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.     

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.     

Continuous monitoring of lactate during exercise in humans using subcutaneous and transcutaneous microdialysis

We have evaluated the possibility of monitoring the plasma lactate concentration in human volunteers during cycle ergometer exercise using subcutaneous and transcutaneous microdialysis. In transcutaneous microdialysis, the relative increase in dialysate lactate concentration exceeded that of plasma lactate concentration by a factor of 6 during exercise due to exercise-induced lactate secretion in sweat. During exercise the subcutaneous microdialysis dialysate lactate concentration underestimated the plasma lactate concentration possibly due to diffusion limitation or adipose tissue lactate production. While it was demonstrated that microdialysis can be used for on-line lactate monitoring, neither subcutaneous nor transcutaneous dialysate lactate concentration were linearly related to the plasma lactate concentration during exercise, and it was found therefore that it was not possible to monitor directly plasma lactate concentration during exercise.     

Identification of aneuploid cells in cytological specimens by combined in situ hybridization and immunocytochemistry

The purpose of our study was the application of non-isotopic in situ hybridization with chromosome-specific repetitive DNA probes for the determination of cytogenetically aberrant cells in routine cytological materials, such as cervical smears and breast tumour aspirates. Hyperdiploid cells in fine needle aspirates (FNA) of breast tumours could be visualized by in situ hybridization with a chromosome l-specific repetitive DNA probe. However, for the evaluation of a specific cell type in heterogeneous cell populations, i.e. cervical smears, a procedure combining immunocytochemistry and in situ hybridization can be required. Therefore, we developed a combination protocol using β-galactosidase/ ferri-ferrocyanide (blue-green) for immunocytochemistry and peroxidase/DAB (brown-black) for detection of the DNA probe. the described protocol enabled us to distinguish squamous epithelial cells within heterogeneous cell populations. By combining the chromosome 1 DNA probe with a specific cytokeratin marker it was possible to identify the chromosomal abnormal cells within cervical smears.     

Mitochondrial DNA control region polymorphisms: genetic markers for ecological studies of marine turtles

We describe a rapid and sensitive method for the detection of population-specific genetic markers in mitochondrial DNA (mtDNA) and the use of such markers to analyse population structure of marine turtles. A series of oligonucleotide primers specific for the amplification of the mtDNA control region in Cheloniid turtles were designed from preliminary sequence data. Using two of these primers, a 384–385-bp sequence was amplified from the 5′ portion of the mtDNA control region of 15 green turtles Chelonia mydas from 12 different Indo-Pacific rookeries. Fourteen of the 15 individuals, including some with identical whole-genome restriction fragment patterns, had sequences that differed by one or more base substitutions. Analysis of sequence variation among individuals identified a total of 41 nucleotide substitutions and a 1-bp insertion/deletion. Comparison with evidence from whole-genome restriction enzyme analysis of the same individuals indicated that this portion of the control region is evolving approximately eight times faster than the average rate and that the sequence analysis detected approximately one fifth of the total variation present in the genome. Restriction enzyme analysis of amplified products from an additional 256 individuals revealed significant geographic structuring in the distribution of mtDNA genotypes among five of the 10 rookeries surveyed extensively. Additional geographic structuring of genotypes was identified through denaturing gradient gel electrophoresis (DGGE) of amplified products. Only two of the 10 rookeries surveyed could not be differentiated, indicating that the Indo-Pacific C. mydas include a number of genetically differentiated populations, with minimal female-mediated gene flow among them. Important applications for genetic markers in the conservation and management of marine turtles include the identification of appropriate demographic units for research and management (i.e. genetically discrete populations) and assessment of the composition of feeding and harvested populations.     

Assignment of five polymorphic ovine microsatellites to bovine syntenic groups

A panel of bovine somatic cell hybrids was used to map ovine microsatellites. Five of seven microsatellites were assigned to five bovine syntenic groups. These microsatellites were designated D5S10 (MAF23), D1S4 (MAF46), D13S1 (MAF18), D4S3 (MAF50), and DXS2 (MAF45), mapped to syntenic groups U3 (chromosome 5), U10 (chromosome 1), U11, U13, and the X chromosome, respectively. Two remaining sheep microsatellites amplified rodent DNA in the hybrid somatic cell panel, and were not assigned to bovine syntenic groups. Assignment of ovine-derived microsatellites to bovine syntenic groups provides additional evidence of the usefulness of microsatellites for mapping closely related species. The use of ovine and bovine microsatellites will aid in development of comparative genomic maps for these two species.