Application of fluorescence in situ hybridization to molecular cytogenetics of wheat

M. Yamamoto¹ and Y. Mukai²

1 Department of Biotechnology, Kansai Women's Junior College, Kashiwara, Osaka, Japan

2 Department of Biology, Osaka Kyoiku University, Ikeda, Osaka, Japan

In recent years it has been possible to recognize specific repeated sequences and even single-copy sequences on metaphase chromosomes using biotin-labeled probes. In our wheat molecular cytogenetic program some efforts have been made towards exploring the possibilities of the useful application of fluorescence in situ hybridization with biotin-labeled probes. The results of such studies are presented here.

Wheat ribosomal RNA gene (rDNA) and the 120-bp repeated DNA family of rye (pSC 119) were used as probes. Probe DNA was labeled with biotin-16-dUTP by random primer method. Hybridization reactions were incubated at 37C for 6 hr. Fluorescent detection of hybridized biotinylated DNA was carreid out using the rabbit anti-biotin antibody and the FITC-goat-rabbit antibody.

The fluorescence hybridization of rDNA to a metaphase spread of monosomic 1A line of Triticum aestivum cv. Chinese Spring is shown in Figure 1. A rDNA locus on chromosome 1A is clearly visible in addition to rDNA loci on chromosome 1B, 6B, and 5D. In situ hybridization using the rDNA probe was also conducted in the 1R(1B) substitution line of wheat, Burgas 2, which was regenerated from immature embryo culture (Fig.2). This line was found lacking in one of the two rDNA loci on the chromosome 1R, although it had a pair of chromosome 1R. In metaphase spread, the NOR present on the wheat chromosome 6B was stretched whereas the rye NOR remained contracted. Interphase nuclei of Burgas showed that the NOR loci of 6B were dispersed and thus were being actively transcribed. On the contrary, the rye NOR locus was condensed, indicating no activity. The activity of the rye rDNA genes always remained suppressed in the wheat background. The degree of rDNA gene expression was visible by fluorescence detection after hybridization. In F₁ hybrid between Chinese Spring wheat and Prolific rye, the chromosomes were readily distinguished from wheat chromosomes on the basis of in situ hybridization patterns to pSC 119 probe (Fig.3). The higlily repeated sequences on both wheat and rye chromosomes were seen as yellow segments. The rye chromosomes were entirely orange in color, whereas the wheat chromosomes appeared red. The individual chromosomes of rye were also easily identified. As shown in Figure 4a, the increased probe concentration resulted in brighter detection signals relative to Figure 3. Rye chromosomes can be strikingly visualized. In Burgas wheat, the two rye chromosomes are clearly seen in the metaphase spread, as are their domains in the interphase nucleus (Fig. 4b). We can call this approach "chromosome or genome painting." Thus, fluorescence in situ hybridization with biotin-labeled probe seems to be a powerful tool for identification of specific chromosomes in wheat and its relatives and for detection of alien chromosomes in their hybrids.

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