Green corolla segments in a wild Petunia species caused by a mutation in FBP2, a SEPALLATA-like MADS box gene.

A Petunia inflata isolate with a novel phenotype of a purple corolla limb with green corolla segments (GCS) was characterized. The GCS have stomata and trichomes on the adaxial side, and resemble calyx segments in epidermal morphology. The GCS phenotype was inherited in a recessive manner. In the GCS plant, a novel inhibitor/defective spm-like transposable element (dPifTp1) was inserted in the second intron of the Floral Binding Protein 2 (FBP2) gene. The sequence of the resulting transcript contained five silent mutations as compared the corresponding open reading frame of P. x hybrida FBP2 mRNA. The GCS phenotype co-segregated with an FBP2 fragment containing a dPifTp1 insertion. The transcript level of the FBP2 gene in GCS flowers was markedly lower than that in wild-type (WT) flowers, suggesting that partially inhibited FBP2 gene expression caused the morphogenesis of calyx-like tissue in the corolla segments of GCS flowers. Gene expression pattern analysis using a full-length Petunia floral cDNA microarray indicated that some photosynthesis-related genes were expressed at significantly higher levels in the GCS of GCS flowers, but the mRNA levels of most other genes in the GCS were similar to those in the WT corolla. Taken together, these data suggest that the partial loss of FBP2 expression does not shift global gene expression in the corolla segments of the GCS flower toward that of calyx, even though calyx-like morphogenesis was established in the corolla segments.

Phylogenetic analysis of the genus Petunia (Solanaceae) based on the sequence of the Hf1 gene.

Polymerase chain reaction fragment length polymorphisms and nucleotide sequences for a cytochrome P450 gene encoding flavonoid-3',5'-hydroxylase, Hf1, were studied in 19 natural taxa of Petunia. Natural Petunia taxa were classified into six groups based on major insertion or deletion events that occurred only in intron II of the locus. The maximum parsimony method was used to calculate strict consensus trees based on nucleotide sequences in selected regions of the Hf1 locus. Petunia taxa were divided into two major clades in the phylogenetic trees. Petunia axillaris (including three subspecies), P. exserta, and P. occidentalis formed a clade with 100% bootstrap support. This clade is associated with a consistently inflexed pedicel, self-compatibility in most taxa, and geographical distribution in southern and western portions of the genus range. The other clade, which comprised the remainder of the genus is, however, less supported (up to 71% bootstrap); it is characterized by a deflexed pedicel in the fruiting state (except P. inflata), self-incompatibility, and a northeastern distribution. A nuclear gene, Hf1, seems to be a useful molecular marker for elucidating the phylogeny of the genus Petunia when compared with the nucleotide sequence of trnK intron of chloroplast DNA.

Distribution of self-compatible and self-incompatible populations of Petunia axillaris (Solanaceae) outside Uruguay.

Petunia axillaris occurs in temperate South America and consists of three allopatric subspecies: axillaris, parodii, and subandina. Previous studies have revealed that subsp. axillaris is self-incompatible (SI), subsp. parodii is self-compatible (SC) in Uruguay, and subsp. subandina is SC in Argentina. The SI/SC status over the entire distribution range is not completely understood, however. The objective of this study was to examine the overall SI/SC status of the respective subspecies in comparison with floral morphology. The results confirmed that subsp. parodii and subsp. subandina were SC throughout the distribution range, and that subsp. axillaris was also SC in Brazil and in most of the Argentinean territory. The SI P. axillaris occurs in the natural population only between 34 and 36 degrees S, along the eastern shore of South America. The Brazilian and Uruguayan subsp. axillaris differed in SI/SC status and floral morphology. We discuss the cause of this difference.

A morphological study of the Petunia integrifolia complex (Solanaceae).

BACKGROUND AND AIMS: Petunia inflata has been treated taxonomically in various ways: it has been described as an independent species, treated as a synonym of P. integrifolia, and also regarded as a subspecies of P. integrifolia. The present study was designed to resolve the ambiguity involving the P. integrifolia complex (P. integrifolia plus P. inflata). METHODS: Tentative identification (either integrifolia group or inflata group) was carried out in the field based on the observation of live specimens at the restricted type localities. The accuracy of the tentative identification was later tested with principal component and cluster analyses of data obtained by measuring 21 morphological characters on cultivated live specimens sourced from 113 natural populations of the P. integrifolia complex in Argentina, Brazil, Paraguay and Uruguay. KEY RESULTS: There was a clear, statistically significant gap between the morphological measurements of the two groups, ensuring the accuracy of identification carried out in the field except for a probable hybrid swarm. Previously, the condition of the pedicel in the fruiting state was considered an important character distinguishing between these two groups; however, the condition of the pedicel was rather variable in the integrifolia group. The two groups were found to have geographically distinct distributions: the integrifolia group occurred in southern regions, whereas the inflata group occurred in northern regions. CONCLUSIONS: Based on the available evidence, it is suggested that the two groups are allopatric species, P. integrifolia and P. inflata, in agreement with the opinion of Fries (1911).

Two novel transposable elements in a cytochrome P450 gene govern anthocyanin biosynthesis of commercial petunias.

The gene Hf1 plays a key role in the expression of floral color in petunias. Hf1 encodes a flavonoid-3',5'-hydroxylase (F3'5'H). The recessive allele (hf1) in an inbred line of petunia is known to be generated by the insertion of a transposable element (Psl). We isolated a novel Mutator-like transposable element (named dTph9) from the hf1 allele of a commercial petunia with red flowers. Another novel transposable element (named rTph1) was found in hf1 of another red petunia. rTph1 shared features with the copia-like retrotransposable element family. These novel elements were inserted independently in the third exon of the Hf1 gene, at different positions. The hf1 allele harboring dTph9 or rTph1 cannot produce an active F3'5'H enzyme because there are stop codons in the dTph9 and rTph1 sequences. Southern analysis showed that these elements were present in relatively low copy numbers and that mutation of the Hf1 locus was associated with the transposition of both elements. We conclude that a loss-of-function mutation of the petunia Hf1 gene is caused by the insertion of at least two different transposable elements, other than Psl, within the Hf1 gene.

Phylogenetic analysis of Petunia sensu Jussieu (Solanaceae) using chloroplast DNA RFLP.

BACKGROUND AND AIMS: The phylogenetic relationships of Petunia sensu Jussieu (Petunia sensu Wijsman plus Calibrachoa) are unclear. This study aimed to resolve this uncertainty using molecular evidence. METHODS: Phylogenetic trees of 52 taxa of Petunia sensu Jussieu were constructed using restriction fragment length polymorphism (RFLP) of chloroplast DNA digested with 19 restriction enzymes and hybridized with 12 cloned Nicotiana chloroplast DNA fragments as probes. KEY RESULTS: In all, 89 phylogenetically informative RFLPs were detected and one 50 % majority consensus tree was obtained, using the maximum parsimony method, and one distance matrix tree, using the neighbour joining method. Petunia sensu Wijsman and Calibrachoa were monophyletic sister clades in both trees. Calibrachoa parviflora and C. pygmaea, previously thought to differ from the other species in terms of their cross-compatibility, seed morphology, and nuclear DNA content, formed a basal clade that was sister to the remainder of Calibrachoa. Several clades found in the phylogenetic trees corresponded to their distribution ranges, suggesting that recent speciation in the genus Petunia sensu Jussieu occurred independently in several different regions. CONCLUSIONS: The separation of Petunia sensu Wijsman and Calibrachoa was supported by chloroplast DNA analysis. Two groups in the Calibrachoa were also recognized with a high degree of confidence.

Nuclear DNA content in the genus Hepatica (Ranunculaceae).

Using flow cytometry, we measured the nuclear DNA contents of all known taxa in Hepatica. Nuclear DNA content of Hepatica falconeri (diploid, crenate leaf lobes) was significantly lower than that of diploid entire species. Among the tetraploid species, crenate species had lower DNA contents than the entire taxon H. nobilis var. pubescens. The DNA content of the tetraploid species was more than double that of the diploid species among the same leaf-type groups.

Delphinidin accumulation is associated with abnormal flower development in petunias.

The relative floral anthocyanidin contents of 195 commercial petunias with floral colours other than white and yellow were determined using HPLC, and the presence of five anthocyanidins (cyanidin, peonidin, delphinidin, petunidin, and malvidin) was confirmed. Pelargonidin was not detected, and delphinidin was not a major component. Using a principal component analysis of the relative anthocyanidin contents, the petunias were classified into three phenotype-groups accumulating cyanidin, peonidin, or malvidin, (plus petunidin) as the major anthocyanidin. A fourth phenotype was segregated in the progeny obtained by self-pollinating an F1 hybrid of the malvidin group; this accumulated delphinidin 3-glucoside in a markedly crumpled corolla-limb (delphinidin group). Such inferior floral traits, associated with the accumulation of delphinidin 3-glucoside, are thought to be the driving force that removed the delphinidin group from commercial petunias. A comparison of flowers of the delphinidin group and those of the other groups may provide a useful tool towards a deeper understanding of how anthocyanin biosynthesis relates to normal development of the corolla.

Breakdown of self-incompatibility in a natural population of Petunia axillaris caused by loss of pollen function.

Although Petunia axillaris subsp. axillaris is described as a self-incompatible taxon, some of the natural populations we have identified in Uruguay are composed of both self-incompatible and self-compatible plants. Here, we studied the self-incompatibility (SI) behavior of 50 plants derived from such a mixed population, designated U83, and examined the cause of the breakdown of SI. Thirteen plants were found to be self-incompatible, and the other 37 were found to be self-compatible. A total of 14 S-haplotypes were represented in these 50 plants, including two that we had previously identified from another mixed population, designated U1. All the 37 self-compatible plants carried either an S(C1)- or an S(C2)-haplotype. S(C1)S(C1) and S(C2)S(C2) homozygotes were generated by self-pollination of two of the self-compatible plants, and they were reciprocally crossed with 40 self-incompatible S-homozygotes (S(1)S(1) through S(40)S(40)) generated from plants identified from three mixed populations, including U83. The S(C1)S(C1) homozygote was reciprocally compatible with all the genotypes examined. The S(C2)S(C2) homozygote accepted pollen from all but the S(17)S(17) homozygote (identified from the U1 population), but the S(17)S(17) homozygote accepted pollen from the S(C2)S(C2) homozygote. cDNAs encoding S(C2)- and S(17)-RNases were cloned and sequenced, and their nucleotide sequences were completely identical. Analysis of bud-selfed progeny of heterozygotes carrying S(C1) or S(C2) showed that the SI behavior of S(C1) and S(C2) was identical to that of S(C1) and S(C2) homozygotes, respectively. All these results taken together suggested that the S(C2)-haplotype was a mutant form of the S(17)-haplotype, with the defect lying in the pollen function. The possible nature of the mutation is discussed.

Differentiation in the status of self-incompatibility among Calibrachoa species (Solanaceae).

The overall status of self-incompatibility, as assessed by the rate of capsule-set after self-pollination, was investigated in the genus Calibrachoa (Solanaceae). Thirty-two species were surveyed using a total of 655 individuals collected in 102 different native populations in Argentina, Brazil, Mexico, and Uruguay. The rate of capsule-set in 278 voucher specimens collected from the same native habitats was also measured to obtain additional information to assess the degree of self-(in)compatibility. Only one species, Calibrachoa parviflora, was self-compatible (SC, autogamous) and the other 31 species were found to be self-incompatible (SI). A mixed population (SI and SC individuals in the same population) was not found. The differentiation of C. parviflora as an autogamous species is associated with a successful occupation of different (riparian) habitats within a larger range of geographic distribution compared to the rest of the species in the principally SI genus of Calibrachoa.