Development of a System for Identification of Hybrid Plants

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A simple and efficient method for identification of hybrids after cell fusion was established by combining a method for the efficient extraction of DNA from very small amounts of plant tissue and a Southern blotting with nonradioactively labeled rDNA from rice as a probe. One hundred milligrams of leaf or ca llus tissue, are sufficient for identification of the hybrid, and the amount of material can be reduced to as little as 10 mg. Since this method permits selection of parasexual hybrids not only at the plantlet stage but also at the callus stage, hybrids can be identified at the early stages of the development of the products of cell fusion.Segments of mitochondrial DNA carrying the gene for the alphasubunit of ATPase (atpA) were detected by Southern hybridization with atpA from pea as probe. In the case of Nicotiana langsdorffii, we identified four fragments that are derived from combinations of two different 5' and two different 3' franking regions of atpA. All of the four types share the coding region, suggesting that the four types can be generated as a result of homologous recombination in the coding region of atpA. By contrast, N. Glauca generated only one fragment which indicated the existence of only a single type of atpA. In the case of somatic hybrids between N. Langsdorffii and N. Glauca, three new fragments were detected in addition to parental fragments. These new fragments can be explained by homologous recombination within the coding region of atpA. These results show that the coding region of atpA is involved not only in intragenomic homologous recombination but can also be involved in homologous recombination between two parental mitochondrial genomes of somatic hybrids.