Multiplex PCR as useful tool for characterization of interspecific hybrids of Petunia and Calibrachoa

Petunia and Calibrachoa are ornamental plants of international economic importance. Commercially, it is advantageous to combine desirable features of Calibrachoa, such as diversity of flower colors, with the pH tolerance and vigorous growth of Petunia. Currently on the floricultural market several commercial hybrids between Petunia and Calibrachoa exist, such as 'Supercal' or 'Calitunia'. However, even though 'Supercal' and 'Calitunia' are recognized as hybrids of Petunia and Calibrachoa, it is not generally known how they were obtained. A variety of different methods is available for hybrid identification between closely related genera. To detect closely related genera, exon sequences are too similar or even identical, whereas intron sequences may differ sufficiently in length and sequence to be used in the amplification of PCR fragments that can distinguish between sequences derived from the parent species. In this study we clearly demonstrate that PCR amplicons from selected introns with flanking exon regions can, in fact, be used to identify and study hybrids of Petunia and Calibrachoa. Four different introns with flanking exon areas were used, each from a different gene and with a different sequence for Petunia and Calibrachoa, as genus specific markers for chromosomes of interspecific hybrids. PCR primers specific for Petunia and Calabrachoa were constructed from determined sequences and tested on a range of presumed hybrids using multiplex PCR, which confirmed that the hybrids were derived from these two species. Similar technology can be used for the molecular characterization of other, new hybrids formed between closely related species or cultivars.