The distribution of genetic diversity in a Brassica oleracea gene bank collection related to the effects on diversity of regeneration, as measured with AFLPs.

The ex situ conservation of plant genetic resources in gene banks involves the selection of accessions to be conserved and the maintenance of these accessions for current and future users. Decisions concerning both these issues require knowledge about the distribution of genetic diversity within and between accessions sampled from the gene pool, but also about the changes in variation of these samples as a result of regenerations. These issues were studied in an existing gene bank collection of a cross-pollinating crop using a selection of groups of very similar Dutch white cabbage accessions, and additional groups of reference material representing the Dutch, and the global white cabbage gene pool. Six accessions were sampled both before and after a standard regeneration. 30 plants of each of 50 accessions plus 6 regeneration populations included in the study were characterised with AFLPs, using scores for 103 polymorphic bands. It was shown that the genetic changes as a result of standard gene bank regenerations, as measured by AFLPs, are of a comparable magnitude as the differences between some of the more similar accessions. The observed changes are mainly due to highly significant changes in allele frequencies for a few fragments, whereas for the majority of fragments the alleles occur in similar frequencies before and after regeneration. It is argued that, given the changes of accessions over generations, accessions that display similar levels of differentiation may be combined safely.

QTL identification for early blight resistance (Alternaria solani) in a Solanum lycopersicum x S. arcanum cross.

Alternaria solani (Ellis and Martin) Sorauer, the causal agent of early blight (EB) disease, infects aerial parts of tomato at both seedling and adult plant stages. Resistant cultivars would facilitate a sustainable EB management. EB resistance is a quantitatively expressed character, a fact that has hampered effective breeding. In order to identify and estimate the effect of genes conditioning resistance to EB, a quantitative trait loci (QTL) mapping study was performed in F2 and F3 populations derived from the cross between the susceptible Solanum lycopersicum (syn. Lycopersicon esculentum) cv. 'Solentos' and the resistant Solanum arcanum (syn. Lycopersicon peruvianum) LA2157 and genotyped with AFLP, microsatellite and SNP markers. Two evaluation criteria of resistance were used: measurements of EB lesion growth on the F2 plants in glasshouse tests and visual ratings of EB severity on foliage of the F3 lines in a field test. A total of six QTL regions were mapped on chromosomes 1, 2, 5-7, and 9 with LOD scores ranging from 3.4 to 17.5. Three EB QTL also confer resistance to stem lesions in the field, which has not been reported before. All QTL displayed significant additive gene action; in some cases a dominance effect was found. Additive x additive epistatic interactions were detected between one pair of QTL. For two QTL, the susceptible parent contributed resistance alleles to both EB and stem lesion resistance. Three of the QTL showed an effect in all tests despite methodological and environmental differences.

A mixed-model approach to association mapping using pedigree information with an illustration of resistance to Phytophthora infestans in potato.

Association or linkage disequilibrium (LD)-based mapping strategies are receiving increased attention for the identification of quantitative trait loci (QTL) in plants as an alternative to more traditional, purely linkage-based approaches. An attractive property of association approaches is that they do not require specially designed crosses between inbred parents, but can be applied to collections of genotypes with arbitrary and often unknown relationships between the genotypes. A less obvious additional attractive property is that association approaches offer possibilities for QTL identification in crops with hard to model segregation patterns. The availability of candidate genes and targeted marker systems facilitates association approaches, as will appropriate methods of analysis. We propose an association mapping approach based on mixed models with attention to the incorporation of the relationships between genotypes, whether induced by pedigree, population substructure, or otherwise. Furthermore, we emphasize the need to pay attention to the environmental features of the data as well, i.e., adequate representation of the relations among multiple observations on the same genotypes. We illustrate our modeling approach using 25 years of Dutch national variety list data on late blight resistance in the genetically complex crop of potato. As markers, we used nucleotide binding-site markers, a specific type of marker that targets resistance or resistance-analog genes. To assess the consistency of QTL identified by our mixed-model approach, a second independent data set was analyzed. Two markers were identified that are potentially useful in selection for late blight resistance in potato.

Unique genomic configuration revealed by microsatellite DNA in polyploid dogroses, Rosa sect. Caninae.

An allopolyploid complex with high genomic integrity has been studied. Dogroses transmit only seven chromosomes (from seven bivalents) through the pollen, whereas 21, 28 or 35 chromosomes (from seven bivalents and 14, 21 or 28 univalents) come from the egg cells. Seedlings derived from two interspecific crosses were analysed with flow cytometry and molecular markers to determine ploidy level, mode of reproduction and genomic constitution. Evidence was obtained for the formation of unreduced male and female gametes, which can take part in fertilization (producing seedlings with higher ploidy than the parental plants) or in apomictic reproduction. Random amplified polymorphic DNA (RAPD) and microsatellite analyses indicated that three seedlings (5%) were derived through apomixis, whereas the other 49 were hybrids. Bivalent formation appears to involve chromosomes that consistently share the same microsatellite alleles. Allele-sharing between the maternally transmitted and highly conserved univalent-forming chromosomes reflected the taxonomic distance between different genotypes. The frequently recombining bivalent-forming chromosomes were taxonomically less informative.

Genetic structure in populations of an ancient woodland sedge, Carex sylvatica Hudson, at a regional and local scale.

Wood sedge (Carex sylvatica) is a well-known ancient woodland species with a long-term persistent seed bank and a caespitose growth habit. All thirteen isolated Carex sylvatica populations in the Dutch Rhine floodplain (including the river branches Waal and IJssel) were mapped in detail and analysed for genetic variation at a large number of AFLP loci and one microsatellite locus. Across all populations, only 40 % of the sampled individuals (n=216) represented a unique genotype. A high number of the studied patches (spatial clusters of tussocks, 2-10 m in diameter) within populations contained only one or a few genotypes. Identical plants (tussocks) were also found 20-500 m apart and in one case even 1000 m apart. Observed heterozygosity levels (H(O)=0.029) were low, indicating low levels of gene flow, which is in agreement with the selfing nature of other caespitose sedges. Although the number of genotypes in populations is low, these genotypes are genetically very distinct and variation within populations accounted for 55% of the total variation. The absence of a correlation between genetic and geographic distances among populations, and the scattered distribution of genotypes among patches within woodlands, support our hypothesis of rare establishments and subsequent local dispersal within woodlands in this forest floor species, which may benefit from and partly depend on human land use and forest management activities.

Microsatellite analysis of Rosa damascena Mill. accessions reveals genetic similarity between genotypes used for rose oil production and old Damask rose varieties.

Damask roses are grown in several European and Asiatic countries for rose oil production. Twenty-six oil-bearing Rosa damascena Mill. accessions and 13 garden Damask roses were assayed by molecular markers. Microsatellite genotyping demonstrated that R. damascena Mill. accessions from Bulgaria, Iran, and India and old European Damask rose varieties possess identical microsatellite profiles, suggesting a common origin. At the same time, the data indicated that modern industrial oil rose cultivation is based on a very narrow genepool and that oil rose collections contain many genetically identical accessions. The study of long-term vegetative propagation of the Damask roses also reveals high somatic stability for the microsatellite loci analyzed.

Past and current gene flow in the selfing, wind-dispersed species Mycelis muralis in western Europe.

The distribution of genetic diversity in Mycelis muralis, or wall lettuce, was investigated at a European scale using 12 microsatellite markers to infer historical and contemporary forces from genetic patterns. Mycelis muralis has the potential for long-distance seed dispersal by wind, is mainly self-pollinated, and has patchily distributed populations, some of which may show metapopulation dynamics. A total of 359 individuals were sampled from 17 populations located in three regions, designated southern Europe (Spain and France), the Netherlands, and Sweden. At this within-region scale, contemporary evolutionary forces (selfing and metapopulation dynamics) are responsible for high differentiation between populations (0.34 < F(ST) < 0.60) but, contrary to expectation, levels of within-population diversity, estimated by Nei's unbiased expected heterozygosity (H(E)) (0.24 < H(E) < 0.68) or analyses of molecular variance (50% of the variation found within-populations), were not low. We suggest that the latter results, which are unusual in selfing species, arise from efficient seed dispersal that counteracts population turnover and thus maintains genetic diversity within populations. At the European scale, northern regions showed lower allelic richness (A = 2.38) than populations from southern Europe (A = 3.34). In light of postglacial colonization hypotheses, these results suggest that rare alleles may have been lost during recolonization northwards. Our results further suggest that mutation has contributed to genetic differentiation between southern and northern Europe, and that Sweden may have been colonized by dispersers originating from at least two different refugia.

Ex-situ conservation of Black poplar in Europe: genetic diversity in nine gene bank collections and their value for nature development.

Populus nigra L. is a pioneer tree species of riparian ecosystems that is threatened with extinction because of the loss of its natural habitat. To evaluate the existing genetic diversity of P. nigra within ex-situ collections, we analyzed 675 P. nigra L. accessions from nine European gene banks with three amplified fragment length polymorphism (AFLP) and five microsatellite [or simple sequence repeat (SSR)] primer combinations, and 11 isozyme systems. With isozyme analysis, hybrids could be detected, and only 3% were found in the gene bank collection. AFLP and SSR analyses revealed effectively that 26% of the accessions were duplicated and that the level of clonal duplication varied from 0% in the French gene bank collection up to 78% in the Belgian gene bank collection. SSR analysis was preferred because AFLP was technically more demanding and more prone to scoring errors. To assess the genetic diversity, we grouped material from the gene banks according to topography of the location from which the accessions were originally collected (river system or regions separated by mountains). Genetic diversity was expressed in terms of the following parameters: percentage of polymorphic loci, observed and effective number of alleles, and Nei's expected heterozygosity or gene diversity (for AFLP). Genetic diversity varied from region to region and depended, to some extent, on the marker system used. The most unique alleles were identified in the Danube region (Austria), the Rhône region (France), Italy, the Rijn region (The Netherlands), and the Ebro region (Spain). In general, the diversity was largest in the material collected from the regions in Southern Europe. Dendrograms and principal component analysis resulted in a clustering according to topography. Material from the same river systems, but from different countries, clustered together. The genetic differentiation among the regions (F(st)/G(st)) was moderate.

Assignment of allelic configuration in polyploids using the MAC-PR (microsatellite DNA allele counting-peak ratios) method.

Polysomic inheritance frequently results in the simultaneous occurrence of several microsatellite DNA alleles on a single locus. The MAC-PR (microsatellite DNA allele counting-peak ratios) method was recently developed for the analysis of polyploid plants and makes use of the quantitative values for microsatellite allele peak areas. To date, this approach has only been used in plants with known genetic relationships. We report here the application of MAC-PR for the first time to random samples of unknown pedigrees. We analysed six microsatellite loci using a set of tetraploid ornamental rose ( Rosa x hybrida L.) varieties. For each locus, all alleles were analysed in pairwise combinations in order to determine their copy number in the individual samples. This was accomplished by calculating the ratios between the peak areas for two alleles in all of the samples where these two alleles occurred together. The allele peak ratios observed were plotted in a histogram, and those histograms that produced at least two well-separated groups were selected for further analysis. Mean allelic peak ratio values for these groups were compared to the relationships expected between alleles in hypothetical configurations of the locus investigated. Using this approach, we were able to assign precise allelic configurations (the actual genotype) to almost all of the varieties analysed for five of the six loci investigated. MAC-PR also appears to be a very effective tool for detecting 'null' alleles in polyploid species.

Microsatellite DNA marker inheritance indicates preferential pairing between two highly homologous genomes in polyploid and hemisexual dog-roses, Rosa L. Sect. Caninae DC.

According to previous cytological evidence, the hemisexual dog-rose species, Rosa sect. Caninae, transmit only seven chromosomes (derived from seven bivalents) through their pollen grains, whereas egg cells contain 21, 28 or 35 chromosomes (derived from seven bivalents and 14, 21 or 28 univalents) depending on ploidy level. Two sets of reciprocal pairwise interspecific crosses involving the pentaploid species pair R. dumalis and R. rubiginosa, and the pentaploid/tetraploid species pair R. sherardii and R. villosa, were analysed for 13 and 12 microsatellite DNA loci, respectively. Single loci were represented by a maximum of three simultaneously occurring alleles in R. villosa, and four alleles in the other three parental plants. In the experimentally derived offspring, the theoretical maximum of five alleles was found for only one locus in the pentaploid progenies. Microsatellite DNA allele composition was identical with that of the maternal parent in 10 offspring plants, which were probably derived through apomixis. Almost all microsatellite DNA alleles were shared with the maternal parent also in the remaining offspring, but 1-4 alleles shared only with the paternal parent, indicating sexual seed formation. Analysis of quantitative peak differences allowed a tentative estimation of allelic configuration in the individual plants, and suggested that bivalent formation preferentially takes place between chromosomes that consistently share the same microsatellite alleles and therefore appear to be highly homologous. Moreover, alleles that were shared between the species in each cross combination comparatively often appear to reside on the bivalent-forming chromosomes, whereas species-specific alleles instead occur comparatively often on the univalent-forming chromosomes and are therefore inherited through the maternal parent only. Recombination then takes place between very similar genomes also in interspecific crosses, resulting in a reproduction system that is essentially a mixture between apomixis and selfing.

Genetic differentiation and trade among populations of peach palm ( Bactris gasipaes Kunth) in the Peruvian Amazon-implications for genetic resource management.

Peach palm ( Bactris gasipaes Kunth) is cultivated for fruit and 'heart of palm', and is an important component of agroforestry systems in the Peruvian Amazon. In this study, AFLP was used to compare genetic diversity among domesticated populations along the Paranapura and Cuiparillo rivers, which are managed by indigenous and colonist farming communities, respectively. Gene diversity was 0.2629 for the populations in indigenous communities and 0.2534 in colonist communities. Genetic differentiation among populations ( G(st)) was 0.0377-0.0416 ( P<0.01) among populations along both rivers. There was no relation between genetic differentiation and the geographical location of populations along the rivers. Since natural seed dispersal by birds and rodents is thought to occur only across relatively short distances (100-200 m), it is likely that exchange of material by farmers and commercial traders is responsible for most of the 'long-distance' (over more than 20 km) gene flow among populations along the two rivers studied. This exchange of material may be important to counteract the effects of selection as well as genetic drift in small groups of trees in farmers' fields, much as in a metapopulation, and may account for the weak genetic differentiation between the two rivers ( G(st)=0.0249, P<0.01). A comparison with samples from other landraces in Peru and Brazil showed the existence of an isolation-by-distance structure up to 3,000 km, consistent with gene flow on a regional scale, likely mediated by trade in the Amazon Basin. Results are discussed with regard to practical implications for the management of genetic resources with farming communities.

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.

Identification of cut rose (Rosa hybrida) and rootstock varieties using robust sequence tagged microsatellite site markers.

In this study a DNA fingerprinting protocol was developed for the identification of rose varieties based on the variability of microsatellites. Microsatellites were isolated from Rosa hybrida L. using enriched small insert libraries. In total 24 polymorphic sequenced tagged microsatellite site (STMS) markers with easily scorable allele profiles, from six different linkage groups, were used to characterize 46 Hybrid Tea varieties and 30 rootstock varieties belonging to different species (Rosa canina L., Rosa indica Thory., Rosa chinensis Jacq., Rosa rubiginosa L., and Rosa rubrifolia glauca Pour.). Clones and known flower color mutants were identified as being identical, all other varieties were differentiated by a unique pattern with as few as three STMS markers. The high discriminating power of the loci suggests that a selection of the most-robust STMS markers may be able to differentiate any two varieties within rootstocks or Hybrid Teas except for mutants. The selected STMS markers will be useful as a tool for reference collection management, for assessing essential derivation of varieties and illegal propagation.

Cloning and characterization of four apple MADS box genes isolated from vegetative tissue.

With the aim of finding genes involved in the floral transition of woody species four MADS box genes containing cDNAs from apple (Malus domestica) have been isolated. Three genes were isolated from vegetative tissue of apple, but were homologues of known genes that specify floral organ identity. MdMADS13 is an AP3-like B class MADS box gene, and was mainly expressed in petals and stamens as demonstrated by Northern blot analysis. MdMADS14 and -15 are AGAMOUS-like genes. They differed slightly in expression patterns on Northern blots, with MdMADS15 mRNA levels equally high in stamens and carpels, but MdMADS14 preferably expressed in carpels. MdMADS14 is likely to be the apple orthologue of one of the Arabidopsis thaliana SHATTERPROOF genes, and MdMADS15 closely resembled the Arabidopsis AGAMOUS gene. It has been shown with RT-PCR that the three floral apple MADS box genes are expressed in vegetative tissues of adult as well as juvenile trees, albeit at low levels. MdMADS12 is an AP1-like gene that is expressed at similar levels in leaves, vegetative shoots, and floral tissues, and that may be involved in the transition from the juvenile to the adult stage.

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.

Construction and testing of a microsatellite database containing more than 500 tomato varieties.

The aim of this study was to evaluate the suitability of sequence tagged microsatellite site (STMS) markers for varietal identification and discrimination in tomato. For this purpose, a set of 20 STMS primer pairs was used to construct a database containing the molecular description of the most common varieties (>500) of tomato grown in Europe. The database was built and tested by a consortium of five European laboratories each using a different STMS detection system. In this way, it could be demonstrated that the STMS markers and database were suitable for use in network activities where a common database is being established on a continuing basis with data from different laboratories.Microsatellite polymorphism in tomato was found to be relatively low. The number of alleles per locus ranged from 2 to 8 with an average of 4.7 alleles per locus. Nevertheless, more than 90% of the varieties had different microsatellite profiles. A "blind testing" exercise showed that in general, identification of unknown samples (or detecting the most similar variety) with the 20 markers and the database was relatively easy for homogeneous varieties but less certain with heterogeneous varieties when using pools of 6 individuals.

Microsatellite markers useful throughout the genus Dianthus.

Using repeats found in sequences from Dianthus species present in the EMBL database, primers for STMS (sequence-tagged microsatellite site) analysis were developed and tested. Five loci were polymorphic and amplified products of sufficient quality in nearly all of the 26 Dianthus species tested, except MS-DINGSTA, which amplified in only one-third of the species. Loci MS-DINMADSBOX and MS-DCDIA30 produced allele series that were mostly two nucleotides (the repeat unit) apart. MS-DCAMCRBSY and MS-DINCARACC also amplified regular series of alleles, but more than two fragments per individual were detected in a number of species. Both loci code for a member of the ACC synthase gene family. The observation that the loci amplified across a wide range of Dianthus species may imply that the different species within the genus are relatively closely related. Alternatively, it may indicate that the regions selected for primer design (some of which are in coding regions) are well conserved. These microsatellites will be useful for the measurement of genetic diversity in natural populations of Dianthus species and the identification of carnation varieties.

Microsatellite retrieval in lettuce (Lactuca sativa L.).

By using enriched genomic libraries, microsatellite-containing sequences were isolated from lettuce (Lactuca sativa) with high efficiency. With this approach, a sizeable fraction (up to 55%) of the clones contained a microsatellite. In about half of these clones, primers could be designed for PCR amplification of the microsatellite. This yielded 28 primer sets amplifying unambiguously scorable products, of which 26 showed polymorphisms in a test set of six lettuce varieties. Practically all microsatellite-amplifying primer sets yielded products in lettuce's nearest relative, L. serriola, but only half of the primer sets yielded products in the more distant species L. saligna and L. virosa. An average polymorphism information content (PIC) value of 0.55 and an average number of 3.5 alleles per locus were in the normal range for a self-fertilizing species like lettuce. In addition, the incidental cloning of a microsatellite-containing repeat family, apparently specific for Lactuca, is reported and the implications for the efficient retrieval of single-locus microsatellite sequences are discussed. The microsatellite loci isolated will be useful for distinguishing lettuce cultivars and for screening diversity of genetic resources.

Phylogenetic relationships among Lactuca (Asteraceae) species and related genera based on ITS-1 DNA sequences.

Internal transcribed spacer (ITS-1) sequences from 97 accessions representing 23 species of Lactuca and related genera were determined and used to evaluate species relationships of Lactuca sensu lato (s.l.). The ITS-1 phylogenies, calculated using PAUP and PHYLIP, correspond better to the classification of Feráková than to other classifications evaluated, although the inclusion of sect. Lactuca subsect. Cyanicae is not supported. Therefore, exclusion of subsect. Cyanicae from Lactuca sensu Feráková is proposed. The amended genus contains the entire gene pool (sensu Harlan and De Wet) of cultivated lettuce (Lactuca sativa). The position of the species in the amended classification corresponds to their position in the lettuce gene pool. In the ITS-1 phylogenies, a clade with L. sativa, L. serriola, L. dregeana, L. altaica, and L. aculeata represents the primary gene pool. L. virosa and L. saligna, branching off closest to this clade, encompass the secondary gene pool. L. virosa is possibly of hybrid origin. The primary and secondary gene pool species are classified in sect. Lactuca subsect. Lactuca. The species L. quercina, L. viminea, L. sibirica, and L. tatarica, branching off next, represent the tertiary gene pool. They are classified in Lactuca sect. Lactucopsis, sect. Phaenixopus, and sect. Mulgedium, respectively. L. perennis and L. tenerrima, classified in sect. Lactuca subsect. Cyanicae, form clades with species from related genera and are not part of the lettuce gene pool.