*Correspondjng author: Jaroslav Dolezel
Institute of Experimental Botany, Laboratory of Molecular Cytogenetics and Cytometry, Sokolovska 6, CZ-77200 Olomouc, Czech Republic
E-mail address: dolezel@ueb.cas.cz

Key words: chromosome addition lines, chromosome purification, cytogenetics, subgenomic DNA libraries, flow cytometry, genomics, physical mapping, telosomes

Introduction

This memorial issue of the Wheat Information Service celebrates not only many years of a successful international journal, but also the immense progress that has been achieved in genetic research on wheat and in wheat breeding. Looking back, it is fascinating to see how much we owe to the work of earlier generations of wheat geneticists who laid the foundations of current research, including genomics. It would certainly give them a rewarding feeling. In this chapter, we discuss the application of flow cytogenetics to simplify and streamline the analysis of the wheat genome. We envisage as a result of the availability of cytogenetic stocks of wheat, many of them created by our predecessors, flow cytogenetics is bound to make a serious impact in many areas of wheat genome mapping, sequencing and gene cloning.

Why can flow cytogenetics be so useful in wheat genetics? Bread wheat has a very large and complex nuclear genome. With the genome comprising about 17 billion bases (Bennett and Smith 1991), wheat is the world leader in genome size among major agricultural crops. The genome, composed predominantly of repetitive DNA (Smith and Flavell 1975), is more than a hundredfold larger than the Arabidopsis genome; each of the wheat chromosomes is larger than the entire rice genome, which has only recently been sequenced (Goff et al. 2002). Being an allohexaploid species (2n = 6x = 42), the wheat chromosome complement consists of three closely related genomes, A, B, and D, resulting in great genetic redundancy. The genome of allotetraploid durum wheat (2n = 4x = 28, AABB) is only slightly less complex. These unique features pose great challenges for gene discovery and genome sequencing.

It would greatly simplify these tasks if the genome could be physically dissected into small and defined parts. Taking into account the genome size and the relative sizes of individual chromosomes (Gill et al. 1991), the DNA content of individual chromosomes ranges from 606 to 995 Mb, representing only 3.6 to 5.9 per cent of the whole genome. Dissection of individual chromosome arms would provide even smaller fractions of the entire genome, ranging from 224 to 582 Mb, or only 1.3 to 3.4 per cent of the total DNA content. This falls within the range of the genome sizes of rice, tomato, barrel clover and other crop species selected, or being considered, for sequencing because of their small genomes. Our work has established that individual chromosomes and chromosome arms of wheat can indeed be purified using flow cytometric sorting (Kubalakova et al. 2002, Vrana et al. 2000). This has opened avenues for various applications of flow cytogenetics in wheat genetics and genomics.

Flow cytogenetics

Flow cytogenetics is the use of flow cytometry for classification and sorting of chromosomes isolated from mitotic cells (Dolezel et al. 2004a). A liquid suspension of metaphase chromosomes is stained by a DNA-specific fluorescent dye and introduced into the instrument, where it is forced to travel in a narrow stream of fluid at high speed. The chromosomes pass individually through a beam of intense light, and short pulses of scattered light and the fluorescence of stained chromosomes are quantified (Fig. 1). The analysis (called flow karyotyping) facilitates classification of large populations of chromosomes according to their size and relative fluorescence intensity. Specific chromosomes may be purified by breaking the liquid stream into droplets, and using an electrostatic field to deflect into a collection tube electrically charged droplets containing chromosomes of interest (Dolezel et al. 2005). Depending on the actual model, commercial flow sorters allow simultaneous sorting of up to four different chromosomes.

Preparation of a liquid suspension of mitotic chromosomes in plants requires cell populations enriched in mitotic cells, and the ability to release intact chromosomes from cells with rigid cell walls. Although different protocols have been used in plants, the protocol developed by Dolezel et al. (1992) has been used almost exclusively (for review see Dolezel et al. 2004b). In this protocol, a chromosome suspension is prepared by mechanical homogenization of formaldehyde-fixed root tips. Prior to fixation, meristematic cells from root tips are accumulated in metaphase by a combined treatment with a DNA synthesis inhibitor and a mitotic spindle poison (Dolezel et al. 1992).
Preparation of the chromosome suspension is the single major obstacle preventing an easy adaptation of the method to new species. This is because many steps of the protocol must be optimized for a given species, which is laborious and time consuming (Dolezel et al. 1999).

The protocol was originally developed for field bean (Dolezel et al. 1992), and was subsequently modified for garden pea (Gualberti et al. 1996; Neumann et al. 1998), another legume. In 1999 we modified it for barley (Lysak et al. 1999), followed by bread wheat (Vrana et al. 2000) and, more recently, durum wheat (Kubalakova et al. 2005). Using the protocol, ∼5 x 10⁵ chromosomes can be prepared from 30 wheat root tips (Vrana et al. 2000). In our laboratory, the chromosomes are analyzed at a speed of about 1,000 per second, using a Becton Dickinson FACSVantage SE flow sorter. Before focusing on chromosome sorting, let us see what may be learned from classification of chromosomes using flow cytometry.

Flow karyotyping

Classification of chromosomes by flow cytometry results in a histogram of relative fluorescence intensity, the flow karyotype. Because of the high speed of analysis, it is easy to evaluate 10⁴ to 10⁵ chromosomes to generate reproducible profiles. Ideally, each chromosome is represented by a single peak on a flow karyotype. In tetraploid and hexaploid wheat, however, many chromosomes do not differ significantly in fluorescence intensity from one another, and their peaks overlap to form several composite peaks (Vrana et al. 2000; Kubalakova et al. 2005). In both species, only chromosome 3B is resolved as a single peak, and only this chromosome can be purified using flow sorting (Figs. 2a, 3a) (Table 1). Various modifications of chromosome staining and analysis were tested without success to resolve the composite peaks (Dolezel et al., unpublished). The ultimate solution came from using cytogenetic stocks, as we shall demonstrate later.

Interpretation of a flow karyotype requires the assignment of individual peaks to particular chromosomes. How this can be done? A simple approach is to compare the experimental karyotype with a model. Taking into account the relative chromosome lengths and the expected coefficients of variation of chromosome peaks, a theoretical flow karyotype can be developed using suitable software (Dolezel 1991). Nevertheless, the chromosome content of individual peaks can be reliably determined only by analyzing the particles sorted from them. One way is to sort several hundred chromosomes into a PCR tube and run the PCR with chromosome-specific primers (Vrana et al. 2000). An approach preferred by us is to sort chromosomes onto a microscope slide, air-dry them, and evaluate them microscopically after FISH with fluorescently labeled probes for DNA sequences that exhibit a chromosome-specific distribution (Vrana et al. 2000; Kubalakova et al. 2002).

In our previous work, we have demonstrated that flow cytometry is sensitive enough to detect variations in chromosome number and structure (reviewed by Dolezel et al. 2004b). Any change in relative frequency of occurrence of a particular chromosome in a population translates into a change in the chromosome peak area. Any change in the chromosome size due to a deletion or a translocation results in a change of the relative fluorescence intensity, and thus in an alteration of the peak position on the flow karyotype. Finally, the presence of an alien chromosome may be detectable provided it differs in size from the chromosomes of the recipient species. All these points have been confirmed for wheat.

For example, wheat cultivars known to carry the 5BL·7BL translocation generate flow karyotypes with an additional peak located to the right of chromosome 3B (Fig. 2b). This is because the 5BL·7BL translocation chromosome is considerably longer than 3B (Kubalakova et al. 2002). This study demonstrated a remarkable sensitivity and reproducibility of flow karyotyping. Not only did this facilitate the detection of translocation chromosomes in cultivars where their presence was known, including the 1RS.1BL translocation, but it also permitted the identification of cultivars where the presence of modified chromosomes had not already been identified. In some cultivars, flow karyotypes differed from the consensus flow karyotype such as that produced for 'Chinese Spring'. For example, in cv. 'Mona' and 'Rexia', the composite peak I is split into two peaks. Although the nature of the structural chromosome change responsible for this alteration is not known, the modified flow karyotype is diagnostic, and was transmitted from the Russian parental cultivar 'lljitschjewka' (Kubalakova et al. 2002).

Alien chromosome addition lines of wheat have been created for gene mapping and chromosome engineering (Gonzalez et al. 2002; Li et al. 2003; Watanabe et al. 2002; Sourdille et al. 1999). Our experience shows that these addition lines can be equally useful in flow cytogenetics. In flow karyotypes of wheat-barley chromosome addition lines the peaks of barley chromosomes overlap with those of wheat and no barley chromosome can be isolated (Dolezel et al., unpublished). However, Suchankova et al. (2005) found that, using wheat- barley telosome addition lines, all armms of barley chromosome 2H-7H could be discriminated (Fig. 2d). This observation represents important progress for the flow cytogenetics of barley, where only chromosome 1H can be discriminated in the wild-type karyotype (Lysak et al. 1999). Additionally, sorting the chromosome arms provides even smaller genome fractions than the intact wild-type chromosomes. The use of wheat-barley telosome addition lines will facilitate purification of twelveout of fourtee barley chromosome arms by flow sorting.

A similar observation was made in rye, where only the smallest chromosome, 1R, can be sorted from some cultivars; the remaining six chromosomes form a composite peak. However, these six chromosomes are easily discriminated, and thus sorted, from wheat-rye chromosome addition lines (Kubalakova et al. 2003). On the flow karyotypes, peaks of rye chromosomes are located to the right of chromosome 3B (Fig. 2c). Flow karyotyping of individual wheat-rye addition lines also helped to determine the frequency of rye chromosomes in the populations. While the analysis of most of the addition lines suggested a disomic status of the added rye chromosome, and hence good stability of the line, reduced frequencies of chromosomes 5R and 7R were observed (Kubalakova et al. 2003). Rye chromosomes are frequently eliminated from addition lines, and chromosome 5R is particularly unstable (Miller 1984). The power of flow cytometry is that it analyses large populations of chromosomes, and thus accurately determines the transmission rates of alien chromosomes.

Due to their polyploid nature, tetraploid durum wheat and hexaploid bread wheat tolerate aneuploidy. This has permitted production of a complete series of telosomic lines (Sears 1966; Joppa 1993). The absence of one of the chromosome arms makes the telosomes much shorter. As a consequence, their peaks on the flow karyotype appear to the left of the peaks representing the remaining bi-armed chromosomes (Fig. 2e). In double ditelosomic lines, the short and long arms can be resolved as separate peaks. From the 42 arms of bread wheat, 40 can be easily discriminated and sorted (Kubalakova et al. 2002). The only two exceptions are the long arms of chromosomes 3B and 5B, whose peaks overlap with the composite peak I, which represents the three smallest chromosomes (1D, 4D, and 6D). As the D-genome chromosomes are absent in durum wheat, the peaks of 3BL and 5BL are easily resolved in this species (Kubalakova et al. 2005) (Fig. 3b). The composite peak II of the durum wheat flow karyotype consists of two chromosomes, 1A and 6A. Once one of these chromosomes is replaced by telocentrics, peak II represents only one chromosome that can be sorted.

Because of the cost, the use of flow cytometry for karyotype analysis can be justified only in specific cases. The most important outcome of our flow karyotyping experiments was the discovery of the immense potential of cytogenetic stocks, namely ditelosomic and double ditelosomic lines, for physically dissecting the wheat genome into small and defined parts. Because polyploid wheat has the capacity to maintain chromosomes of other genomes of the tribe Triticeae, either as addition, substitution or translocation lines, flow cytogenetics of wheat offers a universal flow-based platform for dissecting not only the genomes of hexaploid and tetraploid wheats themselves, but also the genomes of a wide range of related species (Table 2). So far, only barley and rye additions have been analyzed in detail but the list of alien introgressions in wheat is long.

Flow sorting

Provided the wheat chromosome suspension is analyzed at a rate of ∼10³ particles per second, a particular chromosome can be sorted at a rate of about 25 copies per second. The sort rate is lower than expected because the machine classifies all incoming particles which are present in the suspension, including chromatids and chromosome debris. The sort yield depends on the concentration of the chromosome of interest in the suspension. As the sample quality varies to a certain extent, typical daily yields range from 2 to 5 x10⁵ sorted wheat chromosomes.

In addition to sort yield, the purity of the fractions, that is, the absence of any contamination from other parts of the genome, is of paramount importance. In our hands, microscopic analysis of sorted fractions after FISH provides the most complete information on the nature of sorted particles. If telosomes are sorted, dual-color FISH is performed with one of the probes specific for telomeric repeats. This discriminates between the telocentrics with telomeric repeats present on both termini that are the original telocentrics present in the line, and the chromosome arms resulting from breakage of complete chromosomes during sample handling. The purity of sorted fraction ranges from 85 to 99% (Kubalakovaet al. 2002, 2003, 2005) and depends on many factors, with the quality of the chromosome suspension and the speed of sorting being the two most important.

There are two principal applications of flow-sorted chromosomes: microscopic analysisof chromosomes sorted onto glass slides, and the use of DNA from specific chromosomes in molecular analyses. Physical mapping of DNA sequences using PCR with specific primers was one of the first uses of sorted chromosomes in plants. The fact that several hundred chromosomes, which are a sufficient template for PCR, can be sorted in a very short time makes this approach very attractive. It has been used to link genetic and physical maps in field bean, garden pea and chickpea (Macas et al. 1993; Neumann et al. 2002; Vlacilova et al. 2002). In field bean and garden pea, translocation chromosomes were sorted and used for PCR, an approach that facilitated the mapping of specific DNA sequences to subchromosomal regions.

Without any doubt, the most attractive use of sorted chromosomes is the production of chromosome- and chromosome arm-specific DNA libraries. Such libraries provide a permanent and easily accessible molecular resource specific for a particular part of the genome. Although the construction of short-insert DNA libraries is not a demanding task, so far only one library of this type has been created in wheat (Wang et al. 1992). Given the possibility of purifying chromosome arms in wheat, the creation of such libraries and their use for targeted isolation of molecular markers to saturate genetic maps at specific genome regions is worth consideration (Pozarkova et al. 2002; Roman et al. 2004).

A great deal of attention has been paid to the creation of large-insert DNA libraries, namely libraries cloned in a BAC vector. The main interest stems from the importance of large-insert DNA clones for the preparation of physical contig maps in genome sequencing projects, as well as in positional gene cloning (Meyers et al. 2004). Physical maps are usually produced by ordering DNA clones from BAC libraries on the basis of the clone fingerprint pattern (Luo et al. 2003). Due to the complexity of their genomes, BAC libraries of durum and bread wheats with adequate genome coverage comprise 0.5-1 x 10⁶ clones (Wang et al. 1992). Maintenance and handling of such numbers is difficult and expensive. The cost and difficulty are also important factors in the use of such libraries to develop physical contig maps. In this context, chromosome and chromosome-arm specific BAC libraries, which need to consist of only 0.5-1 x 10⁵ clones to provide excellent coverage, represent a powerful alternative.

Construction of a genomic BAC library requires high molecular weight DNA. We have developed a protocol for preparation of intact DNA from flow-sorted plant chromosomes (Simkova et al. 2003). The availability of an efficient protocol for BAC cloning (Chalhoub et al. 2004), then opened the road to the creation of BAC libraries from specific wheat chromosomes. A subgenomic BAC library was created from 10⁷ 'Chinese Spring' chromosomes 1D, 4D arid 6D (about 13 mug DNA) sorted from the composite peak I (Fig. 2a). The library comprises 87,168 clones with an average insert size of 85 kb, and represents a 3.4-fold coverage of the three chromosomes (Janda et al. 2004). A chromosome-specific BAC library was created from 'Chinese Spring' chromosome 3B (Safar et al. 2004). The library was prepared from only 1.8 x 10⁶ chromosomes (about 4 mug DNA) that were sorted from the peak 3B (Fig. 2a) and is the first ever chromosome-specific BAC library created in plants. It consists of 67,968 clones with an average insert size of 103 kb and represents a 6.2-fold chromosome coverage of the chromosome. Finally, a BAC library was constructed from the short arm of chromosome 1B (1BS) of cv. 'Pavon 76'. Six million chromosome arms (equivalent to 4 mug DNA) were used to obtain 65,280 clones with an average insert size of 82 kb, giving a 14.5 times coverage of the arm (Janda et al., in preparation). This last success confirmed that BAC libraries can be created from specific chromosome arms in wheat.

The use of chromosome- and chromosome arm-specific BAC libraries, which represent no more than a few per cent of the bread wheat genome, is expected to have a major impact on wheat genomics (Gill et al. 2004). Preliminary results obtained with the 3B -specific BAC library confirm these expectations. The small size and the specificity of the library greatly facilitate map-based cloning of agronomically important genes, such as the major QTL for Fusarium head blight resistance (Liu et al. 2005) and a durable rust resistance gene (W. Spielmeyer, pers. comm.). The complete library could be fingerprinted in less than six months, and the construction of a physical map of chromosome 3B is advancing rapidly (C. Feuillet, pers. comm.).

There are other potential uses of DNA from flow-sorted chromosomes that merit attention. In order to limit redundant sequencing of repetitive elements from large genomes, some research groups have developed procedures to capture gene-rich portions of the genome. The methylation filtration strategy is based on the notion that, unlike most of the repetitive elements, individual genes are relatively undermethylated (Moore et al. 1993). The filtering relies on the McrBC restriction-modification system of Escherichia coli (Rabinowicz et al. 1999). The so-called Cot fractionation is based on the observation that at a given concentration, repetitive DNA reassociates faster than the low-copy sequences (Peterson et al. 2002). This can be used to reduce the abundance of repetitive DNA and enrich for genic DNA (Yuan et al. 2003).

Methylation filtration has already been tested on wheat (Li et al. 2004) and both the Cot fractionation and methylation filtration should be compatible with flow cytogenetics. Unlike working with total genomic DNA, filtration of chromosome arm-specific DNA would further reduce the complexity of the process. While methylation filtration requires native chromosomal DNA, Cot fractionation can be performed with DNA isolated from a pooled chromosome arm-specific BAC library. An attractive possibility is to use DNA from sorted chromosomes for hybridization on DNA arrays and chips, with the aim of mapping DNA sequences to specific chromosome arms. This application can now be vigorously euxplored in the light of the recent production of the Affyinetrix wheat GeneChip (T. Close, pers. comm.). The list of the potential uses of sorted chromosomes would not be complete without including HAPPY mapping (Thangavelu et al. 2003) and optical mapping (Aston et al. 1999), althogh the potential of these methods for developing physical maps in wheat remains to be shown.

The second large area of application of sorted wheat chromosomes involves analyses of the long-range molecular structure of chromosomes and cytogenetic mapping. An opportunity to analyze microscopically hundreds of copies of a particular chromosome on one microscope slide makes this approach in many ways superior to the traditionally used metaphase spreads. FISH mapping on sorted chromosomes offers higher throughput and a chance to detect chromosome polymorphism. Thus, Kubalakova et al. (2002) revealed a striking variation in the GAA banding pattern of chromosome 3B in several wheat cultivars. Such results would be difficult to obtain on a limited number of conventional metaphase spreads. Because large numbers of chromosomes can be evaluated rapidly, the method facilitates the discovery of rare structural changes. Kubalakova et al. (2003) analyzed by FISH rye B chromosomes sorted onto a microscope slide. Using probes for several DNA repeats, they found that about 0.5% of the B chromosomes carried a translocation with an A chromosome.

We expect that flow-based cytogenetic mapping will be invaluable in the development of physical maps. It will be used to resolve the order of contigs and clones, to confirm the precise physical positions of centromeres, to determine the distances from the actual chromosome termini of markers near the termini of linkage maps, and to estimate the numbers and sizes of gaps (Harper and Cande 2000). Our experiments with BAC FISH showed that most of the wheat BAC clones could not be localized to specific loci. This is probably because of the presence of high proportions of dispersed DNA repeats (Fig. 4a). Thus, a procedure for anchoring BAC clones from contigs should include a subcloning step. An important advantage of FISH mapping on sorted chromosomes is that they are free of cytoplasm and cell-wall remnants that otherwise cause nonspecific probe binding and hinder access to the DNA template. In our hands, BAC subclones as short as 1 kb can be readily localized (Kubalakova et al., in preparation) (Fig. 4b). Finally, flow-sorted chromosomes can be stretched longitudinally to achieve higher linear resolution (Fig. 4c) (Valarik et al. 2004). In short, FISH on sorted chromosomes offers higher throughput, higher sensitivity and higher resolution when compared to mitotic metaphase spreads.

Conclusions

This article describes the current state of art of flow cytogenetics in wheat. Our aim has been to provide a comprehensive overview without too many technical details. For specifics, the readers are referred to the original publications. Since the first successful flow cytometric analysis and sorting, the number of applications of flow cytogenetics in wheat has been growing. In many areas, the flow-based approaches are superior to the traditional ones. They make the analyses more targeted, more cost effective, more productive and more sensitive. The single drawback of flow cytometry is that it is relatively expensive. However, once created, the subgenomic DNA libraries maybe shared with others, and DNAs isolated from sorted chromosomes, and microscope slides with the sorted chromosomes dried on, can be distributed worldwide. Hence, not every laboratory that desires access to these materials needs to go to the trouble and expense of establishing and running its own flow cytometric facility. Finally, we would like to point out that the individual applications of flow-sorted chromosomes should not be seen as isolated approaches. On the contrary, together they provide a unique and powerful flow-based strategy for wheat genomics.

Acknowledgments

We are grateful for the provision of various cytogenetic stocks of wheat, barley and rye by Takashi Endo, Berndt Friebe, Bikram S. Gill, Neil Jones, Shahryar F. Kianian, Tamas Lelley, Adam Lukaszewski, Wojciech Sodkiewicz, and Nobuyoshi Watanabe. This work is supported by the Czech Science Foundation (grant awards 521/04/0607, 521/05/0257, 521/ 05/P257, and 521/05/H013).

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