Development of a System for Identification of Hybrid Plants

将从微量植物组织中高效提取DNA的方法与以水稻非放射性标记rDNA为探针的Southern杂交相结合，建立了一种简单有效的细胞融合后鉴定杂交种的方法。100毫克的叶片或愈伤组织足以鉴定杂交种，材料的数量可以减少到10毫克。由于该方法不仅允许在苗期选择杂交种，而且在愈伤组织阶段也允许选择杂交种，因此可以在细胞融合产物发育的早期阶段对杂交种进行鉴定。以烟草为例，我们鉴定了四个片段，它们来自ATPA的两个不同的5‘和两个不同的3’端印花区的组合。所有这四种类型都有相同的编码区，这表明这四种类型可以作为ATPA编码区同源重组的结果。相比之下，N.Glauca只产生了一个片段，表明只有一种类型的ATPA存在。在Langsdorffii和N.Glauca之间的体细胞杂交中，除了亲本片段外，还检测到了三个新的片段。这些新片段可以通过ATPA编码区内的同源重组来解释。这些结果表明，ATPA的编码区不仅参与了基因组内的同源重组，也参与了体细胞杂种两个亲本线粒体基因组之间的同源重组。

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.