleolus organizing regions (NORs) and centromeres. Centromeres and telomeres are suggested to constitute separate, although perhaps interdependent, centres of heterochromatin amplification. The possible role of nuclear architecture in determining the accumulation, distribution and spread of these sequences is discussed.

Identification of Renner complexes and duplications in permanent hybrids of Gibasis pulchella (Commelinaceae)

Discovery of permanent hybridity in the very large chromosomes of Gibasis pulchella (Commelinaceae) has allowed specific identification of segmental interchanges in complex heterozygotes. The interchanges are confined to terminal regions, are sometimes very small, and may be unequal in size. Breakpoints have occurred close to major C-bands, probably at euchromatin/heterochromatin boundaries. Complete and disjunctional ring formation at meiosis results in the segregation of two Renner complexes, each of which can be specifically identified with C-banding. The complex carrying the interchanges is usually transmitted through the pollen. Certain chromosomes that have undergone more extensive change than the rest of the complement may have some special significance. There is evidence of small duplications within heterozygous genomes. Permanent hybridity in different organisms may have quite different origins, possibly initiated by major karyotype repatterning following the activation of transposons that generate chromosome breakage.

Chromosome evolution in the Gibasis linearis group (Commelinaceae)

Karyotypes, DNA amounts, and meiotic behaviour were examined in population samples of two closely related species, Gibasis venustula and G. heterophylla, and their F1 hybrids. All samples were diploid (2n=12). DNA amount was similar in G. heterophylla, G. venustula ssp. robusta, and some populations of G. venustula ssp. venustula but in 13 samples of the latter, it showed as much as a 60% difference despite karyotypic uniformity. Although DNA levels were related to the altitude of the habitat, there was some variation within two morphological and ecological races of subspecies venustula. Analysis of hybrid meiosis and pollen fertility demonstrated the cytogenetical discreteness of populations, races, and subspecies. Populations and races were concluded still to be evolving following ecological isolation. The significance of quantitative and qualitative genome changes in evolution and their bearing on the classification of these species is discussed.

Autosyndetic pairing in Gibasis (Commelinaceae) hybrids revealed by C-banding

Two closely related species of Gibasis, G. karwinskyana and G. consobrina, and their F1 hybrids were studied cytologically at the diploid and tetraploid level. Despite similarity in their basic karyotype, pairing was extremely limited in the diploid hybrid and almost exclusively autosyndetic in the tetraploid, except for multivalent formation due to interchange heterozygosity. The analysis was considerably facilitated by the use of C-banding techniques at meiosis, by which the chromosomes of each species could be readily identified. In the parents, quadrivalents were formed between homologous but non-identical chromosomes, which also formed autosyndetic bivalents in the hybrids. Meiotic pairing in the hybrids was unaffected by polytypy for C-bands among different populations of the parental species.