Molecular Cytogenetics and Genome Organization

200. Schwarzacher T, Heslop-Harrison JS. 2000. Practical in situ hybridization . Bios: Oxford. pp. 203+xii. (order from bookshops or Sigma Chemical, cost c. US$50/£GBP30).

199. Castilho A, Vershinin A, Heslop-Harrison JS. 2000 . Repetitive DNA and the chromosomes in the genome of oil palm ( Elaeis guineensis ). Annals of Botany 85 : 837-844.

Like most plant genomes, much of the oil palm genome ( Elaeis gunieensis L., 2n=32) consists of repetitive DNA sequences. We aimed to isolate and characterize a range of repetitive sequences from the genome of the crop and analyse the repeats by sequencing, Southern and in situ hybridization. Three unrelated repetitive sequence families, with no homology to known sequences, showed a dispersed distribution along the chromosomes with concentration in the proximal parts of arms, while simple sequence repeats of DNA (GA, GATA and CAC) were clustered in the distal parts. Copia -like retroelements were dispersed throughout the genome, with a concentration in proximal regions, but not as abundant as in species with larger genomes. Among tandemly repeated sequences, a major 18S-25S rDNA site was present on a single pair of chromosomal sites, often on a satellite with no visible connection to its parent chromosome. A major 5S rDNA site was located on another chromosome pair; variable numbers of minor sites of both rDNA families were also detected. The telomeric sequence (CCCTAAA) was located at the ends of all chromosome arms, but no intercalary sites of amplification were detected. No other major families of tandemly repeated sequences were found. The molecular cytogenetic analysis and chromosome amplification patterns of major sequence families provide the reference point for examination of genomic organization of major classes of the repetitive DNA in normal and in tissue culture material including abnormal regenerants.

198. Heslop-Harrison JS, Osuji J, Hull R, Harper G, D'Hont A, Carreel F. 1999 . Fluorescent in situ hybridization of plant chromosomes: illuminating the Musa genome. INIBAP Annual Report 1998 : 26-29. http://www.ipgri.cgiar.org/publications/pubfile.asp?ID_PUB=48

197. Frello S, Heslop-Harrison JS. 2000. Chromosomal variation in Crocus vernus Hill (Iridaceae) investigated by in situ hybridization of rDNA and a tandemly repeated sequence. Annals of Botany 86: 317-322.

196. Chaves R, Heslop-Harrison JS, Guedes-Pinto H. 2000 . Centromeric heterochromatin in the cattle rob(1;29) translocation: alpha satellite I sequences, in situ Msp I digestion patterns, chromomycin staining and C-bands . Chromosome Research 8: 621-626.

195. Schmidt A, Doudrick RL, Heslop-Harrison JS, Schmidt T. 2000. The contribution of short repeats of low sequence complexity to large conifer genomes. Theor.Appl.Genet. 101 : 7-14.

The abundance and genomic organization of six simple sequence repeats consisting of di-, tri-, tetranucleotide sequence motifs and a minisatellite repeat have been analyzed in different gymnosperms by Southern hybridization. Within the gymnosperm genomes investigated, the abundance and genomic organization of micro- and minisatellite repeats largely follows taxonomic groupings. We found that only particular simple sequence repeat motifs are amplified in gymnosperm genomes, while others such as (CAC) 5 and (GACA) 4 are present in only low copy numbers. The variation in abundance of simple sequences motifs reflects a similar situation as found in angiosperms. Species of the two- and three-needle pine section Pinus are relatively conserved and can be distinguished from Pinus strobus which belongs to the five-needle pine section Strobus . The hybridization pattern of Picea species, bald cypress and gingko were different from patterns detected in Pinus species. Furthermore, sequences with homology to the plant telomeric repeat (TTTAGGG) n have been analyzed in the same set of gymnosperms. Telomere-like repeats are highly amplified within two- and three-needle pine genomes such as slash pine ( Pinus elliottii Engelm. var. elliottii ) compared to P. strobus , Picea species, bald cypress and gingko. Pinus elliottii var. elliottii was used as a representative species to investigate the chromosomal organization of telomere-like sequences by fluorescence in situ hybridization (FISH). The telomere-like sequences are not restricted to the ends of chromosomes; they form large intercalary and pericentric blocks showing that they are a repeated component of the slash pine genome. Conifers have genomes larger than 20000 Mbp, and our results clearly demonstrate that repeats of low sequence complexity such to (CA) 8 , (GA) 8 , (GGAT) 4 , (GATA) 4 , and minisatellite- and telomere-like sequences represent a large fraction of the repetitive DNA of these species. The striking differences in abundance and genome organization of the various repeat motifs suggest that these repetitive sequences evolved differently in the gymnosperm genomes investigated.

194. Heslop-Harrison JS. 2000 . RNA, genes, genomes and chromosomes: repetitive DNA sequences in plants. Chromosomes Today 13 : 45-56+ plate VI.

193. Castilho A, Neves N, Rufini-Castiglione M, Viegas W, Heslop-Harrison JS. 1999. 5-Methylcytosine distribution and genome organization in Triticale before and after treatment with 5-azacytidine . J. Cell Sci . 112 : 4397-4404.

Triticale (2n=6x=42) is a hybrid plant including rye (R) and wheat (A and B) genomes. Using genomic in situ hybridization with rye DNA as a probe, we found the chromosomes of the R genome were not intermixed with the wheat chromosomes in 85% of nuclei. After treatment of seedlings with low doses of the drug 5-azacytidine (5-AC), leading to hypomethylation of the DNA, the chromosomes became intermixed in 60% of nuclei; the next generation showed intermediate organization. These results correlate with previous data showing that expression of R-genome rRNA genes, normally suppressed, is activated by 5-AC treatment and remains partially activated in the next generation. The distribution of 5-methylcytosine (5-mC) was studied using an antibody to 5-mC. Methylation was detected along the lengths of all chromosomes; there were some chromosome regions with enhanced and reduced methylation, but these were not located at consistent positions, nor were there differences between R and wheat genome chromosomes. After 5-AC treatment, lower levels of methylation were detected. After 5-AC treatment, in situ hybridization with rye genomic DNA sometimes showed micronuclei of rye-origin and multiple translocations between wheat and rye chromosomes. Genomic DNA was analysed using methylation-sensitive restriction enzymes and, as probes, two rDNA sequences, two tandemly organised DNA sequences from rye (pSc200 and pSc250), and copia and the gypsy group retrotransposon fragments from rye and wheat. DNA extracted immediately after 5-AC treatment was cut more by methylation-sensitive restriction enzymes than DNA from untreated seedlings. Each probe gave a characteristic restriction fragment pattern, but rye- and wheat-origin probes behaved similarly, indicating that hypomethylation was induced in both genomes. In DNA samples from leaves taken 13 to 41 days after treatment, RFLP patterns were indistinguishable from controls and 5-AC treatments with all probes. Surprising differences in hybridization patterns were seen between DNA from root-tips and leaves with the copia -fragment probes.

192. Katsiotis A, Loukas M, Heslop-Harrison JS. 2000 . Repetitive DNA, genome and species relationships in Avena and Arrhenatherum (Poaceae). Annals of Botany 86 : 1135-1142.

Repetitive sequences have been widely used for examining genome and species relationships by in situ and Southern hybridization. In the present study, double-stranded DNA sequences, from denatured DNA reannealed to Cot = 1, from Avena strigosa (2 n = 2 x = 14; A genome; referred to as CotA) and Avena sativa (2 n = 6 x = 42; ACD genome; referred to as CotACD) were isolated with a hydroxyapatite column, and were used for in situ hybridization on hexaploid A. sativa chromosomes. Probe CotACD labelled all chromosomes evenly throughout their length at the same intensity. Probe CotA labelled the 28 A and D genome chromosomes strongly and the 14 C genome chromosomes weakly. Three cloned repetitive sequences, pAvKB9 (126 bp), pAvKB26 (223 bp) and pAvKB32 (721 bp) were characterized in the A, B, C and D Avena genomes and the genus Arrhenatherum using molecular and cytological methods. Clones pAvKB9 and pAvKB26 were absent from the Avena C genome, while both could identify the presence of the D genome by Southern hybridization. In situ hybridization to diploid and tetraploid Avena species revealed that the probes showed a dispersed genomic organization and that they are present on both arms of all chromosomes. These sequences were excluded from areas where tandem repeats, such as rRNA genes and telomeres, are present. These results indicate the close relationship between A and D genomes and the presence of common DNA sequences between A and C Avena genomes. All three clones hybridized to Southern blots containing Arrhenatherum digested genomic DNA, indicating Arrhenatherums close affinity to A, B and D Avena genomes.

191. Taketa S, Ando H, Takeda K, Harrison GE, Heslop-Harrison JS. 2000. The distribution, organization and evolution of two abundant and widespread repetitive DNA sequences in the genus Hordeum . Theor. Appl. Genet . 100: 169-176.

The genomic organization and chromosomal distributions of two abundant tandemly repeated DNA sequences, dpTa1 and pSc119.2, were examined in six wild Hordeum taxa representing the four basic genomes of the genus by Southern and fluorescence in situ hybridization. The dpTa1 probe hybridized to between 30 and 60 sites on chromosomes of all five diploid species studied, but hybridization patterns differed among the species. Southern hybridizations of the pSc119.2 sequence to chromosomes and digested DNA detected signals in H. bulbosum , H. chilense , H. marinum and H. murinum 4x (Y-genome), but not in H. murinum 2x and H. vulgare ssp. spontaneum . Compared to multiple sites of dpTa1 signals, a maximum of one pSc119.2 signal was observed in the terminal or subterminal region of each chromosome arm in the species carrying this sequence. Thus, the species carrying the same I-genome differed in the presence ( H. bulbosum ) or absence ( H. spontaneum ) of pSc119.2. The presence of pSc119.2 in the tetraploid cytotype of H. murinum but its absence in the diploid cytotype suggests that the tetraploid is not likely to be an simple autotetraploid of the diploid. Data about the inter- and intra-specific variation of the two independent repetitive DNA sequences gives information about both the interrelationships of the species and the evolution of the repetitive sequences.

190 . Heslop-Harrison J, Heslop-Harrison JS, Heslop-Harrison Y. 1999 . The structure and prophylactic role of the angiosperm embryo sac and its associated tissues: Zea mays as a model . Protoplasma 209: 256-272.

Various developmental phases can be distinguished in the definition of the archesporium and the early life of the embryo, taking Zea mays (maize) as a model within the Gramineae, and other families where pertinent: (1) the isolation of the megasporocyte and the functional spore derived from it; (2) the maturation of the specialised walls of the embryo sac, and their reinforcement by ensheathments derived from the contiguous nucellar cells during a sequence of phased genetic ablation; (3) the differentiation of the synergids, the associated flange, and the filiform apparatuses; (4) the blocking of the pollen tube pathway by secondary secretions in the micropylar region and the coagulation of the pollen tube cytoplasm within the filiform apparatuses during the process of fertilization; and finally (5) the development of a compound cutinized envelope of four fused layers (six where the outer integument is also involved) after fertilization. For the nascent haploid generation, the period of maximum vulnerability in respect to both pathogen invasion and the transition from diplophase control occurs during these phases. It is concluded that many of the protective features form a prophylactic shield and are key components of the angiosperms in general, which may have contributed to their evolutionary success as a group. Other physiological or biochemical adaptations or barriers may also supplement the mainly structural features described here.

189. Taketa S, Harrison G, Heslop-Harrison JS. 1999 . Comparative physical mapping of the 5S and 18S-25S rDNA in nine wild Hordeum species and cytotypes. Theor. Appl. Genet . 98 : 1-9.

The physical locations of the 5S and 18S-25S rDNA sequences were examined in nine wild Hordeum species and cytotypes by double-target in situ hybridization using digoxigenin-labelled 5S rDNA and biotin-labelled 18S-25S rDNA as probes. H. vulgare ssp. spontaneum (2n=2x=14; I-genome) had a similar composition of 5S and 18S-25S rDNA to cultivated barley ( H. vulgare ssp. vulgare , I-genome), with two major 18S-25S rDNA sites and minor sites on four of the other five chromosomes; three chromosomes had 5S rDNA sites. The closely related H. bulbosum (2x; also I-genome) showed only one pair of 5S rDNA sites and one pair of 18S-25S rDNA sites on different chromosomes. Four wild diploid species, H. marinum (X-genome), H. glaucum and H. murinum (Y-genomes) and H. chilense (H-genome), differed in the number (2 to 3 pairs), location and relative order of 5S and the one or two major 18S-25S rDNA sites, but no minor 18S-25S rDNA sites were observed. H. murinum 4x had three chromosome pairs carrying 5S rDNA, while the diploid had only a single pair. Two other tetraploid species, H.�brachyantherum 4x and H. brevisubulatum 4x, (both considered to have H-type genomes) had minor 18S-25S rDNA sites, as well as the major sites. Unusual double 5S rDNA loci - two sites on one chromosome arm separated by a short distance - were found in the American H-genome species, H. chilense and H. brachyantherum 4x. The results indicate that the species H.�brachyantherum 4x and H. brevisubulatum 4x have a complex evolutionary history, probably involving multiplication of minor rDNA sites (as in H. vulgare sensu lato ), or incorporation of both I and H types of genome. The rDNA markers are useful for investigation of chromosome evolution and phylogeny.

188 . Alkhimova AG, Heslop-Harrison JS, Shchapova AI, Vershinin AV. 1999 . Rye chromosome variability in wheat-rye addition and substitution lines. Chromosome Res . 7 : 205-212.

In a study of polymorphism and stability in rye chromosomes, three rye varieties and the sets of wheat-rye addition and substitution lines were compared using two non-homologous, highly repetitive DNA families, pSc200 and pSc250. The rye varieties, Petkus, Imperial and Onohoiskaya, showed polymorphism for the presence and the size of the pSc200 in situ hybridization signals on chromosome pairs 2R, 4R and 7R, and the pSc250 signals on chromosomes 5R, 6R and 7R. Chromosome 1R was heteromorphic within the Onohoiskaya variety. Differences in the distribution of chromosome polymorphisms imply that intervarietal changes to these highly repetitive DNA families occurred independently, despite their juxtaposition or even overlapping locations in subtelomeric heterochromatic regions. In the set of Saratovskaya 29 wheat/Onohoiskaya substitution lines, only chromosome 2R was altered compared to its counterpart in the parental rye variety due to amplification of the pSc250 signal on the long arm, although this did not exceed intervarietal polymorphism. In the set of Chinese Spring wheat/Imperial addition lines, only two Imperial chromosomes, 4R and 6R, were unchanged. We detected the loss of one or both rye homologous chromosomes, the loss of one arm, and the deletion of subtelomeric heterochromatin accompanied by the loss of the pSc200 signal. The results show that Saratovskaya 29/Onohoiskaya chromosome substitution lines possess increased chromosome stability compared to Chinese Spring/Imperial addition lines.

187. Vaughan HE, Heslop-Harrison JS, Hewitt GM. 1999 . The localisation of mitochondrial sequences to chromosomal DNA in Orthopterans . Genome 42: 874-880.

186. Bardsley D, Cuadrado A, Jack P, Harrison G, Castilho A, Heslop-Harrison JS. 1999 . Chromosome markers in the tetraploid wheat Aegilops ventricosa analysed by in situ hybridization. Theor.Appl.Genet. 99: 300-304.

185. Heslop-Harrison JS. 1999 . Aspects of the cell biology of pollination and wide hybridization. In: Cresti M, Tiezzi A, eds. Sexual Plant Reproduction . Heidelberg: Springer, 139-144.

The study of the cell and molecular biology of pollen is important. It is critical to crops and the development of new crops through plant breeding. For the cereals, the seed, the product of pollination, is the crop; for many other crops, propagation is via the seed. Plant breeding uses meiosis and pollen transmission of the new combinations of genes to generate the variability required by plant breeders. While biotechnology offers the opportunity for direct gene transfer through transformation, further cycles of breeding are normally required to gain stability and multiply seed. Wide hybridization is used to introduce chromosomes, chromosome segments and genes into early stages of breeding programmes, increasing the range of variability available to breeders. A clear understanding of the constraints on pollen viability, its dispersal, fertility and capacity to fertilize different genotypes, and perhaps long-term storage potential, is important to both in situ and ex situ germplasm or biodiversity conservation, and related topics of population ecology and gene-flow. The study of pollen has allowed and will continue to facilitate fundamental understanding of developmental biology and genetics. As a haploid, genetics may be more straightforward, and its relative simplicity combined with defined developmental pathways and easy accessibility means developmental and molecular studies can be carried out without some of the problems encountered in working with sporophytic tissue. Wide hybrids also provide a powerful tool to examine genomic interactions and define critical, often genetically-controlled, aspects of development.

184. Harper G, Osuji JO, Heslop-Harrison JS, Hull R. 1998. Integration of banana streak badnavirus into the Musa genome: molecular and cytogenetic evidence. Virology 255 : 207-213.

183. Lima-Brito J, Guedes-Pinto H, Heslop-Harrison JS. 1998. The activity of nucleolar organizing chromosomes in multigeneric F1 hybrids involving wheat, triticale and tritordeum. Genome 41 : 763-768.

182. Heslop-Harrison JS, Murata M, Ogura Y, Schwarzacher T, Motoyoshi F. 1999 . Polymorphisms and genomic organization of repetitive DNA from centromeric regions of Arabidopsis thaliana chromosomes. Plant Cell 11 : 31-42.

A highly abundant repetitive DNA sequence family of Arabidopsis thaliana , the pAL1 family, is composed of 178 bp tandemly repeated units and is located at the centromeres of all five chromosome pairs. Analysis of multiple copies showed 95% conservation of nucleotides, with some alternative bases, and revealed two boxes, 30 and 24 bp long, that are 99% conserved. Sequences at the 3 end of these boxes showed similarity to yeast CDEI and human CENP-B DNA-protein binding motifs. When oligonucleotides from less conserved regions were hybridized in situ and visualized by using primer extension, they were detected on specific chromosomes. When used for polymerase chain reaction with genomic DNA, single primers, or primer pairs orientated in the same direction showed negligible amplification, indicating a head-to-tail repeat unit organization. Most primer pairs facing in opposite directions gave several strong bands corresponding to their positions within the pAL1 repeat unit. However, some primer pairs showed no amplification which is consistent with the primer extension results, indicating that there are chromosome-specific variants of AtCon. The results are significant because they elucidate the organization, mode of amplification, dispersion, and evolution of one of the major repeated sequence families of Arabidopsis. The evidence presented here suggests that the AtCon family, like human -satellites, plays a role in Arabidopsis centromere organization and function.

181. Goubely C, Arnaud P, Tatout C, Heslop-Harrison JS, Deragon J-M. 1999 . S1 SINE retroelements are methylated at symmetrical and non-symmetrical positions in Brassica napus : identification of a preferred target site for asymmetrical methylation . Plant Mol. Biol. 39 : 243-255.

DNA methylation has been often proposed to operate as a genome defence system against parasitic mobile elements. To test this possibility, the methylation status of a class of plant mobile elements, the S1 Bn SINEs, was analyzed in detail using the bisulfite modification method. We observed that S1 Bn SINE retroposons are methylated at symmetrical and asymmetrical positions. Methylated cytosines are not limited to transcriptionally important regions but are well distributed along the sequence. S1 Bn SINE retroposons are two-fold more methylated then the average methylation level of the Brassica napus nuclear DNA. By in situ hybridization, we showed that this high level of methylation does not result from the association of S1 Bn elements to genomic regions known to be highly methylated suggesting that S1 Bn elements were specifically methylated. A detailed analysis of the methylation context showed that S1 Bn cytosines in symmetrical CpG and CpNpG sites are methylated at a level of 87% and 44% respectively. We observed that 5.3% of S1 Bn cytosines in non-symmetrical positions were also methylated. Of this asymmetrical methylation, 57% occurred at a precise motif (Cp(A/T)pA) that only represented 12% of the asymmetrical sites in S1 Bn sequencessuggesting that it represents a preferred asymmetrical methylation site. This motif is methylated in S1 Bn elements at only half the level observed for the Cp(A/T)pG sites. We show that non-S1 Bn CpTpA sites can also be methylated in DNA from B. napus and from other plant species.

180. Kubis S, Schmidt T, Heslop-Harrison JS. 1998 . Repetitive DNA elements as a major component of plant genomes. Ann. Bot. 82S : 45-55.

A major part of the nuclear genome of most plants is composed of different repetitive DNA elements. Studying these sequence elements is essential for our understanding of the nature and consequences of genome size variation between different species, and for studying the large-scale organization and evolution of plant genomes. Sugar beet ( Beta vulgaris L.) is an important crop and a suitable model for such investigations: with a genome size of 0.8 pg 1C (760 Mbp) it contains significant amounts of all major groups of repetitive sequences among its 9 chromosome pairs, but analysis is not complicated by polyploidy or the huge size of some genomes, and there is valuable genetic data, recombinant DNA libraries and wild relatives to complement studies of sequences contribution to genome size in sugar beet. A sophisticated understanding of the structure of the genome will provide valuable data about the major factors responsible for genome size variation, useful aids in the development of a molecular understanding of genome evolution, and perhaps indicate strategies for crop improvement. Using molecular and cytological approaches, we have characterized a range of differentially organized repetitive DNA sequence elements from the genomes of the cultivated and wild beet species, leading to an extensive model of the repetitive DNA, its organization and evolution.

179. Schmidt T, Kubis S, Katsiotis A, Jung C, Heslop-Harrison JS. 1998 . Molecular and chromosomal organization of two repetitive DNA sequences with intercalary locations in sugar beet and other Beta species. Theor.Appl.Genet. 97: 696-704.

In a search for repetitive DNA sequences in the sugar beet genome, two sequences with repeat unit lenghts of 143 and 434 bp were isolated and characterized. The pSV family showed unusual conservation of restriction sites reflecting homogenization of the analyzed repeats. Members of the family are organized as tandem repeats as revealed by PCR and sequencing of dimeric units. The pSV satellite occurs in a large intercalary array which exists on all chromosome arms of sugar beet. The pSV sequence family is present in different abundance in the section Beta , Corolliflora and Nanae but not detectable by Southern hybridization in the section Procumbentes . The pDRV family is characterized by an interspersed genomic organization. The sequence is detectable in all sections of the genus and is amplified in species of the section Beta but was also detected, although at lower abundance, in the remaining three sections. Fluorescent in situ hybridization has shown that the pDRV sequence family is dispersed over all chromosomes of the sugar beet complement with some regions of clustering and centromeric depletion.

178. Osuji JO, Harrison G, Crouch J, Heslop-Harrison JS. 1998 . Prospects for the application of molecular cytogenetics to Musa breeding. Acta Horticulturae in press.

Characterization of banana and plantain germplasm has mainly been based on the use of phenotypic characters and molecular marker technologies such as RFLP and RAPD. However, molecular cytogenetic studies may offer greater understanding of phylogenetic relationships and clarity of taxonomic discrimination as well as assisting selection and identifying aneuploids. Total genomic DNA from diploid A and B genome accessions has been used for in situ hybridization to mitotic chromosomes of various Musa accessions. Genomic in situ hybridization has offered evidence for a close genomic relationship between the A and B genomes of Musa but enables discrimination of chromosomes originating from the two genomes. It has also provided data on genomic origin and the ratio of chromosome sets in selected accessions of wild and cultivated bananas, plantains and artificial hybrids. Further details of the chromosomal gross structure such as the occurrence of two satellited chromosomes in diploid accessions and three satellited chromosome in triploid accessions was confirmed. There are many applications of double target in situ hybridization in Musa breeding. These include clarifying the complex chromosomal behaviour during interploid and/or interspecific Musa crosses and determining the parental source of chromosome complements of Musa hybrids. This technique also has the potential of verifying the designated genomic complement of Musa germplasm and of detecting chromosomal exchanges in the artificial hybrids.

177 . Schmidt T, Heslop-Harrison JS. 1998 . Genomes, genes and junk: the large scale organization of plant chromosomes. Trends Plant Sci. 3 : 195-199.

176. Kuipers AGJ, Heslop-Harrison JS, Jacobsen E. 1998 . Characterisation and physical localisation of Ty1- copia -like retrotransposons in four Alstroemeria species. Genome 41 : 357-367.

The genus Alstroemeria contains species with very large genomes (2C between 36.5 - 78.9 pg (17,600 and 38,000 Mb) in 2n=2x=16 species). We investigated the diversity, genomic and chromosomal organisation of Ty1- copia -like retrotransposons in four Alstroemeria species. Analysis of 33 PCR-amplified sequences corresponding to a conserved domain of Ty1- copia reverse transcriptase (rt) gene showed high heterogeneity among predicted amino acid sequences, common in plant Ty1- copia -like retrotransposons; no two sequences were identical but most fell into one of five subgroups; levels of inter- and intra-species heterogeneity were similar. Hae III digested genomic DNA of various Alstroemeria species contained distinct bands upon hybridisation with individual rt gene fragments. Hybridisation with the heterogeneous pool of retrotransposon rt fragments from the PCR (retrotransposon pool) revealed additional bands and smears; some minor bands were characteristic of either Brazilian or Chilean-origin species. In situ hybridisation of the retrotransposon pool from three species to metaphase chromosomes from the same species showed a dispersed distribution of the retrotransposon pool with exclusion from rDNA and sometimes other chromosomal sites. A . pelegrina , with no major heterochromatic sites, showed some clustering and small negative bands. The retrotransposon pool was excluded from major, DAPI-staining, bands in A. aurea , but in contrast the sites of the major tandemly repeated sequences in A . inodora showed a similar hybridisation signal to the rest of the chromosome arms. The data are discussed in the context of the contribution of Ty1- copia -like retrotransposons to plant genome size, their evolution and their value for phylogenetic and biodiversity studies.

175. Schmidt T, Kubis S, Doudrick RL, Kamm A, Brandes A, Desel C, Heslop-Harrison JS. 1998 . FISHing DNA sequences - fluorescent in situ hybridization in plant genome analysis. Scanning in press .

Repetitive DNA, organized in defined structural sequence classes, causes variation in genome size of higher plants over several orders of magnitude. In the reference species sugar beet ( Beta vulgaris ), we have characterized major families of repetitive DNA sequences of the cell nucleus including satellite DNA, microsatellites, retrotransposons and multigene families. We demonstrate by fluorescent in situ hybridization how repetitive DNA contributes to the structure of plant chromosomes and defines some of the differences between related genomes. In plant breeding research, highly repetitive DNA sequences were used for the detection of alien wild beet chromatin in Beta and for the correlation of genetic linkage maps and chromosomes. In species varying from Arabidopsis thaliana to slash pines ( Pinus elliottii var. elliottii ) with genome sizes ranging from about 150 Mbp to 25000 Mbp, we show by comparative in situ hybridization general patterns of the organization of repetitive DNA sequences in plants.

174. Osuji JO, Crouch J, Harrison G, Heslop-Harrison JS. 1998 . Molecular cytogenetics of Musa L. species, banana and plantain cultivars, and artificial hybrids: location of 18S-5.8S-25S and 5S rDNA and telomere-like sequences. Ann.Bot. 82: 243-248.

The physical sites of 18S-5.8S-25S and 5S rRNA genes and telomeric sequences in the Musa L. genome were localized by fluorescent in situ hybridization on mitotic chromosomes of selected lines. A single major intercalary site of the 18S-5.8S-25S rDNA was observed on the short arm of the nucleolar organizing chromosome in each genome. AA and BB genome diploids had a single pair of sites, triploids had three sites while a tetraploid hybrid had four sites. The probe is useful for quick determination of ploidy, even using interphase nuclei from slowly growing tissue culture material. Variation in the intensity of signals was observed among heterogeneous Musa lines indicating variation in the number of copies of the 18S-5.8S-25S rRNA genes. Eight subterminal sites of 5S rDNA were observed in Calcutta 4 (AA) while Butohan 2 (BB) had six sites; some were weaker in both genotypes. All triploid lines showed six major sites of 5S rDNA but various numbers of weaker sites. The diploid hybrids had five to eight while the tetraploid hybrid had eleven sites of 5S rDNA while the tetraploid hybrid had eleven sites. The telomeric sequence was detected as pairs of dots at the ends of all the chromosomes analyzed but no intercalary sequences were seen. The molecular cytogenetic studies of Musa using repetitive and single copy DNA probes should yield insight into the genome and its evolution. They and provide data for Musa breeders to tackleing the challenges caused by Banana streak virus and somaclonal variation in tissue culture, as well as generating genetic markers in Musa .

173. Flavell AJ, Pearce SR, Heslop-Harrison JS, Kumar A. 1997. The evolution of Ty1- copia group retrotransposons in eukaryote genomes. Genetica 100 : 185-195.

172. Kubis SE, Heslop-Harrison JS, Desel C, Schmidt T. 1998 . The genomic organization of non-LTR retrotransposons (LINEs) from three Beta species and five other angiosperms. Plant Mol. Biol . 36 : 821-831

We have isolated and characterized conserved regions of the reverse transcriptase gene from non-LTR retrotransposons, also called long interspersed nuclear elements (LINEs), from Beta vulgaris, B. lomatogona and B. nana . The novel elements show strong homology to other non-LTR retrotransposons from plants, humans and animals. LINEs are present in all species of the genus Beta tested, but there was variation in copy number. Analysis by Southern hybridization and fluorescent in situ hybridization revealed the clustered organization of these retroelements in beet species. PCR amplification using new degenerate primers to conserved motifs of the predicted LINE protein sequence enabled the cloning of LINEs from both Monocotyledonae ( Allium cepa, Oryza sativa and Secale cereale ) and Dicotyledonae ( Nicotiana tabacum and Antirrhinum majus ) indicating that LINEs are a universal feature of plant genomes. A dendrogram of fifteen new and six previously isolated sequences showed the high level of sequence divergence while revealing families characteristic of some genera. The genomic organization of non-LTR retrotransposons was examined in more detail in A. majus and O. sativa .

171. Heslop-Harrison JS. 1996 . Book Review: Bananas and plantains by Gowen S, ed. 1995. 612pp. London: Chapman and Hall. Ann.Bot. 77 : 193-195.

170. Galasso I, Harrison GE, Schmidt T, Pignone D, Heslop-Harrison JS. 1999. Location of two repeated DNA sequences of Vigna unguiculata (L.) Walp. on chromosomes and extended DNA fibres by FISH. J.Genet.Breeding 53 : 215-221.

169. Guedes-Pinto H, Ribeiro-Carvalho C, Pinto-Carnide O, Igrejas G, Nascimento M, Carnide VP, Harrison G, Heslop-Harrison, JS, Gateau I, Branlard G. 1997. Studies of Barbela wheat, an old Portuguese landrace with rye introgression. In: Chromosomes in Agriculture, Conference Proceedings, ed. Lelly T. In press.

The Portuguese wheat landrace aggregate known as Barbela is a unique repository of genetic variability. Modern analyses are able to show the diversity and importance of the genes and gene combination present in the landrace. Remarkable variability was found in morphology and yield components. Unusual and novel high and low molecular weight glutenin and gliadin storage proteins were detected with numerous different patterns in SDS-PAGE analysis. Accessions showed a range of tolerance to high aluminium ion levels, associated with acid soils in the field, with some lines more tolerant than the tolerant wheat tester. Genomic in situ hybridization was able to show the introgression of one or two pairs of small chromosome segments of rye-origin, sometimes intercalary and not associated with heterochromatin, into some Barbela accessions. The studies show the potential value of farmer-selected landraces to modern breeding programmes, and the importance of extensive collection, conservation and detailed analysis of such materials.

168. Osuji JO, Harrison G, Crouch J, Heslop-Harrison JS. 1997 . Identification of the genomic constitution of Musa L. lines (bananas, plantains and hybrids) using molecular cytogenetics. Ann.Bot. 80: 787-793.

The genomic constitutions of some Musa L. lines (bananas, plantains and artificial hybrids) were identified using molecular cytogenetic techniques. Double target in situ DNA:DNA hybridization to chromosome spreads using as probes, total genomic DNA isolated from diploid Musa lines of known AA (labelled with biotin-11-dUTP) and BB (labelled with digoxigenin-11-dUTP) genome constitution was carried out. The use of 60% acetic acid combined with heating over a flame gave high quality chromosome spreads free of cytoplasm for in situ hybridization. Total genomic A DNA labelled broad centromeric regions of all 22 chromosomes of the diploid line, Calcutta 4 ( M. acuminata Colla. ssp. burmanniccoides ; A genome) with some chromosomes showing stronger hybridization. Labelled DNA from the B genome hybridized strongly to the centromeric regions of all 22 chromosomes of Butohan 2 ( M. balbisiana Colla; B genome). The two satellited chromosomes of genome B labelled strongly with genomic A DNA. In situ hybridization of labelled A and B genomic DNA to metaphase chromosomes of triploid AAB and ABB cultivars discriminated between A and B genome chromosomes. The plantains Agbagba, Obino l'Ewai and Mbi Egome showed 22 genome A and 11 genome B chromosomes while the cooking bananas Bluggoe and Fougamou showed 11 genome A and 22 genome B chromosomes. Hybridization of labelled A and B genomic DNA to chromosomes of the hybrids showed that TMP2x 2829-62 has all 22 genome A chromosomes while TMPx 4698-1 has 33 genome A and 11 genome B chromosomes. In situ hybridization of labelled total genomic DNA to chromosomes has immense potential for identification of chromosome origin and can be used to characterize cultivars and hybrids produced in Musa breeding.

167. Galasso I, Harrison GE, Pignone D, Brandes A, Heslop-Harrison JS. 1997. The distribution and organization of Ty1- copia -like retrotransposable elements in the genome of Vigna unguiculata (L.) Walp. and its relatives. Ann.Bot. 80 : 333-337.

Ty1- copia -like retrotransposable elements were amplified from Vigna species by the polymerase chain reaction (PCR) with primers derived from the conserved region of the reverse transcriptase (RT) gene. Southern blots of genomic DNA digests from Vigna unguiculata and related species probed with the PCR derived sequences from V. unguiculata (rt-cow), V. unguiculata subsp. dekindtiana (rt-dek), V. luteola (rt-lut) and V. vexillata (rt-vex) showed variable hybridization patterns and intensities, generally correlating with taxonomic relationships. Sequencing of four clones revealed homologies to the reverse transcriptase gene of known Ty1- copia -like elements, although comparison of the predicted peptide sequences with each other and previously isolated reverse transcriptase genes from other legumes typically showed less than 50% identity. Fluorescence in situ hybridization of the PCR products showed that Ty1- copia elements represented a major fraction of the genome and were dispersed relatively uniformly over all chromosomes, with exclusion from centromeric, subtelomeric and nucleolar organizing regions, and a few sites of greater abundance. We are now able to start to understand the organization, variability and evolution of retrotransposons within the Fabaceae.

166. Kumar A, Pearce SR, McLean K, Harrison G, Heslop-Harrison JS, Waugh R, Flavell AJ. 1997. The Ty1- copia group of retrotransposons in plants: genomic organisation, evolution and use as molecular markers . Genetica 100 : 197-204.

The genomic organisation and diversity of the Ty1 -copia group retrotransposons has been investigated in several crop plants and their relatives from both dicotyledonous and monocotyledonous families, including potato ( Solanum tuberosum ), faba beans ( Vicia faba ), Vicia melanops , Vicia sativa , barley ( Hordeum vulgare ), rye ( Secale cereale ) and onion ( Allium cepa ). Extreme heterogeneity in the sequence of the Ty1 -copia retrotransposons from all these plants was revealed following sequence analysis of reverse transcriptase fragments. The estimated copy numbers of the Ty 1 -copia group retrotransposons for the genomes of S. tuberosum , L . esculentum , A. cepa , S. cereale and V. faba is highly variable, ranging from a few hundred to approximately a million copies per genome. In situ hybridisation data from metaphase and prophase chromosomes of V. faba , S. cereale and H. vulgare suggest that retrotransposon sequences are dispersed throughout the euchromatic regions of the genome but are almost undetectable in most heterochromatic regions. In contrast, similar data from metaphase chromosomes of A. cepa suggests that although retrotransposon sequences are dispersed throughout the euchromatic regions of the genome, they are predominantly concentrated in the terminal heterochromatin. These results are discussed in the context of the role played by the Ty1- copia group retrotransposons in the evolution of the plant genome. Lastly, the application of retrotransposon sequences as genetic markers for mapping genomes and for studying genetic biodiversity in plants are discussed.

165. Schmidt T, Kubis S, Desel C, Jung C, Heslop-Harrison JS. 1997. Molekulare cytogenetik von wild- und kulturruben der gattung Beta. Vort.Pflanzenz_cht. in press.

164. Viegas W, Silva M, Neves N, Castilho A, Barao A, Queiroz A, Heslop-Harrison JS, Morais-Cecilio L, Amado L, Delgado M, Carvalho V. 1996. Expression of 1R rDNA loci in Triticale: genetic and developmental controls. In: Guedes-Pinto H, Darvey N, Carnide VP, eds. Triticale: Today and Tomorrow . Amsterdam: Kluwer, 127-134.

Using the silver staining technique, which allows the identification in metaphase cells of rDNA loci active in the previous interphase, it was observed the consistent expression of 1R NOR in hexaploid triticale cultivars with a 2R/2D chromosome substitution, confirmed through genomic in situ hybridization using rye total genomic DNA and different cloned probes of repetitive sequences. A control mediated by non-nucleolar chromosomes on the suppression of rye NOR in triticale was therefore detected since it was possible to observe the expression of those loci in the presence of all wheat major NORs. It was also established that the 1R NOR expression pattern in complete hexaploid triticale cultivars is a developmental-dependent process, since: (i) the epigenetic pattern of 1R NOR inactivation in the presence of wheat genomes is syncronously established in embryos and endosperms between the 4th and the 5th day after fertilization; (ii) the influence of induced DNA demethylation on that pattern, through the incorporation of 5-azacytidine, is also dependent on the seed developmental stage; and (iii) the rye NOR suppression pattern is maintained until meiosis being erased during this process.

163. Roose ML, Schwarzacher T, Heslop-Harrison JS . 1998. The chromosomes of Citrus and Poncirus species and hybrids:Identification of characteristic chromosomes and physical mapping of rDNA loci using in situ hybridization and fluorochrome banding. J. Heredity 89: 83-86.

162. Murata M, Heslop-Harrison JS, Motoyoshi F. 1997 . Physical mapping of the 5S ribosomal RNA genes in Arabidopsis thaliana by multi-color fluorescence in situ hybridization with cosmid clones. Plant Journal 12 : 31-37.

The 5S ribosomal RNA genes were mapped to mitotic chromosomes of Arabidopsis thaliana by fluorescence in situ hybridization (FISH). In ecotype Landsberg erecta , hybridization signals appeared on three pairs of chromosomes, two of which were metacentric and the other acrocentric. Hybridization signals on a pair of metacentric chromosomes were much stronger than those on acrocentric and on another pair of metacentric chromosomes, probably reflecting the number of copies of the genes on the chromosomes. Other ecotypes, Columbia and Wassilewskija had similar chromosomal distribution of the genes, but the hybridization signals on a pair of metacentric chromosomes were very weak, and detectable only in chromosomes prepared from young flower buds. The chromosomes and arms carrying the 5S rDNA were identified by multi-color FISH with cosmid clones and a centromeric 180-bp repeat as co-probes. The metacentric chromosome 5 and its L arm arries the large cluster of the genes, and the short arm of acroncentric chromosome 4 carries the smaller cluster in all three ecotypes. Chromosome 3 had another small cluster of the genes on its L arm. Using the 5S rDNA as a probe, all chromosome components of A. thaliana could be identified by FISH. Chromosome 1 is a large metacentric, chromosome 2, an acrocentric carrying the 18S-5.8S-25S rDNA clusters on its short arm, chromosome 3, a metacentric carrying a small cluster of 5S rDNA genes on its L arm, chromosome 4, an acrocentric carrying both 18S-5.8S-25S and 5S rDNAs on its short (L) arm, chromosome 5, a metacentric carrying the large cluster of 5S rDNA on a centromeric region of the L arm.

161. Heslop-Harrison JS, Brandes A, Taketa S, Schmidt T, Vershinin AV, Alkhimova EG, Kamm A, Doudrick RL, Schwarzacher T, Katsiotis A, Kubis S, Kumar A, Pearce SR, Flavell AJ, Harrison GE. 1997. The chromosomal distributions of Ty1- copia group retrotransposable elements in higher plants and their implications for genome evolution. Genetica 100 : 197-204.

Retrotransposons make up a major fraction - sometimes more than 40% - of all plant genomes investigated so far. We have isolated the reverse transcriptase domains of the Ty1- copia group elements from several species, ranging in genome size from some 100�Mbp to 23,000�Mbp, and determined the distribution patterns of these retrotransposons on metaphase chromosomes and within interphase nuclei by DNA:DNA in situ hybridization. With some exceptions, the reverse transcriptase domains were distributed over the length of the chromosomes. Exclusion from rDNA sites and some centromeres (e.g. Slash pine, 23,000 Mbp, or barley, 5,500 Mbp) is frequent, while many species exclude retrotransposons from other sites of heterochromatin (e.g. intercalary and centromeric sites in broad bean). In contrast, in the plant Arabidopsis thaliana , widely used for plant molecular genetic studies because of its small genome (c. 100�Mbp), the Ty1- copia group reverse transcriptase gene domains are concentrated in the centromeric regions, collocalizing with the 180bp long satellite sequence pAL1. Unlike the pAL1 sequence, though, the Ty1- copia signal is also detectable as weaker, diffuse hybridization along the lengths of the chromosomes. Possible mechanisms for evolution of the contrasting distributions are discussed. Understanding the physical distribution of retrotransposons, and comparisons of the distribution between species, is critical to understanding their evolution and the significance or generation of the new patterns of variability and in speciation. \journals\genet.doc

160. Schmidt T, Jung C, Heslop-Harrison JS, Klein M. 1997. Detection of alien chromatin conferring resistance to the beet cyst nematode ( Heterodera schachtii Schm.) in cultivated beet ( Beta vulgaris L.) using in situ hybridization. Chromosome Res. 5 : 186-193.

Chromatin originating from the wild beet ( Beta procumbens Chr. Sm.) has been investigated in nematode resistant hybrid-derived lines of sugar beet ( Beta vulgaris L.) by in situ hybridization using satellite, telomeric and ribosomal DNA repeats, a yeast artificial chromosome (YAC) and total genomic DNA as probes. The alien chromosome was detected in three monosomic addition lines (2n=18+1) by genomic in situ hybridization. Fluorescent in situ hybridization with a genome-specific satellite repeat and YAC-DNA enabled the visualization of B. procumbens chromosomes, and in double-target hybridization it was shown that they do not carry 18S-5.8S rRNA and 5S rRNA genes. The wild beet-specific satellite repeat and the telomere sequence from Arabidopsis thaliana were used to perform a structural analysis of wild beet chromosome fragments of two resistant fragment addition lines. It was shown that one physical end of the chromosome fragments consists of telomeric repeats. Comparison of fragment sizes indicated that the small chromosome fragments harbouring the resistance gene resulted most likely from the loss of one wild beet chromosome arm and an internal deletion of the remaining arm.

159. Taketa S., Nakazaki T, Schwarzacher T, Heslop-Harrison JS. 1997. Detection of a 4DL chromosome segment translocated to rye chromosome 5R in an advanced hexaploid triticale line Bronco 90 . Euphytica 97 : 91-96.

Bronco 90 is an advanced line of hexaploid triticale and was reported to be a 2D(2R) chromosome substitution type. In F 1 hybrids of this triticale with bread wheat, however, a meiotic configuration of 16 bivalents and 10 univalents was frequently observed indicating the presence of an additional D(R) chromosome substitution or D/R translocation. To determine the chromosome constitution of Bronco 90, C-banding and fluorescent in situ hybridization techniques were applied to somatic and meiotic metaphase chromosomes. These analyses revealed that in Bronco 90, the terminal 7% of the long arm of rye chromosome 5R is derived from the long arm of chromosome 4D. It is known that the terminal segment of 5RL is homoeologous to that of 4DL. This translocated chromosome (5RS.5RL-4DL) paired in its long arm with telosome 4DL at a frequency of 71%, demonstrating the significance of small terminal chromosome segments for pairing. The origin of this novel rye-wheat translocation and its possible association with the absence of hairy peduncle character in Bronco 90 are discussed.

158. Heslop-Harrison Y, Heslop-Harrison JS. 1997. The pollen-tube activated abscission zone in the stigma of pearl millet: structural and physiological aspects. Can.J.Bot. 75 : 1200-1207.

Abscission of the stigmas of pearl millet [ Pennisetum typhoides (Burman) Stapf et Hubbard] after pollination results from the activation of a specialized zone of tissue near the base. This abscission zone comprises a well-defined stretch of the basal column made up of thin-walled cells lacking a cuticularized epidermis. The penetration of the first few pollen tubes following a compatible pollination results in loss of turgor throughout the tissue, followed by loss of adhesion between the cells and ultimately disruption of the whole zone. Pectolytic enzymes are released concomitantly, but the cellulosic skeletons of the cells persist. Numerous calcium oxalate druses, identified cytochemically and by energy-dispersive X-ray analysis, are present in the cells of the abscission zone; these undergo dissolution after activation of the zone, while simultaneously the druse population in the stigma stump and the upper ovary wall increases. The barrier formed by the degenerating abscission zone tissues excludes pollen tubes arriving after the initial activation, and also, as shown by Willingale et al. (1986), prevents the penetration of pathogens such as ergot and so has a prophylactic role. We suggest that the collapsed abscission zone not only forms a physical barrier but also a chemical one. Dissolution products of the numerous calcium oxalate druses during the breakdown of the tissue are likely to raise the calcium ion concentrations beyond the optimum for pollen tube growth, so preventing further penetration. Inhibited tubes swell at the tips and may show growth disorientation before arrest, responses similar to those seen in pearl millet tubes in culture when exposed to higher than normal calcium ion concentration. \journals\penn.doc

157. Heslop-Harrison J, Heslop-Harrison Y, Heslop-Harrison JS. 1997. Motility in ungerminated grass pollen:association of myosin with polysaccharide-containing wall-precursor bodies (P-particles). Sex. Plant Reprod. 10 : 65-66.

156. Neves N, Silva M, Heslop-Harrison JS, Viegas W. 1997. Nucleolar dominanace in triticales: control by unlinked genes. Chromosome Res. 5: 125-131.

Hybrid plants and animals often show suppression of activity of ribosomal genes (rDNA) originating from one of the parental or ancestral species. In the wheat x rye amphiploid triticale, containing 28 chromosomes of wheat-origin and 14 from rye, rDNA of rye-origin (on chromosome 1R) is not normally expressed, while the 1B and 6B origin rDNA from wheat show strong expression. Expression of rDNA can be accurately assessed by the silver staining method that stains both interphase nucleoli and metaphase rDNA sites that were actively expressed at the previous interphase. We show here that substitution of another rye chromosome, 2R, by a chromosome from hexaploid wheat, 2D, (triticale-2D(2R)) prevents suppression of the rye-origin rDNA, and leads to activity of all six major rDNA loci. These results were found in two different triticales and supported by rDNA behaviour in wheat-rye chromosomal addition lines. Models for chromosomal interactions leading to control of rDNA expression are presented. \journals\bacum.doc bacumtab

155. Pearce SR, Harrison G, Heslop-Harrison JS, Flavell AJ, Kumar A. 1997. Characterisation and genomic organisation of Ty1- copia group retrotransposons in rye ( Secale cereale ). Genome 40 : 617-625.

The genomic organisation of the Ty1 -copia retrotransposons in rye ( Secale cereale ) has been studied. We have used the polymerase chain reaction (PCR) to amplify sequences from a conserved domain of the reverse transcriptase gene of the Ty1 -copia retrotransposons in this plant. Sequence analysis of 26 of these PCR products shows them to be a highly heterogeneous population, a feature which is common in plants. Slot blot analysis shows there to be about 100,000 individual Ty1 -copia retrotransposons in rye. In situ hybridisation of a heterogeneous probe, representing the whole population of rye Ty1 -copia retrotransposon sequences, to chromosome spreads of triticale ( Triticosecale ) a rye-wheat hybrid shows that these sequences are present throughout all the rye chromosomes, but are absent from the centromeric regions and in particular from the terminal heterochromatin. Southern analysis of oat, barley, wheat and rye, using as a probe R9, one of the rye sequences which is closely similar to the BARE- 1 element of barley, shows that close relatives of this retrotransposon subgroup are present in all these species in high copy number. Northern analysis on RNAs from seedlings shows that the BARE- 1 subgroup is transcribed in somatic leaf tissue of all these cereal plants but at variable amounts; high in barley, moderate in wheat and rye and extremely low in oat.

154. Vershinin AV, Heslop-Harrison JS. 1998 . Comparative analysis of the nucleosomal structure of rye, wheat and their relatives . Plant Mol.Biol. 36 : 149-161.

The structure of bulk chromatin and separate chromosomal regions of some cereal species were compared using micrococcal nuclease (MNase) digestion and a set of DNA probes localized in the authentic telomeres, subtelomeric regions and bulk chromatin. We revealed discrepancies in the patterns of MNase kinetics between species, most extreme between rye and wheat. Rye bulk chromatin showed the fastest kinetics and rye telomeric repeat arrays were shortest, ranging from 8 kb to 50 kb; in contrast, wheat chromatin displayed the highest MNase resistance and longest telomeric repeat arrays from 15 kb up to 175 kb. The results suggest a higher sensitivity of rye chromatin, perhaps due to the higher content of hairpin structures which are more accessible for MNase cleavage. The telomeric regions of both species have shorter nucleosomal repeats than that of bulk chromatin and cleaved more quickly with MNase, implying an altered higher-order structure of telomeric chromatin. A strictly defined spacing arrangement (phasing) of nucleosomes was demonstrated along arrays of tandemly organized DNA sequences with different monomer lengths. Rye chromatin showed a gradient of sensitivity to MNase along the chromosomes. We propose that along with usual length of nucleosomal repeat, subtelomeric regions of rye chromosomes contain the nucleosomes with altered length corresponding to the shorter arrays of telomeric repeat.

153. Leitch AR, Maluszynska J, Leitch IJ, Anamthawat-J¢nsson K, Schwarzacher T, Heslop-Harrison JS. 1996. In situ hybridization of chromosomes using DNA probes labelled with digoxigenin and detected by epifluorescence. In: Jahier J, ed. Techniques of Plant Cytogenetics . Paris: INRA, 82-86+plate V.

152. Linde-Laursen I, Heslop-Harrison JS, Shepherd KW, Taketa S. 1997. The barley genome and its relationship with the wheat genomes: a survey with a recommendation for barley chromosome nomenclature. Hereditas 126 : 1-16.

The paper reviews recent literature on the cytology, cytogenetics, molecular cytogenetics and gene structure of barley, and on the relationship of the barley and wheat genomes. The information collected demonstrates largely similar genetic contents and collinearity of gene loci in the seven chromosomes of barley and in those of other members of the Triticeae. The high level of synteny indicates that the whole group can be considered as a single gene pool. On this basis the 7th International Barley Genetics Symposium recommended that (i) the currently used designations of the barley chromosomes (the BURNHAM and HAGBERG system) should become secondary to the use of the Triticeae system; i.e. each of the seven barley chromosomes is designated by a numeral between 1 and 7 according to its homoeologous relationship with the chromosomes of the wheat genomes followed by the genomic symbol H, e.g., 2H; (ii) the genomes of H. vulgare and H. bulbosum are symbolized with the letter H; and (iii) the arms of the barley chromosomes are designated by the capital letters S and L (cf. SINGH and TSUCHIYA 1982b). Further it was recommended that the barley genome present in the barley cultivar 'Betzes' should become the reference genome in the Triticeae.

151. Galasso I, Heslop-Harrison JS, Perrino P, Pingone D. 1997 . Locations and organization of major repetitive DNA sequence families in Vigna unguiculata (L.) Walp. In: Singh BB, Mohan Raj DR, Dashiell KE, Jackai LEN, eds. Advances in Cowpea Research . Ibadan, Nigeria: International Institute of Tropical Agriculture, 372-375.

150. Brandes A, Thompson H, Dean C, Heslop-Harrison JS. 1997. Multiple repetitive DNA sequences in the paracentromeric regions of Arabidopsis thaliana L. Chromosome Res. 5 : 238-246.

Nine repetitive DNA sequences present in the haploid Arabidopsis thaliana genome in 7 to 300 copies were hybridized in situ to metaphase and interphase chromosomes. Every sequence was detected on all five chromosome pairs, but was not evenly dispersed over the genome. Clusters of signals were found in particular regions of the centromeric heterochromatin and each sequence showed a characteristic distribution pattern. Some sequences hybridized more strongly on different chromosomes reflecting chromosome specific amplification or presence of homologous sequences. No hybridization signals could be detected on euchromatic regions. In situ hybridization on extended chromatin fibres showed that the pAL1 repeats are interrupted by another repetitive DNA sequence. A cosmid subclone (74A) contained a (GA) 38 microsatellite motif, and hybridization with a (GA) oligonucleotide revealed that most of the hybridization sites of 74A correspond to the distribution of this microsatellite motif. The results show that the paracentromeric heterochromatin of A. thaliana chromosomes is composed not only of the tandemly arranged 180 bp repeat family pAL1/pAtMr, but also of some other repetitive sequences, thus giving a better understanding of the organization of sequences at the centromeres of A. thaliana .

149. Lima-Brito JE, Guedes-Pinto H, Harrison GE, Heslop-Harrison JS. 1997. Molecular cytogenetic analysis of durum wheat x tritordeum hybrids. Genome 40 : 362-369.

Southern and in situ hybridization were used to examine the chromosome constitution, genome relationships, repetitive DNA sequences and nuclear architecture in durum wheat x tritordeum hybrids (2n=5x=35), where tritordeum is the fertile amphiploid (2n=4x=28) between Hordeum chilense and durum wheat. Using in situ hybridization, H. chilense total genomic DNA hybridized strongly to the H. chilense chromosomes and weakly to the wheat chromosomes, which showed some strongly labelled bands. pHcKB6, a cloned repetitive sequence isolated from H. chilense , enabled the unequivocal identification of each H. chilense chromosome at metaphase. Analysis of chromosome disposition in prophase nuclei, using the same probes, showed the H. chilense -origin chromosomes were in individual domains with only limited intermixing with wheat origin chromosomes. Six major sites of 18S-26S rDNA genes were detected on the chromosomes of the hybrids. Hybridization to Southern transfers of restriction enzyme digests using genomic DNA showed some variants of tandem repeats, perhaps due to methylation. Both techniques gave complementary information extending that available from phenotypic, chromosome morphology or isozyme analysis, and perhaps are useful to follow chromosomes or chromosome segments during further crossing of the lines in plant breeding programs. \journals\zeedthw.doc

148. Schondelmaier J, Schmidt T, Heslop-Harrison JS, Jung C. 1997. Genetic and chromosomal localization of the 5S rDNA locus in sugar beet ( Beta vulgaris L.). Genome 40 : 171-175.

147. Heslop-Harrison JS. (Ed) 1996. Unifying Plant Genomes. SEB Symposium 50 . Cambridge: Company of Biologists.

146. Schwarzacher T, Heslop-Harrison JS. 1996 & 1997. Fluorescent in situ hybridization. In: Sigma Chemical Catalog . St Louis: Sigma, 1424-1425. (1997 Catalog pages 1536-1537.)

An outline protocol for in situ hybridization using Sigma reagents and CY3 fluorochromes.

145. Anamthawat-J¢nsson K, Heslop-Harrison JS, Schwarzacher T. 1996. Genomic in situ hybridization for whole chromosome and genome analysis. In: Clark M, ed. In situ Hybridization: A Laboratory Companion . London: Chapman and Hall, 1-23.

144. Heslop-Harrison JS. 1996. Electron microscopy and plant chromosomes. In: Fukui K, Nakayama S, eds. Plant Chromosomes: Laboratory Methods . Boca Raton: CRC Press, pp. 219-239.

143. Brandes A, Heslop-Harrison JS, Kamm A, Kubis S, Doudrick RL, Schmidt T. 1997. Comparative analysis of the chromosomal and genomic organization of Ty1-copia -like retrotransposons in pteridophytes, gymnosperms and angiosperms . Plant Mol.Biol. 33: 11-21.

We have investigated the physical distribution of the reverse transcriptase genes of Ty 1 -copia -like retrotransposable elements from 12 plant species belonging to different subdivisions by hybridization in situ on chromosome preparations. Ty 1 -copia -like elements showed different and non-random hybridization patterns. In some species they were evenly distributed over most of the length of all chromosomes, whereas other species showed exclusion of retroelements at centromeric regions, nucleolus-organizing regions and regions known to be mainly composed of tandemly repeated sequences. In the fern Pteris cretica the retroelements showed a dispersed genomic organization with clustering at some chromosomal regions and whole chromosomes showing little signal. In Arabidopsis thaliana and Cicer arietinum, Ty 1 -copia -like elements were found in clusters at the paracentromeric heterochromatin, a novel organization for a repetitive element in A. thaliana . New retroelement families were isolated from A. thaliana and from Beta vulgaris . Alignment of the deduced peptide sequences with Ty 1 -copia -like elements from other plants showed considerable divergence which was used to calculate their relationships, indicating the value of reverse transcriptase gene analysis in phylogenetic and biodiversity studies.

142. Galasso I, Blanco A, Katsiotis A, Pignone D, Heslop-Harrison JS. 1997. Genome organization and phylogenetic relationships in the genus Dasypyrum (Triticeae, Poaceae) analysed by Southern and in situ hybridization of total genomic and cloned DNA probes. Chromosoma 106 : 53-61.

Molecular cytogenetic methods have been used to study the controversial phylogenetic relationships between the two species Dasypyrum villosum (L.) Candargy (2n=2x=14) and D. breviaristatum (Lindb. f.) Frederiksen (2n=4x=28). Using total genomic DNA from the species as probes for in situ hybridization to chromosomes, we found that the peri-centromeric regions of the chromosome arms of both species are similar, while distal regions show substantial differences. Two dispersed repetitive DNA sequences were isolated; pDbKB45 is distributed along the chromosomes but amplified in the sub-telomeric regions of D. breviaristatum chromosomes, while pDbKB49, in both species, is less amplified in terminal regions. Size separated restriction enzyme digests of DNA showed many repetitive fragments, but few in common between the two species. After probing Southern transfers with genomic DNA for D. breviaristatum , all tracks showed similar hybridization patterns although one extra small band was evident in the D. breviaristatum tracks. In contrast, probing with D. villosum showed very substantial differences between the two species. Genomic in situ hybridization to meiotic metaphases from an interspecific hybrid showed seven bivalents of D. breviaristatum origin and seven univalents from D. villosum . We also analysed the physical organization of 5S rDNA, 18S-25S rDNA and a tandemly repeated sequence from rye. Taken together, our data support the autotetraploid origin of D. breviaristatum , but its genomes and that of D. villosum show extensive differences, so the tetraploid is unlikely to be directly derived from D. villosum . \journals\dasy.doc

141 . Jones GH, Heslop-Harrison JS. 2000. Classical and molecular cytogenetics of Arabidopsis . In: Wilson ZA ed. Arabidopsis: A Practical Approach . Oxford: Oxford University Press, 105-124.

140. Katsiotis A, Hagidimitriou M, Heslop-Harrison JS. 1997. The close relationship between the A and B genomes in Avena L. determined by molecular cytogenetic analysis of total genomic, tandemly and dispersed repetitive DNA sequences. Annals of Botany 79 : 103-109.

The genus Avena L. (Poaceae) consists of diploid, tetraploid, and hexaploid species, with the B genome known only in tetraploid species and the D genome in the hexaploid species. DNA:DNA in situ hybridization, using total genomic DNA from diploid Avena strigosa Schreb. (A s genome) as a probe, labelled all 28 chromosomes of the AB tetraploid Avena vaviloviana (Malz.) Mordv. strongly and uniformly, revealing the close relationship between these two genomes. Comparison of patterns of size-separated DNA restriction fragments between the diploid A. strigosa and the tetraploid A. vaviloviana , using 32 different restriction enzymes, revealed no differences. Southern hybridization using total AB genomic DNA as a probe also gave no differences in banding patterns between the two genomes, even when a large excess of A genomic DNA was used as a block. From an A. vaviloviana genomic library, 1,800 colonies were blotted and probed sequentially with A and AB genomic DNA, but no colony was identified to be B genome specific. DNA digests of AB genome tetraploids with restriction enzyme Hae III gave a strong band at 4.2 kb. Clone pAbKB3, derived from the 4.2 kb band, was found to be part of a Ty1-copia -like retrotransposon present in A and B genome chromosomes. Cloned rRNA genes were used for in situ hybridization and showed that diploid A. strigosa has four major sites for 18S-25S rDNA and two pairs of sites for 5S rDNA (two pairs on the same satellited chromosome, on different chromosome arms), while 4 x A. vaviloviana has eight major sites for 18S-25S rDNA and four pairs of sites for 5S rDNA (four pairs on the same satellited chromosome, on different chromosome arms). A repetitive sequence from rye, pSc119.2, showed dispersed hybridization, while the telomeric sequence in clone pLT11 hybridized to telomeres. Again no discrimination was possible between A and B genome chromosomes. The molecular similarities between the diploid A. strigosa and the barbata group tetraploids clearly indicate that the barbata group of tetraploids arose from A s diploids through autotetraploidization. \journals\avena2.doc

139 . Kubis S, Heslop-Harrison JS, Schmidt T. 1997. A family of differentially amplified repetitive DNA sequences in the genus Beta reveals genetic variation in Beta vulgaris subspecies and cultivars. J. Mol. Evol. 44 : 310-320.

Members of a highly abundant restriction satellite family have been isolated from the wild beet species Beta nana . The satellite DNA sequence is characterized by a conserved Rsa I restriction site and is present in three of four sections of the genus Beta , namely Nanae , Corollinae and Beta . It was not detected in species of the evolutionary old section Procumbentes , suggesting its amplification after separation of this section. Sequences of eight monomers were aligned revealing a size variation from 209 - 233 bp and an AT-content ranging from 56.5% to 60.5%. The similarity between monomers in B. nana varied from 77.7% to 92.2%. Diverged subfamilies were identified by sequence analysis and Southern hybridization. A comparative study of this repetitive DNA element by fluorescent in situ hybridization and Southern analyses in three representative species was performed showing a variable genomic organization and heterogeneous localizations along metaphase chromosomes both within and between species. In B. nana the copy number of this satellite, with some 30000 per haploid genome, is more than 10-fold higher than in Beta lomatogona and up to 200 times higher than in Beta vulgaris , indicating different levels of sequence amplification during evolution in the genus Beta . In sugar beet ( B. vulgaris ), the large scale organization of this tandem repeat was examined by pulsed field gel electrophoresis. Southern hybridization to genomic DNA digested with Dra I demonstrated that satellite arrays are located in AT-rich regions and the tandem repeat is a useful probe for the detection of genetic variation in closely related B. vulgaris cultivars, accessions and subspecies. \papers\betanana.doc

138. Heslop-Harrison JS. 1996. Comparative analysis of plant genome architecture. In: Heslop-Harrison JS, ed. Unifying Plant Genomes: Comparison, Collinearity and Conservation . Cambridge: Company of Biologists, pp 17-23.

Many genes are similar in most plants and it is clear that the ordering of genes is highly conserved across wide taxonomic groupings. Repetitive DNA, consisting of sequence motifs between 2 and 10,000 base pairs long, repeated many hundreds or thousands of times in the genome, represents the majority of most plant genomes and defines some of the differences between species. Some sequences are highly conserved in many species, while other sequences show species or even chromosome specificity. Different types of sequences have markedly contrasting genomic distributions; even among tandem repeats, some are sub-terminal, some paracentromeric and others intercalary. The reasons for these different distributions are largely unknown, and mechanisms of homogenization, dispersion and amplifications are the subject of much speculation. Aspects of plant genome architecture - the organization of repetitive and single-copy DNA sequences along the chromosomes, and the positioning of those sequences within the nucleus at interphase - have important consequences for plant genetics. Models of large scale genome organization may be useful in learning the function of different components of the genome, in evolutionary studies and in plant breeding. \journals\seb.doc

137. Heslop-Harrison Y, Heslop-Harrison JS. 1996. Lodicule function and filament extension in the grasses: Potassium ion movement and tissue specialization. Ann.Bot. 77 : 573-582.

Flowering in the grasses has been the subject of a great deal of previous research, yet much remains to be learned concerning the role of environmental and endogenous factors in controlling the actual events of anthesis. The objective of this study has been to investigate further two key processes, namely lodicule function and stamen extension. The lodicules are the two diminutive bodies lying between the lemma and the ovary base in the grass floret which, by expanding rapidly at the time of anthesis, lever away the rigid lemma allowing anthers and stigmas to emerge. Expansion results from the swelling of a cushion of tissue at the base of each lodicule. We show that this is driven by the influx of water associated with the accumulation of K + in specialized distensible cells of the basal cushion. The stamen filaments normally elongate synchronously with the enlarging lodicules, again with the passage of K + into the extending cells. The walls of distensible cells of the lodicule bear lattice-like cellulose thickenings with transverse bars in an outer layer and longitudinal bars in an inner, a basket-like disposition allowing rapid expansion while preserving the integrity of the protoplast. The microfibrils in the walls of the cylindrical filament cells are disposed helically, constraining expansion to the longitudinal axis. The principal extraneous factors initiating lodicule function in mature florets of rye are mechanical disturbance and light. In the field radiant heat is unlikely to be a dominant factor. In a customarily cleistogamous cultivar of wheat, sporadic florets open chasmogamously in intense light, both lodicules and stamen filaments enlarging. In cleistogamous flowers in the same fluorescence the lodicules fail to enlarge while the filaments extend, suggesting that the influx of osmoticum into the two sets of organs is independently controlled.

136. Kosina R, Heslop-Harrison JS. 1996. Molecular cytogenetics of an amphiploid trigeneric hybrid between Triticum durum , Thinopyrum distichum and Lophopyrum elongatum . Ann.Bot. 78 : 583-589.

In situ hybridization of total genomic DNA was used to analyse lines derived from an amphiploid between tetraploid wheat, Triticum durum Desf. (2n=4x=28), and the wheatgrasses Thinopyrum distichum (Thunb.) A. Lve (2n=4x=28) and Lophopyrum elongatum (Host) A. Lve (2n=2x=14). A range of chromosome numbers was detected, arising from loss or gain of chromosomes. Total genomic DNA probes from Thinopyrum species, L. elongatum and Triticum monococcum L. were able to discriminate chromosomes from the A and B genomes of tetraploid wheat and those of wheatgrass-origin. The method did not discriminate the two wheatgrass genomes, J and E, indicating their close similarity. Chromosomal aberrations - including telocentric and ring chromosomes - were frequent. Distal inter-genomic translocations of parts of A and B genome chromosome arms, unusual in wheat itself, were more frequent than translocations between T. durum and wheatgrass. In situ hybridization of an rDNA probe most frequently revealed four sites associated with secondary constrictions on T. durum chromosomes and four on Th. distichum or L. elongatum chromosomes, although there was variation in the number of loci between and within plants. Within interphase and prophase nuclei, the three genomes were not intermixed and often lay in distinct sectors.

135. Heslop-Harrison JS. 1998. Cytogenetic Analysis of Arabidopsis. In: Martinez-Zapater JM, Salinas J, eds. Arabidopsis Protocols . Totowa, New Jersey: Humana Press. pp 119-127.

134. Schwarzacher T, Wang ML, Leitch AR, Miller N, Moore G, Heslop-Harrison JS. 1997. Flow cytometric analysis of the chromosomes and stability of a wheat cell culture line. Theor.Appl.Genet. 94 : 91-97.

A rapidly growing, long term suspension culture derived from Triticum aestivum L. (wheat) was synchronized using hydroxyurea and colchicine and a chromosome suspension with 2 to 3�x�10 6 chromosomes ml -1 was made. After staining with the DNA-specific fluorochromes Hoechst 33258 and Chromomycin A 3 , univariate and bivariate flow cytometry histograms showed fifteen clearly resolved peaks corresponding to individual chromosome types or groups of chromosomes with similar DNA contents. The flow karyotype was closely similar to a histogram of DNA content measurements of Feulgen-stained chromosomes made by microdensitometry. We were able to show the stability of the flow karyotype of the cell line over a year while a parallel subculture had a slightly different, stable, karyotype following different growth conditions. The data show that flow cytometric analysis of plant karyotypes enables accurate, statistically precise chromosome classification and karyotyping of cereals. There was little overlap between individual flow histogram peaks, so the method is useful for flow sorting and the construction of chromosome-specific recombinant DNA libraries. Using bivariate analysis, the AT:GC ratio of all the chromosomes was remarkably similar, in striking contrast to mammalian flow karyotypes. We speculate about a fundamental difference in organization and homogenization of DNA sequences between chromosomes within mammalian and plant genomes.

133 . Castilho A, Miller TE, Heslop-Harrison JS. 1997. Analysis of a set of homoeologous group 1 wheat- Aegilops umbellulata recombinant chromosome lines using genetic markers. Theor.Appl.Genet. 94 : 293-297.

Eleven wheat- Ae. umbellulata recombinant lines involving chromosome 1U, including an important high molecular weight gluentin locus, were characterized by protein and RFLP markers. Four 1U-1A recombinants, one 1U-1B recombinant, two 1U deletions with either nullisomy for chromosome 1A or 1B, and a 1U ditelosomic addition line were detected, while three recombinant lines involved 1U and non-homoeologous wheat chromosomes. Similar recombination events were found in independent lines, and no small segmental translocations of Ae. umbellulata chromatin were detected. Correlation of the markers with physical maps of the wheat- Ae. umbellulata breakpoints obtained using in situ hybridization enabled the marker order to be established on chromosomes 1A, 1B and 1U. The short arm of chromosome 1A probably differs from both 1U and 1B by one, different, inversion. As now being found to be universal in the Triticeae, clustering of the genetical map in the distal physical regions of the group 1 chromosomes was found.

132. Ribeiro-Carvalho C, Guedes-Pinto H, Harrison G, Heslop-Harrison JS. 1996. Wheat-rye chromosome translocations involving small terminal and intercalary rye chromosome segments in the Portuguese wheat landrace Barbela. Heredity 78 : 539-546.

131. Thompson H, Schmidt R, Brandes A, Heslop-Harrison JS, Dean C. 1996. A novel repetitive sequence associated with the centromeric regions of Arabidopsis thaliana chromosomes. Mol.Gen.Genet. 253 : 247-252.

The middle repetitive fraction of the Arabidopsis genome has been relatively poorly characterized. We describe here a novel repetitive sequence cloned in the plasmid mi167, and present in ~90 copies in the genome of Arabidopsis thaliana ecotype Columbia. Hybridization analysis to physically mapped YAC clones representing Arabidopsis chromosome 4 revealed four mi167-hybridizing loci, all clustered near the centromere. No other loci were detected on YAC clones covering chromosome 4. In situ hybridization experiments to Arabidopsis chromosome spreads showed that mi167-hybridizing sequences are clustered at the centromeric heterochromatin of all five chromosomes; on two chromosomes the hybridization appeared to be localised on one arm. Additional mi167 hybridizing loci were detected, one of which was adjacent to a non-centromeric, heterochromatic region. This work supports the idea that the majority of middle repetitive DNA in the Arabidopsis genome is clustered. It also adds to our understanding of the organization of the centromere of Arabidopsis chromosome 4.

130. Vershinin AV, Alkhimova EG, Heslop-Harrison JS. 1996. Molecular diversification of tandemly organized DNA sequences and heterochromatic chromosome regions in some Triticeae species. Chromosome Res. 4 : 517-525.

The subtelomeric heterochromatin of rye ( Secale cereale ) chromosomes makes up 12-18% of the genome and consists largely of a small number of tandemly organized DNA sequence families. The genomic organization, chromosomal locations and the structural organization of monomer units of the major DNA sequences from these regions were investigated and compared in other Triticeae species from the genera Secale, Agropyron, Dasypyrum, Triticum and Hordeum . Southern hybridization and PCR analysis established that all studied species preserve the tandem type of sequence organization but the copy number is altered drastically between species. In the pSc200 family, a fraction of the tandem arrays is present with a head-to-head orientation of dimers in S.cereale and S.montanum . Members of the same family are more heterogeneous and present as head-to-head monomers in the Dasypyrum species and A.cristatum. In situ hybridization demonstrates different organizations of the sequence families in the various species: pSc200 and pSc250 are are concentrated in major blocks at the ends of most rye chromosome arms, while they are more dispersed and in smaller blocks in Dasypyrum and Agropyron , indicating accumulation is not simply due to the sequence itself. In contrast to rye, D.villosum has major blocks of only pSc200 while D.breviaristatum shows greater amplification of pSc250. These data indicate that each repetitive family is an independent unit of evolution, and suggest that the two Dasypyrum species are not closely related. The data are discussed in terms of existing evolutionary models for repetitive DNA sequences. The contribution of random events, through molecular drive and selection, to the evolution of heterochromatic regions is considered.

129 . Schmidt T, Heslop-Harrison JS. 1996. The physical and genomic organization of microsatellites in sugar beet. Proc.Natl.Acad.Sci.USA 93 : 8761-8765.

128. Pearce SR, Pich U, Harrison G, Flavell AJ, Heslop-Harrison JS, Schubert I, Kumar A. 1996. The Ty1-copia group retrotransposons of Allium cepa are distributed throughout the chromosomes but are enriched in the telomeric heterochromatin. Chromosome Res. 4 : 357-364.

The genomic organization and diversity of the Ty1-copia group retrotransposons has been investigated in a monocotyledonous plant, Allium cepa . We used the polymerase chain reaction (PCR) to generate sequences corresponding to a conserved domain of the reverse transcriptase gene of Ty1-copia retrotransposons in this plant. Sequence analysis of 27 of these PCR products shows that they are a highly heterogeneous population, a feature which is common in plants but not in yeast and Drosophila . Slot-blot analysis shows there are 100 000-200 000 copies of Ty1-copia group retrotransposons within the A. cepa genome (2c=31.7pg), indicating that they are a significant component of the genome of this plant. In situ hybridization to metaphase chromosomes reveals that Ty1-copia retrotransposons are distributed throughout the euchromatin of all chromosomes of A. cepa but are enriched in the terminal heterochromatic regions, which contain tandem arrays of satellite sequences. This is the first clear evidence for the presence of Ty1-copia retrotransposons in the terminal heterochromatin of plants and contrasts with the distribution of these elements in other plant species.

127. Kamm A, Doudrick RL, Heslop-Harrison JS, Schmidt T. 1996. The genomic and physical organization of Ty1-copia -like sequences as a component of large genomes in Pinus elliottii var. elliottii and other gymnosperms. Proc.Natl.Acad.Sci.USA 93 : 2708-2713.

A DNA sequence, TPE 1, representing the internal domain of a Ty1-copia retroelement, was isolated from genomic DNA of Pinus elliottii Engelm. var. elliotti (slash pine). Genomic Southern analysis showed that this sequence, carrying partial reverse transcriptase and integrase gene sequences, is highly amplified within the genome of slash pine and part of a dispersed element >4.8kbp. Fluorescent in situ hybridization to metaphase chromosomes shows that the element is relatively uniformly dispersed over all 12 chromosome pairs and is highly abundant in the genome. It is largely excluded from centromeric regions and intercalary chromosomal sites representing the 18S-5.8S-25S rRNA genes. Southern hybridization with specific DNA probes for the reverse transcriptase gene shows that TPE 1 represents a large sub-group of heterogeneous Ty1-copia retrotransposons in Pinus species. Because no TPE 1 transcription could be detected, it is most likely an inactive element - at least in needle tissue. Further evidence for inactivity was found in recombinant reverse transcriptase and integrase sequences. The distribution of TPE 1 within different gymnosperms that contain Ty1-copia group retrotransposons, as shown by a PCR assay, was investigated by Southern hybridization. The TPE 1 family is highly amplified and conserved in all Pinus species analyzed, showing a similar genomic organization in the three-and five-needle pine species investigated. It is also present in spruce, bald cypress (swamp cypress), and in gingko but in fewer copies and a different genomic organization.

126. Heslop-Harrison JS. 1995. Chromosome function. Chromosome Res. 3(S1) : 85-86.

125 . Neves N, Castilho A, Silva M, Heslop-Harrison JS, Viegas W. 1996. Genomic interactions: gene expression, DNA methylation and nuclear architecture. Chromosomes Today 12 : 182-200 +plate 6a, 6b.

124. Schmidt T, Heslop-Harrison JS. 1996. High resolution mapping of repetitive DNA by in situ hybridization - molecular and chromosomal features of prominent dispersed and discretely localized DNA families from the wild beet species Beta procumbens . Plant Mol.Biol. 30 : 1099-1114.

Members of three prominent DNA families of Beta procumbens have been isolated as Sau 3A repeats. Two families consisting of repeats of about 158bp and 312bp are organized as satellite DNAs ( Sau 3A satellites I and II), whereas the third family with a repeat length of 202bp is interspersed throughout the genome. Multi-colour fluorescence in situ hybridization was used for physical mapping of the DNA families, and has shown that these tandemly organized families occur in large heterochromatic and DAPI positive blocks. The Sau 3A satellite I hybridized exclusively around or near the centromeres of 10, 11 or 12 chromosomes. The Sau 3A satellite family I showed high intraspecific variability and high-resolution physical mapping was performed on pachytene chromosomes using differently labelled repeats. The physical order of satellite subfamily arrays was visualized and provided evidence that large arrays of plant satellite repeats are not contigous and consist of distinct subfamily domains. Re-hybridization of a heterologous rRNA probe to mitotic metaphase chromosomes revealed that the 18S-5.8S-25S rRNA genes are located at subterminal position on one chromosome pair missing repeat clusters of the Sau 3A satellite family I. It is knowsn that arrays of Sau 3A satellite I repeats are tightly linked to a nematode ( Heterodera schachtii ) resistance gene and our results show that the gene might be located close to the centromere. Large arrays of the Sau 3A satellite II were found in centromeric regions of 16 chromosomes, and in addition, a considerable interspersion of repeats over all chromosomes was observed. The family of interspersed 202bp repeats is uniformly distributed over all chromosomes and largely excluded from rRNA gene cluster but shows local amplification in some regions. Southern hybridization has shown that all three families are specific for genomes of the section Procumbentes of the genus Beta .

123. Heslop-Harrison JS. 1995. Comparative analysis of plant genome architecture. In: Maluszynski M, ed. Induced mutations and molecular techniques for crop improvement . Vienna: International Atomic Energy Authority, 159-169.

It is clear that there are close, family-wide similarities between different crop species in both the genes (often with only allelic differences) and the gene order along chromosomes. However, there are extensive differences in both the types and organization of the repetitive DNA, even between related species, which may be of importance for genome changes and the exchange of genes in both long (evolutionary) and short (plant breeding) timescales. There is additional non-genic information in a genome, related to methylation and coiling of sequences, and to the three-dimensional organization of those sequences in the nucleus. Highly repetitive DNA makes up the majority of most plant genomes. Some sequences, such microsatellites, are similar in every organism, while others repeat units are specific to one species or a small group of species. Different sequences have characteristic genomic distribution, and most can be identified by their chromosomal distribution. Knowledge of genome architecture - both organization and nature of repetitive sequences, and the three dimensional organization in the interphase nucleus - is likely to be helpful for applied research and plant breeding. We have little knowledge of why repetitive sequences have particular characteristics. Is the three dimensional architecture of the nucleus important for function? Do repetitive sequences put coding or regulatory sequences in particular nuclear positions? Why are different sequences located in particular sites in the genome?

We are now constructing a comprehensive and quantitative model of variable and constant parts of the plant genome. Such integrated models of large scale genome organization may be useful in learning the function of different components of the genome, and in evolutionary studies. Since repetitive DNA changes are frequent, perhaps we can learn more about what manipulations are possible in plant genomes by examining the changes already made between related species during evolution or already during plant breeding programmes.

121. Lima-Brito J, Guedes-Pinto H, Harrison GE, Heslop-Harrison JS. 1995. Chromosome identification and nuclear architecture in Triticale x Tritordeum F1 hybrids. J.Exp.Bot. 47 : 583-588.

In situ hybridization with cloned, repetitive DNA probes and total genomic DNA enables the parental origin of all chromosomes to be established in metaphases of Triticale X Tritordeum F1 hybrids (2n=6x=42). Nuclei contain seven chromosomes of Hordeum chilense origin, seven from Secale cereale and 28 of wheat origin. When used as a probe, total genomic rye DNA labelled the rye chromosomes strongly and uniformly along their lengths, with brighter regions coincident with the terminal heterochromatin. The probe labelled the wheat-origin chromosomes weakly and was almost undetectable on the H. chilense origin chromosomes. In contrast, under the same conditions, H. chilense DNA hybridized strongly to the H. chilense and with intermediate strength to the S. cereale origin chromosomes, excluding the subtelomeric heterochromatin: it hybridized only weakly to the wheat chromosomes, in some experiments revealing characteristic bands on wheat chromosomes. Cloned repetitive DNA probes from rye and H. chilense were used as probes to identify the linkage groups of all of their own-species chromosomes. Analysis of hybridization patterns of various probes to prophase and interphase nuclei indicated that there are many non-random features in the localization of both repetitive DNA and whole chromosomes, although general patterns of nuclear organization have yet to emerge. Both the particular lines used and the techniques developed here are likely to be valuable for production and characterization of plant breeding material.

122. Lima-Brito J, Guedes-Pinto H, Heslop-Harrison JS. 1996. Molecular cytogenetic analysis of Triticale x Tritordeum F1 hybrids. In: Triticale: Today and Tomorrow . Eds. Guedes-Pinto H, Darvey N, Carnide VP. Amsterdam: Klewer, 183-188.

120. Vershinin A, Schwarzacher T, Heslop-Harrison JS. 1995. The large scale genomic organization of repetitive DNA families at the telomeres of rye chromosomes. Plant Cell 7 : 1823-1833.

Repetitive DNA sequences in the terminal heterochromatin of rye ( Secale cereale ) chromosomes have consequences for the structural and functional organization of chromosomes. The large-scale genomic organization of these regions was studied using the telomeric repeat from Arabidopsis and clones of three nonhomologous, tandemly repeated, subtelomeric DNA families with complex but contrasting higher order structural organizations. Polymerase chain reaction analysis with a single primer shows a fraction of the repeat units of one family are organized in a "head-to-head" orientation. Such structures suggests evolution of chromosomes by chromatid-type breakage-fusion-bridge cycles. In situ hybridization and pulse field gel electrophoresis showed the order of the repeats and the heterogeneity in the lengths of individual arrays. After Xba I digestion and pulse field gel electrophoresis, all three subtelomeric clones showed strong hybridization signal from 40 to 100 kb with a maximum at 50-60 kb. We suggest these fragments define a basic higher-order structure and DNA loop-domains of regions of rye chromosomes consisting of arrays of tandemly organized sequences.

119. Heslop-Harrison JS. 1995. Flow cytometry and genome analysis. Probe 5(1) : 14-17.

118. Castilho A, Miller TE, Heslop-Harrison JS. 1996. Physical mapping of translocation breakpoints in a set of wheat- Aegilops umbellulata recombinant lines using in situ hybridization. Theor. Appl. Genet. 93 : 816-825. DOI: 10.1007/BF00224081

Aegilops umbellulata Zhuk. carries genes at Glu-U1 loci that code for a pair of high molecular weight glutenin subunits not found in common wheat, Triticum aestivum . Wheat- Ae. umbellulata recombinant lines were produced with the aim of transferring genes coding for glutenin subunits from Ae. umbellulata into wheat with minimal flanking material. We used fluorescent genomic in situ hybridization to evaluate the extent of recombination and to map physically the translocation breakpoints on 11 wheat- Ae. umbellulata recombinant lines. In situ hybridization was able to identify alien material in wheat and showed breakpoints not only near the centromeres but also along chromosome arms. To characterize and identify chromosomes further, including deletions along the 1U chromosome, we used simultaneous multiple target in situ hybridization to localize a tandemly repeated DNA sequence (pSc119.2) and the 18S-25S and 5S rRNA genes. One line contained an Ae. umbellulata telocentric chromosome and another two had different terminal deletions, mostly with some wheat chromosome rearrangements. Although from six independent original crosses, the other eight lines included only two types of intercalary wheat- Ae. umbellulata recombination events. Five occurred at the 5S rRNA genes on the short arm of the Ae. umbellulata chromosome with a distal wheat-origin segment, and three breakpoints were proximal to the centromere in the long arm, so most of the long arm was of Ae. umbellulata -origin. The results allow characterization of recombination events in the context of the karyotype. They enable design of crossing programmes to generate lines where smaller Ae. umbellulata chromosome segments are transferred to wheat with the potential to improve breadmaking quality by incorporating novel glutenin subunits without undesirable linked genes.

117. Pearce SR, Harrison G, Wilkinson M, Li D, Heslop-Harrison JS, Flavell AJ, Kumar A. 1995. The Ty1-copia group retrotransposons in Vicia species: copy number, sequence heterogeneity and chromosomal localisation. Mol.Gen.Genet. 250 : 305-315.

We present an in-depth study of the Ty1-copia group of retrotransposons within the plant genus Vicia , which contains species with widely differing genome sizes. We have compared the numbers and sequence heterogeneities of these genetic elements in three diploid Vicia species chosen to represent large ( Vicia faba , 1C=13.3pg), medium ( V. melanops , 1C=11.5pg) and small ( Vicia sativa , 1C=2.3pg) genomes within the genus. The copy numbers of the transposons are all high but vary greatly, with V. faba containing approximately 1,000,000 copies, V. melanops with about 1000 copies and V. sativa 5000 copies. The degree of sequence heterogeneity of Ty1-copia group elements correlates with their copy number within each genome, but neither heterogeneity or copy number are related to the genome size of the host. In situ hybridisation to metaphase chromosomes shows that the retrotransposons in V. faba are distributed throughout all chromosomes but are much less abundant in certain heterochromatic regions. These results are discussed in the context of plant retrotranposon evolution.

116. Galasso I, Schmidt T, Pignone D, Heslop-Harrison JS. 1995. The molecular cytogenetics of Vigna unguiculata (L.) Walp: the physical organization and characterization of 18S-5.8S-25S rRNA genes, 5S rRNA genes, telomere-like sequences, and a family of centromeric repetitive DNA sequences. Theor. Appl. Genet. 91 : 928-935.

A knowledge of genome organization is important for understanding how genomes function and evolve, and provide information likely to be useful in plant breeding programmes involving hybridization and genetic manipulation. Molecular techniques, including in situ hybridization, molecular cloning and DNA sequencing, are proving valuable tools to investigate the structure, organization, and diversity of chromosomes in agricultural crops. Heterologous labelled 18S-5.8S-25S (pTa71) and 5S rDNAs (pTa794) were used for in situ hybridization on Vigna unguiculata (L.) Walp. chromosomes. Hybridization with 18S-5.8S-25S rRNA gene probes occurred at the same chromosomal sites which were positive to the CMA fluorochrome. Silver staining of nucleolar-organizing regions indicated that all the rDNA sites detected using the 18S-5.8S-25S rRNA gene probe possessed active genes. Degenerate telomeric repeats gave hybridization signals at the telomeres of most chromosomes and no intercalary sites were detected at metaphase; the sequences appear to have not preferential distribution in interphase nuclei. A repetitive Dra I family from V. unguiculata was cloned (pVuKB1) and characterized. The Dra I repeat is 488 nucleotides long. AT rich (74%), and hybridized on all chromosomes in the centromeric areas. The presence of this sequence family was investigated by Southern hybridization in different Vigna species and other Leguminoseae . It was only detected in V. unguiculata , and hence represents a species-specific DNA sequence.

115. Castilho A, Heslop-Harrison JS. 1995. Analysis of plant genomes by in situ hybridization. In: Coulton GR, de Belleroche J, eds. In situ hybridization: Handbooks on Laboratory Methods . Heidelberg: Springer, in press.

114. Schwarzacher T, Heslop-Harrison JS. 1995. Molecular cytogenetic investigations of meiosis. In: Brandham PE, Bennett MD eds Kew Chromosome Conference IV . 407-416+xl.

113. Cuadrado A, Jouve N, Heslop-Harrison JS. 1995. Physical mapping of the 5S rRNA multigene family in 6x triticale and rye: identification of a new rye locus. Genome 38 : 623-626.

112. Katsiotis A, Schmidt T, Heslop-Harrison JS. 1995 . Chromosomal and genomic organization of Ty1-copia -like retrotransposon sequences in the genus Avena . Genome 39 : 410-417.

A cloned repetitive sequence, pAvKB30, obtained from an Avena vaviloviana (AB- genome) genomic library, along with two polymerase chain reaction (PCR) products, derived from primers of the conserved region of the reverse transcriptase (RT) gene of retrotransposons, were characterized molecularly and cytologically. The cloned DNA fragment was a dispersed repeat present in all Avena species used in this study ( A. strigosa, A. clauda, A. vaviloviana, A. magna, and A. sativa ). The fragment was sequenced (210bp) and found to be 69.5 percent homologous to part of WIS-2-1A, and 60.5 percent homologous to the leader sequence of BARE-1; both of these elements have been characterized as Ty1-copia like retrotransposons, in wheat and barley, respectively. In situ hybridization of pAvKB30 to diploid, tetraploid, and hexaploid oat species, revealed that the probe is present on both arms of all chromosomes (A-, B-, C-, and D- genomes), but excluded from their centromeric and nucleolar organizer regions. By using double in situ hybridization in hexaploid A. sativa (ACD-genome) pAvKB30 was found to be present in lower copy numbers in C-genome chromosomes compared to A- and D-genome chromosomes. Furthermore, under low stringency conditions, pAvKB30 hybridized on Southern blots containing barley, wheat, rye and Arrhenatherum DNA, which is considered the most closely related genus to Avena . However, under high stringency conditions, it hybridized only on Arrhenatherum DNA. All Avena species included in this study yielded a PCR product when the primers from the RT domain of retrotransposons were used. Two products rtA , obtained by using A. strigosa (A s -genome) as template, and rt C, obtained by using A. clauda (C p -genome) as template, gave similar Southern and in situ hybridization results to pAvKB30, but each was more abundant in its genome-of origin.

111. Orgaard M, Jacobsen N, Heslop-Harrison JS. 1995. Molecular cytogenetics in the genus Crocus . In: Brandham PE, ed. Kew Chromosome Conference IV . 291-299+xxxv.

110. Schmidt T, Kubis S, Heslop-Harrison JS. 1995. Analysis and chromosomal localization of retrotransposons in sugar beet ( Beta vulgaris L.): LINEs and Ty1-copia -like elements as major components of the genome. Chromosome Res. 3 : 335-345.

DNA sequences of the reverse transcriptase gene of LTR and non-LTR (non-viral) retrotransposons have been isolated and cloned from the genome of sugar beet ( Beta vulgaris ). Both retrotransposon types are highly amplified in sugar beet and may account for 2-5% of the genome. The BNR1 family, representing the first non-viral retrotransposon reported from a dicotyledonous species, shows homology to the mammalian L1 family of long interspersed repeated sequences (LINE) and to retrotransposable elements from maize and lily. Sequences of the Tbv family are homologous to the Ty1-copia class of LTR retrotransposons. The BNR1 and Tbv retrotransposon families are characterized by sequence heterogenity and are probably defective. The deduced peptide sequences were used to investigate the relation to other retroelements from plants, insects and mammals. Fluorescence in situ hybridization was used to investigate the physical distribution and revealed that both retrotransposon families are present on all sugar beet chromosomes and largely excluded from chromosomal regions habouring the 18S-5.8S-25S rRNA genes. The BNR1 family is organized in discrete clusters while the Tbv family of Ty1-copia-like retrotransposons shows a more uniform distribution along chromosome arms and is absent from some chromosomal regions. These contrasting distributions emphasize the differences in evolutionary amplification and dispersion mechanisms between the two types of retrotransposons. The in situ results of both elements reflect significant features of a higher order structure of the genome, as it is known for both SINEs and LINEs in human.

109. Anamthawat-Jonsson K, Heslop-Harrison JS. 1995. Establing relationships between closely related species using total genomic DNA as a probe. In: Clapp JP, ed. Methods in Molecular Biology - Species Identification Protocols . Totowa, New Jersey: Humana Press, 209-225.

108. Heslop-Harrison JS, Schwarzacher T. 1996. Genomic Southern and in situ hybridization. In: Jauhar PP, ed. Methods of Genome Analysis in Plants . Boca Raton: CRC, 163-179.

107. Gazdova B, Siroky J, Fajkus J, Brzobohaty B, Kenton A, Parokonny A, Heslop-Harrison JS, Palme K, Bezdek M. 1995. Characterization of a new family of tobacco highly repetitive DNA, GRS, specific for the N. tomentosiformis genomic component. Chromosome Res. 3 : 245-254.

Members of a new family of highly repetitive DNA sequences called GRS were isolated from Nicotiana tabacum L. genomic DNA and characterized. Cloned, sequenced monomeric units (180-182bp) of GRS exhibit properties characteristic of molecules that possess a stable curvature. The GRS family represents about 0.15% of total genomic DNA (10 4 copies per haploid genome) and could be derived from either Nicotiana tomentosiformis or Nicotiana otophora , two possible ancestors of the T genome of the amphidiploid N. tabacum . Sequence homology between the HRS60 (Koukalova et al. 1989) and the GRS family has been estimated to be 57%. In situ hybridization was used to localize GRS on mitotic chromosomes. Hybridization signals were obtained on five pairs of chromosomes at intercalary sites of the longer chromosome arms. The majority of GRS sequences appeared to be organized in tandem arrays and a minority were found to be dispersed through the genome in short clusters, interspersed with other types of DNA repeats, including 25S rDNA sequences. Several loci containing both GRS and HRS60 were also found. Such hybrid loci may indicate intergenomic transfer of the DNA in the amphidiploid N. tabacum . GRS sequences, like HRS50 (Fajkus et al. 1992), were found to specify the location of nucleosomes. The position of the nucleosome core has been mapped with respect to a conserved Mbo l site in the GRS sequence and an oligo A/T tract is a major centre of the DNA curvature.

106. Schwarzacher T, Mann S, Heslop-Harrison JS. 1995. Fluorescent in situ hybridization (FISH) using Cy3. Sigma ImmuNotes 13 : 1-3.

105. Castilho A, Heslop-Harrison JS. 1995. Physical mapping of 5S and 18S-25S rDNA and repetitive DNA sequences in Aegilops umbellulata. Genome 38: 91-96.

An accurate physical map of the location of the 5S and the 18S-5.8S-25S rRNA genes and a repetitive DNA sequence has been produced on Aegilops umbellulata Zhuk., (2 n = 2 x = 14) chromosomes by in situ hybridization. Chromosome morphology together with the hybridization pattern of pSc119.2, a DNA sequence from rye, allowed identification and discrimination of different chromosomes; pSc119.2 hybridizes with all Ae. umbellulata chromosomes at the telomeres, except for the short arm of chromosome 6U, and shows intercalary sites on the long arms of chromosomes 6U and 7U. The 5S and 18S-25S rDNA have been mapped physically only on the short arms of chromosomes 1U and 5U. On chromosome 1U the order of the genes is 5S rDNA subterminal and 18S-25S rDNA more proximal, while on chromosome 5U the position of the genes is reversed. The relative order of the genes, together with the hybridization pattern of the pSc119.2, is useful in identifying whole chromosomes or chromosome segments from Ae. umbellulata in recombinant or addition lines with wheat. The data help link the physical organization of chromosomes to the genetic map. Other members of the Triticeae vary in the prescence and order of the 5S and 18S-25S rDNA sequences on groups 1 and 5, indicating multiple and complex evolutionary rearrangements of the chromosome arms.

104. Castilho A, Heslop-Harrison JS. 1995. Molecular cytogenetics of wheat- Aegilops umbellulata recombinant lines. In: Wang RRC, Jensen KB, Jaussi C, eds. Proceedings of the 2nd International Triticeae Conference (June 1994) . Logan, Utah: Utah State University, 150-154.

103. Orgaard M, Jacobsen N, Heslop-Harrison JS. 1995. The hybrid origin of two cultivars of Crocus (Iridaceae) analysed by molecular cytogenetics including genomic Southern and in situ hybridization. Ann.Bot. 76 : 253-262.

102. Heslop-Harrison JS, Schwarzacher T. 1995. Molecular cytogenetics and the wheat genome. In: Tsunewaki K, ed. Plant Genome and Plastome: Their Structure and Evolution . Tokyo: Kodansha, 53-60+ix.

The methods of molecular cytogenetics are combining information about the molecular organization and structure of the genome of wheat (and its wild or cultivated relatives) with data about the chromosomes and their genetics. Genomic DNA:DNA in situ hybridization using total genome DNA as a probe to chromosome is proving particularly useful to paint chromosomes originating from different genomes in hybrids, or to detect alien chromosomes in a wheat background. Both the numbers and morphology of alien chromosomes can be detected, and the method gives considerable information about the distribution, structure and relationships of repetitive DNA elements in the different genomes. The use of defined DNA probes for in situ hybridization is enabling the physical locations of particular DNA sequences to be discovered along chromosomes. The generation of an integrated genetical and physical map of the wheat genome will be useful for understanding the structure of the genome and localization of recombination, and may also help target generation of new recombinant and other genetic stocks for plant breeding programs.

101. Heslop-Harrison JS. 1995. 50th SEB Symposium, Unifying Plant Genomes. Programme and Abstracts . London: Society for Experimental Biology.

100. Heslop-Harrison JS, Flavell RB. 1994. The Chromosome. ISBN 1 872748 64 3 (Paperback edition) . Ed. 2 Oxford: Bios.

99. Heslop-Harrison JS, Schwarzacher T. 1996. Flow cytometry and chromosome sorting. In: Fukui K, Nakayama S, eds. Plant Chromosomes: Laboratory Methods . Boca Raton: CRC Press. pp. 85-108.

98. Kamm A, Galasso I, Schmidt T, Heslop-Harrison JS. 1995. Analysis of a repetitive DNA family from Arabidopsis arenosa and relationships between Arabidopsis species. Plant Molecular Biology 27; 853-862.

We have analysed a family of highly repetitive DNA from Arabidopsis arenosa (L.) Lawalree [syn. Cardaminopsis arenosa (L.) Hayck] composed of AT-rich tandem repeats of 166-179 bp in head to tail organization. Sequence comparison between several repeat units revealed a high level of divergence of 4.5% to 25%. The sequence family shows more than 58% homology to satellite sequences described in Arabidopsis thaliana (L.) Heynh. but no homology to other satellite repeats in the Cruciferae. Within the genus Arabidopsis the satellite sequences was found to be present in A. Thaliana and Arabidopsis suecica (Fries) Norrlin, but not in Arabidopsis griffithiana (Boiss.) N. Busch and Arabidopsis pumila (Stephan) N. Busch. In situ hybridization to metaphase chromosomes of A. arenosa (2 n =4 x =32) showed the sequence to be localized at the centromeres of all 32 chromosomes with substantial hybridization along the chromosome arms. Using Southern hybridization and in situ hybridization, we give evidence that A. suecica is a hybrid of A. thaliana and A. arenosa. A considerable reorganization of the A. thaliana satellite sequence pAL1 occurred in the hybrid genome while no molecular change of the A. arenosa repeat was observed in the hybrid. Analysis of related repeats enabled differentiation between closely related genomes and are useful for the investigation of hybrid genomes.

97. Harrison GE, Heslop-Harrison JS. 1995. Centromeric repetitive DNA in the genus Brassica . Theor. Appl. Genet. 90 : 157-165. DOI: 10.1007/BF00222197

96. Doudrick RL, Heslop-Harrison JS, Nelson CD, Schmidt T, Nance WL, Schwarzacher T. 1995. Karyotype of Slash Pine ( Pinus elliottii var. elliottii ) using patterns of fluorescence in situ hybridization and fluorochrome banding. J.Heredity 86: 289-296.

A karyotype and idiogram were prepared for slash pine ( Pinus elliotti Engelm. var. elliotti ;2n=2x=24) using mitotic metaphase cells from root-rips of seedlings; each metaphase had 11 pairs of long metacentric chromosomes and one shorter pair of submetacrentric chromosomes. Fluorescent in situ hybridization showed seven pairs of chromosomes with intercalary sites for genes of 18S-5.8S-25S rRNA (18S-25S rDNA) and one pair with a paracentromeric site. Probes of 5S rDNA localized a major site of hybridization on another pair of chromosomes, whereas two minor sites occurred on other chromosomes, one with and one without a site of 18S-25S rDNA. After in situ hybridization, chromosomes were stained with the GC-base-specific fluorochrome chromomycin A 3 (CMA). Positive bands at intercalary sites were detected only on two pairs of chromosomes, paracentromeric sites only on three pairs of chromosomes, and bands at both regions on four pairs. Many bands of CMA showed at sites of hybridization of 18S-25S rDNA but one major band occured at a centromere without an rDNA site. After staining with 4',6-diamidino-2-phenylindole (DAPI), bands appeared at AT-rich intercalary and/or centromeric regions of nearly all chromosomes. A characteristic double band of DAPI occured near the centromere of one pair of chromosomes. Patterns of fluorescence in situ hybridization and flurochrome banding allowed us to identify all 12 pairs of chromosomes and to establish a standard karyotype. We propose that the numbers assigned to the chromosomes be used for homologous group designations in slash pine and as the framework for homoeologous group designations of chromosomes in other species of pine and perhaps other genera of conifers.

95. Neves N, Heslop-Harrison JS, Viegas W. 1995. rRNA gene activity and control of expression mediated by methylation and imprinting during embryo development in wheat x rye hybrids. Theor.Appl.Genet. 91 : 529-533.

94. Heslop-Harrison JS. 1995. Molecular cytogenetics and the organization of plant chromosomes. In: Berville A, Tersac M, eds. Techniques and Utilisation of Molecular Markers . Paris: INRA. 107-115.

92. Heslop-Harrison JS, Schwarzacher T. 1991. The ultrastructure of Arabidopsis thaliana chromosomes. In: Schweizer D, Peuker K, Loidl J, eds. Fourth International Conference on Arabidopsis Research . Vienna: University of Vienna, 3.

91. Schmidt T, Schwarzacher T, Heslop-Harrison JS. 1994. Physical mapping of rRNA genes by fluorescent in situ hybridization and structural analysis of 5S rRNA genes and intergenic spacer sequences in sugar beet ( Beta vulgaris ). Theor.Appl.Genet. 88 : 629-636.

A digoxigenin-labelled 5S rDNA probe (pTa794) and a rhodamine-labelled 18S-5.8S-25S rDNA probe (pTa71) were used for double-target in situ hybridization to root-tip metaphase, prophase and interphase chromosomes of cultivated beet, Beta vulgaris L. After in situ hybridization with the 18S-5.8S-25S rDNA probe, one major pair of sites was detected which corresponded to the secondary constriction at the end of the short arm of chromosome 1. The two rDNA chromosomes were often associated and the loci only contracted late in metaphase. In the majority of the metaphase plates analyzed, we found a single additional minor hybridization site with pTa71. One pair of 5S rRNA gene clusters was localized near the centromere on the short arm of one of the three largest chromosomes which does not carry the 18S-5.8S-25S genes. Because of the difficulties in distinguishing the very similarly sized B.�vulgaris chromosomes in metaphase preparations, the 5S and the 18S-5.8S-25S rRNA genes can be used as markers for chromosome identification. Two Xba I fragments (pXV1 and pXV2), comprising the 5S ribosomal RNA gene and the adjacent intergenic spacer, were isolated. The two 5S rDNA repeats were 349 bp and 351 bp long, showing considerable sequence variation in the intergenic spacer. The use of fluorescent in situ hybridization complemented by molecular data for gene mapping and for integrating genetic and physical maps of beet species is discussed.

90. Anamthawat-J¢nsson K, Heslop-Harrison JS. 1995. Molecular cytogenetics of Icelandic birch species: physical mapping by in situ hybridization and rDNA polymorphism. Can.J.Forest Res. 25 : 101-108.

The physical mapping of genes can reveal the organization of a genome and identify relationships of plant species, especially where they are involved in interspecific hybridization and polyploidy. Here we determine the chromosomal locations of th major ribosomal gene family (18S-5.8S-26S) by fluorescent in situ hybridization in two Icelandic birch species, Betula pubescens Ehrh. and Betula nana L. In the tetraploid birch ( B. pubescens ), the rDNA was localized on four major and two minor sites, while the diploid dwarf birch ( B. nana ) had four major sites. The major loci in both species were in nucleolus organizer regions, close to the centromeres of a pair of metacentric and a pair of sub-metacentric chromosomes. The dispersed interphase in situ hybridization pattern showed gene expression at all major sites. The two additional loci in B. pubescens , when detected, appeared to be sub-telomeric and inactive at interphase. Southern analysis of rDNA showed considerable restriction fragment length polymorphism in B. pubescens . Some polymorphism may reflect gene flow among populationsand between two co-existing birch species. The understanding of genome relationships, gene introgression, and evolution of birch species will be important to the breeding programmes steered towards environmental conservation and forestry.

89. Kamm A, Schmidt T, Heslop-Harrison JS. 1994. Molecular and physical organization of highly repetitive undermethylated DNA from Pennisetum glaucum . Mol.Gen.Genet. 244 : 420-425.

A Hae III monomer of a repetitive DNA family from Pennisetum glaucum (L.) R. Br. cv. Massue has been cloned and characterized. The repeat is 137 bp long and organized in head to tail orientation in tandem arrays. The Hae III monomer contains 55% A+T residues. The distribution of this highly repetitive sequence in different Pennisetum species and in other cereals was investigated and showed that the Hae III satellite is present in all Pennisetum species investigated but absent from other genera examined. In situ hybridization revealed a centromeric localization of this sequence on all seven chromosome pairs and indicated chromosome-specific differences in copy number. Methylation was investigated by comparative restriction enzyme analysis (MspI/HpaII) which showed a greater extent of methylation of the internal C of the enzyme recognition site 5'-CCGG. A South-Western analysis, using an anti-methylcytosine antibody to examine the methylation status in P. glaucum confirmed that the sequence is not highly methylated.

88. L¢pez-Le¢n MD, Neves N, Schwarzacher T, Heslop-Harrison JS, Hewitt GM, Camacho JPM. 1994. Possible origin of a B chromosome deduced from its DNA composition using double FISH technique. Chromosome Res. 2 : 87-92

Double fluorescent in situ hybridization (FISH) with two DNA probes (a 180 bp tandemly repeated DNA and ribosomal DNA) was performed in embryo cells of the grasshopper Eyprepocnemis plorans . Repetitive DNA was present in most standard chromosomes (excepting 7,8 and 10) and in the proximal two-thirds of the B chromosome, which was its major location in the complement. Ribosomal DNA was present distallyon the B, and in the active nucleolar organizer regions (NORs) of the X, 9, 10 and 11 chromosomes. A small number of rRNA gene clusters was also observed in the pericentromeric regions of chromosomes1-8. The double FISH technique showed that the B chromosome (B 2 ) is mainly composed of a 180 bp tandem repeat and ribosomal DNA, the minute short arm being the only region that does not hybridize with them. The location and order of the centromere and both the DNA sequences on the B chromosome coincide only with those in the X chromosome, indicating that the B most probably derives from the X.

87. Leitch AR, Brown JKM, Mosgller W, Schwarzacher T, Heslop-Harrison JS. 1994. The spatial localization of homologous chromosomes in human fibroblasts at mitosis. Hum.Genet. 93 : 275-280.

Chromosomes from ten human male fibroblast metaphases were completely reconstructed from electron micrographs of serially sectioned material. Chromosome centromere positions were determined by finding the three-dimensional coordinates of the centromere mid-point. The data set showed the identity of nine chromosome types (chromosome 1, 2, 3, 6, 9, 16, 17, 18 and the Y chromosome) preserved as they are positioned in vivo. The results indicate that there is (1) no significant association of the homologous chromosomes examined, (2) a significant tendency for a central location of the Y chromosome and of chromosome 18, (3) a significant tendency for a peripheral location of chromosome 6, (4) no significant tendency for homologous chromosomes to reorganize as metaphase advances and (5) no significant differential condensation across the metaphase plate. Therefore, the only organization pattern observed for the centromeres of the homologous chromosomes studied is some sorting by size across the metaphase plate. These results may be typical of dividing cell types. Different chromosome arrangements are found in some non-dividing cell types (e.g. mammalian brain cells). The different distributions of chromosomes in different cell types can be considered as forms of "nuclear differentiation". It is postulated that nuclear differentiation may be related to cell differentiation.

86. Schmidt T, Heslop-Harrison JS. 1994. Variability and evolution of highly repeated DNA sequences in the genus Beta . Genome 36 : 1074-1079.

Satellite DNA from wild beet species was separated from restriction endonuclease digested genomic DNA by polyacrylamide gel electrophoresis. Two non-homologous Hae III satellite DNA repeats were cloned from the wild beet Beta trigyna . The typeI repeat is 140 - 149 bp long and AT-rich, while the typeII is 162bp in size and GC-rich. A third repetitive Hae III element cloned from the related wild beet B. corolliflora was shown to be organized as a Hin fI satellite DNA family in the cultivated beet B. vulgaris ssp.vulgaris and the wild beet B. vulgaris ssp.maritima. This typeIII satellite monomer is 149 bp long and contains a high number of short direct subrepeats. The monomer was found in different genomic organizations and copy numbers in all sections of the genus Beta indicating an amplification early in the phylogeny. The Hae III repeats from B. trigyna are characterized by a lower variability and form long tandem arrays in the genomes of Corollinae species. The investigation of the distribution of all three sequence families provided data which may contribute to the solution of taxonomic problems of the genus Beta and be useful in the characterization of hybrids and derived lines with alien wild beet chromosomes.

85. Heslop-Harrison JS. 1994. Mechanical interactions with plant cells: a selective overview. In: Lyall F, El Haj AJ, eds. Biomechanics and Cells: SEB Seminar Series 54 . Cambridge: Cambridge University Press, 107-120.

84. Heslop-Harrison JS, Maluszynska J. 1994. The molecular cytogenetics of Arabidopsis. In: Meyerowitz EM, Sommerville CR, eds. Arabidopsis . Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 63-87.

83. Chen DF, Dale PJ, Heslop-Harrison JS, Snape JW, Harwood W, Bean S, Mullineaux PM. 1994. Stability of transgenes and presence of N6 methyladenine DNA in transformed wheat cells. Plant J. 5 : 429-436.

A number of stably transformed wheat cell lines were obtained using two different direct gene transfer techniques. When integration of the foreign genes was investigated in DNA samples taken at 10 months postselection, complicated profiles of transgene bands were observed in Southern blot analysis. Among these, a number of common bands were identified showing similar hybridization patterns between independently transformed cell lines. This type of hybridization pattern has been a common observation and is usually interpreted as concatameric rearrangement of intergrated DNA. However, when integration was reinvestigated with DNA samples taken at 30 months postselection, the hybridization pattern changed and most of the common band had disappeared. Further analysis using a set of methylation-sensitive enzymes revealed that the DNA represented by the common bands was N 6 -adenine methylated ( m6 A DNA), and there was even m6 A DNA in some 30 month samples. Although the source of m6 A DNA in the wheat cultures was not clearly established, the data indicate that transformation of an endophyte (e.g. a mycoplasma-like organism) may have occurred at the same time as the transformation of wheat cells. The integration pattern of undermethylated transgenes in the transformed cells became simpler and clearer after treating the DNA preparations with Dpn l, which only cuts m6 A DNA. The implications of these data for other methods of inoculating cereals with DNA are discussed.

82. Anamthawat-J¢nsson K, Heslop-Harrison JS. 1993. Isolation and characterization of genome-specific DNA sequences in Triticeae species. Mol.Gen.Genet. 240 : 151-158.

Two contrasting genome-specific DNA sequences were isolated from Aegilops speltoides (wild goat grass) and Hordeum chilense (wild barley), both representing more than 1% of the genomes. The repetitive DNA fragments were identified as being genome-specific before cloning by genomic Southern hybridization (using total genomic DNA as a probe), and hence extensive screening of clones was not required. For each fragment, up to six recombinant plasmid clones were screened and about half were genome-specific. Clone pAesKB52 from Ae.�speltoides was a 763 bp Eco RI fragment, physically organized in simple tandem repeats and shown to localize to sub-telomeric chromosome regions of species with the Triticeae S-genome by in situ hybridization to chromosomes. The sequence data showed an internal duplication of some 280�bp which presumably occurred before sequence amplification and dispersion, perhaps by unequal crossing over and reciprocal translocation. In situ hybridization showed the sequence distribution varied between closely related (S-genome) species. Clone pHcKB6 was a 339 bp Dra I fragment from H. chilense , also tandemly repeated but more variable (loosing the Dra I site) resulting in a ladder-pattern in Southern blots which had little background smear. In situ hybridization showed that the tandem repeats were present as small clusters dispersed along all chromosome arms except at a few discrete regions including the centromeres and telomeres. The clone hybridized essentially specifically to the H-genome of H.�chilense and hence was able to identify the origin of chromosomes in H. chilense x S. africanum hybrid by in�situ hybridization. It has a high AT content (66%), small internal duplications, and a 50 bp degenerate inverted repeat. We speculate that it has dispersed by retrotransposition in association with other sequences carrying coding domains. The organization and evolution of such sequences are important to understand long-range genome organization, and the types of change that can occur during evolutionary and plant breeding timescales. Genome-specific sequences are also useful as markers for alien chromatin in plant breeding.

81. Orgaard M, Heslop-Harrison JS. 1994. Investigations of genome relationships in Leymus, Psathyrostachys and Hordeum by genomic DNA:DNA in situ hybridization. Annals of Botany 73 : 195-203.

80. Orgaard M, Heslop-Harrison JS. 1994. Relationships between species of Leymus, Psathyrostachys and Hordeum (Poaceae, Triticeae) inferred from Southern hybridization of genomic DNA and cloned DNA probes. Plant Syst.Evol. 189 : 217-231. DOI: 10.1007/BF00939728

79. Maluszynska J, Heslop-Harrison JS. 1993. Physical mapping of rDNA in Brassica species. Genome 36 : 774-781.

78. Heslop-Harrison JS, Flavell RB. 1993. The Chromosome. ISBN 1 872748 32 5 . Oxford: BIOS.

77. Heslop-Harrison JS, Leitch AR, Schwarzacher T. 1993. The physical organization of interphase nuclei. In: Heslop-Harrison JS, Flavell RB, eds. The Chromosome . Oxford: BIOS, 221-232.

76. Leitch IJ, Heslop-Harrison JS. 1993. Physical mapping of four sites of 5S rDNA sequences and one site of the alpha-amylase-2 gene in barley ( Hordeum vulgare ). Genome 36 : 517-523.

75. Heslop-Harrison JS. 1992. Nuclear architecture in plants. Curr.Op.Genet.Devel. 2 : 913-917.

74. Heslop-Harrison JS, Schwarzacher T. 1993. Molecular cytogenetics - biology and applications in plant breeding. Chromosomes Today 11 : 191-198.

Molecular cytogenetic studies allow us to examine and understand genome organization in species with large genomes - including wheat, the world's most important crop. Many generations of inbreeding and selection of crops have reduced their genetic base, but it can be expanded by crossing with alien species and selecting lines with recombinant chromosomes. Indeed, the highest yielding wheat varieties grown in Europe include one pair of chromosomes with a wheat-rye translocation. In situ hybridization is proving valuable for identifying the origin of chromosomes in first generation and backcrossed hybrids between wheat and its wild and cultivated relatives. We are screening lines for the presence of alien chromosomes, and, particularly, small alien segments that may carry important characters but are difficult to detect by other methods. We are also examining the physical organization of the genome using repetitive and low copy sequences, and relating sequence distribution along chromosomes and within the nucleus to behaviour and gene expression.

73. Leitch AR, Heslop-Harrison JS. 1993. Ribosomal RNA gene expression and localization in cereals. Chromosomes Today 11 : 91-100.

The rRNA genes are the most accessible genes for the study of gene expression at a molecular, structural, and ultrastructural level. This paper describes the occurrence and distribution of rRNA in wheat, barley and rye. These cereal species are closely related and intercrossable but the rRNA genes show only a very loose conservation to chromosomes of the same homoeologous group which is surprising considering the close co-linearity of single copy markers. The cereals have an apparent excess in the number of genes required to sustain ribosome synthesis. Some of the "redundant" genes may occur in inactive rDNA loci that are not associated with a nucleolus. Factors affecting locus suppression are reviewed. Loci involved in rDNA transcription include regions that contain actively transcribing genes and regions that contain inactive genes. The active genes are probably associated with the dense fibrillar component of the nucleolus, while the inactive genes remain condensed. In rye, the condensed, inactive genes occur in perinucleolar chromatin and the active genes are decondensed at the distal end of the rDNA locus. In wheat condensed genes occur inside and outside the nucleolus with each active locus showing several sites with decondensed genes separated by condensed, intranucleolar presumably inactive genes. In rye and wheat active rDNA loci extend through the nucleolus and active rDNA sequences do not loop out from perinucleolar chromatin.

72. Leitch AR, Maluszynska J, Leitch IJ, Anamthawat-J¢nsson K, Schwarzacher T, Heslop-Harrison JS. 1992. Hybridation in situ sur chromosomes utilisant des sondes d'ADN marqu_es _ la digoxig_nine et d_tect_es par _pifluorescence. In: Jahier J, ed. Techniques de Cytog_n_tique V_g_tale . Paris: INRA, 85-90.

71. Heslop-Harrison JS, Harrison GE, Leitch IJ. 1992. Reprobing of DNA:DNA in situ hybridization preparations. Trends Genet. 8 : 372-373.

Membranes used for Southern hybridization can be probed many times. For DNA:DNA in situ hybridization to animal and plant chromosomes, several differently labelled probes can be used simultaneously but problems with co-location of probes or wide differences in signal strength make such experiments impossible to analyse, while the extra detection reagents may increase background signal. It is useful to be able to reuse preparations which are hard to obtain - for example from meiotic and zygotic material - or to map newly available probes with respect to previously localized markers. We have found that slides used for in situ hybridization can be reprobed with high reliability after both single and double target probing. After photography of the first probing, coverslips are removed and preparations re-denatured, hybridized, and the sites of hybridization detected using standard in situ hybridization protocols. Further rounds of reprobing suffer from increased background and chromatin loss, but still give probe localization data.

70. Heslop-Harrison JS. 1992. Book review: Electron Microscopy of Plant Cells by Hall and Hawes. Ann.Bot. 70 : 388-389.

69. Schwarzacher T, Leitch AR, Heslop-Harrison JS. 1994. DNA:DNA in situ hybridization - methods for light microscopy. In: Harris N, Oparka KJ, eds. Plant Cell Biology: A Practical Approach . Oxford: Oxford University Press, 127-155.

The following three manuscripts are distributed together as a lab. schedule/protocol.

68. Leitch IJ, Heslop-Harrison JS. 1994. Detection of digoxigenin-labeled DNA probes hybridized to plant chromosomes in situ . In: Isaac PG, ed. Protocols for nucleic acid analysis by nonradioactive probes. Methods in molecular biology. Volume 28 . Totowa, New Jersey: Humana, 177-186.

67. Schwarzacher T, Heslop-Harrison JS. 1994. Direct fluorochrome labelled DNA probes for direct fluorescent in situ hybridization to chromosomes. In: Isaac PG, ed. Protocols for nucleic acid analysis by nonradioactive probes. Methods in molecular biology. Volume 28 . Totowa, New Jersey: Humana, 167-176.

Number: TS39 . Schwarzacher T, Leitch AR. 1993. Enzymatic treatment of plant material to spread chromosomes for in situ hybridization. In: Isaac PG, ed. Protocols for nucleic acid analysis by nonradioactive probes. Methods in molecular biology. Volume 28 . Totowa, New Jersey: Humana, 153-160

66. Maluszynska J, Heslop-Harrison JS. 1993. Molecular cytogenetics of the genus Arabidopsis : In situ localization of rDNA sites, chromosome numbers and diversity in centromeric heterochromatin. Ann.Bot. 71 : 479-484.

65. Heslop-Harrison JS. 1993. Applications of molecular cytogenetics in the Triticeae. In: Damania A, ed. Biodiversity and wheat improvement . Chichester: Wiley and Sons, 31-38.

Molecular cytogenetics enables information from molecular biology to be linked directly to chromosomes and cytogenetics. Particular DNA sequences can be localized along chromosomes, in particular using the technique of DNA:DNA in situ hybridization. There are four major areas where current molecular cytogenetics methods can be of direct use to the Triticeae breeder: 1) The evaluation of genome relationships, including identification of parents or ancestors in unknown crosses or in polyploid species. 2) Identification of alien chromosomes and chromosome segments in breeding lines including alien chromatin. 3) Mapping of genes to examine recombination, evolution, and to aid with molecular gene cloning and mapping strategies; and 4) The physical sorting of chromosomes by flow cytometry or microdissection to generate chromosome or chromosome region specific libraries. The present paper reviews current applications of these techniques for the evaluations of relationships and germplasm, and discusses the future prospects of such techniques.

64. Leitch IJ, Heslop-Harrison JS. 1992. Physical mapping of the 18S-5.8S-26S rRNA genes in barley by in situ hybridization. Genome 35 : 1013-1018.

The 18S-5.8S-26S rRNA genes have been located on five pairs of barley ( Hordeum vulgare L., 2n=2x=14) chromosomes (in descending order of copy number, barley chromosome numbers 6, 7, 5, 1 and 2; homoeologous groups 6H, 5H, 1H, 7H and 2H) by in situ hybridization followed by C-banding. All sites were at intercalary positions. The pairs of major sites on chromosomes 6 (6H) and 7 (5H) are well known. Silver staining of nuclei and meiotic analysis have previously indicated that additional rDNA sites may be present, but the presence of sites on a further three chromosome pairs was unexpected. Within the tribe Triticeae, few species have more than two pairs of rDNA sites, and they have not been reported on group 2 chromosomes. We propose calling the new sites Nor -H1 [on chromosome 5 (1H)], Nor -H4 [on chromosome 1 (7H)] and Nor -H5 [on chromosome 2 (2H)], and that any further rDNA sites on homoeologous group 2 chromosomes should be called Nor -5. In situ hybridization is valuable for gene mapping, since it can detect the presence of genes with a very wide range of copy numbers at different sites.

63. Leitch IJ, Anamthawat-J¢nsson K, Schwarzacher T, Leitch AR, Harrison GE, Shi M, Heslop-Harrison JS. 1992. ECL advances studies in plant genetics and evolution. Life Sci.News 8 : 7-8.

62. Wang ML, Leitch AR, Schwarzacher T, Heslop-Harrison JS, Moore G. 1992. Construction of a chromosome-enriched Hpa II library from flow-sorted wheat chromosomes. Nucl.Acids Res. 20 : 1897-1901.

61. Leitch AR, Schwarzacher T, Wang ML, Leitch IJ, ëurlan-Momirovic G, Moore G, Heslop-Harrison JS. 1993. Molecular cytogenetic analysis of repeated sequences in a long term wheat suspension culture. Plant Cell,Tissue and Organ Culture 33 : 287-296.

A rapidly growing Triticum aestivum L. (wheat) derived long term suspension culture (named TaKB1), that is probably not regenerable, was analysed for karyotype rearrangements, stability and changes in repetitive DNA. The cell line has an average chromosome number of 21 and the DNA amount of unreplicated cells of TaKB1 measured by flow cytometry is about 30% lower than an unreplicated (1C) bread wheat genome. In situ hybridization of a repetitive DNA sequence (pSc119.2), which occurs as tandemly repeated blocks (heterochromatin) in wheat, shows that chromosomes from the TaKB1 line have fewer and weaker subtelomeric locations of the sequence than wheat, suggesting deletions of distal chromosome segments and a reduction in the sites and copy number of the sequence. The in situ hybridization pattern and chromosome morphology allowed 27 chromosome types to be identified in the cell line. No two analysed cells contained the same chromosome complement, although some chromosome types were present in every cell. Using Southern hybridization the structure and copy number of a retroelement (Wis-2) and its flanking sequence was shown to be the same in the TaKB1 cell line and wheat. An in situ analysis of rDNA in the TaKB1 cell line (using the probe pTa71) showed a reduction in number of sites and rRNA genes in each cell from that in wheat. Interphase cells of the cell line showed dispersed signal throughout the nucleolus with no evidence for clusters of condensed and inactive rRNA genes.

60. Anamthawat-J¢nsson K, Schwarzacher T, Heslop-Harrison JS. 1993. Behavior of parental genomes in the hybrid Hordeum vulgare x H. bulbosum . J.Heredity 84 : 78-82.

59. Schwarzacher T, Anamthawat-J¢nsson K, Harrison GE, Islam AKMR, Jia JZ, King IP, Leitch AR, Miller TE, Reader SM, Rogers WJ, Shi M, Heslop-Harrison JS. 1992. Genomic in situ hybridization to identify alien chromosomes and chromosome segments in wheat. Theor.Appl.Genet. 84 : 778-786.

Genomic in situ hybridization was used to identify alien chromatin in chromosome spreads of wheat, Triticum aestivum L., lines incorporating chromosomes from Leymus multicaulis (Kar. & Kir.) Tzvelev and Thinopyrum bessarabicum (Savul. & Rayss) Lve, and chromosome arms from Hordeum chilense Roem. & Schult, H. vulgare L. and Secale cereale L. Total genomic DNA from the introgressed alien species was used as a probe together with excess amounts of unlabelled blocking DNA from wheat for DNA:DNA in situ hybridization. The method labelled the alien chromatin yellow-green, while the wheat chromosomes showed only the orange-red fluorescence of the DNA counterstain. Nuclei were screened from seedling root tips (including those from half-grains) and anther wall tissue. The genomic probing method identified alien chromosomes and chromosome arms and allowed counting in nuclei at all stages of the cell cycle, so complete metaphases were not needed. At prophase or interphase, two labelled domains were visible in most nuclei from disomic lines, while only one labelled domain was visible in monosomic lines. At metaphase, direct visualization of the morphology of the alien chromosome or chromosome segment was possible and allowed identification of the relationship of the alien chromatin to the wheat chromosomes. The genomic in situ hybridization method is fast, sensitive, accurate and informative. Hence it is likely to be of great value for both cytogenetic analysis and in plant breeding programmes.

58. Heslop-Harrison JS. 1992. Pollen capture, adhesion and hydration. In: Cresti M, Tiezzi A, eds. Sexual Plant Reproduction . Heidelberg: Springer, 81-88.

57. Heslop-Harrison JS. 1992. Cytological techniques to assess pollen quality. In: Cresti M, Tiezzi A, eds. Sexual Plant Reproduction . Heidelberg: Springer, 41-48.

56. Heslop-Harrison JS. 1992. The angiosperm stigma. In: Cresti M, Tiezzi A, eds. Sexual Plant Reproduction . Heidelberg: Springer, 59-68.

Download three chapters from Sexual Plant Reproduction: Pollen techniques, Pollen behaviour, and Stigma characteristics

55. Leitch AR, Mosgller W, Shi M, Heslop-Harrison JS. 1992. Different patterns of rDNA organization at interphase in nuclei of wheat and rye. J.Cell Sci. 101 : 751-757.

The physical location of the rDNA repeating units (25S, 18S and 5.8S rRNA genes and the intergenic spacer sequences) was investigated in rye ( Secale cereale L.) and wheat ( Triticum aestivum L.) root tip meristematic cells by in situ hybridization using light and electron microscopy. The rDNA sequences are organized differently in the two related and intercrossable species. In rye, (2n=14, one pair of chromosomes with nucleolar organizing regions, NORs), two condensed blocks of rDNA-containing chromatin occurred in each interphase nucleus. The blocks were associated with the periphery of nucleoli and a single labelled, decondensed rDNA fibre extended into the nucleolus from the block. We term this expression pattern terminal decondensation. In wheat (2n=6x=42, five pairs of chromosomes with NORs), inactive condensed labelled chromatin was found unassociated with nucleoli. Active NORs had some condensed rDNA associated with the nucleolar periphery, but, in contrast to rye, condensed rDNA was also found within the nucleolus. The condensed labelled rDNA in wheat nucleoli was visible as fluorescent foci in the light microscope and labelled condensed chromatin in the electron microscope. Its absence in rye shows that condensed rDNA need not be present in active plant nucleoli. Diffuse labelled sites of rDNA, likely to represent actively transcribed rDNA, were found in both rye and wheat. Active rDNA loci in wheat have many expressed segments separated by unexpressed, condensed, rDNA - fragmented decondensation - while each locus in rye has a single, unexpressed perinucleolar condensed block of rRNA genes. Thus the positions of actively transcribed genes within the tandem arrays of rDNA at each locus are fundamentally different in the two cereals. The NOR chromosome appeared to extend through the nucleolus, and active rDNA sequences did not loop out from chromatin into the nucleolus as is frequently described in nucleolar models.

54. Heslop-Harrison JS. 1992. Molecular cytogenetics, cytology and genomic comparisons in the Triticeae. Hereditas 116 : 93-99.

Within the Triticeae, many useful genes have been transferred between genera, and the transferred characters are found widely in cereal crops. The subject matter of this paper has provided the basis for our understanding of gene transfer and chromosome behaviour. Genomes can be studied by cytogenetic examination of the chromosomes in species, natural and synthetic hybrids. Work on the molecular biology can demonstrate directly differences between their DNA sequences - in the code itself, and in the length and copy number of particular sequences. Molecular cytogenetics, particularly using DNA:DNA in situ hybridization, enables the two technologies to be linked by allowing direct observation of the dispersal of different types of DNA within chromosomes. Co-evolution of sequence families within genomes is a major feature of species evolution and divergence within the tribe. Thus, genome-specific sequences can be isolated and cloned relatively easily. Many such sequences are tandemly repeated and co-localize with C-bands on chromosomes; each C-band may consist of several different sequence classes. Information about band sizes and their constitution assists genome studies. Other families of middle and highly repetitive DNA sequences are dispersed within a genome, and may show retrotransposon-like modes of co-evolution. I give examples of the types of difference between chromosomes in various species, and relate the differences to DNA sequence, chromosome morphology and behaviour. Despite, or perhaps because of, the wide range of aneuploids, hybrids and translocations that can be made within the Triticeae, in natural populations, the basic chromosome number and morphology is remarkably stable; even translocations are uncommon. What constraints may restrict genome evolution? What limits, or can advance, the manipulation and transfer of genes within the Triticeae?

53. Snape JW, Laurie DA, Leitch IJ, Heslop-Harrison JS. 1992. Prospects in barley cytogenetics. Barley Genetics VI : 833-848.

52. Heslop-Harrison JS. 1992. Book Review: Chromosome Engineering by Gupta and Tsuchiya. J.Exp.Bot. 43 : 435-437.

51. Leitch IJ, Leitch AR, Schwarzacher T, Maluszynska J, Anamthawat-J¢nsson K, Shi M, Harrison GE, Heslop-Harrison JS. 1991. Two-colour mapping of plant DNA sequences using digoxigenin and biotin. Boehringer Mannheim Update 4 : 10-11.

50. Heslop-Harrison JS, Schwarzacher T, Anamthawat-J¢nsson K, Leitch AR, Shi M, Leitch IJ. 1991. In situ hybridization with automated chromosome denaturation. Technique 3 : 109-115.

49. Schwarzacher T, Heslop-Harrison JS, Anamthawat-J¢nsson K, Finch RA, Bennett MD. 1992. Parental genome separation in reconstructions of somatic and premeiotic metaphases of Hordeum vulgare x H. bulbosum . J.Cell Sci. 101 : 13-24.

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. Serial section reconstructions of metaphases and comparison with light micrograph karyotypes enabled chromosomes to be identified from their morphology. The three dimensional positions of their centromeres was established and analysed in the reconstructions of somatic root tip metaphases and cells at mitotic metaphase near their entry into meiosis. Parental genomes tended to lie in spatially separated domains in both tissues. Although varying in morphology, the two sets of chromosomes had similar mean sizes, so size differences did not cause the separation observed. The ultrastruture of centromere-associated structures of the chromosomes originating from the two genomes were very different. Those of the more central genome, originating from H. vulgare, were larger than those of the more peripheral genome of H. bulbosum origin.

48. Anamthawat-J¢nsson K, Heslop-Harrison JS. 1992. Species specific DNA sequences in the Triticeae. Hereditas 116 : 49-54.

Species specific DNA sequences are desirable markers for studying relationships between species and for plant breeding. This paper describes two aspects of molecular cytogenetic analysis of essentially species specific DNA sequences in the tribe Triticeae: sequences that differ substantially in their abundance and homology between species or genomes. Firstly, total genomic DNA can be used as a probe which is made highly specific to species by blocking with DNA sequences common with other species. Examples are given from studies of intergeneric and interspecific hybrids: Hordeum chilense x Secale africanum and H. vulgare x H. bulbosum. Secondly, species specific probes can be obtained from isolated and cloned DNA sequences. Both in situ and Southern hybridization techniques have proven important; in situ hybridization is required for localization and finding the distribution of sequences in the genomes while Southern hybridization gives information about the nature and structural variants of the sequences. The understanding of sequence behaviour, evolutionary patterns and organization in the genomes may lead to more effective and directed production of new recombinant lines, wide hybrids or alien gene transfer.

47. Leitch AR, Schwarzacher T, Mosgller W, Bennett MD, Heslop-Harrison JS. 1991. Parental genomes are separated throughout the cell cycle in a plant hybrid. Chromosoma 101 : 206-213.

The positions of the genomes originating from each parent were analysed in root tip nuclei of the mature, sexual F1 hybrid plant Hordeum vulgare (barley) x Secale�africanum (a wild rye). The two genomes of the hybrid were identified in both spread and sectioned material by non-radioactive DNA:DNA in situ hybridization using labelled total genomic DNA from one parent as a probe and unlabelled total genomic DNA from the other parent to block non-specific hybridization. Complete three-dimensional reconstructions of sets of labelled sections enabled detailed analysis of the position of the genomes at interphase. The parental genomes lay in various non-intermixed configurations, including lateral and concentric arrangements. On spread preparations, the two parental genomes were found to be spatially separated throughout the cell cycle; the genome originating from H. vulgare tended to be located more centrally than that from S.�africanum . The results show that the nucleus is spatially organized above the level of the DNA and chromosome at the genome level.

46. Heslop-Harrison JS. 1992. Natural and artificial hybrids in the grasses. In: Mulcahy D, Bergamini-Mulcahy G, eds. Angiosperm Pollen and Ovules . New York: Springer, 408-413.

Conclusions, not abstract: Many ways have been devised to overcome the barriers to wide hybridization between agriculturally important crops and their relatives. The production of hybrids has had immense economic benefits: many cereals include genes introduced by breeders who have overcome barriers to hybridization. Direct gene transfer might conceivably make crossing technology obsolete, although such molecular gene transfer is only now being used to move genes into major crops. Many hybridization barriers have been overcome by empirical methods, but scientific understanding has followed. For example, bud pollination in the Brassicas was later shown to involve the incompatibility proteins being produced late in maturation. The genetics of crossing barriers between closely related species, particularly within the Triticeae, has enabled elucidation of some genes related to the breeding systems. Further understanding of the subject of this volume, pollen and ovules, will lead to the production of even wider hybrids and wider gene transfer through sexual methods, along with new insights into the genetics and physiology of the life of sporophytes, fertilization and embryology.

45. Anamthawat-J¢nsson K, Heslop-Harrison JS, T¢masson T. 1993. Genetics, cytogenetics and molecular genetics of Icelandic birch: implications for breeding and reforestation. In: Alden JN, Mastrantonio L, eds. Forest Development in Cold Climates . New York: Plenum, 357-368.

44. Heslop-Harrison JS, Mosgller W, Schwarzacher T, Leitch AR. 1991. Volumes and positions of chromosomes in reconstructions of fibroblasts. Am.J.Hum.Genet. Supplement 49 : 380.

Ten divisions from human male fibroblasts have been completely reconstructed in three dimensions using electron micrographs of serial thin sections. All 46 chromosomes could be identified to a group from their volumes and centromere indicies. Chromosome morphology was clearly visible, and the ultrastructure of centromeres, satellite constrictions and one heterochromatic site can be visualized. The positions of the centromeres and telomeres of all chromosomes were analysed. The metaphase plate tended to be flat and an oval shape, which did not reflect cell outline. Large chromosomes tended to be more peripheral than the smaller chromosomes. In many cells the chromosomes of the G group were associated, but other groups showed few significant associations. In the cells in which the metaphase plate was most flat, centromeres of small chromosomes (groups E, F and G) were central and those of large chromosomes (groups A and B) were more peripheral. The non-random arrangement of the chromosomes might relate to errors in division or gene activity.

43. Heslop-Harrison JS. 1991. The molecular cytogenetics of plants. J.Cell Sci. 100 : 15-21.

Recent results show that the molecular genetics of defined systems can be tackled directly, and allow the directed manipulation of gene expression during development. In the rapid acquisition of information about DNA sequences, a gap has appeared between our knowledge of the genome at the molecular level and at the chromosome level. Molecular cytogenetics is now enabling the linking of molecular genetics with cytogenetics, using a combination of tools from both disciplines, and, in particular, direct in situ hybridization of labelled probes to interphase nuclei and chromosomes prepared for light or electron microscopy. The work is providing novel answers about genome and gene organization, gene activities and even meiotic recombination. Here, I aim to discuss some of the recent progress in this area from the viewpoint of a plant cytogeneticist. Many of my examples are taken from two contrasting groups of species: The small-grained cereals are grasses, and include barley, rye and wheat, while Arabidopsis thaliana , usually referred to as Arabidopsis, is a small rapidly growing species in the Brassica family.

42. Mosgller W, Leitch AR, Brown JKM, Heslop-Harrison JS. 1991. Chromosome arrangements in human fibroblasts at mitosis. Hum.Genet. 88 : 27-33.

The positions of the centromeres of all 46 human chromosomes were analysed in three dimensional reconstructions of electron micrographs of ten serially sectioned unpretreated human male fibroblast cells. The reconstructions show that the spatial positioning of the chromosomes during division is not random. The centromeres were arranged on a metaphase plate, which was ellipsoidal and tended to be flat. The distance of centromeres from the centre of the mitotic figure was correlated with chromosome size; small chromosomes tended to be central in all the metaphases. Large chromosomes were more peripheral, especially in cells which were more advanced in mitosis. Thus there is a tendency for larger chromosomes to move outwards as metaphase advances. In many cells the A group centromeres were overdispersed, while G group centromeres tended to be clustered. The acrocentric chromosomes (D and G groups) also tended to be clustered when analysed together, probably reflecting associations in nucleoli at the previous interphase. The results show that chromosome disposition is non-random and changes during division.

41. Maluszynska J, Heslop-Harrison JS. 1991. Localization of tandemly repeated DNA sequences in Arabidopsis thaliana . Plant J. 1 : 159-166.

In situ hybridization to interphase nuclei and chromosomes of Arabidopsis thaliana (2n�=�10) shows that there are four sites of rDNA in a diploid nucleus. The sites are located on chromosomes�2 and 4, and the strength of hybridization indicates that copy number is similar at both pairs of sites. Hybridization to trisomic line 4 revealed five hybridization sites. Silver staining of nucleoli demonstrates that all four loci can be active in diploid interphase nuclei. The tandemly repeated probe pAL1 hybridizes near to the centromeres of all five chromosome pairs. In diploid interphase nuclei, 10 sites of hybridization are detected, while 15 are seen in triploid nuclei. The sites of hybridization co-localize with the centromeric heterochromatin visualized by staining DNA with the fluorochrome DAPI. The results demonstrate that molecular cytogenetics can be applied to A.�thaliana , and high resolution physical chromosome maps can be generated. Both probes may be useful for interphase cytogenetics, where they enable chromosome number and aneuploidy to be examined in tissues without divisions. The physical localization of these hybridization sites provide a starting point for linking RFLP and physical chromosome maps.

40. Leitch AR, Schwarzacher T, Wang ML, Moore G, Heslop-Harrison JS. 1991. Flow cytometry of cereal chromosomes. Cytometry Supplement 5 : 39.

Flow cytometry of plant chromosomes is important because of the economic interest in understanding and manipulating genetic material of crop species. Flow cytometry will substantially enhance karyotype analysis, gene mapping and construction of chromosome specific libraries. Root tips from barley and bread wheat and a suspension cell line from Triticum�monococcum , a wild wheat, [now known to be T.�aestivum , culture cTaKB1] were synchronized using 2.5 mM hydroxyurea. After treatment with colchicine, a metaphase index of 15-30% was observed. Chromosomes were isolated from the T. monococcum [ T. aestivum ] line using enzyme digestion of the cell walls and a polyamine isolation buffer. The chromosome suspension was stained with Hoechst 33258 and Chromomycin A 3 and analysed on a FacStar Plus cytometer. The flow histograms showed a bimodal distribution with seven peaks with high DNA content and several less defined peaks with about half the DNA content. Feulgen DNA measurements of spread metaphase chromosomes were compared with the flow data. The seven main peak might correspond to the seven chromosome types of T. monococcum . These peaks are clear of background and would be suitable for sorting.

39. Anamthawat-J¢nsson K, Schwarzacher T, Leitch AR, Heslop-Harrison JS. 1991. Genomic probing for differentiating closely related species and detection of alien chromosome transfers. J.Exp.Bot. 42, Supplement : 34.

Hybrids are often used to broaden the genetic background of crops such as barley, rye and wheat. Through backcrossing, chromosome segments including desirable genes from wild species can be transferred into the cereal crop. To distinguish DNA originating from closely related species in hybrid plants and plants containing alien chromosomes or chromosome segments, total genomic DNA is labelled and used as a probe. The probe can identify the genomic origin of DNA by in situ hybridization to chromosome preparations and by Southern hybridization to extracted DNA. The species specificity is improved by blocking common repetitive sequences with unlabelled DNA from one of the parents. We have used the total genomic DNA probing technique to confirm the parental origin of chromosomes in the hybrids Hordeum chilense x Secale africanum and H.�vulgare x H.�bulbosum. The method was also used to identify chromosome segments of S.�cereale and H.�chilense in bread wheat ( Triticum aestivum ). In situ localization seems more sensitive than Southern hybridization in detecting alien chromosome substitutions. The genomic probing method is rapid and highly applicable since it does not involve cloning and screen strategies, and it can be used with many species. It also gives information about genome evolution through introgression, chromosome recombination, and the dispersal of repetitive sequences.

38. Leitch IJ, Leitch AR, Heslop-Harrison JS. 1991. Physical mapping of plant DNA sequences by simultaneous in situ hybridization of two differently labelled fluorescent probes. Genome 34 : 329-333.

37. Schwarzacher T, Heslop-Harrison JS. 1991. In situ hybridization to plant telomeres using synthetic oligomers. Genome 34 : 317-323.

The distribution of telomeric repeats in Hordeum vulgare (barley) and Secale cereale (rye) was studied by DNA:DNA in situ hybridization to root tip chromosome preparations. Biotinylated synthetic oligomers, (TTTAGGG) 6 and (CCCTAAA) 6 , homologous to the consensus sequence of the Arabidopsis thaliana telomeric repeat, were used as probes, and hybridization sites were detected by Texas Red fluorescence or horseradish peroxidase-catalysed precipitation of diaminobenzidine. After examination in the light microscope, the same preparations were transferred to the electron microscope for high resolution analysis. Sites of hybridization were visualized as single or double dots at the end of most chromosome arms. The sizes of the signal dots varied widely, indicating that individual telomeres may contain different numbers of repeats of the telomeric sequence. In contrast to many mammals, no telomere repeats were detected other than at the ends of the chromosomes. At interphase, signals were concentrated in one region of the nucleus, as would be expected from the Rabl orientation typical for dividing cells from cereal root tips.

36. Heslop-Harrison JS, Bennett MD. 1990. Nuclear architecture in plants. Trends Genet. 6 : 401-405.

35. Schwarzacher T, Leitch AR, Heslop-Harrison JS. 1990. In situ hybridization and the architecture of the nucleus. Trans.Roy.Microscop.Soc. 1 : 669-674.

34. Heslop-Harrison JS. 1990. Gene expression and parental dominance in hybrid plants. Development 21-28.

33. Heslop-Harrison JS, Leitch AR, Schwarzacher T, Anamthawat-J¢nsson K. 1990. Detection and characterization of 1B/1R translocations in hexaploid wheat. Heredity 65 : 385-392.

Total genomic DNA from rye was labelled with biotin and used as a probe for in situ hybridization to show the size and translocation point of the rye chromosome segments in five wheat varieties which carry a translocation between wheat chromosome 1B and the short arm of rye chromosome 1R (1B/1R). All the translocation breakpoints were at, or very near to, the centromere. Using genomic DNA to block some cross-hybridization, little signal from hybridization between the rye probe and wheat chromosomes was observed despite the 78% sequence homology between the species. The translocation was identified in cells at all stages of the cell cycle. Total genomic rye DNA used as a probe was also able to distinguish rye, triticale and wheat varieties carrying the translocation, by Southern hybridization. The techniques using genomic probes are useful for detecting, characterizing and following alien chromosomes or chromosome segments through breeding programmes, by both in situ and Southern hybridization.

32. Anamthawat-J¢nsson K, Heslop-Harrison JS. 1990. Centromeres, telomeres and chromatin in the interphase nucleus of cereals. Caryologia 43 : 205-213.

31. Leitch AR, Mosgoller W, Schwarzacher T, Bennett MD, Heslop-Harrison JS. 1990. Genomic in situ hybridization to sectioned nuclei shows chromosome domains in grass hybrids. J.Cell Sci. 95 : 335-341.

In situ hybridization using biotinylated total genomic DNA and avidin detection systems was adapted for examination of thin sectioned plant material in the light and electron microscopes. Root tip material was preserved prior to sectioning so that the in vivo disposition of the chromatin was maintained. Use of total genomic DNA from Secale africanum as a probe enabled the chromatin from the two parental genomes in the grass hybrid Hordeum chilense x S. africanum to be distinguished. The biotinylated probe preferentially labelled the chromosomes of S. africanum origin. DNA-DNA hybrids were visualized at the light-microscope level by Texas Red fluorescence and at the electron-microscope level by the enzymic precipitation of DAB (diaminobenzidine) of by colloidal gold particles. The use of thin sections allowed the location of probe hybridization to be established unequivocally in both metaphase and interphase nuclei. Analysis of interphase nuclei showed that chromatin originating from the two parental genomes did not intermix but occupied distinct domains.

30. Anamthawat-J¢nsson K, Schwarzacher T, Leitch AR, Bennett MD, Heslop-Harrison JS. 1990. Discrimination between closely related Triticeae species using genomic DNA as a probe. Theor.Appl.Genet. 79 : 721-728.

Labelled total genomic DNA was used as a probe in combination with blocking DNA to discriminate between taxonomically closely related species in the Hordeum and Secale genera. Discrimination was possible both by Southern hybridization to size fractionated restriction enzyme digests of genomic DNA and by in situ hybridization to chromosome preparations. To distinguish between two species (e.g. H. vulgare and H. bulbosum ) genomic DNA from one species was used as the labelled probe, while unlabelled DNA from the other species was applied at a much higher concentration as a block. The blocking DNA presumably hybridized to sequences in common between the block and DNA sequences on the membrane or chromosomes in situ. If so, mainly species specific sequences would remain as sites for probe hybridization. These species specific sequences are dispersed and represent a substantial proportion of the genome (unlike many cloned, species specific sequences). Consequently, rapid, non-radioactive methods detected hybridization sites satisfactorily. The method was able to confirm the parentage of hybrid plants. It has potentially wide application in plant breeding for the detection of alien DNA transfer, and it can be easily adapted to many species.

29. Heslop-Harrison JS, Schwarzacher T, Leitch AR, Anamthawat-J¢nsson K, Bennett MD. 1988 . A method of identifying DNA sequences in chromosomes of plants European Patent Application Number 8828130.8. December 2. Patent .

The patent makes 19 claims. The first and major claim is for "A method of identifying the origin of one or more DNA sequences in the nuclear genome of a target eukaryotic plant containing genetic material from at least two different sources comprises hybridizing DNA from the target plant with labelled total genomic DNA fragments selected to hybridize to DNA from one of the sources, while preventing hybridization of the probe to sequences common to more than one source with unlabelled total genomic DNA fragments selected to block such common sequences by hybridization with them in the target plant DNA and/or in the labelled DNA, and detection of the sites of hybridization of the labelled probe to the DNA of the target plant." The patent application describes the use of large amounts of competitive, blocking DNA, in combination with labelled genomic DNA, as a probe to identify and characterize alien genomes, chromosomes or chromosome segments in situ or by membrane hybridization. The method has wide application in detecting the parentage of hybrids and for detecting, characterizing and following alien chromosome introgression in plant breeding lines.

28. Heslop-Harrison JS. 1990. Energy dispersive X-ray analysis. In: Linskens HF, Jackson JF, eds. Modern Methods of Plant Analysis New Series, Volume 11 . Berlin: Springer, 244-277.

27. Atkinson MD, Heslop-Harrison JS. 1990. A simple method of hidden-line removal in three-dimensional modelling: hiding the problem behind filled polygons. Comp.Appli.Biosci. 6 : 55-56.

26. Heslop-Harrison JS, Leitch AR, Schwarzacher T, Smith JB, Atkinson MD, Bennett MD. 1989 . The volumes and morphology of human chromosomes in mitotic reconstructions. Hum.Genet. 84 : 27-34.

Ten unpretreated normal human male fibroblast cells in mitosis were completely reconstructed from micrographs of between 82 and 119 consecutive serial sections. All 46 of the chromosomes and their centromeres could be reconstructed in every cell. Measurements of chromosome volumes and centromere indicies are presented. The data enabled allocation all chromosomes to their groups (A to G), and chromosomes 1, 2, 3, 6, 9, 16, 17, 18 and Y were individually identified. Comparisons with published karyotypes showed that volume measurements correlated well with measurements of DNA content and chromosome length. Centromere indicies also showed good correlations, but the acrocentric chromosomes were more unequally armed than found by length measurement. Secondary constrictions at the nucleolar organizing region were visible on about a third of the acrocentric chromosomes. One chromosome of the C group, number 9, had a diffuse subcentromeric region (DSR) on the long arm, at the position of the constitutive heterochromatin and (in meiotic cells) the paramere.

25. Schwarzacher T, Leitch AR, Bennett MD, Heslop-Harrison JS. 1989 . In situ localization of parental genomes in a wide hybrid. Ann.Bot. 64 : 315-324.

Biotinylated total genomic DNA from Secale�africanum Rapf used as probe in hybridization experiments, distinguishes between DNA from Hordeum and Secale , both in chromosome spreads in situ and on isolated DNA bound to filters. On hybridization filters (dot blots), the dots containing H.�chilense Roem. und Schult and H.�vulgare L.DNA were significantly less labelled than the dots containing S.�africanum DNA. In root tip metaphases of the hybrid H.�chilense (X S.�africanum , after in situ hybridization with biotinylated S.�africanum DNA, the seven larger chromosomes from S.�africanum were labelled positively, while the seven smaller chromosomes from H.�chilense remained unlabelled. At different stages during the cell cycle of the same hybrid root tips, the chromatin originating from the two parental genomes does not mix. The sets of chromosomes tend to remain spatial separated, and individual chromosomes occupy distinct domains. These results show that the genome separation, previously reported for metaphase is maintained at interphase.

24. Heslop-Harrison JS, Huelskamp M, Wendroth S, Atkinson MD, Leitch AR, Bennett MD. 1988 . Chromatin and centromeric structures in interphase nuclei. In: Brandham PE, ed. Kew Chromosome Conference III . London: Allen and Unwin, 209-217.

23. Heslop-Harrison JS, Smith JB, Bennett MD. 1988 . The absence of the somatic association of centromeres of homologous chromosomes in grass mitotic metaphases. Chromosoma 96 : 119-131.

Root-tip metaphases from Hordeum vulgare (19 cells) , H. marinum (11 cells) , Aegilops umbellulata (10 cells) and Zea mays (10 cells) four species were completely reconstructed from electron micrographs of serially sectioned nuclei. The identity of each chromosome was found by measuring the volumes of its two arms and the presence of absence of a secondary constriction at the nucleolar organizing region. With the positions of the centromere in three dimensions, these data were used to analyse the relative positions of homologous and heterologous centromeres. In 31 of the 50 cells analysed, homologues were on average further apart than heterologues. Except for two nucleolar organising chromosomes, there was no evidence of any tendency for the distances between different homologous types to be differently distributed from distances between heterologues. Average distances between homologues of the single nucleolar organizing chromosome (linkage group 6) of Zea (2 n =20) were lower than the average for heterologues and the interhomologue distances were distributed significantly differently from the separation distances of chromosome 6 to other chromosomes. Presumably this association occurred because of nucleolar fusion in the previous interphase. Homologues of one of the two nucleolar organizing chromosomes of A. umbellulata were also distributed significantly differently from heterologues, with a tendency for homologues to lie further apart than the average heterologous pair. These results do not support previous work using squashed and spread metaphase preparations (some including abnormal, marked chromosomes) for these species.

22. Heslop-Harrison JS, Heslop-Harrison Y, Reger BJ. 1987 . Anther-filament extension in Lilium : potassium ion movement and some anatomical features . Annals of Botany 59 : 505-515.

21. Heslop-Harrison J, Heslop-Harrison JS, Heslop-Harrison Y. 1986 . The comportment of the vegetative nucleus and generative cell in the pollen and pollen tubes of Helleborus foetidus L. Annals of Botany 58 : 1-12.

It has been reported that in species of Plumbaginaceae, Chenopodiaceae, Cruciferae and Amaryllidaceae a 'male germ unit' is formed in which the two male gametes remain inter-connected, with one of the pair linked intimately to the vegetative nucleus. In two species the unit has been shown to remain intact in the pollen tube, and some accounts imply that it is polarized in its movement, the vegetative nucleus leading in the tube. Evidence given in this paper indicates that such a unit is unlikely to be present in Helleborus foetidus L. (Ranunculaceae). Application of an optical sectioning technique has shown that at no time is there a persistent linkage between the generative cell and the vegetative nucleus in unhydrated, hydrated and germinating pollen, nor is one present in the early pollen tube. Furthermore, no inter-connections between the two entities were seen in protoplasts from living, hydrated and incipiently germinating grains isolated mechanically in an osmotically balancing medium. Following germination, the vegetative nucleus leaves the grain in advance of the generative cell in most instances, but in the samples examined the generative cell led in about 30 per cent of the tubes. Assembling a polarised male germ unit in these circumstances would require (a) the formation of an inter-connection between the vegetative nucleus and the generative cell or one of the gametes derived from it during passage through the tube, and (b) where the generative cell initially leads in the tube, an exchange in relative positions. It is considered improbable that these conditions could consistently be met. Mature, incipiently germinating pollen of H. foetidus releases a fibrillar component when extruded into suitable media. Webs or clusters of fibrils are commonly seen to be associated with both the vegetative nucleus and the generative cell. The possibility that the fibrils are composed of aggregates of microfilaments is considered.

20. Heslop-Harrison Y, Heslop-Harrison JS, Heslop-Harrison J. 1986 . Germination of Corylus avellana L. pollen: hydration and the function of the oncus. Acta Bot.Neerl. 35 : 265-284.

As reported by SCHOCH-BODMER fifty years ago, the onci play an important part in accommodating volume changes in the 3-porate pollen of Corylus avellana L. during cycles of dehydration and rehydration. The onci, which are rich in pectins judging from their cytochemical properties, possess a remarkable fine-structure, characterised by densely packed, convoluted lamellae. Staining responses suggest that the protein content of the body of the oncus is low, although esterase activity is distributed throughout. During germination in a granular sporopollenin seal at the preferred aperture is disrupted by the gelatinisation of underlying pectins, and the pollen tube emerges as an outgrowth from the inner cellulosic layer of the intine. This process is preceded by the release of proteins from each aperture site. The source of this outflow is a protein-rich umbonate cap, 1-3 �m in thickness, lying beneath the pore over the body of the oncus and extending under the encompassing exine annulus. The nexine is absent from the annulus, so that the apertural proteins are continuous with the interbacular material. This implies that in this pollen type there are no clearly separate "gametophytic" and "sporophytic" domains. As in species of Compositae and Cruciferae with a similar sporophytic type of self-incompatibility (SI) system, incompatible pollen of C. avellana is inhibited on the stigma surface, the stigma papillae responding rapidly to the presence of the incompatible pollen of synthesis of internal callose. This suggests that the S-factors are held in the pollen wall, and it is possible that they form on component of the poral proteins. In the light of recent reports of sporophytic proteins held at the apertural sites in various angiosperm pollens, the possibility is considered that the poral proteins of C. avellana are also of sporophytic origin, at least in part. Should they contain tapetum-derived -factors a ready explanation of the sporophytic control of the SI system is at hand.

19. Heslop-Harrison JS, Reger BJ. 1986 . Chloride and potassium ions and turgidity in the grass stigma. J.Plant Physiol. 124 : 55-60.

18. Heslop-Harrison JS, Reger BJ. 1986 . X-ray microprobe mapping of certain elements in the ovary of Zea mays L. Ann.Bot. 57 : 819-822.

17. Heslop-Harrison JS. 1986 . Ion localisation in the stigma and pollen tube of cereals. In: Mulcahy D, Bergamini-Mulcahy G, Ottaviano E, eds. Biotechnology and ecology of pollen . Massachusetts: Springer, 351-356.

16. Heslop-Harrison JS, Heslop-Harrison J, Heslop-Harrison Y, Reger BJ. 1985 . The distribution of calcium in the grass pollen tube. Proc.R.Soc.Lond.B 225 : 315-327.

The distribution of calcium in the terminal regions of actively extending pollen tubes of two Gramineae, Zea mays and Pennisetum americanum , has been investigated by chlorotetracycline (CTC) fluorescence and by energy-dispersive X-ray analysis. Neither method reveals a concentration gradient declining from the tip towards the older parts of the tube comparable with that reported from the pollen tubes of Lilium longiflorum . The difference evidently arises from dissimilarities in pollen-tube growth physiology and the apical organization of the tube. Growth is achieved by the insertion of dictyosome-derived vesicles carrying wall-precursor materials at the tube tip. In L. longiflorum these are produced in a subapical zone of the tube, and a characteristic zonation of cytoplasmic organelles in the distal region develops during growth. In the grasses, large numbers of the wall precursor bodies ('P-particles') are produced before pollen dispersal, and are stored in the grain; they are distributed throughout the tube during early growth, and the organelle zonation in the apex is less pronounced. CTC-induced fluorescence is strongly associated with mitochondria, membranes and P-particles, suggesting that the observed distribution of calcium may reflect mainly the distribution of the element held in organelles and membranes rather than mobile Ca2+ in the cytosol.

15. Heslop-Harrison JS, Chapman V, Bennett MD. 1985 . Heteromorphic bivalent association at meiosis in bread wheat. Heredity 55 : 93--103.

14. Heslop-Harrison JS, Bennett MD. 1985 . Interlocked bivalents in reconstructed metaphase I cells of bread wheat. J.Cell Sci. 75 : 85-92.

13. Heslop-Harrison JS, Bennett MD. 1984 . Chromosome order - possible implications for development. J.Embryol.Exp.Morph. 83 Supplement : 51-73.

12. Heslop-Harrison JS. 1984 . Image quantifiers - comprehensive capabilities and wide choice. Optical Elect.Microsc. 11 : 12-17.

11. Bennett MD, Smith JB, Ward JP, Heslop-Harrison JS. 1984 . Predictable mean order of heterologues in four simple grass genomes. Chromosomes Today 8 : 299.

10. Heslop-Harrison JS, Bennett MD. 1984 . Centromere positions in undistorted reconstructed metaphases of four grass species. Chromosomes Today 8 : 315.

9. Heslop-Harrison JS. 1983 . Spatial Disposition of Chromosomes in Wheat Species . Cambridge: University of Cambridge.

8. Bennett MD, Heslop-Harrison JS, Smith JB, Ward JP. 1983 . DNA density in mitotic and meiotic metaphase chromosomes of plants and animals. J.Cell Sci. 63 : 173-179.

7. Heslop-Harrison JS. 1983 . Chromosome disposition in Aegilops umbellulata. In: Brandham PE, Bennett MD, eds. Kew Chromosome Conference II . London: Allen and Unwin, 63-70.

6. Heslop-Harrison JS, Bennett MD. 1983 . The positions of centromeres on the somatic metaphase plate of grasses. J.Cell Sci. 64 : 163-177.

5. Heslop-Harrison JS, Bennett MD. 1983 . The spatial order of chromosomes in root-tip metaphases of Aegilops umbellulata . Proc.R.Soc.Lond.B 218 : 225-239.

4. Heslop-Harrison JS, Bennett MD. 1983 . Prediction and analysis of spatial order in haploid chromosome complements. Proc.R.Soc.Lond.B 218 : 211-223.

3. Bennett MD, Smith JB, Heslop-Harrison JS. 1982 . Nuclear DNA amounts in angiosperms. Proc.R.Soc.Lond.B 216 : 179-199.

2. Dodds JH, Heslop-Harrison JS, Hall MA. 1980 . Metabolism of ethylene to ethylene oxide by cell-free preparations from Vicia faba L. cotyledons: effects of structural analogues and inhibitors. Plant Sci.Lett. 19 : 175-180.

1. Shivanna KR, Heslop-Harrison Y, Heslop-Harrison JS. 1978 . Inhibition of the pollen tube in the self-incompatibility response of grasses. Incompat.Newsl. 5-7.

Number: TS33. Albini SM, Schwarzacher T. 1992. In situ localization of two repetitive DNA sequences to surface-spread pachytene chromosomes of rye. Genome 35 : 554-563.

Surface spread pollen mother cells at meiotic prophase from Secale cereale (rye) were used for fluorescent DNA:DNA in situ localization of two tandemly repeated DNA sequences: pTa71, a wheat rDNA clone, and pSc119.2, a cloned 120bp repeat from rye heterochromatin. The fluorescent hybridization signal, consisting of many yellow-green points, was closely associated with the bivalent axis, corresponding to the synaptonemal complexes, and located in the surrounding chromatin. The rDNA signal was associated with one bivalent, the smallest of the seven, at a distance about 13% of the bivalent length from the telomere. This corresponded to the position of the nucleolar organizing region of silver-stained synaptonemal complexes analyzed in the electron microscope and published data for somatic metaphase chromosomes. The relative length of the axis covered with rDNA signal is less than expected from somatic metaphases, but corresponds more closely to the proportion of the sequences in the genome. Hybridization signal with the 120bp repeat was located mainly at telomeric regions of several bivalents that showed thickenings of the axis after DAPI staining probably corresponding to somatic C-bands. These major and some minor intercalary sites agree with the distribution of the 120bp repeat in somatic metaphase. Fluorescent in situ hybridization to plant surface spread pachytene chromosomes which can be obtained in large numbers has great potential for studying meiotic prophase, high resolution mapping of DNA sequences and investigating the relationship of DNA sequences to the synaptonemal complex.

Number: Moore G, Cheung W, Foote T, Gale MD, Koebner RMD, Leitch AR, Leitch IJ, Money T, Stancombe P, Yano M, Flavell R. 1993. Some key features of cereal genome organisation. Genomics 15: 472-482.

Cereal (Gramineae) genomes unlike those of mammals exhibit little suppression of CpG dinucleotides (Russell et al., 1971), and therefore have many potential CpG recognition sites for methylation-sensitive restriction enzymes. However most of these sites are methylated. Analysis of cereal genomic libraries and regions flanking genes indicates that unmethylated Not I sites are useful landmarks for regions containing genes/single copy sequences. Studies of a rye chromosome arm indicate that its peri-centromeric region has a reduced density of unmethylated Not I (and Mlu I) sites and therefore of genes. Unmethylated Mlu I and Nru I sites are distributed non-randomly in the genomes of wheat, barley and rice. Analysis of the genomic blocks defined by these sites in the genomes of wheat and barley indicates that members of families of repetitive sequences are distributed in equivalent proportions between the blocks. These blocks of interspersed repeats appear to be in multiple copies and are likely to have arisen by amplification, although alternative explanations are possible. These features of the organisation and evolution of the wheat, barley and rice genomes are discussed.

Number: TS41 . Schlegel R, Kynast R, Schwarzacher T, Rmheld V, Walter A. 1992. Mapping of genes for copper efficiency in rye and the relationship between copper and iron efficiency. In Proceedings of the VIIIth International Colloquium for the Optimization of Plant Nutrition , Lisbon, Portugal.

TS24. Moore G, Cheung W, Schwarzacher T and Flavell R. 1991. BIS1, a major component of the cereals genome and a tool for studying genomic organization. Genomics 10 : 469-476.

Schwarzacher T. 1994. Mapping in Plants: Progress and Prospects. Curr.Op.Genet.Devel. 4 : 868-874.

Comprehensive genetic maps now are available for all the world's important crop species. Remarkable data show conservation of types and order of markers over family-wide taxonomic groupings, and illuminate species relationships and mechanisms of genome evolution. Comparison of genetic and physical maps revealed differences in genetic distance throughout genomes with implications for genome organization, gene isolation and transformation.

In situ hybridization of labelled total genomic DNA with unlabelled blocking DNA enabled the parental origin of all chromosomes to be established in root tips of the mature sexual hybrid plant Hordeum�vulgare x H.�bulbosum. The parental genomes tended to remain spatially separated throughout the cell cycle with the chromosomes of H.�vulgare origin lying in a more central domain than those of H.�bulbosum origin. During anaphase and telophase, chromosomes of H.�bulbosum�origin tended to lag. Although the chromatids usually separated, they did not have V-shape the characteristic of anaphase chromatids. Aneuploid nuclei, missing H.�bulbosum origin chromosomes, arose when the lagging chromatids were not incorporated into the daughter nuclei, although most cells remained diploid. Some interphase cells contained micronuclei, all of which were of H. bulbosum origin. Information about chromosome disposition and movement is important to enable the understanding of chromosome stability.

In situ hybridization of labelled total genomic DNA with unlabelled blocking DNA enabled the parental origin of all chromosomes to be established in root tips of the mature sexual hybrid plant Hordeum vulgare x H. bulbosum. The parental genomes tended to remain spatially separated throughout the cell cycle with the chromosomes of H. vulgare origin lying in a more central domain than those of H. bulbosum origin. During anaphase and telophase, chromosomes of H. bulbosum origin tended to lag. Although the chromatids usually separated, they did not have V-shape the characteristic of anaphase chromatids. Aneuploid nuclei, missing H. bulbosum origin chromosomes, arose when the lagging chromatids were not incorporated into the daughter nuclei, although most cells remained diploid. Some interphase cells contained micronuclei, all of which were of H. bulbosum origin. Information about chromosome disposition and movement is important to enable the understanding of chromosome stability.