Journal of Experimental Botany, Vol. 51, No. 346, pp. 965-970,
May 2000
© 2000 Oxford University Press
A new FISH protocol with increased sensitivity for physical mapping with short probes in plants
University of Verona, Dipartimento Scientifico e Tecnologico, Strada le Grazie 15, Cà Vignal 1, 37134 Verona, Italy
Received 25 November 1999; Accepted 21 January 2000
 |
Abstract |
|---|
 Top Abstract Introduction>Materials and methodsResultsDiscussionReferences |
|---|
Fluorescence in situ hybridization (FISH) is a well-established technique used for the detection of specific DNA regions, that has been applied to interphase nuclei, pachytene and metaphase chromosomes as well as to extended DNA fibres. This technique allows the physical mapping of specific DNA sequences both on individual chromosomes and extended fibres. A new FISH protocol is described here that enhances the sensitivity of the method. Probes for small unique DNA sequences of less than 2 kb give high signal-to-noise ratio with this method, and can be visualized easily by means of conventional fluores cence microscopy.
Key words: FISH, Asparagus officinalis, physical mapping.
|
Introduction |
|---|
|
TopAbstractIntroduction>Materials and methodsResultsDiscussionReferences |
|---|
Fluorescence in situ hybridization (FISH) is a well-established technique to detect specific DNA regions, which can be used for many purposes. Large probes, such as those derived from BAC and YAC libraries, have been successfully used to mark chromosomes in species such as barley (Lapitan et al., 1997
) and rice (Ohmido et al., 1998). Unfortunately, the use of large probes on plants with a large and complex genome (11–40 pg of DNA) resulted in a very high background, probably because of the presence, in the plant genome, of dispersed repetitive sequences (Fuchs et al., 1996).
FISH has also been used extensively in plants to analyse interphase nuclei, pachytene and metaphase chromosomes (Morais-Cecilio et al., 1996; Heslop-Harrison et al., 1993; Shen et al., 1987; Zhong et al., 1996), in order to investigate the disposition of chromatin in interphase nuclei, and to map DNA sequences on chromosomes. Mapping resolution is higher on pachytene chromosomes than on metaphase chromosomes (Shen et al., 1987; Zhong et al., 1996), due to their less contracted structure; it is even higher in interphase nuclei (Lawrence et al., 1988, 1990; Trask et al., 1989, 1991). In the latter case, however, the possibility of identifying individual chromosomes is lost. The use of FISH on extended DNA fibres further enhances the physical mapping resolution (de Jong et al., 1999), due to the high linear stretching degrees of chromatin, evaluated to be around 3.27 kb µm-1, according to Fransz et al. (Fransz et al., 1996a).
The sensitivity of the FISH techniques so far developed has allowed the identification of single DNA sequences as small as 0.25 kb on human chromosomes (Richards et al., 1994). However, FISH on plants with small probes (less than 10 kb) seems to be more difficult to perform.
The reports on FISH with probes for small single copy DNA targets (1–2 kb) are limited in number. The best results have been obtained by Fransz and coworkers (Fransz et al., 1996b), who were able to recognize a 1.4 kb DNA fragment on Petunia metaphase chromosomes; by Ten Hoopen and coworkers (Ten Hoopen et al., 1996), who recognized a 1.18 kb unique sequence (a flavonol synthase gene) on Petunia chromosomes and by Ohmido and coworkers (Ohmido et al., 1998), who mapped a 1.29 kb RFLP marker on rice chromosomes. In these works, images were mainly captured with a cooled CCD camera and often digitally imaged to enhance the faint signal intensity; direct microscopy images showed low signal-to-noise ratio.
Within a project devised to study sex determinants in Asparagus officinalis L., a dioecious species in which males are heterogametics and sex determinants are located on the L5 long homomorphic chromosome pair (Loptien, 1979; Biffi et al., 1995), a FISH method was set up which led to the detection of probes shorter than 2 kb, directed to single copy DNA sequences, on chromosomes and interphase nuclei, without the help of special detection systems (such as CCD camera and digital imaging).
Three probes were used, named 
23 (1.2 kb), 
47 (1.4 kb) and 
9 (1.7 kb), generated as RFLP fragment; the latter ones (47 and 9) are supposed to be linked to the sex locus (Spada et al., 1998).
The method described in this paper enabled strong, specific and reproducible signals, to be obtained with very low background, which led to a preliminary physical mapping of short nucleotidic sequences related to traits of great agronomic and biological importance on Asparagus officinalis.
|
>Materials and methods |
|---|
|
TopAbstractIntroduction>Materials and methodsResultsDiscussionReferences |
|---|
Plant material
Eight-day-old seedlings from two diploid cultivars of Asparagus officinalis L. (EROS, all male seeds, XY, and SIRIO, 50% male seeds, XY, and 50% female seeds, XX) and from one tetraploid cultivar (VIOLETTO DI ALBENGA, 50% male seeds, XYXY, and 50% female seeds, XXXX) were used. Seeds were kindly provided by Dr Agostino Falavigna.
Nuclei preparation and prehybridization
2 mm long root tips were excised and fixed in 4% methanol- free formaldehyde ( Polyscience) in PBS on ice for 15 min and rinsed three times with the same buffer. Nuclei were extracted as previously described (Levi et al., 1994); they were spread on slides and air-dried, post-fixed for 10 min with 4% formaldehyde in PBS, rinsed three times in PBS and twice in 2x SSC. Slides were then treated with 200 µg ml-1 RNase-A (Fluka) in 2x SSC at 37 °C for 1.5 h, washed in the same buffer, then treated for 10 min with 1 µg ml-1 Proteinase K (Boehringer Mannheim) in PBS at 37 °C, washed again in PBS, and finally dehydrated in ethanol series up to ethanol 100% and air-dried.
Probes
Three pUC19 plasmids were used, containing three different DNA fragments, 47 (1.4 kb), 9 (1.7 kb) and 23 (1.2 kb), isolated as RFLP fragments, the former two supposed to be linked to the sex locus. The DNA probes were labelled with digoxigenin-11-dUTP (Boehringer Mannheim) by Random Priming (DIG DNA Labelling Kit, Boehringer Mannheim) and by PCR.
In situ hybridization
Denaturation of nuclear DNA was performed by soaking slides in 70% deionized formamide in 2x SSC at 72 °C for 5 min, followed by dehydration with chilled ethanol series (70%, 90%, 100%), 5 min each, and air-drying. Slides were afterward incubated with 15 µl of previously denatured (3 min at 80 °C) hybridization mix (2 ng µl-1 Dig-DNA probe, 100 ng µl-1 herring DNA, 100 ng µl-1 yeast RNA, 10% (w/v) dextran sulphate, 50% deionized formamide in 2x SSC), for 16 h at 42 °C, in a moist chamber. After hybridization, slides were washed three times for 5 min in 50% formamide in 4x SSC at 45 °C, and three times for 5 min in 2x SSC at room temperature.
The hybridization signal was revealed by the ‘Fluorescent Antibody Enhancer Set for Dig detection’ (Boehringer Mannheim), according to the instructions of the supplier.
Subsequently, slides were air-dried and mounted with Vectashield antifade containing 5 µg ml-1 DAPI (4',6-diamidino-2-phenylindole) or 1 µg ml-1 propidium iodide for counterstaining of DNA.
Analysis of the hybridization signal
Hybridization signals were observed with a Leica DM RB fluorescence microscope, which had been equipped with filter blocks for DAPI (excitation filter: 340–380 nm; beam splitter: 400 nm; barrier filter: 430 nm), for fluorescein (excitation filter: 470–490 nm; beam splitter: 510 nm; barrier filter: 520 nm) and for simultaneous detection of fluorescein and propidium iodide (excitation filter 1: 490/20 nm; beam splitter 1: 505 nm; barrier filter 1: 525/20 nm; excitation filter 2: 575/30 nm; beam splitter 2: 600 nm; barrier filter 2: 635/40 nm). Photographs were taken with Scotch 650-T-ASA colour slide film. When DAPI counterstaining was used, two photographs of the same field were taken with the two different filters for DAPI and for fluorescein.
Flow cytometry
Nuclei extracted from root tips as described above were stained in suspension with 0.5 µg ml-1 DAPI and analysed for their relative DNA content with a Bryte HS flow cytometer. The following filter blocks were used: excitation block: excitation 365 nm, beam splitter 400 nm, emission >420 nm; separator block (BR): emission 1 450–490 nm, beam splitter 560 nm, emission 2 >590 nm.
Fluoresbryte fluorescent beads (Polyscience) with a 4.5 µm diameter were used as the inner standard.
|
Results |
|---|
|
TopAbstractIntroduction>Materials and methodsResultsDiscussionReferences |
|---|
Setting up the FISH technique
After some initial attempts with traditional cell squashes which gave unsatisfactory results (high background and poor signal), nuclei isolated from root tips and smeared on slides (Levi et al., 1986
) were used in order to reduce the penetrability problems of probes and antibodies and their non-specific binding. Fixation in formaldehyde instead of ethanol/acetic acid was necessary for good isolation of nuclei. Dig-11-dUTP-labelled probes were initially produced by Random DNA Priming, but great improvement of the labelled probe/template ratio was obtained when labelled probes were produced by PCR (1000 ng of labelled probe from 0.01 ng of template, compared with 20 ng of labelled probe from 20 ng of template obtained with Random DNA Priming).
The hybridization buffer, the timing and temperature of the probe and target denaturation were optimized, and, particularly crucial for signal enhancement was the strict control of the duration and temperature of target DNA denaturation (slides had to be treated for at least 5 min at a temperature not lower then 72 °C, followed by an ethanol dehydration to prevent DNA target renaturation). Different signal detection systems were tested: neither anti-Dig Rhodamin or anti-Dig alkaline phosphat ase followed by the HNPP Fluorescent Detection set (Boehringer Mannheim) gave visible signals.
When HNPP was used after a cascade of antibodies it gave a very high background.
The best results were obtained with the ‘Fluorescent Antibody Enhancer Set for Dig detection’ (Boehringer Mannheim), which uses a cascade amplification signal by three different antibodies, the third one conjugated with fluorescein.
Detection of the single copy sequences 9, 47 and 23 with FISH
The hybridization experiments were set up with nuclei from seedling root tips of two different diploid and one tetraploid Asparagus cultivars. Two different probes were used, 9 (1.7 kb) and 47 (1.4 kb). 9 and 47 are two DNA fragments present in single-copy in the Asparagus genome (A Spada, personal communication). The labelled nuclei showed a variable number of signals, visible as brilliant spots; they could be documented by regular photomicroscopy (Figs 1, 2). The background was very low; negative controls, in which the probe was omitted, showed very few spots or none at all (Fig. 2; Table 2). Mitotic chromosomes were occasionally present among the nuclei; some of the long ones were labelled, the spots being located towards the distal ends of the chromosomes (Fig. 3). Hybridization with the 23 probe lpar;1.2 kb) also gave strong signals (not shown).
|
|
|
|
Determination of specificity of hybridization
To determine the hybridization efficiency and specificity, the number of spots/nucleus was determined on nuclei from both diploid and tetraploid cultivars hybridized with 47 and 9 (Tables 1, 2
). Such determination was achieved by excluding the minor areas on the slide where the hybridization failed entirely (no signal at all), and the minor areas in which too many signals were present both on and out of the nuclei (Morais-Cecilio et al., 1997). Table 1, which shows the percentage of labelled nuclei, gives an estimation of the hybridization efficiency, that is between 45% and 70% depending on the probe and on the material.
|
Table 2
shows the distribution of the number of spots per nucleus and the percentage of nuclei showing the number of signals within the expected range (two in nuclei from diploid, four in nuclei from tetraploid plants). The data suggest that a number of nuclei, between 7% and 16% depending on probe and on plant material, could show non-specific spots. Negative controls, where the probe was omitted, failed to show any hybridization, suggesting that a non-specific signal within the labelled nuclei occurred because of non-specific hybridization rather than non-specific antibody attachment.
Flow cytometry
Since DNA endoreduplication phenomena are quite common in plants and could be responsible for additional spots, flow cytometry analysis was performed on nuclei from diploid and tetraploid plants used for in situ experiments. Cytograms of nuclei extracted and labelled with DAPI, with fluorescent beads as the internal standard, are shown in Fig. 4. In nuclei from diploid material about 8% of nuclei had a DNA content higher than 4C, and about 4% an 8C DNA content, while in tetraploid mat erial about 2% of the nuclei had a DNA content higher than 8C.
|
|
Discussion |
|---|
|
TopAbstractIntroduction>Materials and methodsResultsDiscussionReferences |
|---|
Hybridization conditions
FISH detection sensitivity, which can be defined as the smallest DNA sequence to be detected unambiguously (de Jong et al., 1999
), can be enhanced by increasing the power of detection tools, such as CCD cameras and digital imaging, and/or by increasing the signal intensity. This study worked on enhancing signal intensity, and within this parameter on optimizing target accessibility, labelling and hybridization conditions. The optimization of the technique was made possible because, under non-optimal conditions, few spots were detectable and their number and intensity increased slightly when the individual steps were improved. This allowed better experimental conditions to be chosen one by one, but only when all the optimal conditions were used simultaneously was it possible to observe a dramatic increase in the number and intensity of the signals. For this reason it was not possible to assign a relative importance to any single step of the procedure. What was definitively clear is that some steps (denaturation of target DNA, use of probes produced by PCR, use of an antibody cascade signal amplification) were particularly crucial for enhancing the intensity and number of signals, whereas other steps (use of nuclei isolated from root tips instead of cell squashes, the kind of antibody and of detection system used in the cascade signal amplification) proved to be of particular importance in decreasing the background. This new FISH protocol gave a very high signal-to-noise ratio. Most of the positive results reported so far with probes shorter than 2 kb (Fransz et al., 1996b; Ten Hoopen et al., 1996; Ohmido et al., 1998) were obtained by means of capturing images with a cooled CCD camera which enhanced signal intensity and, occasionally, by resorting to digital imaging which enhanced the contrast. Fransz et al. also reported images taken with conventional photomicroscopy, but their signal-to-noise ratio was much lower than the one revealed in these experiments (Fransz et al., 1996b). Ohmido et al. emphasized the fact that effective detection equipment, such as a cooled CCD camera and digital imaging, is essential in capturing the faint signal given by short probes (Ohmido et al., 1998).
PCR-produced probes proved to be better than probes produced by Random Priming. An efficient hybridization involving a short single-copy DNA should probably require that both DNA strands are capable of hybridizing for the entire length with the labelled probe. This is probably better achieved with the PCR labelled probes, since they have a very high labelled probe/template DNA ratio, and a constant length, rather than with Random Priming labelled probes. The use of isolated nuclei instead of squashed cells as starting material enabled the problems of penetrability of such longer probes to be overcome. This probably also decreased background problems, since it is common knowledge that cytoplasm displays a sticky and trapping property in any in situ reaction. Furthermore, as some authors have discovered, in some conditions the presence of the cytoplasm not only increases background signal, but also prevents hybridization or signal detection (Fuchs et al., 1996). The strict control of target denaturation conditions was also critical for signal detection. The temperature as well as the duration of the treatment had to be strictly controlled. The immersion of the slides in the denaturation solution caused a transient decrease of the temperature that had to be accurately checked by an efficient temperature probe directly dipped in the solution; the 5 min of treatment were calculated when 72 °C were re-achieved. A shorter period at 72 °C caused a dramatic decrease in the number of spots. Ethanol dehydration was performed to prevent the target DNA renaturation.
FISH efficiency and specificity, and 9 and 47 mapping
The efficiency of the method devised, calculated as the percentage of labelled nuclei, was roughly between 50% and 70%. Unfortunately, these data cannot be compared with those of other protocols, since their efficiency is not normally reported. The lack of visible signals on part of the nuclei could depend on various factors, such as: lack of hybridization on both DNA target strands; hybridization on one single filament, that could result in a too faint signal; excess of counterstaining fluorescence of the nuclei that could mask signal fluorescence.
A number of spots lower than expected could depend as well on a reduced hybridization efficiency; in the interphase nuclei a single spot could also depend on signal overlay.
The specificity of the FISH signals was suggested by the absence of spots on negative controls and by the number of spots/nucleus in the labelled nuclei. Since FISH, in interphase nuclei, does not allow discrimination between individual chromatids, and hence between G1 and G2 nuclei (Morais-Cecilio et al., 1997), in principle, two spots were expected in nuclei from the diploid cultivar and four in nuclei from the tetraploid cultivar. The percentage of labelled nuclei with the number of spots within the expected range was always higher than 80%. The specificity could be even higher since a certain percentage of nuclei with additional spots might be due to nuclei which had undergone endoreduplication events as shown by flow cytometry.
This high specificity was confirmed by the pattern of hybridization which occurred on metaphase chromosomes. Both with 47 and 9 signals were only found towards the end of the long chromosomes: in the integrated genetic map of Asparagus officinalis published recently (Spada et al., 1998) the two markers are located close together (1.6 cM) and close to the putative sex locus, which maps toward one end of the linkage group 1, corresponding to the sexual chromosome 5, which belongs to the group of the long chromosomes.
The use of this method for the detection of unique short sequences in material enriched in metaphase plates will allow association between markers of different linkage groups and their chromosomes. This will also allow the study of the correlation between genetic recombina tion frequencies and physical distances within the chromosomes.
|
Acknowledgments |
|---|
This research was supported by MIPA (Ministero per le Politiche Agricole). The authors are grateful to Dr A Falavigna (Istituto Sperimentale per l'Orticoltura, Montanaso Lombardo), Professor G Marziani and Dr A Spada (Dipartimento di Biologia, Università di Milano), for kindly providing seeds and probes, and to Dr P Portaluppi for helpful discussions and revision of the manuscript.
|
Notes |
|---|
1 To whom correspondence should be addressed. Fax: +39 45 8027929. E-mail:levi{at}sci.univr.it
|
References |
|---|
|
TopAbstractIntroduction>Materials and methodsResultsDiscussionReferences |
|---|
Biffi R, Restivo FM, Tassi F, Caporali E, Carboni A, Marziani GP, Spada A, Falavigna A.1995. A restriction fragment length polymorphism probe for early diagnosis of gender in Asparagus officinalisL. Horticultural Science 30, 1463–1464.
de Jong JH, Fransz P, Zabel P.1999. High resolution FISH in plants–techniques and applications. Trends in Plant Science 4, 258–263.[Web of Science][Medline]
Fransz PF, Alonso-Blanco C, Liharska TB, Peeters AJM, Zabel P, de Jong HJ.1996a. High resolution physical mapping in Arabidopsis thaliana and tomato by fluorescence in situ hybridization to extended DNA fibres. The Plant Journal 9, 421–430.[Web of Science][Medline]
Fransz PF, Stam M, Montijn B, Ten Hoopen R, Wiegant J, Kooter JM, Oud O, Nanninga N.1996b. Detection of single- copy genes and chromosome rearrangements in Petunia hybrida by fluorescence in situ hybridization. The Plant Journal 9, 767–774.
Fuchs J, Houben A, Brandes A, Schubert I.1996. Chromosome ‘painting’ in plants—a feasible technique? Chromosoma 104, 315–320.[Web of Science][Medline]
Heslop-Harrison JS, Leitch AR, Schwarzacher T.1993. The physical organization of interphase nuclei. In: Heslop-Harrison JS, Flavell RB, eds. The chromosome. Oxford: BIOS Publishers, 221–232.
Lapitan NLV, Brown SE, Kennard W, Stephens JL, Knudson DL.1997. FISH physical mapping with barley BAC clones. The Plant Journal 11, 149–156.
Lawrence JB, Singer RH, McNeil JA.1990. Interphase and metaphase resolution of different distances within the human dystrophin gene. Science 249, 928–932.
Lawrence JB, Villnave CA, Singer RH.1988. Sensitive high-resolution chromatin and chromosome mapping in situ: presence and orientation of two closely-integrated copies of EBV in a lymphoma line. Cell 52, 51–61.[Web of Science][Medline]
Levi M, Sparvoli E, Corbetta N.1994. An antibody against a sequence of human topoisomerase II gives different immunofluorescence patterns in quiescent and proliferating nuclei of Pisum sativum L. Journal of Experimental Botany 45, 1157–1162.
Levi M, Tarquini F, Sgorbati S, Sparvoli E.1986. Determination of DNA content by static cytofluorimetry in nuclei released from fixed plant tissue. Protoplasma 132, 64–68.
Loptien H.1979. Identification of the sex chromosome pair in asparagus (Asparagus officinalis L.). Zeitschrift für Pflanzenzuchtung 82, 162–173.
Morais-Cecílio L, Delgado M, Jones RN, Viegas W.1996. Painting rye B chromosomes in wheat: interphase chromatin organization, nuclear disposition and association in plants with two, three or four Bs. Chromosome Research 4, 195–200.[Web of Science][Medline]
Morais-Cecílio L, Delgado M, Jones RN, Viegas W.1997. Interphase arrangement of rye B chromosomes in rye and wheat. Chromosome Research 5, 177–181.[Web of Science][Medline]
Ohmido N, Akiyama Y, Fukui K.1998. Physical mapping of unique nucleotide sequences on identified rice chromosomes.Plant Molecular Biology 38, 1043–1052.[Web of Science][Medline]
Richards F, Vogt N, Muleris M, Malfoy B, Dutrillaux B.1994. Increased FISH efficiency using APC probes generated by direct incorporation of labelled nucleotides by PCR. Cytogenetics and Cell Genetics 65, 169–171.[Web of Science][Medline]
Shen DL, Wan ZF, Wu M.1987. Gene mapping on maize pachytene chromosomes by in situ hybridization. Chromosoma 95, 311–314.
Spada A, Caporali E, Marziani G, Portaluppi P, Restivo FM, Tassi F, Falavigna A.1998. A genetic map of Asparagus officinalis based on integrated RFLP, RAPD and AFLP molecular markers. Theoretical and Applied Genetics 97, 1083–1089.
Ten Hoopen R, Robbins TP, Fransz PF, Montijn BM, Oud O, Gerats AGM, Nanninga N.1996. Localization of T-DNA insertions in Petunia by fluorescence in situ hybridization: physical evidence for suppression of recombination. The Plant Cell 8, 823–830.[Abstract]
Trask B, Massa H, Kenwrick S, Gitschier .1991. Mapping of human chromosome Xq28 by two-color fluorescence in situ hybridization of DNA sequences to interphase cell nuclei. American Journal of Human Genetics 48, 1–15.[Web of Science][Medline]
Trask B, Pinkel D, van den Engh G.1989. The proximity of DNA sequences in interphase cell nuclei correlated to genomic distance and permits ordering of cosmids spanning 250 kilobase pairs. Genomics 5, 710–717.[Web of Science][Medline]
Zhong X, de Jong JH, Zabel P.1996. Preparation of tomato meiotic pachytene and mitotic metaphase chromosomes suitable for fluorescence in situ hybridization (FISH). Chromosome Research 4, 24–28.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||