POLLEN-TUBE COMPETITION AND MALE FITNESS IN HIBISCUS MOSCHEUTOS

The stigmas of animal-pollinated flowers often capture more pollen than is needed to fertilize all available ovules, and mixed-donor pollen loads are probably common. When this is the case, variation in average pollen-tube growth rates can potentially affect the number of seeds sired by a given plant. Despite considerable interest in effects of postpollination processes on male fitness, little is known about the extent of variation in pollen performance among plants from natural populations. To examine this question in Hibiscus moscheutos (rose mallow), we conducted mixed-donor hand-pollination experiments with 39 pollen donors bearing distinctive isozyme markers. Pairs of competing donors were compared on sets of 11 to 15 recipient plants per pair. These donors often differed in the proportions of seeds they sired, with the maximum deviation from an expected ratio of 50:50 being 68:32. Furthermore, three intensively studied plants exhibited consistent trends in relative pollen performance when each was tested against (1) the same three competitors, and (2) groups of 14 competitors chosen at random from the study population. In a separate experiment, we investigated the effects of salinity stress and high soil nutrients on pollen performance. These environmental factors had anticipated effects on leaf production, flower production, and petal length, but style length and (most importantly) the number of seeds sired relative to a standard pollen donor were not affected. In summary, this study provides the strongest evidence to date that pollen-tube competitive ability varies among coexisting plants and may be an important component of male fitness in plants.     

The bacterial phleomycin resistance geneble as a dominant selectable marker inChlamydomonas

A chimeric gene composed of the coding sequence of theble gene fromStreptoalloteichus hindustanus fused to the 5 and 3 untranslated regions of theChlamydomonas reinhardtii nuclear geneRBCS2 has been constructed. Introduction of this chimeric gene into the nuclear genome ofC. reinhardtii by co-transformation with theARG7 marker yields Arg⁺ transformants of which approximately 80% possess theble gene. Of these co-transformants, approximately 3% display a phleomycin-resistant (Pm^R) phenotype. Western blot analysis using antibodies against theble gene product confirms the presence of the protein in the Pm^R transformants and genetic analysis demonstrates the co-segregation of theble gene with the phenotype in progeny arising from the mating of a Pm^R transformant to wild-type strains. Direct selection of Pm^R transformants was achieved by allowing an 18-h period for recovery and growth of transformed cells prior to selection. This work represents the first demonstration of stable expression and inheritance of a foreign gene in the nuclear genome ofC. reinhardtii and provides a useful dominant marker for nuclear transformation.     

Measuring the toxic effects of high gene dosage on yeast cells

 A novel method, which is rapid, reliable and quantitative, is presented for measuring the toxic effects on yeast cells of high dosage of any given gene. It is based on the possibility of monitoring the presence in cells of a plasmid carrying the ADE2 gene from Saccharomyces cerevisiae by direct observation of colonies, the construction of this particular plasmid being easily made by marked homologous recombination in yeast. Four yeast regulatory genes tested were found to result in various degrees of toxicity at high dosage. Possible implications of the measurement of gene toxicity for eukaryotic cell regulatory mechanisms and for the use of novel general approaches to gene selection, such as the gene-gene interference method, are discussed. Received: 18 March 1996 / Accepted: 23 August 1996     

Use of chicken microsatellite markers in turkey: a pessimistic view

Eighty-eight chicken microsatellite markers, previously developed in our laboratory, were tested for their ability to amplify polymorphic fragments using turkey genomic DNA. Amplification products were obtained for 61 chicken microsatellite markers (69.1%) whereas 27 (30.9%) did not give rise to any products, even when different polymerase chain reaction conditions were employed. From the 61 markers that gave a product, only eight showed a length polymorphism while 37 were monomorphic on the three divergent commercial turkey lines used. The remaining 16 markers yielded many unspecific bands and no specific amplification product could be obtained. Five polymorphic and eleven monomorphic products contained a detectable microsatellite repeat. Furthermore, of the markers that detected a polymorphism in turkey, the observed heterozygosity (15–50%) and allelic variation (only 2 in most cases) was very low. Therefore, on the basis of our results, we think that chicken microsatellite markers are not very useful for mapping purposes in turkey.     

Estimation of the position and effect of a lethal factor locus on a molecular marker linkage map

In the mapping of DNA markers the distortion of segregation of marker genotypes is often observed, which may be caused by a lethal factor acting in filial generations derived from distant crosses. A method is presented for estimating the recombination values between a lethal factor locus and neighboring molecular markers, and the relative viability or fertilization ability of gametes or zygotes affected by the lethal factor in an F₂ population using the maximum likelihood method and the expectation conditional maximization (ECM) algorithm. Three selection models of gamete or zygote were considered, and the most likely one was determined by goodness of fit of the observed frequency of the phenotypes to the expected ones under the models. The method was applied to segregation data of molecular markers of an F₂ population consisting of 144 individuals derived from a cross between an Indica and a Japonica rice variety. The presence of a lethal factor locus (L) located on chromosome III that caused partial gametic selection in both the male and female sides was suggested. The locus L was tightly linked to RFLP marker number 23 of the RFLP linkage map of Saito et al. (1991a), and the fertilization chance of a male or female gamete possessing the lethal factor was, on average, 41.5% of that of the normal gamete.     

Screening apples for OPD20/600 using sequence-specific primers

Apple scab, caused by Venturia inaequalis (Cke.) Wint., is the most serious disease of apple trees in many areas of the world. Resistance to V. inaequalis, derived from the small-fruited species Malus floribunda 821, is determined by a major dominant gene, Vf. Using random decamer primers, we identified a RAPD marker, OPD20/600, which is linked to the Vf gene. OPD20/600 was then cloned and sequenced. Sequence-specific primers based on the marker were used to further screen M. floribunda 821, 7 scab-susceptible apple cultivars, 10 scab-resistant apple cultivars, and 28 scab-resistant Coop selections. The sequence-specific primers allowed identification of polymorphisms of OPD20/600 based on the presence or absence of a single band. The advantages of sequence-specific primers over decamer primers for developing genetic markers are discussed.     

The inheritance and chromosomal localization of AFLP markers in a non-inbred potato offspring

AFLP^TM is a new technique to generate large numbers of molecular markers for genetic mapping. The method involves the selective amplification of a limited number of DNA restriction fragments out of complex plant genomic DNA digests using PCR. With six primer combinations 264 segregating AFLP amplification products were identified in a diploid backcross population from non-inbred potato parents. The identity of an AFLP marker was specified by the primer combination of the amplification product and its size estimated in bases. The segregating AFLP amplification products were mapped by using a mapping population with 217 already known RFLP, isozyme and morphological trait loci. In general, the AFLP markers were randomly distributed over the genome, although a few clusters were observed. No indications were found that AFLP markers are present in other parts of the genome than those already covered by RFLP markers. Locus specificity of AFLP markers was demonstrated because equally sized amplification products segregating from both parental clones generally mapped to indistinguishable maternal and paternal map positions. Locus specificity of AFLP amplification products will allow to establish the chromosomal identity of linkage groups in future mapping studies.Since AFLP technology is a multi-locus detection system, it was not possible to identify the AFLP alleles which belong to a single AFLP locus. The consequences of a genetic analysis based on single alleles, rather than on loci with two or more alleles on mapping studies using progenies of non-inbred parents are discussed.     

Regulation of choline acetyltransferase/iacZ fusion gene expression in putative cholinergic neurons of drosophila melanogaster

We have analyzed the distribution of putative cholinergic neurons in whole-mount preparations of adult Drosophila melanogaster. Putative cholinergic neurons were visualized by X-gal staining of P-element transformed flies carrying a fusion gene consisting of 5′ flanking DNA from the choline acetyltransferase (ChAT) gene and a lacZ reporter gene. We have previously demonstrated that cryostat sections of transgenic flies carrying 7.4 kb of ChAT 5′ flanking DNA show reporter gene expression in a pattern essentially similar to the known distribution of ChAT protein. Whole-mount staining of these same flies by X-gal should thus represent the overall distribution of ChAT-positive neurons. Extensive staining was observed in the cephalic, thoracic, and stomodeal ganglia, primary sensory neurons in antenna, maxillary palps, labial palps, leg, wing, and male genitalia. Primary sensory neurons associated with photoreceptors and tactile receptors were not stained. We also examined the effects of partial deletions of the 7.4 kb fragment on reporter gene expression. Deletion of the 7.4 kb fragment to 1.2 kb resulted in a dramatic reduction of X-gal staining in the peripheral nervous system (PNS). This indicates that important regulatory elements for ChAT expression in the PNS exist in the distal region of the 7.4 kb fragment. The distal parts of the 7.4 kb fragment, when fused to a basal heterologous promoter, can independently confer gene expression in subsets of putative cholinergic neurons. With these constructs, however, strong ectopic expression was also observed in several non-neuronal tissues. © 1995 John Wiley & Sons, Inc.     

Specific monitoring by PCR amplification and bioluminescence of firefly luciferase gene-tagged bacteria added to environmental samples

Abstract The firefly luciferase gene, luc , was demonstrated to hold promise as a specific marker for monitoring of genetically modified bacteria in the environment. PCR amplification and bioluminescence procedures were modified and compared for environmental monitoring of luc -tagged bacteria, using Escherichia coli as a model. The methods were used to track luc -tagged bacterial cells added to intact sediment core microcosms. Detection limits for the luc -tagged cells were the following, expressed as cells per 0.5 g of sediment: 10², by PCR amplification; 10³, by whole cell luminescence; and 10³−10⁴, by measurement of luminescence in cell extracts.     

Histochemical and immunocytochemical study of the migration of neurons from the rat olfactory placode

Immunocytochemical and histochemical methods have been used to describe the neuronal population migrating from the rat olfactory placode and to analyze the spatio-temporal evolution of this neuronal migration during development. Several neuronal markers, such as binding to the lectin Ulex europaeus (UEA I) and the presence of neuron-specific enolase (NSE), olfactory marker protein (OMP), and luteinizing hormone-releasing hormone (LHRH), have been tested in order to determine whether migrating neurons originate from both the medial and the lateral parts of the placode and whether they all express LHRH. Our data show that a large population of differentiated migrating neurons can be identified with an antibody against NSE from the 14th day of gestation and with UEA I one day later. Migrating neurons are closely associated with both the vomeronasal axon fascicles emerging from the medial pit and the olfactory axons originating from the lateral pit. However, the neuron migration from the lateral pit appears to be more discrete than that from the medial pit. No LHRH immunoreactivity has been detected among neurons migrating from the lateral pit. Some neurons accompanying the olfactory axon fascicles exhibit a high level of maturation as shown by their OMP-positivity. Numerous neurons positive for both NSE and UEA I have also been observed within the presumptive olfactory nerve layer in early embryonic stages.