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AFLP mapping in Petunia hybrida was undertaken with the intention of building a high-density genetic map suitable for applications such as map-based gene cloning. In total five maps were constructed from two mapping populations, with placement of more than 800 markers. Despite the large number of markers the resulting map is roughly ten-fold smaller than those of other plant species, including the closely related tomato. Low levels of recombination are reflected in clusters of tightly linked markers, both AFLPs and RFLPs, in all the maps. Clustering patterns vary between mapping populations, however, such that loci tightly linked in one population may be separable in another. Combined with earlier reports of aberrant meiotic pairing and recombination, our results suggest that, for species like petunia, map-based cloning may be more complex than in model species such as arabidopsis and tomato. [ABSTRACT FROM AUTHOR]

S-RNase has been identified to be an S-allele-specific stylar determinant contributing to the self-incompatibility response in Solanaceae. In order to examine the physical location of the S-RNase gene, multi-color fluorescence in situ hybridization (FISH) using the SB1 -RNase cDNA probe and ribosomal RNA gene (rDNA) probe was performed on an SB1 SB2 heterozygote of Petunia hybrida. The SB1 -RNase gene was detected as a doublet signal close to the centromere of chromosome III. Next, we performed FISH using a large genome probe prepared from a λSB1–311 clone (20 kb) which contains the SB1 -RNase gene and its 3´ flanking region. This probe hybridized to the centromeric regions of all P. hybrida chromosomes. Sequence analysis of the λSB1–311 clone revealed the presence of a repetitive sequence consisting of a novel 666 bp unit sequence. A subclone (pBS-SB1B5) containing this unit sequence also hybridized to all of the centromeric regions, confirming that this unit is the centromeric specific repetitive sequence. These data suggested that the S B1 -RNase gene is located very close to (within a distance of 12 kb from) the centromeric-specific repetitive sequence. Likewise, the pBS-SB1B5 probe hybridized to the centromeric regions of all chromosomes in P. littoralis, another Petunia species. However, the probe did not hybridize to the centromere of the chromosomes from other species in Solanaceae. These results suggested that this centromeric repetitive sequence might be a genus-specific one. [ABSTRACT FROM AUTHOR]

Somatic hybrid plants were regenerated following electrofusion between leaf mesophyll protoplasts of P. hybrida (2n = 14) and a wild sexually incompatible species, P. variabilis (2n = 18). The selection of hybrids was based on the hybrid vigour, expressed both in the growth of the callus and at the shoot formation stage, resulting from the combination of parental genomes. Calli exhibiting vigorous growth were selected, and upon transfer to regeneration medium gave rise to shoots. Four regenerated plants from three calli had morphological characteristics intermediate between those of the parents. The hybrid nature of these plants was confirmed by chromosome counts as well as isozyme and DNA analyses. They had amphidiploid chromosome numbers (2n = 32) and were fertile. Following self-pollination and backcrossing with P. variabilis, large numbers of F and BC seedlings were obtained. [ABSTRACT FROM AUTHOR]

The ovaries of two different Petunia species: Petunia hybrida (hort) and Petunia parodii (Steere) were irradiated with τ-ray doses ranging from 50 to 1,000 Gy before pollination. Seed setting occurred after 4 days preculture on a non-sterile medium. Ovaries transformed into fruits were then cultivated aseptically with the following results: (1) τ-ray doses ranging from 200 to 1,000 Gy led to the development of two types of plants: haploids 2n=x=7 and overdiploids 2n>2x=14. (2) The androgenetic origin of haploids was ascertained by using genetic markers. The origin of overdiploids is discussed. (3) Androgenetic haploids contained the chloroplasts of the irradiated female parent. No visible change of cp DNA patterns was observed after irradiation. (4) The four possible androgenetic events were successfully obtained between the two Petunia species: hybrida haploids with hybrida or parodii cytoplasm, and parodii haploids with parodii or hybrida cytoplasm. [ABSTRACT FROM AUTHOR]

To study the control of differential gene expression during plastid biogenesis in Petunia hybrida, we have investigated the in vivo translation and transcription of the rbc L gene, coding for the large subunit of ribulose bisphosphate carboxylase (LSU), and the psa A gene, coding for P700 chlorophyll- a apoprotein (AP700). Differential expression of these plastid-encoded genes was studied in two developmentally different plastid systems, proplastid-like organelles from the green cell suspension AK2401 and mature chloroplasts from green leaves. In vivo translation of rbc L and psa A transcripts was analysed using specific antibodies. Specific transcript levels were analysed using internal fragments of the rbc L and psa A genes. A standardization procedure was used so that a direct correlation could be made between the amount of products and gene copy number. In Petunia hybrida the amount of LSU polypeptides present in both plastid types does not correspond to the amount of specific mRNA for the gene. Although the rbc L transcripts are present in both plastid types, the LSU protein is only present in green leaf plastids and not in cell culture plastids. In vitro translation of isolated rbc L transcripts give similar results, thereby suggesting that differences in the primary structure of the transcripts are responsible for the observed discrepancy. In contrast to this, the amount of AP700 polypeptides does correspond to the amount of the psa A transcripts. Therefore, our results indicate that the expression of chloroplast genes during plastid biogenesis takes place on at least two different levels: expression of the rbc L gene is regulated post-transcriptionally while expression of the psa A gene is regulated at the transcriptional level. [ABSTRACT FROM AUTHOR]

Proteins extracted from the pistils of several clones of Petunia hybrida carrying differing pairs of S alleles were examined by gel electrophoresis. The major protein of pistils, a basic glycoprotein of relatively low molecular weight, showed properties which varied in a simple manner with the S genotype. For each S allele we were able to assign a specific molecular weight (ranging from 27,000 to 33,000) and isoelectric point (in the range 8.3 to 8.7) for this putative S protein. Pistils homozygous at the S locus showed only one major protein on two-dimensional gel electrophoresis, while pistils from plants heterozygous at the S locus showed two. No evidence was obtained for the presence of this putative S protein in pollen extracts. [ABSTRACT FROM AUTHOR]

Chemogenetic investigations and precursor experiments on flowers of Petunia hybrida suggest that recessive alleles of the gene An3 block the biosynthetic pathway of flavonols and anthocyanins between the flavanone and dihydroflavonol step. In confirmation of this hypothesis, activity of the enzyme flavanone 3-hydroxylase, which catalyses the conversion of flavanones to dihydroflavonols, was readily demonstrated in enzyme preparations from flowers of lines with the dominant allele An3, whereas no or very low activity could be found in extracts from lines with recessive alleles ( an3an3). A second genetic factor is described which clearly reduces the amount of flavonols in the flowers but not the amount of anthocyanins. Crossing experiments revealed that this factor represents a third allele of the An3 gene. It is referred to as an3-1. As expected, a residual flavanone 3-hydroxylase activity of about 10% could be found in enzyme extracts from plants with the an3-1 allele. The decreased level of dihydroflavonol formed under this condition is obviously still sufficient for anthocyanin formation but not for flavonol synthesis. Similar to flavanone 3-hydroxylases from other plants, the enzyme of Petunia is a soluble enzyme and belongs according to its cofactor requirements to the 2-oxoglutarate-dependent dioxygenases. The residual flavanone 3-hydroxylase activity found in plants with the an3-1 allele is identical to the activity extracted from An3-genotypes with regard to cofactors, substrate specificity and most of the inhibitors. The difference observed in the respective pH-optima and the genetic data suggest that the mutation providing the an3-1 phenotype is localized in the structural gene for flavanone 3-hydroxylase. [ABSTRACT FROM AUTHOR]

In flower extracts of defined genotypes of Petunia hybrida, an enzyme activity was demonstrated which catalyses the hydroxylation of naringenin and dihydrokaempferol in the 3′-position. Similar to the flavonoid 3′-hydroxylases of other plants, the enzyme activity was found to be localized in the microsomal fraction and the reaction required NADPH as cofactor. A strict correlation was found between 3′-hydroxylase activity and the gene Ht1, which is known to be involved in the hydroxylation of flavonoids in the 3′-position in Petunia. Thus, the introduction of the 3′-hydroxyl group is clearly achieved by hydroxylation of C-intermediates, and the concomitant occurrence of the 3′,4′-hydroxylated flavonoids quercetin and cyanidin (paeonidin) in the presence of the functional allele Ht1 is due to the action of one specific hydroxylase catalysing the hydroxylation of common precursors for both flavonols and anthocyanins. [ABSTRACT FROM AUTHOR]

The effect of environmental factors on the reversion rates of several unstable alleles in Petunia hybrida was investigated. It is demonstrated that the reversion frequency of three unstable alleles, viz. an allele of gene An1 and of gene An11, both involved in anthocyanin synthesis, and of gene Yg3 for leaf colour, is drastically reduced when the temperature is raised from 18 °C to 25 °C. For two of the alleles it was established that this temperature effect is reversible. Changing the light period or light intensity did not have an effect on the reversion rate of the unstable allele of gene An11 at 18 °C or at 25 °C. The results found are in contrast with those obtained in earlier experiments, in which a rise in temperature resulted in an increase in the reversion rate of another unstable allele of gene An1. [ABSTRACT FROM AUTHOR]

In Petunia hybrida frequent mutations of unstable alleles give rise to different types of periclinal chimeras. If genes expressed in the epidermis, such as the gene An1 for flower colour, are concerned, mutations in the dermal layer of the shoot apex will result in changes in the phenotype but not in the offspring. Mutations in the subdermal layer will not lead to an altered phenotype, but to changes in the sporogenous tissues and, thus, to deviating segregations in progenies. Therefore, in crossing experiments with such an unstable mutant, it is always necessary to take the possibility into account that the plant may be a chimera, so as to prevent an incorrect interpretation of the recorded segregational ratios. Mutations of unstable alleles expressed in the mesophyll, such as gene Yg3 for leaf colour, also give rise to chimeras. In such instances, however, a change in phenotype always involves a change in segregational ratios as well, since both the mesophyll and the sporogenous tissues are derived from the subdermal layer of the shoot apex. [ABSTRACT FROM AUTHOR]

Four genes controlling anthocyanin methylation in flowers of Petunia hybrida have been described. Three of them, Mt2, Mf1 and Mf2, caused a dosage effect on anthocyanin methyltransferase activity and degree of methylation of anthocyanins. Antiserum raised against partially purified Mf2-enzyme precipitated three of the four anthocyanin methyltransferases. In two subspecies of one of the ancestral species of P. hybrida: Petunia integrifolia, different anthocyanin methyltransferases were found as determined by immunoprecipitation. The methyltransferase isozymes showed no differences in subcellular or tissue location, and had no physiologically important difference in time course of activity during bud development. The methylation-system in Petunia is discussed with regard to anthocyanin methylation in other plant species. [ABSTRACT FROM AUTHOR]

In crossing experiments with Petunia hybrida, new mutations, some unstable, have been found in descendants of plants having an unstable allele of the anthocyanin gene An1. One of the unstable mutations affecting the new anthocyanin gene An11 was genetically analyzed, and it was subsequently established in which step of anthocyanin synthesis that An11 is involved. The discovery of new, unstable mutations at other loci indicates that in Petunia also a relation exists between unstable mutations and the presence of transposable elements in the genome. It was demonstrated that reverted alleles ( an1) originating from unstable An1 alleles are less stable than the original wild-type allele An1, and that reversions do not increase the chances of occurrence of new, stable or unstable mutations at other loci. These results provide additional arguments in favour of the hypothesis posed in an earlier paper that reversions of unstable An1 alleles are not the result of excision of the inserted transposable element, but are due to the repair of secondary mutations induced by the insert in the regulatory region of the locus. Consequently, a reverted allele still contains the inserted element that may again induce mutations leading to inactivation of An1. [ABSTRACT FROM AUTHOR]

A hypothesis is proposed to interpret the results of crossing experiments with unstable mutants of Petunia hybrida having variegated flowers and showing variation in the colour intensity and the degree of spotting of the corolla. It is postulated that the An1 locus, which is involved in anthocyanin synthesis, is composed of a structural gene with an adjoining regulatory region, the latter in turn comprising two components, viz., the 'mutator', responsible for the activation of the structural gene, and the 'expressor', controlling the rate of activity. Unstable An1 alleles originate from deletions induced by a transposable element inserted within the regulatory region. Such deletions extend from one of the ends of the inserted element across the adjacent DNA and thus may include parts of the 'expressor' and the 'mutator'. Reversions result from repair of the deletions, the inserted element not necessarily becoming lost in the process. [ABSTRACT FROM AUTHOR]

In Petunia hybrida four complementary genes are present, each having, if homozygous recessive a blueing effect on the flower colour. These genes have no qualitative or quantitative effect on anthocyanins and flavonols. In mutants homozygous recessive for one (or more) of the Ph genes the pH of aqueous flower limb homogenates is increased. It is assumed that the Ph genes in Petunia are involved in maintaining the pH in the vacuole fluid in the flower. [ABSTRACT FROM AUTHOR]

The relation between four methylation genes ( Mt1, Mt2, Mf1 and Mf2) in flowers of Petunia hybrida and anthocyanin-methyltransferase activity was investigated in vitro. All genes controlled methyltransferase activity. This activity was measured with cyanidinnd petunidin-derivatives as substrates. A cross provided evidence that the Mf-genes regulate methyltransferases which are distinct from those controlled by the Mtgenes. Different effects of the two Mf-genes in vivo are shown. The results suggest that the four methylationgenes control four different methyltransferases. [ABSTRACT FROM AUTHOR]

Four genes controlling the conversion of dihydroflavonols into anthocyanins have been investigated for their effect on UDP-Glucose: 3-0-flavonoïd glucosyltransferase activity, one of the enzymes involved in this conversion. An1 and An2 control the bulk of UFGT activity; a homozygous recessive for one of these genes shows an activity of 5-20% of the wildtype value. In a homozygous double recessive some 5% activity is still found while in mutants homozygous recessive for An6 or An9, UFGT activity is lower. In F2 progenies segregating for An6 or An9, however, no difference in UFGT activity was found between homozygous recessive and dominant plants. Mutants blocked in a biosynthesis step preceding the formation of dihydroflavonols show normal UFGT activity levels, indicating that no anthocyanidins are needed for UFGT induction. In addition to delphinidin, myricetin was used as a substrate. The results obtained indicate the probability that both substrates can be glucosylated by the same UFGT enzyme. [ABSTRACT FROM AUTHOR]

In Petunia clones with different S-alleles, self- or cross-pollinated excised-styles of 5, 10, 15, 20 or 25 mm were incubated on a standard agar medium for 24, 48 or 72 h. The length and number of protruding incompatible pollen tubes were compared with those of compatible ones. Throughout the experimental period, the length and number of incompatible pollen tubes of pollen from the SS-clone were always less than those of compatible ones. In pollen from SS-, SS- and SS-clones the incompatible pollen tube growth was barely arrested in 5 and 10 mm excised-styles during the first 24 h of incubation. However, inhibition of incompatible pollen tube growth was strengthened with the increase of both excised-style length and incubation period: this was clearly evident in 15 mm or longer excised-styles incubated for 48 h. Ratios of incompatible to compatible pollen tube length in excised-styles incubated for 72 h, were for SS pollen tubes = 0.28, SS=0.48, SS = 0.50, and SS = 0.60, and ratios on tube numbers were SS = 0.01, SS = 0.1, SS = 0.21, and SS = 0.21. These results were in agreement with those of in vivo self-pollination. The incompatibility reaction seemed strongest in SS-, weaker in SS - and weakest in SS- and SS{3}-clones, and therefore the intensity of S-allele expression would be S> S> S. [ABSTRACT FROM AUTHOR]

In three clones of Petunia hybrida with different incompatibility genes, phytic acid is detected exclusively in pollen, stigma and style. These are all parts of the floral structure involved in the incompatibility reaction. Phytase activity was detected in these tissues as well as in the ovary. The level of phytic acid and phytase activity varied between clones with different S alleles. This difference was most evident in stigma and style. The pattern of phytic acid breakdown following pollination depends on whether pollen and style form a compatible or incompatible combination. Incompatible pollination results in a higher rate of degradation. Consideration is given to the relationship between breakdown of phytic acid to myo-inositol and cell wall thickening and plug formation, which occurs to a greater extent in the incompatible combination. [ABSTRACT FROM AUTHOR]

A gene termed gpiB, coding for one of the two isoenzyme zones of glucose phosphate isomerase in Petunia, has been mapped to a locus on chromosome VII by means of linkage to the marker An4, and by an allelic dosage effect on enzyme activity in trisomics. The high degree of linkage of electrophoretic alleles of gpiB to the pollen colour allele pair An4/an4, as demonstrated in the ancestral species, P. axillaris s.l. and P. integrifolia s.l., has been conserved in all cultivars of P. hybrida investigated. Another gene, coding for the enzyme leucyl-aminopeptidase could also be mapped to chromosome VII and the gene order An4 - lapB - gpiB determined. Apparently, distribution of lapB alleles is not related to the hybrid descent of P. hybrida. [ABSTRACT FROM AUTHOR]

The difference in colour intensity between flowers of sporogenic revertants of the white flowering lines W17 and W28 is caused by an incompletely dominant gene Inl. This gene is not linked to the anthocyanin gene Anl. In the dominant state Inl causes a 50% decrease in colour intensity of selfcoloured red flowers. Chromatographic analysis of anthocyanins of plants homozygous recessive or dominant for Inl showed that the same anthocyanins are produced in both genotypes (cyanidin-3-glucoside and cyanidin-3-diglucoside). Anthocyanin synthesis starts at the same stage of development of the flower in both genotypes. When the bud reaches a length of approximately 45 mm, however, anthocyanin synthesis in the Inl Inl line slows down. No influence of the gene Inl on the concentration of dihydroquercetin-7-glucoside in buds and flowers could be observed, which indicates that the influence of Inl on flower colour development is restricted to the last part of the biosynthesis of anthocyanins, i.e. the conversion of dihydroflavonols into anthocyanins. In addition to Inl having a decreasing effect on flower colour intensity, evidence is produced that the gene Inl also influences the reversion frequency of unstable alleles of the gene Anl. [ABSTRACT FROM AUTHOR]

Full coloured Petunia hybrida flowers lose their colour and become completely white if the gene Fa is dominant. This gene is only expressed in mutants with the pH gene Ph4 homozygous recessive. It is shown by genetical experiments that the fading is restricted to the 3-rutinosido(p-coumaroyl)-5glucoside glycosylation pattern of anthocyanins. The 3-glucosides and 3-rutinosides show a weak fading only. [ABSTRACT FROM AUTHOR]

Forty stable an2-n alleles, derived from the unstable system an2-1, have been tested for anthocyanin synthesis. All of them proved to be different from both the An2 and an2 natural alleles. Only two were distinct from the others which according to Duncan's multiple range test formed a group of overlapping populations. Amongst the variants isolated there was a large majority of light-coloured types. Regulation-like effects of the an2-n alleles on the subsequent genes of the anthocyanin pathway have been observed. A hypothesis concerning the nature of the genetic events occurring at the An2 locus is discussed. [ABSTRACT FROM AUTHOR]

A cross between a 0% pseudo-self-compatible (PSC) plant (S) and a 100% PSC plant (S) yielded an F population which, when selfed, produced a high mean seed set which was not significantly different than that produced when the F was backcross pollinated by the 100% PSC parent. Backcross pollinating the F with the 0% PSC parent yielded no seed. No S plants were recovered in the F populations, indicating that pollen tubes containing the S allele were inhibited during pollen tube growth of the selfed F plants. Apparently stylar-conditioned PSC does not remove all discriminatory power from these petunia styles. Crossing the F (S) with an self-incompatible (SI) plant (S) produced plants which were used for computation of a standard linkage test. An approximate map distance of 28 units was found between the S specificity locus and the major gene(s) which influenced its expression. Other generalized PSC modifying genes apparantly were not linked with the S locus. [ABSTRACT FROM AUTHOR]

An S self-incompatible (SI) petunia plant which showed atypical seed set was found in an I population. This plant showed a strong SI reaction when selfed but produced varying amounts of seed when used as the seed parent in crosses with unrelated individuals homozygous for the same S allele. Reciprocal crosses yielded no seed indicating that the reaction was a stylar response. Self seed obtained by high temperature treatments produced 18 plants, all of which exhibited the parental characteristics, the ability to reject self pollen but accept, to varying degrees, pollen bearing the same S allele from unrelated plants. Several petunias homozygous for S, and exhibiting various levels of PSC as determined by self seed set, progeny tests and temperature treatments, were used as pollen parents. The mean seed set of these crosses produced a ranking of the pollen parents which reflected the PSC levels obtained by other methods. The behavior of the F and F populations suggests that the pollen discriminating ability may be a simply inherited, dominant character in these plants. The styles of these unusual petunias illustrate the participation of the pollen tube in determining PSC. [ABSTRACT FROM AUTHOR]

In a progeny of a selfed individual of the dark red-flowered cultivar 'Roter Vogel' some white-flowered plants appeared as the result of a mutation of the genetic factor Anl involved in anthocyanin synthesis. The white flowers of these plants had red spots owing to back-mutations in the dermal cells of the young corolla. Owing to a striking unstability of the new allele of Anl, a number of mutants originated which differ mutually in the frequency of reversion, which expressed itself in the very substantial differences in the spot density of the limb of the corolla. Between a mean number of less than one spot per cm of the limb and a mean number of over 10.000 spots/cm, a series of transitions was found. The reversions did not remain restricted to the young epidermis but also occurred in sporogenous tissues. This resulted in the appearance of selfcoloured red descendants of plants with red-spotted white flowers. There is a positive correlation between the spot density of the parent plants and the percentage of plants with completely red corollas. The red spots on the corolla usually have the same colour as the wild type ('Roter Vogel'), but occasionally mutants occur with paler spots, the colour varying from a very pale pink to a red nearly as deep as in the wild type. The selfcoloured descendants of such mutants also show this colour variation from pale pink to red. On the grounds of these observations a theory was formulated which postulates that the Anl locus consists of a structural gene responsible for an enzyme active during anthocyanin synthesis and a regulatory element built up from intermediate repetitive DNA. This regulatory element in turn is built up of two components, one of which, the 'mutator', decides the activation of the structural gene while the other, the 'expressor', modifies the rate of activation. The mutations must be considered representative of larger or smaller deletions within one or both of these components. Reversions are the result of the restoration of the deletions by means of an amplification of the repetitive DNA in dividing cells of the developing flower buds. [ABSTRACT FROM AUTHOR]