Hyperexpansion of wheat chromosomes sorted by flow cytometry

ABSTRACT

Despite remarkable recent progress in the analysis of plant genome organization and chromosome structure, there is a need for methods enabling DNA sequences to be mapped by fluorescence in situ hybridization (FISH) at high spatial resolution. We sorted mitotic metaphase chromosomes of wheat by flow cytometry and observed the occurrence of hyperexpanded chromosomes among them. However, this phenomenon was not reproducible in subsequent experiments. An investigation into the procedures of flow cytometry revealed that the hyperexpansion of chromosomes became reproducible when the concentration of formaldehyde used in sample fixation was reduced. We conducted FISH analysis with 45S rDNA, 5S rDNA and wheat centromeric repeat sequences as probes on flow-sorted chromosomes and also on chromosomes from squash preparations. We measured the length of chromosomes 1B and 6B, identified by FISH. On average, the hyperexpanded 1B and 6B chromosomes were 7.26 and 7.53 times longer, respectively, than the same chromosomes from the squash preparations. The most stretched 1B and 6B chromosomes both exceeded 100 micrometers.

MAIN

Very large chromosomes are useful because they provide improved spatial resolution, for example in constructing detailed cytological maps of individual chromosomes. Two types of giant chromosomes, polytene chromosomes and lampbrush chromosomes, are well known in animals. Polytene chromosomes are commonly found in the salivary glands of certain insects of the order Diptera, and the individual chromosomes are as long as several hundred micrometers; in the case of Drosophila melanogaster (2n = 8) the total length of the moderately stretched salivary chromosomes exceeds one millimeter (Bridges, 1935). Lampbrush chromosomes are found in the growing oocytes of many animals including amphibian species; in the case of certain newts, the absolute lengths of the lampbrush chromosomes are between approximately 200 and 900 micrometers (Barsacchi et al., 1970). In plants, however, no such extremely large chromosomes are known. The giant chromosomes that Brink and Cooper (1944) and Nishikawa (1954) found in the antipodal cells of an intergeneric hybrid between Hordeum and Secale and in those of the wild wheat species Aegilops squarrosa, respectively, are about twice as long as mitotic metaphase chromosomes observed in root tip cells. Only meiotic chromosomes at the pachytene stage are substantially larger than mitotic metaphase chromosomes, and pachytene analysis has been conducted for detailed cytogenetic studies in plants. Nevertheless, pachytene analysis has never become prevalent because it is not always easy to have a continuous supply of flowering plants, is cumbersome to obtain anthers at the right stage, and is difficult to observe entangled pachytene chromosomes in species with many chromosomes.

Flow cytometry is a unique technique for sorting microscopic particles in fluid. The method has been used to sort mitotic metaphase chromosomes (Doležel et al., 2014). One of its applications is to make chromosome preparations in which thousands of separate chromosomes are sorted onto a glass slide. While sorting wheat chromosomes by flow cytometry, we noticed the occurrence of greatly expanded chromosomes that were several times larger than mitotic metaphase chromosomes prepared by the squash method. Apart from that, their overall morphology remained intact. In subsequent experiments, however, we failed to reproduce such hyperexpanded chromosomes. We then began to investigate the procedures of flow cytometry, including sample preparation, and succeeded in obtaining hyperexpanded chromosomes reproducibly. In this paper we report on the hyperexpanded chromosomes in terms of the flow cytometry conditions and of the degree of expansion compared with mitotic metaphase chromosomes from regular flow cytometry and from squash preparations.

Procedure of flow cytometry

To prepare aqueous suspensions of intact mitotic metaphase chromosomes, we followed the protocol of Vrána et al. (2012). We germinated seeds of hexaploid bread wheat (Triticum aestivum L., 2n = 6x = 42) cv. Chinese Spring at 25℃, and, after about 2.5 days, when the roots of the seedlings reached a length of 2–3 cm, we incubated the root part in 2 mM hydroxyurea solution for 18 h to inhibit DNA synthesis and to synchronize the cell cycle. We next incubated the seedlings in hydroxyurea-free Hoagland’s nutrient solution for 5.5 h, treated them in 2.5 μM amiprophos-methyl solution for 2 h, and then immersed them in ice water for 16 h to accumulate mitotic cells at metaphase. We then cut off the root tips (about 1 cm long) and fixed them in 2% (v/v) formaldehyde at 5℃ for 20 min. After that, we washed the roots in Tris buffer (pH 7.5) three times for 5 min each at 5℃. We excised meristem root tips (about 3 mm long) and placed them in a sample tube containing 1 ml LB01 lysis buffer, where we homogenized them using a Polytron PT1200 homogenizer (Kinematica AG, Littau, Switzerland) at 20,000 rpm for 13 s. We filtered the crude homogenate through 50-μm nylon mesh into a polystyrene tube. Prior to flow cytometric analysis, we passed the chromosome suspension through a 20-μm nylon mesh to remove smaller clumps and cell debris, and stained chromosomes with DAPI (Invitrogen, Eugene, OR) at a final concentration of 2 μg/ml. We conducted flow cytometry and chromosome sorting experiments with a FACSAria II SORP flow cytometer (BD Biosciences, San Jose, CA). We sorted chromosomes into a 10-μl drop of P5 buffer sucrose (2%, w/v) on slides (Vrána et al., 2012), air-dried the slides at room temperature, and stored them in a refrigerator until use. We counterstained chromosome preparations with a mounting medium containing DAPI for microscopic observation.

Occurrence of highly stretched chromosomes

We used the above standard protocol for the initial flow cytometry experiment in which we observed the occurrence of hyperexpanded chromosomes in one of the 10 preparations. We tried to reproduce the hyperexpanded chromosomes of bread wheat using the standard protocol, but without success. After modifying some of the conditions of flow cytometry, such as the duration of formaldehyde treatment, the concentration of formaldehyde and the sucrose content of the P5 buffer, we found that fixing in a lower concentration of formaldehyde (1%, v/v), without changing the other conditions of the standard protocol, was effective for reproducible hyperexpansion of wheat chromosomes. As shown in Supplementary Fig. S1, hyperexpanded chromosomes, which were stained faintly with DAPI, were interspersed with less expanded chromosomes in the same preparations. We regarded chromosomes exceeding approximately 50 μm as hyperexpanded chromosomes, and counted preparations as having hyperexpanded chromosomes when we observed one or more such chromosomes per field of view at 100x magnification (about 2.2 mm). We repeated flow cytometry experiments using 1% formaldehyde fixation three times, and each time found some preparations having hyperexpanded chromosomes: five out of ten preparations in the first experiment, six out of 20 in the second and 17 out of 30 in the third.

Measurement of hyperexpanded chromosomes

Figure 1 shows hyperexpanded chromosomes together with chromosomes from the standard flow cytometry (2% formaldehyde fixation) and from squash preparations. We used two satellite chromosomes, 1B and 6B, easily identifiable by morphology, to study the magnitude of the enlargement in the hyperexpanded chromosomes. To identify chromosomes 1B and 6B unequivocally, we conducted fluorescence in situ hybridization (FISH) using three repetitive probes: biotin-labeled 5S rDNA (amplified by PCR using the primer set reported by Fukui et al., 1994), digoxigenin-labeled 45S rDNA (from a DNA clone reported by Gerlach and Bedbrook, 1979) and biotin-labeled wheat centromeric repeats (amplified by PCR using the primer set reported by Ito et al., 2004). Prior to FISH, we fixed chromosome preparations from flow cytometry in a 3:1 ethanol:acetic acid fixative for 4–5 days at room temperature and air-dried them. We followed the FISH procedures described by Sakai et al. (2009) with the following modification: after denaturation in 0.15 N NaOH/ethanol, we replaced three ethanol treatments with a single treatment of 2x SSC for 3 min at room temperature. The FISH signals of 45S rDNA were localized in the nucleolus organizer regions of both chromosomes 1B and 6B, and that of 5S rDNA was only on the satellite of chromosome 1B (Fig. 2).

Fig. 1.

Comparison of mitotic metaphase chromosomes from flow cytometry and a squash preparation. (A) Part of a stage micrometer (one scale unit = 10 μm). (B) Mitotic metaphase chromosomes from flow cytometry (1% formaldehyde fixation). (C) Mitotic metaphase chromosomes from flow cytometry (2% formaldehyde fixation). (D) Two mitotic metaphase cells in a squash preparation. We converted digital images of DAPI-stained chromosomes into inverted grayscale images using Adobe Photoshop Ver. 12.0. Chromosomes and the micrometer scale were photographed at the same magnification with an identical 10x objective lens.

Fig. 2.

Comparison of mitotic metaphase chromosomes 1B and 6B from flow cytometry and squash preparations. (A) Part of a stage micrometer (one scale unit = 10 μm). (B) FISH images of mitotic metaphase chromosomes 1B and 6B from flow cytometry (1% formaldehyde fixation). Note that fine chromatin fibers are visible between the chromatids. (C) FISH images of mitotic metaphase chromosomes 1B and 6B from flow cytometry (2% formaldehyde fixation). (D) FISH images of mitotic metaphase chromosomes 1B and 6B from squash preparations. FISH probes: green, 45S rDNA; red, 5S rDNA and wheat centromeric repeats. Chromosomes and the micrometer scale were photographed at the same magnification with an identical 40x objective lens.

We digitally recorded FISH photomicrographs of relatively long and straight 1B and 6B chromosomes from flow-cytometry and squash preparations. Using Adobe Photoshop Ver. 12.0, we converted DAPI color images into inverted grayscale images for length measurement and chose ten longest ones for each chromosome to calculate the average (Supplementary Figs. S2–S5). The average lengths of these hyperexpanded 1B and 6B chromosomes were 95.1 μm and 93.4 μm, respectively, with the longest ones exceeding 100 μm (Table 1). On average, these hyperexpanded 1B and 6B chromosomes were, respectively, 4.40 and 4.1 times longer than those from the standard flow cytometry (2% formaldehyde fixation), and 7.26 and 7.53 times longer than those from the squash preparations. In spite of their great expansion, the hyperexpanded chromosomes well retained their overall morphology and structure, showing fine chromatin fibers (Fig. 2B). We observed no hyperexpanded chromosomes in the chromosome suspension prior to flow cytometry (Supplementary Fig. S6), and found hyperexpanded chromosomes in a sucrose droplet on a glass slide immediately after flow sorting. These observations suggested that some physical force stretched chromosomes uniformly along their length during the flow cytometry process, but we do not know when this happened; nor do we know why highly stretched chromosomes occurred in the initial standard flow cytometry involving 2% formaldehyde fixation. The cause may be attributable to uneven fixation of chromosomes in a formaldehyde solution: the strength of fixation is probably affected by the total volume of root tissue and also by the position of mitotic metaphase cells within root apical meristems. This might also explain why not all preparations from the flow cytometry using 1% formaldehyde fixation had highly stretched chromosomes, and why hyperexpanded chromosomes were interspersed with smaller chromosomes in the same preparations.

Table 1. Length measurement (μm) of mitotic metaphase chromosomes 1B and 6B from flow-cytometry and squash preparations

	Chomosome3)										Mean
	1	2	3	4	5	6	7	8	9	10
Flow cytometry (1%)1)
1B	110.7	99.0	98.0	96.1	94.9	94.7	92.4	92.0	89.3	83.6	95.1
6B	118.2	105.6	103.6	98.7	92.4	86.2	84.6	84.1	83.3	77.7	93.4
Flow cytometry (2%)2)
1B	25.4	23.6	23.2	22.5	22.2	21.7	20.6	20.0	19.1	17.2	21.6
6B	25.0	21.6	20.9	20.4	19.2	18.7	18.5	18.0	15.9	15.3	19.4
Squash preparation
1B	17.6	14.4	13.3	13.1	12.5	12.4	12.2	12.2	12.2	11.5	13.1
6B	14.8	13.4	12.9	12.9	12.7	11.9	11.9	11.8	11.7	10.3	12.4

1)  Flow cytometry using 1% formaldehyde fixation.

2)  Flow cytometry using 2% formaldehyde fixation.

3)  Numbered in descending order of length.

Use of the hyperexpanded chromosomes

The hyperexpanded1B and 6B chromosomes were over seven times longer than the same chromosomes from squash preparations. Singh and Tsuchiya (1975) reported the length of one (6H) of the barley pachytene chromosomes as seven times longer than that at mitotic metaphase. Since wheat and barley chromosomes are similar in size, the hyperexpanded chromosomes were comparable to pachytene chromosomes in terms of length. In addition to the size, the advantage of the hyperexpanded chromosomes is that their morphology remained intact. This feature will make them very valuable in studies on chromosome structure and function. Hyperexpanded chromosomes should also be useful for the construction of detailed cytological maps using single-copy FISH. This hyperexpansion technique, which should be applicable to other species, will bring new developments to chromosome research.

ACKNOWLEDGMENTS

This work has been supported by grant LO1204 from the National Program of Sustainability I. Takashi R. Endo was supported by the Operational Program Education for Competitiveness — European Social Fund (project CZ.1.07/2.3.00/20.0165).

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