The cultivars of Petunia hybrida Vilm. (hereafter referred to as garden petunias) have been bred since the early 1830s (Paxton, 1836). The progenitors of garden petunias have been presumed to be P. axillaris [=P. axillaris (Lam.) Britton, Sterns & Poggenb. subsp. axillaris, after current treatment] with white flowers and P. violacea Lindl. [=P. integrifolia (HOOK.) Schiz & Thell.] with purple flowers (Ferguson and Ottley, 1932). Petunia inflata R. E. Fr with purple flowers and P. parodii Steere [= P. axillaris subsp. parodii (Steere) Cabrera ] with white flowers have also been proposed as a likely parent of garden petunias (Sink, 1975). Thus, it was a common idea that the garden petunias have been bred through cross-hybridizations between the P. axillaris complex and P. integrifolia complex. Recent detailed studies on wild Petunia species revealed that the P. axillaris complex consists of three subspecies, i.e., subsp. axillaris, subsp. parodii, and subsp. subandina T. Ando (Ando, 1996). These three subspecies are morphologically similar to each other except for flower characters, and it is difficult to identify which subspecies of P. axillaris has been used for the initial breeding of garden petunias. Moreover, another fundamental question still remains on the parental species used as a partner of P. axillaris complex because of the existence of several species closely related to P. integrifolia (Griesbach et al., 2000; Koes et al., 1987; Sink, 1975; Wijsman, 1982).
At present, comparison or analysis of sequences in fragments of organelle DNA amplified with the use of universal primers is widely used in species identification, genetic diversity, and phylogenetic studies in many plant species. Several attempts have been made to understand the phylogeny of Petunia sensu Jussieu using molecular data obtained by various methods of DNA analysis such as restriction fragment length polymorphism (RFLP) analysis of ribosomal DNA (Kabbaj et al., 1995), DNA amplification fingerprinting (Cerny et al., 1996), and RFLP analysis of chloroplast DNA (Ando et al., 2005a). As an alternative, polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis, which is simple, rapid, reliable, and cost saving, has also been used as a powerful tool for identifying plant varieties and species in many plant species such as those in the genus Gossypium (Martsinkovskya et al., 1996), Beta (Shen et al., 1998), and Glycine (Xu et al., 2001). To our knowledge, however, PCR-RFLP analysis has not been used for determining the parents of garden petunias.
Chalcone synthase is a key enzyme of the anthocyanin biosynthesis and encoded by the structural Chs gene (Heller and Hahlbrock, 1980). In the inbred line of Petunia hybrida ‘Violet 30’, the Chs genes form a small multigene family consisting of 12 Chs members, among which eight complete genes (Chs-A, B, D, F, G, H, J, and L) have been cloned (Koes et al., 1987, 1989a). Each complete Chs gene consisted of two exons separated by an intron of variable sequence (Koes et al., 1989a) and only two (Chs-A and Chs-J) were expressed mainly in floral tissues (Koes et al., 1989b). Based on these studies, Griesbach et al. (2000) analyzed the Chs-A gene intron in some wild species and cultivars of Petunia and found that some genetic heterogeneity existed in wild species but not in garden petunias.
In this study, we applied PCR-RFLP analysis to the intron with adjacent part of exons in the Chs-J gene of several Petunia taxa and garden petunias for discriminating two taxonomic groups used as progenitors of garden petunias and for identifying the subspecies of P. axillaris that played a role as one of the progenitors of garden petunias.
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