Use of short microsatellites from database sequences to generate polymorphisms among Lycopersicon esculentum cultivars and accessions of other Lycopersicon species

A search of nearly 2000 sequences from Solanaceae species in the EMBL and Genbank databases yielded 220 microsatellites. Among these were 80 microsatellites from 675 Lycopersicon entries. Dinucleotide repeats, as well as (CAA)_n and (TAA)_n repeats, were over-represented in non-coding DNA. The other trinucleotide repeats were predominantly found in exonic DNA. PCR analysis of 44 of the microsatellite-containing Lycopersicon loci identified 36 primer pairs that yielded well-scorable fragments, or groups of fragments, in L. esculentum cultivars and accessions of Lycopersicon species. Twenty-nine of these amplified bands that were polymorphic among the four Lycopersicon species. Ten primer pairs generated polymorphic bands among seven tomato cultivars. Upon examining the number of microsatellites and the degree of polymorphisms in relation to the repeat type and motif, the type of DNA the microsatellite resided in, the length of the microsatellite, and the presence of imperfections in the microsatellite, only two significant correlations were found. (i) Imperfect repeats were less polymorphic among species than perfect repeats. (ii) The percentage of loci polymorphic among cultivars increased from 6% for the shortest loci (with eight or less repeat units) to 60% for the group with the longest repeats (12 repeat units or longer). Among the species, however, all length classes contained about 83% polymorphic loci. In general, 2–4 alleles were found for each locus among the samples of the test set. In a few cases, up to eight alleles were found. A combination of these microsatellite loci can therefore be useful in distinguishing cultivars of tomato, which are genetically very closely related to each other. Received: 9 August 1996 / Accepted: 23 August 1996     

Reproducibility testing of RAPD,AFLP and SSR markers in plants by a network of European laboratories

A number of PCR-based techniques can be used to detect polymorphisms in plants. For their wide-scale usage in germplasm characterisation and breeding it is important that these marker technologies can be exchanged between laboratories, which in turn requires that they can be standardised to yield reproducible results, so that direct collation and comparison of the data are possible. This article describes a network experiment involving several European laboratories, in which the reproducibility of three popular molecular marker techniques was examined: random-amplified fragment length polymorphism (RAPD), amplified fragment length polymorphism (AFLP) and sequence-tagged microsatellites (SSR). For each technique, an optimal system was chosen, which had been standardised and routinely used by one laboratory. This system (genetic screening package) was distributed to different participating laboratories in the network and the results obtained compared with those of the original sender. Different experiences were gained in this exchange experiment with the different techniques. RAPDs proved difficult to reproduce. For AFLPs, a single-band difference was observed in one track, whilst SSR alleles were amplified by all laboratories, but small differences in their sizing were obtained.     

S-specific proteins in styles of self-incompatible Nicotiana alata

Summary A comparison of the stigma protein patterns of individual plants of the inbred- and cross-progenies in Nicotiana alata by isoelectric focusing revealed the presence of S-specific proteins. The S allele-protein relationship was found for three different S alleles. The S-specific proteins occurred in both stigma and stylar parts of the pistil whereas they were absent in leaves. In clone OWL the concentration of S-specific proteins in the stigma increased gradually during floral development. The shift from compatibility to incompatibility was not accompanied by an abrupt increase in concentration of the S-proteins.     

Mechanism of peroxidase isoenzyme induction in pollinated Nicotiana alata styles

Summary A comparative study on the induction of peroxidase isoenzymes, specifically number 10 (P-10) in Nicotiana alata styles revealed significant differences between the various plants of an inbred progeny. In some plants the ageing-induced increase in P-10 activity was very low, whereas in some others, it was relatively high. Pollination accelerated this increase, independent of the pollen genotype. Fertilization was followed by a considerable increase in the activity of several peroxidase isoenzymes, including P-10 in all the plants.Two plants that differed greatly with regard to P-10 induction were used in additional experiments in order to ascertain the mechanism involved in the induction of P-10. The increase in P-10 activity due to pollination or fertilization can partly be explained on the basis of auxin and auxin-induced ethylene activity. The differences in P-10 induction between various plants of the inbred progeny were probably due to differences in their sensitivity to ethylene.     

Microsatellite genotyping of carnation varieties

A set of 11 sequence-tagged microsatellite markers for carnation (Dianthus caryophyllus) was developed using a DNA library enriched for microsatellites. Supplemented with three markers derived from sequence database entries, these were used to genotype carnation varieties using a semi-automated fluorescence-based approach. In a set of 82 cultivars, the markers amplified 4-16 alleles each. The effective number of alleles varied from 1.9 to 6.0. For the eight best scorable markers, heterozygosity was between 0.51 and 0.99. The markers were able to distinguish all cultivars with a unique combination of alleles, except for sport mutants, which were readily grouped together with the original cultivar. In addition, one group of three and one group of six cultivars each had the same combination of 'allelic peaks'. The cluster of three varieties concerned original cultivars and their mutants. The cluster of six consisted of four mutants from the same cultivar and two other varieties.     

Do S allele-specific peroxidase isoenzymes exist in self-incompatible Nicotiana alata?

Summary In order to test Pandey's hypothesis that peroxidase isoenzymes determine S-gene specificity in Nicotiana alata, peroxidase isoenzymes in styles and pollen from various plants of an inbred- and a cross progeny were compared by means of starch gel electrophoresis and electrofocusing.No relation between the S-genotype and the peroxidase isoenzyme patterns of pollen or of styles could be established. The differences between the isoenzyme patterns of different S-genotypes were ascribed to differences in the genetic background of various plants that had the same S-genotype.     

The use of semi-automated fluorescent microsatellite analysis for tomato cultivar identification

 The objectives of this study were to evaluate the usefulness of a fluorescent-analysis method for genotyping PCR-based tomato microsatellite markers (or STMSs) and to establish the value of these markers to generate unique DNA profiles of tomato cultivars. The analyses were performed using forward primers labelled with a fluorochrom and using an ALF express DNA sequencer. In general, analysis of the tomato STMSs revealed distinct allelic peaks. PCR artefacts like stuttering and differential amplification were observed for several tomato STMS markers, but in most cases these artefacts did not seriously hamper allele designation. Comparison of fluorescent and silver-stained allelic profiles revealed a similar distribution of alleles among the test cultivars. Sixteen tomato cultivars were DNA-typed for 20 selected STMS markers using the fluorescent approach. Length polymorphism among the PCR products was detected with 18 of these markers, yielding gene diversity values from 0.06 to 0.74. The number of alleles per microsatellite locus ranged from 2 to 8. As few as four STMSs were sufficient to differentiate between all 16 cultivars, indicating that these markers are especially suitable for a species like tomato which has low levels of variation as detected by other types of markers. Received: 5 February 1998 / Accepted: 7 April 1998     

Mentor pollen effects on gametophytic incompatibility in Nicotiana,Oenothera and Lycopersicum

Summary Attempts were made, through mentor pollen techniques, to overcome self-incompatibility in species belonging to the genera Nicotiana and Oenothera and in a hybrid of Lycopersicum, which are characterized by a gametophytic system of incompatibility. While radiation-killed incompatible pollen did not generate mentor effects in any of the material tested, radiation-killed compatible pollen was found to promote a high level of illegitimate fertilizations by incompatible pollen in N. alata. No evidence was obtained that radiation-killed compatible pollen could induce mentor effects in strictly self-incompatible clones of O. organensis and of the interspecific hybrid L. esculentum x L. peruvianum.This publication is contribution no 1563 from the Biology Radio-protection and Medical Research programme of the Directorate General XII of the Commission of the European Communities     

Construction and analysis of a microsatellite-based database of European wheat varieties

A database of 502 recent European wheat varieties, mainly of winter type, was constructed using 19 wheat microsatellites and one secalin-specific marker. All datapoints were generated in at least two laboratories using different techniques for fragment analysis. An overall level of >99.5% accuracy was achieved. The 199 alleles detected allowed discrimination between all of the varieties except duplicates, and varieties derived from identical parents. Approximately 25% of the varieties showed some heterogeneities, with the highest level of heterogeneity in south-eastern European material. The highest genetic diversity and the highest number of rare alleles were found in varieties from southern Europe. The relative allele frequencies varied for most microsatellites in different geographical regions.