EXPERIMENTAL CELL RESEARCH 239, 179–182 (1998) ARTICLE NO. EX973892 SHORT NOTE A Method for Fluorescence in Situ Hybridization against Synaptonemal Complex-Associated Chromatin of Plant Meiocytes N. Cun ˜ ado and J. L. Santos 1 Departamento de Gene ´tica, Facultad de Biologı B a, Universidad Complutense, 28040-Madrid, Spain [13]. In the other one, two repetitive DNA clones, the An improved method of preparing two-dimen- cDNA clone pTa71 and the 120 base pairs repeat clone sional surface spreads of plant synaptonemal com- pSc119.2, were used for FISH against rye SCs, while plexes (SCs) associated with fluorencent in situ hy- the chromatin associated with the lateral elements bridization is described. This technique produces were counterstained with DAPI (4, 6-diamidino-2-phe- clear preparations of SCs and, in addition, consis- nylindole) and propidium iodide [14]. We report in this tently reveals the organization and location of differ- paper a reliable and accurate method for in situ local- ent repetitive DNA sequences in plant meiotic pro- ization of different repetitive DNA sequences to sur- phase chromosomes.  1998 Academic Press face-spread pachytene chromosomes from three plant genera. This technique involves a novel combination of fluorescence in situ hybridization and silver staining of the synaptonemal complexes. INTRODUCTION Fluorescence in situ hybridization (FISH) has widely MATERIALS AND METHODS been applied in past years to different organisms in- cluding yeast [1, 2], mammals [3, 4], and plants [5, 6]. The plant materials employed included two cereal species, Secale The technical details vary but the procedures deter- cereale and Aegilops uniaristata, both 2n Å 14, and a liliaceous spe- cies, Muscari comosum, 2n Å 18. mine essentially the nuclear or chromosomal locations Preparation of SCs spreads. Fresh unfixed anthers containing of DNA sequences, unique or repetitive, in interphase pollen mother cells potentially in prophase I of meiosis were removed nuclei and in mitotic or meiotic chromosomes. How- from inflorescences. One anther of each floret (Secale and Aegilops) ever, whole mount surface-spread pachytene chromo- or flower bud (Muscari) was stained and squashed in acetic orcein somes are rarely used despite their organization is par- to establish the approximate stage of the remaining anthers, which, ticularly suitable for the detection of in situ hybridiza- in the case of Secale and Aegilops, were then prepared for synaptone- mal-complex isolation as indicated by Holm [15], with minor modifi- tion since it includes distinct decondensed chromatin cations: namely, the presence of 0.03% ‘‘Triton X-100’’ detergent in loops anchored to chromosome cores [7, 8]. the swelling medium, and with the fixative solution containing 4% Nevertheless, there are a few reports in rodents [9, paraformaldehyde and 1.7% sucrose in distilled water, adjusted to 10] and man in which immunostaining of synaptone- pH 8.9 with borate buffer. Anthers of Muscari were squashed out onto a cavity slide with 50 ml of digestion medium (0.05 g snail gut mal complex (SCs) proteins and FISH techniques were enzyme ‘‘Cytohelicase,’’ Sigma, 0.125 g polyvinyl pyrrolidone, and combined in order to obtain further insights into both 0.19 g sucrose in 12.5 ml sterile distilled water). After about 6 min the normal meiosis I pairing process and meiotic chro- enzymatic treatment, single drops of meiocyte suspension were mosome structure. In addition, there are a couple of transferred onto single drops of 0.03% ‘‘Triton X-100’’ detergent on papers on the use of nonisotopic in situ hybridization slides. The meiocytes were allowed to spread for about 5 min and then about five drops of the fixative solution mentioned earlier were against SCs at the transmission electron microscopy added. The preparations were dried overnight on a warm plate at level in chicken [11] and mouse [12], respectively. 30°C, rinsed, and air dried. For silver impregnation, a few drops of To date only two examples of in situ hybridization 30% AgNO 3 solution were placed on the preparations which were to plant SCs have been reported. One of them used a then covered with a patch of nylon cloth at 30 – 40°C until they turned colorimetric method to detect DNA sequences at the a pale yellow color. Slides were rinsed, air dried, and stored at 4°C until in situ hybridization. nucleolar organizing regions of Lolium longiflorum DNA probes and labeling. The probes employed in FISH analyses included pSc119.2, pSc74, and pSc34, containing, respectively, the 1 To whom correspondence and reprint requests should be ad- highly repeated DNA sequences of 120, 350 – 480, and 610 base pairs (bp) derived from S. cereale [16–18]. A rDNA probe, pTa71, con- dressed. Fax: 34-1-394 4844. E-mail: jlsc53@eucmax.sim.ucm.es. 179 0014-4827/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved.