M. De Loose

AFLP markers reveal high polymorphic rates in ryegrasses (Lolium spp.)

An evaluation was performed of the potential use of AFLP markers to reveal polymorphisms among Lolium perenne plants with different degrees of kinship. Radioactive and fluorescent detection techniques were applied. The use of a fluorescent detection approach contributed greatly to the speed and ease of conducting and interpreting the AFLP patterns. The great discriminative power of AFLP markers and their capacity to represent genetic relationships among ryegrass plants was shown. Despite the high polymorphic value of the AFLP markers, standard statistical tests could not differentiate between two gene pools derived from different breeding programmes. It proved also impossible to correlate fodder and turf phenotypes with AFLP distance data. A very important point revealed by our data is the high degree of genetic diversity within commercial ryegrass varieties. Our findings are relevant to any outcrossing crop with a breeding strategy based on the production of synthetic populations.

A comparative study of molecular and morphological methods of describing relationships between perennial ryegrass (Lolium perenne L.) varieties

Abstract A sample set of registered perennial ryegrass varieties was used to compare how morphological characterisation and AFLP® (AFLP® is a registered trademark of Keygene N.V.) and STS molecular markers described variety relationships. All the varieties were confirmed as morphologically distinct, and both the STS and AFLP markers exposed sufficient genetic diversity to differentiate these registered ryegrass varieties. Distances obtained by each of the approaches were compared, with special attention given to the coincidences and divergences between the methods. When correlations between morphological, AFLP and STS distances were calculated and the corresponding scatter-plots constructed, the variety relationships appeared to be rather inconsistent across the methods, especially between morphology and the molecular markers. However, some consistencies were found for closely related material. An implication could be that these molecular-marker techniques, while not yet suited to certain operations in the traditional registration of new varieties, could be suitable methods for investigating disputable distinctness situations or possible EDV (EDV= essentially derived variety. An EDV is a variety being clearly distinct from, but conforming in the expression of the essential characteristics of, an ’initial variety’ (IV) from which it is found to have been predominantly derived) relationships, subject to establishing standardised protocols and statistical techniques. Some suggestions for such a protocol, including a statistical test for distinctness, are given.

AFLP based alternatives for the assessment of Distinctness, Uniformity and Stability of sugar beet varieties

Abstract  Three approaches for addressing criteria for Distinctness, Uniformity and Stability (DUS) assessment by means of AFLP data are presented. AFLP data were obtained for three consecutive seed deliveries of 15 sugar beet varieties that were under investigation for the official Belgian list (’93, ’94 and ’95). In total, 696 AFLP markers were scored on 1350 plants. As a first approach, a cluster analysis based on Nei’s standard genetic distances between varieties and/or seed deliveries was made. Three major groups put together varieties belonging to corresponding breeding programmes. Statistical procedures, involving bootstrapping and random sampling of subsets of markers, were applied to test the reproducibility of the ordinations and the redundancy present in the data set. In a second approach, the genetic structure inferred by varieties and seed deliveries was submitted to an Analysis of Molecular Variance (AMOVA). Major genetic variation was attributed to individual plant differences within seed deliveries. Differences among seed deliveries seemed to be as important as differences among varieties or breeding programmes. Individual plant data were used for assignment tests. The computation of the assignment was based on the ranking of individual genotypes to one other (based on Jaccard similarity coefficients). The distribution over the accessions for each variety or seed delivery was used to check what group of plants each individual is genetically most similar to. Varieties were classified according to the degree to which the distribution over the different accessions was mainly allocated to their appropriate seed deliveries (from the same variety) or cross- allocated to other varieties. Criteria for DUS-evaluation could be set by each of the approaches; it is discussed in what way the result obtained differs and agrees.

Single-copy T-DNAs integrated at different positions in the Arabidopsis genome display uniform and comparable β-glucuronidase accumulation levels

Abstract.This study aimed at determining whether transgene expression variability is observed in single-copy T-DNA plants and whether it can be correlated with the T-DNA integration position. Among a population of 135 Arabidopsis thaliana transformants, selected on the basis of antibiotic resistance marker expression, 21 single-copy T-DNA transformants were identified and characterized. In 19 of these 21 lines, 35S-β-glucuronidase transgene expression, measured in two subsequent generations, was similar. This observation means that the intra-transformant variability was as high as the inter-transformant variability. Integration into an intergenic or genic region, into an exon or intron, in sense or antisense orientation, did not result in differential transgene expression. Remarkably, single-copy transformants were not always the highest expressers, implying that low transgene expression is not always induced by multicopy transformants. In only 2 of the 21 single-copy plants was the transgene expression more than 20-fold lower. However, characteristics of the insertion position in one of these lines did not differ significantly when compared to high-expressing lines. In the remaining line, methylation of the transgene was clearly demonstrated. In conclusion, screening for single-copy T-DNA transformants greatly enriches for stable and high transgene expression, because the integration position is not a major determinant of transgene expression variability in Arabidopsis.

AFLP markers demonstrate local genetic differentiation between two indigenous oak species [Quercus robur L. and Quercus petraea (Matt.) Liebl.] in Flemish populations

Abstract. The nuclear genetic variation within and between four sessile (Q. petraea) and six pedunculate (Q. robur) autochthonous Flemish oak populations was investigated with AFLP markers. One sessile and one pedunculate oak population were additionally screened for detailed leaf characteristics using an image analysis system. Principal coordinate analysis on the AFLP data classified the oaks in two main groups, according to their taxonomic status. No species-specific AFLP markers were found using four primer combinations, but marker frequency differences up to 71% were recorded between both species. Analysis of the genetic structure showed that the divergence between species, as observed by ordination, was significant. Both species revealed similar diversity levels. A smaller though significant differentiation was also revealed for both species among populations within species. Molecular and morphology based approaches showed a high degree of consistency. Screening of 60 AFLP primer combinations using a bulking strategy did not allow identifying species-specific markers, which supports the conclusions reached in previous studies. The distribution of genetic variability at the species and at the population level is discussed.

Validation of criteria for the selection of AFLP markers to assess the genetic variation of a breeders' collection of evergreen azaleas

Abstract Fluorescent AFLP and automated data analysis were employed to assess the genetic conformity within a breeders’ collection of evergreen azaleas. The study included 75 genotypes of Belgian pot azaleas (Rhododendron simsii Planch. hybrids), Kurume and Hirado azaleas and wild ancestor species from the Tsutsusi subgenus. Fluorescent detection and addition of an internal size standard to each lane enabled the automated scoring of each fragment arising from a single AFLP primer combination (PC). The use of three PCs generated an initial data set with a total of 648 fragments ranging from 70 bp to 450 bp. Different marker selection thresholds for average fluorescent signal intensity and marker frequency were used to create eight extra restricted data subsets. Pairwise plant genetic similarity was calculated for the nine data sets using the Simple Matching coefficient (symmetrical, including double-zeros) and Jaccard coefficient (asymmetrical, excluding double zeros). The averages, the ranges and the correlation to one other (Mantel analysis) were compared for the obtained similarity matrices. This revealed the sensitivity of ordinations obtained by both similarity coefficients for the presence of weak or intensive markers or for the degree of polymorphism of the markers. For 34 cultivars, pedigree information (at maximum to the fifth ancestor generation) was available. Genetic similarity by descent (kinship coefficient) was turned into a genetic distance and correlated to the genetic conformity, as revealed by the different selections of AFLP markers (Mantel analysis). Use of a Simple Matching coefficient with no or moderate selection to signal intensity and excluding rare and abundant markers gave the best correlation with pedigree. Finally, the ordination of the studied genotypes by means of dendrograms and principal co-ordinate analysis was confronted with known or accepted relationships based on geographical origin, parentage and morphological characters. Genotypes could be assigned to three distinct groups: pot azaleas, Kurume azaleas and Hirado azaleas. Wild ancestor species appeared to be more related to the Japanese azaleas. Intermediate cultivars could be typified as crossings with Kurume or Hirado azaleas or with wild species.

PLASMA CONCENTRATIONS OF PRALIDOXIME METHYLSULPHATE IN ORGANOPHOSPHORUS POISONED PATIENTS

Using pharmacokinetic data from healthy human volunteers in a bicompartmental pharmacokinetic model, a repeated dose scheme for pralidoxime methylsulphate (Contrathion®) was developed producing plasma levels remaining above the assumed “therapeutic concentration” of 4 mg·1−1. Using the same data, it was found that a concentration of 4 mg · 1−1 could also be obtained by a loading dose of 4.42 mg · kg−1 followed by a maintenance dose of 2.14 mg · kg−1 · h−1. In order to study the pharmacokinetic behaviour of pralidoxime in poisoned patients, this continuous infusion scheme was then applied in nine cases of organophosphorus poisoning (agents: ethyl parathion, ethyl and methyl parathion, dimethoate and bromophos), and the pralidoxime plasma levels were determined. The mean plasma levels obtained in the various patients varied between 2.12 and 9 mg·1−1. Pharmacokinetic data were calculated, giving a total body clearance of 0.57±0.271· kg−1· h−1 (mean ± SD), an elimination half-life of 3.44±0.90 h, and a volume of distribution of 2.77±1.451 ·kg−1.

An improved mass spectrometric method for identification and quantification of phenolic compounds in apple fruits

Thirty-nine phenolic compounds were analysed using ultra high performance liquid chromatography (UHPLC) coupled with diode array and accurate mass spectrometry detection using electrospray ionisation (DAD/ESI-am-MS). Instrumental parameters such as scan speed, resolution, and mass accuracy were optimised to establish accurate mass measurements. The method was fully validated in terms of model deviation (r(2)>0.9990), range (typically 10-3500 ngg(-1)), intra/inter-day precision (<6% and <8%, respectively) and accuracy (typically 100 ± 10%). The mass accuracy of each selected phenolic compound was below 1.5 ppm. The results confirmed that the UHPLC-DAD/ESI-am-MS method developed here was convenient and reliable for the determination of phenolic compounds in apple extracts.

Identification and characterization of T-DNA inserts by T-DNA fingerprinting

A T-DNA fingerprinting method is presented based on amplified fragmentlength polymorphism with an anchored polymerase chain reaction step. Thismethod allows discrimination between different T-DNA inserts in stablytransformed plants. The technique was evaluated by analyzing 51 transgenicArabidopsis lines that had been characterized in detail by genomicblotting. Comparison of the obtained fingerprints with the availableintegration information demonstrated that fingerprints were correlated tothe predicted patterns, except for the inverted repeat junctions and forthose inserts with large deletions at the left or right border. Ourexperiments show that by using T-DNA fingerprinting multi-copy transgeniclines can be eliminated efficiently so that the technique can be used toenrich a population of transgenic plants for putative single-copytransformants.

Thermal degradation of cloudy apple juice phenolic constituents

Although conventional thermal processing is still the most commonly used preservation technique in cloudy apple juice production, detailed knowledge on phenolic compound degradation during thermal treatment is still limited. To evaluate the extent of thermal degradation as a function of time and temperature, apple juice samples were isothermally treated during 7,200s over a temperature range of 80-145 °C. An untargeted metabolomics approach based on liquid chromatography-high resolution mass spectrometry was developed and applied with the aim to find out the most heat labile phenolic constituents in cloudy apple juice. By the use of a high resolution mass spectrometer, the high degree of in-source fragmentation, the quality of deconvolution and the employed custom-made database, it was possible to achieve a high degree of structural elucidation for the thermolabile phenolic constituents. Procyanidin subclass representatives were discovered as the most heat labile phenolic compounds of cloudy apple juice.