DNA content of heterochromatin and euchromatin in tomato (Lycopersicon esculentum) pachytene chromosomes.

Lycopersicon esculentum (tomato) has a small genome (2C = 1.90 pg of DNA) packaged in 2n = 2x = 24 small acrocentric to metacentric chromosomes. Like the chromosomes of other members of the family Solanaceae, tomato chromosomes have pericentromeric heterochromatin. To determine the fraction of the tomato genome found in euchromatin versus heterochromatin, we stained pachytene chromosomes from primary microsporocytes with Feulgen and analyzed them by densitometry and image analysis. In association with previously published synaptonemal complex karyotype data for tomato, our results indicate that 77% of the tomato microsporocyte genome is located in heterochromatin and 23% is found in euchromatin. If heterochromatin is assumed to contain few active genes, then the functional genes of the tomato must be concentrated in an effective genome of only 0.22 pg of DNA (1C = 0.95 pg x 0.23 = 0.22 pg). The physical segregation of euchromatin and heterochromatin in tomato chromosomes coupled with the small effective genome size suggests that tomato may be a more useful subject for chromosome walking and gene mapping studies than would be predicted based on its genome size alone. Key words : tomato, Lycopersicon esculentum, genome size, heterochromatin, euchromatin, pachytene chromosomes, synaptonemal complex.     

High-resolution chromosome mapping of BACs using multi-colour FISH and pooled-BAC FISH as a backbone for sequencing tomato chromosome 6

Within the framework of the International Solanaceae Genome Project, the genome of tomato (Solanum lycopersicum) is currently being sequenced. We follow a 'BAC-by-BAC' approach that aims to deliver high-quality sequences of the euchromatin part of the tomato genome. BACs are selected from various libraries of the tomato genome on the basis of markers from the F2.2000 linkage map. Prior to sequencing, we validated the precise physical location of the selected BACs on the chromosomes by five-colour high-resolution fluorescent in situ hybridization (FISH) mapping. This paper describes the strategies and results of cytogenetic mapping for chromosome 6 using 75 seed BACs for FISH on pachytene complements. The cytogenetic map obtained showed discrepancies between the actual chromosomal positions of these BACs and their markers on the linkage group. These discrepancies were most notable in the pericentromere heterochromatin, thus confirming previously described suppression of cross-over recombination in that region. In a so called pooled-BAC FISH, we hybridized all seed BACs simultaneously and found a few large gaps in the euchromatin parts of the long arm that are still devoid of seed BACs and are too large for coverage by expanding BAC contigs. Combining FISH with pooled BACs and newly recruited seed BACs will thus aid in efficient targeting of novel seed BACs into these areas. Finally, we established the occurrence of repetitive DNA in heterochromatin/euchromatin borders by combining BAC FISH with hybridization of a labelled repetitive DNA fraction (Cot-100). This strategy provides an excellent means to establish the borders between euchromatin and heterochromatin in this chromosome.     

FISH to meiotic pachytene chromosomes of tomato locates the root-knot nematode resistance gene Mi-1 and the acid phosphatase gene Aps-1 near the junction of euchromatin and pericentromeric heterochromatin of chromosome arms 6S and 6L, respectively

 The root-knot nematode resistance gene Mi-1 in tomato has long been thought to be located in the pericentromeric heterochromatin region of the long arm of chromosome 6 because of its very tight genetic linkage (approx. 1 cM) to the markers Aps-1 (Acid phosphatase 1) and yv (yellow virescent). Using Mi-BAC clones and an Aps-1 YAC clone in fluorescence in situ hybridisation (FISH) to pachytene chromosomes we now provide direct physical evidence showing that Mi-1 is located at the border of the euchromatin and heterochromatin regions in the short arm (6S) and Aps-1 in the pericentromeric heterochromatin of the long arm (6L) close to the euchromatin. Taking into account both the estimated DNA content of hetero- and euchromatin regions and the compactness of the tomato chromosomes at pachytene (2 Mb/μm), our data suggest that Mi-1 and Aps-1 are at least 40 Mb apart, a base pair-to-centiMorgan relationship that is more than 50-fold higher than the average value of 750 kb/cM of the tomato genome. An integrated cytogenetic-molecular map of chromosome 6 is presented that provides a framework for physical mapping. Received: 24 July 1998 / Accepted: 14 August 1998     

Cross-species bacterial artificial chromosome-fluorescence in situ hybridization painting of the tomato and potato chromosome 6 reveals undescribed chromosomal rearrangements

Ongoing genomics projects of tomato (Solanum lycopersicum) and potato (S. tuberosum) are providing unique tools for comparative mapping studies in Solanaceae. At the chromosomal level, bacterial artificial chromosomes (BACs) can be positioned on pachytene complements by fluorescence in situ hybridization (FISH) on homeologous chromosomes of related species. Here we present results of such a cross-species multicolor cytogenetic mapping of tomato BACs on potato chromosomes 6 and vice versa. The experiments were performed under low hybridization stringency, while blocking with Cot-100 was essential in suppressing excessive hybridization of repeat signals in both within-species FISH and cross-species FISH of tomato BACs. In the short arm we detected a large paracentric inversion that covers the whole euchromatin part with breakpoints close to the telomeric heterochromatin and at the border of the short arm pericentromere. The long arm BACs revealed no deviation in the colinearity between tomato and potato. Further comparison between tomato cultivars Cherry VFNT and Heinz 1706 revealed colinearity of the tested tomato BACs, whereas one of the six potato clones (RH98-856-18) showed minor putative rearrangements within the inversion. Our results present cross-species multicolor BAC–FISH as a unique tool for comparative genetic studies across Solanum species.     

Chromatin structure and physical mapping of chromosome 6 of potato and comparative analyses with tomato

Potato (Solanum tuberosum) has the densest genetic linkage map and one of the earliest established cytogenetic maps among all plant species. However, there has been limited effort to integrate these maps. Here, we report fluorescence in situ hybridization (FISH) mapping of 30 genetic marker-anchored bacterial artificial chromosome (BAC) clones on the pachytene chromosome 6 of potato. The FISH mapping results allowed us to define the genetic positions of the centromere and the pericentromeric heterochromatin and to relate chromatin structure to the distribution of recombination along the chromosome. A drastic reduction of recombination was associated with the pericentromeric heterochromatin that accounts for ~28% of the physical length of the pachytene chromosome. The pachytene chromosomes 6 of potato and tomato (S. lycopersicum) share a similar morphology. However, distinct differences of heterochromatin distribution were observed between the two chromosomes. FISH mapping of several potato BACs on tomato pachytene chromosome 6 revealed an overall colinearity between the two chromosomes. A chromosome inversion was observed in the euchromatic region of the short arms. These results show that the potato and tomato genomes contain more chromosomal rearrangements than those reported previously on the basis of comparative genetic linkage mapping.     

Molecular characterization of Vulmar1, a complete mariner transposon of sugar beet and diversity of mariner- and En/Spm-like sequences in the genus Beta.

Transposons of the Tc1-mariner superfamily are widespread in eukaryotic genomes. We have isolated the mariner element Vulmar1 from Beta vulgaris L., which is 3909 bp long and bordered by perfect terminal inverted repeats of 32 bp with homology to terminal inverted repeats of transposons from soybean and rice. According to a characteristic amino acid signature, Vulmar1 can be assigned to the DD39D group of mariner transposons. Vulmar1 is flanked by a 5'-TA-3' target site duplication that is typical for mariner transposons. Southern hybridization revealed that mariner-like copies are highly abundant in Beta species, and sequence analysis of 10 transposase fragments from representative species of the four Beta sections revealed an identity between 34% and 100% after conceptual translation. By fluorescent in situ hybridization, Vulmar1 was detected in distal euchromatin as well as in some intercalary and pericentromeric regions of all B. vulgaris chromosomes. In addition, using PCR, we were able to amplify fragments of the transposase gene of En/Spm-like transposons in the genus Beta. En/Spm-like transposase sequences are highly amplified in four Beta sections and showed a considerable degree of conservation (88.5-100%) at the protein level, while the homology to corresponding regions of En/Spm transposons of other plant species ranges from 49.5% to 62.5%. By fluorescent in situ hybridization, En/Spm-like transposon signals of strong intensity were detected on all chromosomes of B. vulgaris.     

Abundance and chromosomal distribution of six Drosophila buzzatii transposons: BuT1, BuT2, BuT3, BuT4, BuT5, and BuT6

The abundance and chromosomal distribution of six class-II transposable elements (TEs) of Drosophila buzzatii have been analyzed by Southern blotting and in situ hybridization. These six transposons had been previously found at the breakpoints of inversions 2j and 2q ⁷ of D. buzzatii. These two polymorphic inversions were generated by an ectopic recombination event between two copies of Galileo, a Foldback element. The four breakpoints became hotspots for TE insertions after the generation of the inversion and the transposons analyzed in this work are considered to be secondary invaders of these regions. Insertions of the six transposons are present in the euchromatin but show an increased density in the pericentromeric euchromatin–heterochromatin transition region and the dot chromosome. They are also more abundant in the inverted segments of chromosome 2 rearrangements. We further observed that the accumulation of TE insertions varies between elements and is correlated between dot, proximal regions, and inverted segments. These observations fully agree with previous data in Drosophila melanogaster and support recombination rate as the chief force explaining the chromosomal distribution of TEs.Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.Sequence data from this article have been deposited in the EMBL/GenBank Data Libraries under accession number DQ402469.     

Young but not relatively old retrotransposons are preferentially located in gene‐rich euchromatic regions in tomato (Solanum lycopersicum) plants

Long terminal repeat (LTR) retrotransposons are the major DNA components of flowering plants. They are generally enriched in pericentromeric heterochromatin regions of their host genomes, which could result from the preferential insertion of LTR retrotransposons and the low effectiveness of purifying selection in these regions. To estimate the relative importance of the actions of these two factors on their distribution pattern, the LTR retrotransposons in Solanum lycopersicum (tomato) plants were characterized at the genome level, and then the distribution of young elements was compared with that of relatively old elements. The current data show that old elements are mainly located in recombination‐suppressed heterochromatin regions, and that young elements are preferentially located in the gene‐rich euchromatic regions. Further analysis showed a negative correlation between the insertion time of LTR retrotransposons and the recombination rate. The data also showed there to be more solo LTRs in genic regions than in intergenic regions or in regions close to genes. These observations indicate that, unlike in many other plant genomes, the current LTR retrotransposons in tomatoes have a tendency to be preferentially located into euchromatic regions, probably caused by their severe suppression of activities in heterochromatic regions. These elements are apt to be maintained in heterochromatin regions, probably as a consequence of the pericentromeric effect in tomatoes. These results also indicate that local recombination rates and intensities of purifying selection in different genomic regions are largely responsible for structural variation and non‐random distribution of LTR retrotransposons in tomato plants.     

S Elements: A Family of Tc1-like Transposons in the Genome of Drosophila Melanogaster

The S elements form a diverse family of long-inverted-repeat transposons within the genome of Drosophila melanogaster. These elements vary in size and sequence, the longest consisting of 1736 bp with 234-bp inverted terminal repeats. The longest open reading frame in an intact S element could encode a 345-amino acid polypeptide. This polypeptide is homologous to the transposases of the mariner-Tc1 superfamily of transposable elements. S elements are ubiquitous in D. melanogaster populations and also appear to be present in the genomes of two sibling species; however, they seem to be absent from 17 other Drosophila species that were examined. Within D. melanogaster strains, there are, on average, 37.4 cytologically detectable S elements per diploid genome. These elements are scattered throughout the chromosomes, but several sites in both the euchromatin and β heterochromatin are consistently occupied. The discovery of an S-element-insertion mutation and a reversion of this mutation indicates that S elements are at least occasionally mobile in the D. melanogaster genome. These elements seem to insert at an AT dinucleotide within a short palindrome and apparently duplicate that dinucleotide upon insertion.     

Characteristics of the tomato nuclear genome as determined by sequencing undermethylated EcoRI digested fragments

A collection of 9,990 single-pass nuclear genomic sequences, corresponding to 5 Mb of tomato DNA, were obtained using methylation filtration (MF) strategy and reduced to 7,053 unique undermethylated genomic islands (UGIs) distributed as follows: (1) 59% non-coding sequences, (2) 28% coding sequences, (3) 12% transposons—96% of which are class I retroelements, and (4) 1% organellar sequences integrated into the nuclear genome over the past approximately 100 million years. A more detailed analysis of coding UGIs indicates that the unmethylated portion of tomato genes extends as far as 676 bp upstream and 766 bp downstream of coding regions with an average of 174 and 171 bp, respectively. Based on the analysis of the UGI copy distribution, the undermethylated portion of the tomato genome is determined to account for the majority of the unmethylated genes in the genome and is estimated to constitute 61±15 Mb of DNA (~5% of the entire genome)—which is significantly less than the 220 Mb estimated for gene-rich euchromatic arms of the tomato genome. This result indicates that, while most genes reside in the euchromatin, a significant portion of euchromatin is methylated in the intergenic spacer regions. Implications of the results for sequencing the genome of tomato and other solanaceous species are discussed.     

Different types of plant chromatin associated with modified histones H3 and H4 and methylated DNA

Eukaryotic chromosomes are organized into two large and distinct domains, euchromatin and heterochromatin, which are cytologically characterized by different degrees of chromatin compaction during interphase/prophase and by post-synthesis modifications of histones and DNA methylation. Typically, heterochromatin remains condensed during the entire cell cycle whereas euchromatin is decondensed at interphase. However, a fraction of the euchromatin can also remain condensed during interphase and appears as early condensing prophase chromatin. 5S and 45S rDNA sites and telomere DNA were used to characterize these regions in metaphase and interphase nuclei. We investigated the chromosomal distribution of modified histones and methylated DNA in the early and late condensing prophase chromatin of two species with clear differentiation between these domains. Both species, Costus spiralis and Eleutherine bulbosa, additionally have a small amount of classical heterochromatin detected by CMA/DAPI staining. The distribution of H4 acetylated at lysine 5 (H4K5ac), H3 phosphorylated at serine 10 (H3S10ph), H3 dimethylated at lysine 4 or 9 (H3K4me2, H3K9me2), and 5-methylcytosine was compared in metaphase, prophase, and interphase cells by immunostaining with specific antibodies. In both species, the late condensing prophase chromatin was highly enriched in H4K5ac and H3K4me2 whereas the early condensing chromatin was very poor in these marks. H3K9me2 was apparently uniformly distributed along the chromosomes whereas the early condensing chromatin was slightly enriched in 5-methylcytosine. Signals of H3S10ph were restricted to the pericentromeric region of all chromosomes. Notably, none of these marks distinguished classical heterochromatin from the early condensing euchromatin. It is suggested that the early condensing chromatin is an intermediate type between classical heterochromatin and euchromatin.     

Molecular cytogenetic maps of sorghum linkage groups 2 and 8

To integrate genetic, physical, and cytological perspectives of the Sorghum bicolor genome, we selected 40 landed bacterial artificial chromosome (BAC) clones that contain different linkage map markers, 21 from linkage group 2 (LG-02) and 19 from linkage group 8 (LG-08). Multi-BAC probe cocktails were constructed for each chromosome from the landed BACs, which were also preevaluated for FISH signal quality, relative position, and collective chromosome coverage. Comparison to the corresponding linkage map revealed full concordance of locus order between cytological and prior segregation analyses. The pericentromeric heterochromatin constituted a large quasi-uniform block in each bivalent and was especially large in the bivalent corresponding to LG-08. Centromere positions in LG-02 and LG-08 were progressively delimited using FISH to identify landed BACs for which the FISH signals visibly flanked the centromere. Alignment of linkage and cytological maps revealed that pericentromeric heterochromatin of these sorghum chromosomes is largely devoid of recombination, which is mostly relegated to the more distal regions, which are largely euchromatic. This suggests that the sorghum genome is thus even more amenable to physical mapping of genes and positional cloning than the C-value alone might suggest. As a prelude to positional cloning of the fertility restorer, Rf1, FISH of BAC clones flanking the Rf1 locus was used to delimit the chromosomal position of the gene. FISH of BACs that contain the most proximal linkage markers enabled localization of Rf1 to a approximately 0.4-Mbp euchromatic region of LG-08. Cytogenetic analyses of Rf1 and other trait loci will aid in assessing the feasibility of positional cloning and help formulate strategies required for cloning this and other agriculturally critical genes.     

Reinterpreting pericentromeric heterochromatin

In fission yeast, pericentromeric heterochromatin is directly responsible for the sister chromatid cohesion that assures accurate chromosome segregation. In plants, however, heterochromatin and chromosome segregation appear to be largely unrelated: chromosome transmission is impaired by mutations in cohesion but not by mutations that affect heterochromatin formation. We argue that the formation of pericentromeric heterochromatin is primarily a response to constraints on chromosome mechanics that disfavor the transmission of recombination events in pericentromeric regions. This effect allows pericentromeres to expand to enormous sizes by the accumulation of transposons and through large-scale insertions and inversions. Although sister chromatid cohesion is spatially limited to pericentromeric regions at mitosis and meiosis II, the cohesive domains appear to be defined independently of heterochromatin. The available data from plants suggest that sister chromatid cohesion is marked by histone phosphorylation and mediated by Aurora kinases.     

Pilot sequencing of onion genomic DNA reveals fragments of transposable elements,low gene densities,and significant gene enrichment after methyl filtration

Sequencing of the onion (Allium cepa) genome is challenging because it has one of the largest nuclear genomes among cultivated plants. We undertook pilot sequencing of onion genomic DNA to estimate gene densities and investigate the nature and distribution of repetitive DNAs. Complete sequences from two onion BACs were AT rich (64.8%) and revealed long tracts of degenerated retroviral elements and transposons, similar to other larger plant genomes. Random BACs were end sequenced and only 3 of 460 ends showed significant (e < -25) non-organellar hits to the protein databases. The BAC-end sequences were AT rich (63.4%), similar to the completely sequenced BACs. A total of 499,997 bp of onion genomic DNA yielded an estimated mean density of one gene per 168 kb, among the lowest reported to date. Methyl filtration was highly effective relative to random shotgun reads in reducing frequencies of anonymous sequences from 82 to 55% and increasing non-organellar protein hits from 4 to 42%. Our results revealed no evidence for gene-dense regions and indicated that sequencing of methyl-filtered genomic fragments should be an efficient approach to reveal genic sequences in the onion genome.     

HP1gamma associates with euchromatin and heterochromatin in mammalian nuclei and chromosomes

Heterochromatin protein 1 (HP1) is a nonhistone chromosomal protein, first identified in Drosophila, that plays a dose-dependent role in gene silencing. Three orthologs, HP1alpha, HP1beta, and HP1gamma, have been characterized in mammals. While HP1alpha and HP1beta have been unambiguously localized in heterochromatin by immunocytochemical methods, HP1gamma has been found either exclusively associated with euchromatin or present in both euchromatin and heterochromatin. Here, using an antibody directed against a peptide epitope at the carboxyl-terminal end of the molecule, we localize HP1gamma in both euchromatin and heterochromatin compartments of interphase nuclei, as well as in the pericentromeric chromatin and arms of mitotic chromosomes of 3T3 cells. This dual location was also observed in nuclei expressing HP1gamma as a fusion protein with green fluorescent protein. In contrast, when the distribution of HP1gamma was analyzed with antibodies directed against an amino-terminal epitope, the protein was detectable in euchromatin and not in heterochromatin, except for transient heterochromatin staining during the late S phase, when the heterochromatin undergoes replication. These data suggest that the controversial immunolocalization of HP1gamma in chromatin is due to the use of antibodies directed against topologically distinct epitopes, those present at the amino-terminal end of the molecule being selectively masked in nonreplicative heterochromatin.     

Comprehensive molecular cytogenetic analysis of sorghum genome architecture: distribution of euchromatin, heterochromatin, genes and recombination in comparison to rice

Cytogenetic maps of sorghum chromosomes 3-7, 9, and 10 were constructed on the basis of the fluorescence in situ hybridization (FISH) of approximately 18-30 BAC probes mapped across each of these chromosomes. Distal regions of euchromatin and pericentromeric regions of heterochromatin were delimited for all 10 sorghum chromosomes and their DNA content quantified. Euchromatic DNA spans approximately 50% of the sorghum genome, ranging from approximately 60% of chromosome 1 (SBI-01) to approximately 33% of chromosome 7 (SBI-07). This portion of the sorghum genome is predicted to encode approximately 70% of the sorghum genes ( approximately 1 gene model/12.3 kbp), assuming that rice and sorghum encode a similar number of genes. Heterochromatin spans approximately 411 Mbp of the sorghum genome, a region characterized by a approximately 34-fold lower rate of recombination and approximately 3-fold lower gene density compared to euchromatic DNA. The sorghum and rice genomes exhibit a high degree of macrocolinearity; however, the sorghum genome is approximately 2-fold larger than the rice genome. The distal euchromatic regions of sorghum chromosomes 3-7 and 10 are approximately 1.8-fold larger overall and exhibit an approximately 1.5-fold lower average rate of recombination than the colinear regions of the homeologous rice chromosomes. By contrast, the pericentromeric heterochromatic regions of these chromosomes are on average approximately 3.6-fold larger in sorghum and recombination is suppressed approximately 15-fold compared to the colinear regions of rice chromosomes.     

Characterization of the Sol3 family of nonautonomous transposable elements in tomato and potato

Sol3 transposons are mobile elements defined by long terminal inverted repeats which are found in tomato and potato. Members of the Sol3 family have been isolated from a variety of solanaceous species including Solanum tuberosum (potato), S. demissum, S. chacoense, Lycopersicon esculentum (tomato), and L. hirsutum. While highly conserved elements are found within different species, Sol3 terminal inverted repeats can also flank unrelated sequences. Southern blot analysis indicates that Sol3 elements are less prevalent in the potato (approximately 50 copies) than in the tomato (>100 copies) genome. No Sol3-hybridizing sequences were observed in tobacco. While a number of Sol3 elements ranging in size from 500 bp to 2 kbp were sequenced, no transposase coding domains could be identified within the internal regions of the elements. The data suggest that the Sol3 represent a heterogeneous family of nonautonomous transposable elements associated with an as-yet-unidentified autonomous transposon. Received: 18 September 1996 / Accepted: 11 March 1997