Reciprocal and multi-species chromosome BAC painting in crucifers (Brassicaceae).

In crucifer cytogenomics, BAC contigs of Arabidopsis thaliana have been used as probes for comparative chromosome painting among species. Here we successfully tested chromosome-specific BAC contigs of A. thaliana (n = 5) and A. halleri (n = 8) as probes for reciprocal BAC painting. Furthermore, BAC contigs of both Arabidopsis species were applied as multi-species painting probes to a third crucifer species, Noccaea caerulescens (n = 7), revealing their shared chromosome homeology. Specifically, we found homeology across portions of chromosomes At2, Ah4, and Nc4, which reflects their shared common origin with chromosome AK4 of the Ancestral Crucifer Karyotype (n = 8). We argue that multi-species and multi-directional painting will significantly expedite comparative cytogenomics in Brassicaceae and other plant families.

Punctuated genome size evolution in Liliaceae.

Most angiosperms possess small genomes (mode 1C = 0.6 pg, median 1C = 2.9 pg). Those with truly enormous genomes (i.e. > or = 35 pg) are phylogenetically restricted to a few families and include Liliaceae - with species possessing some of the largest genomes so far reported for any plant as well as including species with much smaller genomes. To gain insights into when and where genome size expansion took place during the evolution of Liliaceae and the mode and tempo of this change, data for 78 species were superimposed onto a phylogenetic tree and analysed. Results suggest that genome size in Liliaceae followed a punctuated rather than gradual mode of evolution and that most of the diversification evolved recently rather than early in the evolution of the family. We consider that the large genome sizes of Liliaceae may have emerged passively rather than being driven primarily by selection.

Localisation of DNA sequences on plant chromosomes using PRINS and C-PRINS.

Localisation of DNA sequences to plant chromosomes in situ has traditionally been accomplished using fluorescence in situ hybridisation (FISH). Although the method is suitable for most applications it is time-consuming and requires labelled probes. Recently, primed in situ labelling (PRINS) has been developed as an alternative to FISH. PRINS is based on annealing of unlabelled oligonucleotide primer(s) to chromosome DNA and its elongation by DNA polymerase in the presence of labelled nucleotide(s). The method was found useful to detect high-copy tandem repeats on plant chromosomes. Low copy repeats were detected after a more sensitive variant of PRINS called cycling PRINS (C-PRINS), which involves a sequence of thermal cycles analogous to polymerase chain reaction. This paper describes protocols of PRINS and C-PRINS, which have been optimised for chromosome spreads and for chromosomes purified using gradient centrifugation and/or flow sorting. The methods result in clear signals with negligible non-specific labelling. Further work is needed to improve the sensitivity to allow for reliable detection of single- copy DNA sequences.

Heterogeneity of rDNA distribution and genome size in Silene spp.

Genus Silene L. (Caryophyllaceae) contains about 700 species divided into 44 sections. According to recent taxonomic classification this genus also includes taxa previously classified in genera Lychnis and Melandrium. In this work, four Silene species belonging to different sections were studied: S. latifolia (syn. Melandrium album, Section Elisanthe), S. vulgaris (Inflatae), S. pendula (Erectorefractae), and S. chalcedonica (syn. Lychnis chalcedonica, Lychnidiformes). Flow cytometric analysis revealed a genome size of 2.25 and 2.35 pg/2C for S. vulgaris and S. pendula and of 5.73 and 6.59 pg/2C for S. latifolia and S. chalcedonica. All four species have the same chromosome number including the pair of sex chromosomes of the dioecious S. latifolia (2n = 2x = 24). Double target fluorescence in-situ hybridization revealed the chromosomal locations of 25S rDNA and 5S rDNA. A marked variation in number and localization of rDNA loci but no correlation between the numbers of rDNA clusters and genome size was found. FISH and genome size data indicate that nuclear genomes of Silene species are highly diversified as a result of numerous DNA amplifications and translocations.

Chromosome painting in Arabidopsis thaliana.

Chromosome painting, that is visualisation of chromosome segments or whole chromosomes based on fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes is widely used for chromosome studies in mammals, birds, reptiles and insects. Attempts to establish chromosome painting in euploid plants have failed so far. Here, we report on chromosome painting in Arabidopsis thaliana (n = 5, 125 Mb C(-1)). Pools of contiguous 113-139 BAC clones spanning 2.6 and 13.3 Mb of the short and the long arm of chromosome 4 (17.5 Mb) were used to paint this entire chromosome during mitotic and meiotic divisions as well as in interphase nuclei. The possibility of identifying any particular chromosome region on pachytene chromosomes and within interphase nuclei using selected BACs is demonstrated by differential labelling. This approach allows us, for the first time, to paint an entire autosome of an euploid plant to study chromosome rearrangements, homologue association, interphase chromosome territories, as well as to identify homeologous chromosomes of related species.

Flow sorting of mitotic chromosomes in common wheat (Triticum aestivum L.).

The aim of this study was to develop an improved procedure for preparation of chromosome suspensions, and to evaluate the potential of flow cytometry for chromosome sorting in wheat. Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity (flow karyotypes) obtained after the analysis of DAPI-stained chromosomes were characterized and the chromosome content of all peaks on wheat flow karyotype was determined for the first time. Only chromosome 3B could be discriminated on flow karyotypes of wheat lines with standard karyotype. Remaining chromosomes formed three composite peaks and could be sorted only as groups. Chromosome 3B could be sorted at purity >95% as determined by microscopic evaluation of sorted fractions that were labeled using C-PRINS with primers for GAA microsatellites and for Afa repeats, respectively. Chromosome 5BL/7BL could be sorted in two wheat cultivars at similar purity, indicating a potential of various wheat stocks for sorting of other chromosome types. PCR with chromosome-specific primers confirmed the identity of sorted fractions and suitability of flow-sorted chromosomes for physical mapping and for construction of small-insert DNA libraries. Sorted chromosomes were also found suitable for the preparation of high-molecular-weight DNA. On the basis of these results, it seems realistic to propose construction of large-insert chromosome-specific DNA libraries in wheat. The availability of such libraries would greatly simplify the analysis of the complex wheat genome.

Rapid identification and determination of purity of flow-sorted plant chromosomes using C-PRINS.

BACKGROUND: Flow-sorted plant chromosomes are being increasingly used in plant genome analysis and mapping. Consequently, there is a need for a rapid method for identification of sorted chromosomes and for determination of their purity. We report on optimization of procedures for primed in situ DNA labeling (PRINS) and cycling-PRINS (C-PRINS) for fluorescent labeling of repetitive DNA sequences on sorted plant chromosomes suitable for their identification. METHODS: Chromosomes of barley, wheat, and field bean were sorted onto microscope slides, dried, and subjected to PRINS or C-PRINS with primers for GAA microsatellites (barley and wheat) or FokI repeat (field bean). The following parameters were optimized to achieve the highest specificity and intensity of fluorescent labeling: ratio of labeled versus unlabeled nucleotides, nucleotide concentration, and the number and concentration of primers. RESULTS: Under optimal conditions, C-PRINS resulted in strong and specific labeling of GAA microsatellites on sorted barley and wheat chromosomes and FokI repeats on sorted field bean chromosomes. The labeling patterns were characteristic for each chromosome and permitted their unequivocal identification as well as determination of purity after sorting, which ranged from 96% to 99%. A standard polymerase chain reaction (PCR) with chromosome-specific primers was not sensitive enough to detect low-frequency contamination. CONCLUSIONS: The results indicate that a single C-PRINS assay with primers that give chromosome-specific labeling pattern is sufficient not only to determine chromosome content of peaks on flow karyotype but also to determine the purity of sorted chromosome fractions. The whole procedure can be performed in less than 3 h on the next day after sorting. Numerous applications are expected in the area of plant flow cytogenetics.

Flow karyotyping and sorting of mitotic chromosomes of barley (Hordeum vulgare L.).

A high-yield method for isolation of barley chromosomes in suspension, their analysis and sorting using flow cytometry is described. To accumulate meristem root tip cells at metaphase, actively growing roots were subjected to subsequent treatment with 2 mmol/L hydroxyurea for 18 h, 2.5 micromol/L amiprophos methyl for 2 h, and ice water (overnight). This treatment resulted in metaphase indices exceeding 50%. Synchronized root tips were fixed in 2% formaldehyde for 20 min and chromosomes were released into a lysis buffer by mechanical homogenization, producing, on average, 5 x 10(5) chromosomes from 50 root tips. The isolated chromosomes were morphologically intact and suitable for flow cytometric analysis and sorting. While it was possible to discriminate and sort only one chromosome from a barley cultivar with standard karyotype, up to three chromosomes could be sorted in translocation lines with morphologically distinct chromosomes. The purity of chromosome fractions, estimated after PRINS with primers specific for GAA microsatellites, reached 97%. PCR with chromosome-specific primers confirmed the purity and suitability of flow-sorted chromosomes for physical mapping of DNA sequences.