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Flow-sorted chromosomes have been used to simplify analyses of complex plant genomes. In bread wheat, majority of studies involve cultivar Chinese Spring, a genotype chosen for sequencing. Telosomic lines developed from this cultivar enable isolation by flow sorting chromosome arms, which represent less than 3.4 % of the genome. However, access to other wheat cultivars is needed to allow mapping and cloning useful genes. In these cultivars, cytogenetic stocks are not readily available and only one chromosome (3B) can be sorted. Remaining chromosomes form composite peaks on flow karyotypes and cannot be sorted. In order to overcome this difficulty, we tested a pragmatic approach in which composite chromosome peaks are dissected to smaller sections. The analysis of chromosome composition in sorted fractions confirmed feasibility of obtaining sub-genomic fractions comprising only a few chromosomes. Usually one of the chromosomes was more abundant and the frequencies of dominant chromosomes in sorted fractions ranged from 16 % (chromosome 7B) to 80 % (chromosome 2B). The enrichment factor, calculated as the relative proportion of chromosomal DNA in the wheat genome to the proportion of chromosomal DNA in a sorted fraction, ranged from 3.2-fold (7B) to 16.4-fold (5D). At least a 5-fold enrichment can be obtained for 17 out of 21 wheat chromosomes. Moreover, we show that 15 out of the 21 chromosomes can be sorted without being contaminated by their homoeologs. These observations provide opportunities for constructing sub-genomic large-insert DNA libraries, optical mapping, and targeted sequencing selected genome regions in various cultivars of wheat. The availability of fractions enriched for chromosomes of interest and free of contaminating homoeologs will increase the efficiency of research projects and reduce their costs as compared to whole genome approaches. The same methodology should be feasible in other plants where single chromosome types cannot be sorted.

Barley (Hordeum vulgare L.) genome has been difficult to analyze, map, and sequence due to its size (~5 Gb/1C) and high repeat content (over 80%). Flow cytometric sorting of plant mitotic chromosomes, which was first reported in 1984, provided an approach for reduction of complexity to simplify genome analysis and laid the basis of a new discipline—chromosomal genomics. Chromosome flow sorting in barley was established in 1999 and since that time played an important role in genomics of this crop. Initially, flow-sorted chromosomes facilitated assignment of DNA markers to chromosomes or their regions, providing a complement and validation to genetic mapping. A protocol for multiple displacement amplification of chromosomal DNA, established for mapping on Illumina genotyping platform, was shown compatible with next-generation sequencing. This enabled identification of gene content of individual barley chromosomes and establishment of their putative order by an innovative approach, which was later followed also in wheat and rye. Preparation of high-molecular-weight DNA from flow-sorted chromosomes, while developed in barley, opened avenues to constructing chromosome-specific BAC libraries in bread wheat and, more recently, generating optical maps in several cereal species. Thus, the development of a chromosome genomics toolbox contributed to the advances in genomics of other important crops, in addition to barley. Chromosomal genomics plays an important role also in the post-sequencing era by enabling cost-efficient gene cloning, analyzing chromatin structure by chromatin conformation capture approaches, and characterizing the proteome of mitotic chromosomes.

The sequencing of individual chromosomes of common wheat is in progress. The molecular size of wheat chromosome 5B is nearly 870 Mb (5BL = 580 Mb and 5BS = 290 Mb). We produced the first low coverage 454-sequencing of the long and short arms of wheat chromosome 5B (110,793 and 39,695 reads, which compose 8 and 6% of total 5BL and 5BS length, respectively) and calculated the ratios of the different families of repetitive sequences, including transposable elements (TEs), satellite repeats (Afa, pSc119.2 , 5S rDNA and 45S rDNA), and microsatellites, as well as direct and inverted repeat motifs. The TEs accounted for 70% of the total analyzed nucleotide sequences. The content of the Cereba retrotransposon family differed between the two arms of chromosome 5B. Comparing the reads of chromosome 5B with the data from chromosome 5A, we found the retrotransposons Fatima and Sakura and DNA transposon Jorge were prevalent in 5B. The hypothetical coding sequences accounted for 2.0% of the short arm and 2.07% of the long arm. Using in silico mapping, we identified the regions of synteny with rice and Brachypodium chromosomes (1,073,526 and 1,767,298 bp aligned, respectively), and the result was consistent with the data from the expressed sequence tag (EST) mapping of wheat 5B chromosome to the genomes of these grasses. Thus, these results show that low coverage survey sequencing can provide useful information about the composition and evolution of wheat chromosome 5B. T he common wheat (Triticum aestivum L., 2n = 6x = 42) is an allohexaploid composed of three differ ent genomes (AABBDD) that originated from T. urartu Thum. (AA), an Aegilops species related to the Sitopsis section (presumably Ae. speltoides Tausch) (BB), and Ae. tauschii Coss. (DD). Triticum aestivum is a relatively young polyploid derived from two successive interspecific crosses dated approximately 0.5 million years ago and 8,000 to 10,000 yr ago. Bread wheat has a large genome of 17,000 Mb, which mainly consists of repetitive DNA sequences of different origin, and 90% of the genome is reiterative, 80% of which is composed of TEs (Paux et al., 2006; Choulet et al., 2010; Brenchley et al., 2012). For these reasons, sequencing the wheat genome is a challenge, for which the international scientific community has established the International Wheat Genome Sequencing Consortium. To solve the problem of polyploidy in the wheat genome, the

A novel approach for simultaneous localization of two DNA sequences on plant chromosomes is described. The approach is based on a combined use of primed in situ DNA labelling (PRINS) with fluorescent in situ hybridization (FISH). Traditionally, this has been done using FISH with two probes labelled by two different marker molecules. Compared to this method, the combined PRINS-FISH procedure is faster. Furthermore, because one of the DNA sequences is localized by PRINS with specific primers, only one labelled probe is needed.

Primed in situ labelling (PRINS) of nucleic acids was developed as an alternative to traditionally used fluorescence in situ hybridization (FISH). Compared to FISH, PRINS is faster and does not require preparation of labelled probes. Nevertheless, the number of applications for physical mapping of DNA sequences on plant chromosomes remains low. This is due to the fact that there are a number of factors which influence the specificity and sensitivity of the reaction. The purpose of this work was to analyse the effect of some of them, including the age of slides, type of Taq DNA polymerase, number and concentration of primers, the presence and concentration of bovine serum albumine and MgCl2 in the reaction mixture. Furthermore, the effect of various pre-treatments on signal intensity and non-specific fluorescence was studied. A consensus Arabidopsis-type telomeric sequence and Vicia faba mitotic chromosomes were used as a model system. We have found that the age of slides was critical and that under optimal conditions it was possible to achieve relatively high signal to noise ratio.

Analyses of large genomes are hampered by high proportions of repetitive DNA, that make the assembly of short sequence reads difficult. This is also the case for meadow fescue (Festuca pratensis Huds.), one of predominant grass species in temperate and Northern regions with the genome size estimated at 1C = 3,175 Mbp. This species is known for its ability to survive under freezing conditions and it has been used widely in intergeneric hybridization with various ryegrass species to produce superior Festulolium cultivars. Here we describe attempts to dissect the meadow fescue’s genome into smaller fractions—individual chromosomes and groups of chromosomes. Following the methods of flow cytogenetics developed for legumes and cereals, we have developed a chromosome sorting protocol for grasses and currently we are able to sort F. pratensis chromosome 4 (the largest in the genome) and two groups of three chromosomes each: 2, 3, 7 and 1, 5, 6. As the first step we sequenced chromosome 4 by Illumina with 50x coverage and assembled low copy and genic regions. This facilitated detailed comparative analysis with sequenced genomes of rice, Brachypodium and sorghum and provided the first insight into the genome composition of this species. The possibility to purify chromosome 4 opens the way for a more efficient analysis of genetic loci on this chromosome that control important agronomic traits, such as freezing tolerance. Moreover, purified chromosomes are excellent templates for PCR screening as well as cytogenetic and physical mapping.

The large bread wheat genome (1C ∼ 17 Gbp) contains a preponderance of repetitive DNA and the species is polyploid. These characteristics together serve to hamper the molecular analysis of the wheat genome. Its complexity can, however, be reduced by using flow cytometry to isolate individual chromosomes, and these can be exploited to construct chromosome-specific BAC libraries. Such libraries simplify the task of physical map construction, positional cloning and the targeted development of genetic markers. Rapid improvements in the efficiency and cost of DNA sequencing provide an opportunity to contemplate sequencing the wheat genome by preparing sequence-ready physical maps for each chromosome or chromosome arm in turn. The quality of the chromosome-specific libraries depends on their chromosome coverage and the mean insert size. First-generation libraries suffered from a relatively low mean insert size, but improvements to the protocol have generated a second wave of libraries with a significantly increased mean insert size and better chromosome coverage. Each chromosome (arm)-specific library is composed of a manageable number of clones, and so represents a practical tool in the area of wheat genomics.

Procedures for flow cytometric analysis and sorting of mitotic chromosomes (flow cytogenetics) have been developed for chickpea (Cicer arietinum). Suspensions of intact chromosomes were prepared from root tips treated to achieve a high degree of metaphase synchrony. The optimal protocol consisted of a treatment of roots with 2 mmol/L hydroxyurea for 18 h, a 4.5-h recovery in hydroxyurea-free medium, 2 h incubation with 10 micromol/L oryzalin, and ice-water treatment overnight. This procedure resulted in an average metaphase index of 47%. Synchronized root tips were fixed in 2% formaldehyde for 20 min, and chromosome suspensions prepared by mechanical homogenization of fixed root tips. More than 4 x 10(5) morphologically intact chromosomes could be isolated from 15 root tips. Flow cytometric analysis of DAPI-stained chromosomes resulted in histograms of relative fluorescence intensity (flow karyotypes) containing eight peaks, representing individual chromosomes and/or groups of chromosomes with a similar relative DNA content. Five peaks could be assigned to individual chromosomes (A, B, C, G, H). The parity of sorted chromosome fractions was high, and chromosomes B and H could be sorted with 100% purity. PCR on flow-sorted chromosome fractions with primers for sequence-tagged microsatellite site (STMS) markers permitted assignment of the genetic linkage group LG8 to the smallest chickpea chromosome H. This study extends the number of legume species for which flow cytogenetics is available, and demonstrates the potential of flow cytogenetics for genome mapping in chickpea.

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.

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.

Embryogenic callus cultures were established from immature cucumber(Cucumis sativus L.) embryos on E20A (Dumas de Vaulxet al. 1981) or MS (Murashige and Skoog 1962) media supplemented with 6-benzylaminopurine (BAP), α-naphthylacetic acid (NAA) and/or 2,4-dichlorophenoxyacetic acid (2,4-D). Regeneration of plants was observed after a transfer to culture media either without growth regulators or supplemented with kinetin and NAA. Flow cytometry was employed to estimate DNA ploidy levels. Most of cell nuclei in young leaf tissues were found in G1 phase with 2C DNA content. Callus cultures were mixoploid with DNA content ranging from 2C to 32C. The frequency of polyploid cells was increasing with the age of culture and the polyploidization was accompanied by a gradual loss of regeneration ability. Plants regenerated from callus cultures were classified as diploid (57 %), tetraploid (18 %), octoploid (4 %) and mixoploid (2n/4n, 4 %) and (4n/8n, 17 %). The results of this study confirmed a close link between the polyploidization and the loss of totipotencyin vitro. Tetraploid plants obtained in this study have a potential to be used in interspecific crosses where their tetraploid status could help in overcoming existing breeding barriers due to differences in chromosome number.