Search

Next Generation Sequencing (NGS) may enable estimating relationships among genotypes using allelic variation of multiple nuclear genes simultaneously. We explored the potential and caveats of this strategy in four genetically distant Lilium cultivars to estimate their genetic divergence from transcriptome sequences using three approaches: POFAD (Phylogeny of Organisms from Allelic Data, uses allelic information of sequence data), RAxML (Randomized Accelerated Maximum Likelihood, tree building based on concatenated consensus sequences) and Consensus Network (constructing a network summarizing among gene tree conflicts). Twenty six gene contigs were chosen based on the presence of orthologous sequences in all cultivars, seven of which also had an orthologous sequence in Tulipa, used as out-group. The three approaches generated the same topology. Although the resolution offered by these approaches is high, in this case there was no extra benefit in using allelic information. We conclude that these 26 genes can be widely applied to construct a species tree for the genus Lilium.

Background: Plants have developed a variety of mechanisms to counteract aphid attacks. They activate their defences by changing the expression of specific genes. Previously we identified an activation tag mutant of Arabidopsis thaliana on which Myzus persicae population development was reduced. Activation tag mutants are gain-of-function in which the expression of a gene is increased by the insertion of the Cauliflower mosaic virus 35S enhancer that acts on the natural promoter. By further characterizing this previously identified mutant we identified a gene that reduces performance of M. persicae and also provided clues about the mechanism involved. Results: We show that SKU5 SIMILAR 13 (SKS13), a gene whose expression in wild type plants is restricted to pollen and non-responsive to M. persicae attack, is overexpressed in the A. thaliana mutant showing reduced performance of M. persicae. Monitoring M. persicae feeding behaviour on SKS13 overexpressing plants indicated that M. persicae have difficulties feeding from the phloem. The constitutive expression of SKS13 results in accumulation of reactive oxygen species, which is possibly regulated through the jasmonic acid pathway. The enhanced resistance is not aphid species specific as also the population development of Brevicoryne brassicae was affected. Conclusions: We demonstrate that constitutive expression in leaves of the pollen-specific gene SKS13 can enhance plant defence, resulting in a reduction of M. persicae population development and also decreases the transmission of persistent viruses. Overexpression of SKS13 in A. thaliana also affects B. brassicae and possibly other phloem feeding insects as well. Identifying genes that can enhance plant defence against insects will be important to open up new avenues for the development of insect resistant crop plants.

Plants growing in their natural habitats are often challenged simultaneously by multiple stress factors, both abiotic and biotic. Research has so far been limited to responses to individual stresses, and understanding of adaptation to combinatorial stress is limited, but indicative of non-additive interactions. Omics data analysis and functional characterization of individual genes has revealed a convergence of signaling pathways for abiotic and biotic stress adaptation. Taking into account that most data originate from imposition of individual stress factors, this review summarizes these findings in a physiological context, following the pathogenesis timeline and highlighting potential differential interactions occurring between abiotic and biotic stress signaling across the different cellular compartments and at the whole plant level. Potential effects of abiotic stress on resistance components such as extracellular receptor proteins, R-genes and systemic acquired resistance will be elaborated, as well as crosstalk at the levels of hormone, reactive oxygen species, and redox signaling. Breeding targets and strategies are proposed focusing on either manipulation and deployment of individual common regulators such as transcription factors or pyramiding of non- (negatively) interacting components such as R-genes with abiotic stress resistance genes. We propose that dissection of broad spectrum stress tolerance conferred by priming chemicals may provide an insight on stress cross regulation and additional candidate genes for improving crop performance under combined stress. Validation of the proposed strategies in lab and field experiments is a first step toward the goal of achieving tolerance to combinatorial stress in crops.

WOS: 000335066300001, PubMed ID: 24581289, Background: Breeders in the allo-octoploid strawberry currently make little use of molecular marker tools. As a first step of a QTL discovery project on fruit quality traits and resistance to soil-borne pathogens such as Phytophthora cactorum and Verticillium we built a genome-wide SSR linkage map for the cross Holiday x Korona. We used the previously published MADCE method to obtain full haplotype information for both of the parental cultivars, facilitating in-depth studies on their genomic organisation. Results: The linkage map incorporates 508 segregating loci and represents each of the 28 chromosome pairs of octoploid strawberry, spanning an estimated length of 2050 cM. The sub-genomes are denoted according to their sequence divergence from F. vesca as revealed by marker performance. The map revealed high overall synteny between the sub-genomes, but also revealed two large inversions on LG2C and LG2D, of which the latter was confirmed using a separate mapping population. We discovered interesting breeding features within the parental cultivars by in-depth analysis of our haplotype data. The linkage map-derived homozygosity level of Holiday was similar to the pedigree-derived inbreeding level (33% and 29%, respectively). For Korona we found that the observed homozygosity level was over three times higher than expected from the pedigree (13% versus 3.6%). This could indicate selection pressure on genes that have favourable effects in homozygous states. The level of kinship between Holiday and Korona derived from our linkage map was 2.5 times higher than the pedigree-derived value. This large difference could be evidence of selection pressure enacted by strawberry breeders towards specific haplotypes. Conclusion: The obtained SSR linkage map provides a good base for QTL discovery. It also provides the first biologically relevant basis for the discernment and notation of sub-genomes. For the first time, we revealed genomic rearrangements that were verified in a separate mapping population. We believe that haplotype information will become increasingly important in identifying marker-trait relationships and regions that are under selection pressure within breeding material. Our attempt at providing a biological basis for the discernment of sub-genomes warrants follow-up studies to streamline the naming of the sub-genomes among different octoploid strawberry maps., Technological Top Institute Green Genetics [1C004RP]; Socrates-Erasmus Student Exchange Programme, The Socrates-Erasmus Student Exchange Programme is acknowledged for the fellowship awarded to Hulya Yilmaz Temel. Tom Davis, University of New Hampshire, is acknowledged for sharing sequence information for the design of the primers TDFM1, -2, and -6, and Paul Arens, Wageningen UR Plant Breeding is acknowledged for the design of these specific primers. Funding for this research was provided through the Technological Top Institute Green Genetics, project number 1C004RP.

We analysed metabolic diversity in apples from wild species, elite material and a F1 population, using liquid chromatography–mass spectrometry (LC-QTOF-MS). The evaluated elite material appeared to have strongly reduced levels of phenolic compounds, down to 1% of the concentrations in the investigated wild germplasm. In one quarter of the F1 population, the concentrations of phenolic compounds such as quercetin derivatives, procyanidin, catechin and epicatechin were further significantly reduced, due to accumulation of recessive alleles of putatively leucoanthocyanidin reductase, a structural gene that is located at the top of LG16. In another part of F1 progeny, putatively glycosylated forms of s-glycols were up to 50 times more abundant compared to both parents. These metabolites were mapped with high logarithm of odds (LOD) scores at the top of LG8, and progeny that was homozygous recessive for the candidate gene showed the elevated levels. We hypothesize that this was caused by inheritance of non-functional alleles of enoyl-CoA hydratase gene. Both examples of

Progenies of ‘Schmidt's Antonovka’ (SA) have been widely used in Western breeding programs as a source of scab resistance. The identity of SA has remained obscure, especially due to the existence of a series of ‘Antonovka’ cultivars with different origins. In this paper we show Schmidt's Antonovka to be identical to ¿¿¿¿´¿¿¿¿¿ ¿¿¿¿¿¿¿¿¿¿¿¿ or ‘Common Antonovka’ (CA), an old Russian cultivar of unknown origin, by comparing simple sequence repeat (SSR) and SNP genotyping data from several first-generation descendants of SA from two European collections and a CA accession from the germplasm collection held at VNIISPK (The All-Russian Research Institute of Horticultural Breeding, Orel, Russia). The use of CA in Russian breeding programs is also briefly reviewed.

Background Phytophthora infestans, causing late blight in potato, remains one of the most devastating pathogens in potato production and late blight resistance is a top priority in potato breeding. The introduction of multiple resistance (R) genes with different spectra from crossable species into potato varieties is required. Cisgenesis is a promising approach that introduces native genes from the crops own gene pool using GM technology, thereby retaining favourable characteristics of established varieties. Results We pursued a cisgenesis approach to introduce two broad spectrum potato late blight R genes, Rpi-sto1 and Rpi-vnt1.1 from the crossable species Solanum stoloniferum and Solanum venturii, respectively, into three different potato varieties. First, single R gene-containing transgenic plants were produced for all varieties to be used as references for the resistance levels and spectra to be expected in the respective genetic backgrounds. Next, a construct containing both cisgenic late blight R genes (Rpi-vnt1.1 and Rpi-sto1), but lacking the bacterial kanamycin resistance selection marker (NPTII) was transformed to the three selected potato varieties using Agrobacterium-mediated transformation. Gene transfer events were selected by PCR among regenerated shoots. Through further analyses involving morphological evaluations in the greenhouse, responsiveness to Avr genes and late blight resistance in detached leaf assays, the selection was narrowed down to eight independent events. These cisgenic events were selected because they showed broad spectrum late blight resistance due to the activity of both introduced R genes. The marker-free transformation was compared to kanamycin resistance assisted transformation in terms of T-DNA and vector backbone integration frequency. Also, differences in regeneration time and genotype dependency were evaluated. Conclusions We developed a marker-free transformation pipeline to select potato plants functionally expressing a stack of late blight R genes. Marker-free transformation is less genotype dependent and less prone to vector backbone integration as compared to marker-assisted transformation. Thereby, this study provides an important tool for the successful deployment of R genes in agriculture and contributes to the production of potentially durable late blight resistant potatoes.

Selective lignin degrading fungi can be used as pre-treatment to make cellulose in plant cell walls accessible for rumen microbes. According to previous studies, Ceriporiopsis subvermispora and Lentinula edodes can increase the in vitro rumen degradability of lignocellulosic biomass in 7 to 8 weeks. To shorten this treatment three additives, urea (1 µg/g or 10 µg/g), manganese (15 µg/g or 150 µg/g) and linoleic acid (0.5 mM or 1 mM), were tested during C. subvermispora or L. edodes treatment of wheat straw and wood chips. After 2 and 4 weeks samples were taken to analyse the in vitro rumen degradability and fibre content. Two weeks of fungal treatment did not increase the in vitro rumen degradability of wheat straw or wood chips and additives did not have an effect on this. Addition of manganese (both concentrations) to C. subvermispora on wood chips did result in more lignin degradation. Linoleic acid (1 mM) and manganese (15 µg/g) enhanced lignin degradation of wood chips by L. edodes after 2 weeks of treatment. Fungal treatment for four weeks increased the in vitro rumen degradability. However additives did not enhance this. Urea and manganese did result in higher lignin degradation after 4 weeks of treatment of wheat straw with C. subvermispora. The cellulose content was not changed during the fungal treatment, without or with either additive.

Clavibacter michiganensis subsp. michiganensis (Cmm) is the pathogen causing bacterial canker in tomato. The disease was described for the first time in 1910 in Michigan, USA. Cmmis considered the most harmful bacteria threatening tomato. Disease transmission occurs via seed and symptoms become visible at least 20 days after infection. Due to its complex strategy and transmission, Cmm is under quarantine regulation in EU and other countries. There is no method to stop disease progress in plants after infection. Thus, disease management consists usually of chemical treatments as protection and by careful clean cultural practices. However, the use of resistant varieties is the most effective and environmentally friendly method. Unfortunately, there is no cultivar harboring effective resistance on the market although efforts to get resistant varieties already started in the 60s. Our aim in this thesis was to develop valuable genetic material for breeders in order to enable them to release resistant cultivars and provide comprehensive scientific knowledge for further detailed research about Cmm. Our scientific activity in this thesis started with the identification of new Cmm resistance sources and confirmation of existent ones. In Chapter 3 we have screened a collection of wild tomatoes for resistance to Cmm. We made use of Real Time TaqMan PCR for intensive phenotyping. Using wilting and bacterial concentration as parameters for evaluation of the sources, we have identified new sources and confirmed existent ones. We have decided to continue further with one new source, S. pimpinellifolium, and one existent source, S . arcanum. We continued our research in Chapter 4 with a genetic analysis of the new source coming from S. pimpinellifolium. A recombinant inbred line population between the resistant parent, S. pimpinellifolium, and the susceptible parent S. lycopersicum was evaluated in three different environments. Wilting, bacterial concentration, and stem discoloration were the scored parameters. Responses of resistance in different environments were determined and genomic regions responsible for different responses were mapped. In Chapter 5, we have continued our research by fine mapping of previously identified genomic regions and developing nearly isogenic lines containing those genomic regions. By doing fine mapping, we made use of old stock DNA and recently developed different types of SNP marker technology. Previously identified Quantitative Trait Loci(QTLs)could be more precisely delimited. During isogenic line development, embryo rescue was used in order to break the genetic barrier between our S. arcanum source and tomato. Marker assisted backcrossing was applied to obtain lines with a minimum of donor parent in a faster way. By using this method we gained two generations of backcrossing. In order to obtain comprehensive information about different Cmm isolates inTurkey, we have performed multi locus sequence analysis (MLST) analysis on a Cmm collection, which was collected in 20 years of time in different part of Turkey. In Chapter 6 a statistical analysis of this collection revealed that measurement of clonality of this collection was possible as well as it was possible to predict the virulence level of strainsusing a subset of housekeeping genes. All knowledge gained by our experiments and knowledge coming from literature about Cmm have led a Review paper, Chapter 2, by which comprehensive information about Cmm resistance sources, genetic analysis of these sources, detection methods of Cmm, infection strategies of Cmm and interaction with its host was discussed. In conclusion, two good Cmm resistance sources and several tools and methods are available for breeders. Genomic regions of these sources associated with resistance were determined. Wider knowledge about Cmm detection, Cmm infection and Cmm interaction with its host are available for further research.

Tomato (Solanum lycopersicum) is affected by a wide range of biotic stresses, of which Bemisia tabaci is one of the most important.Bemisia tabaci affects tomato directly through phloem sap feeding, and indirectly through its ability to be the vector of a large number of viruses. Different methods are available for whitefly control, and although several biological control agents are used against whiteflies in greenhouse cultivation, chemical control still is an essential component in open field tomato production. Breeding for host plant resistance is considered as one of the most promising methods in insect pest control in crop plants, and especially it is a promising alternative in whitefly control. Resistance to whiteflies was found in several wild relatives of tomato like Solanum peruvianum, S. pennellii, S. habrochaites, S. lycopersicum var. cerasiforme, S. pimpinellifolium andS. galapagense. In spite of previous breeding efforts, whiteflies are still a problem in tomato cultivation. The aim of my research was to identify and understand resistance mechanisms targeting specific stages of the whitefly life cycle in order to provide breeders with tools for developing whitefly resistant varieties. I assessed the natural variation and whitefly resistance in Solanum galapagense and S. cheesmaniae, two wild tomato species endemic to the Galapagos Islands. Previously, Solanum galapagense and S. cheesmaniae were classified as two species based on a morphological species concept, but with molecular markers no clear separation could be made. So far, only a limited number of accessions/populations of S. galapagense and S. cheesmaniae have been evaluated for insect resistance and therefore it was unknown if the insect resistance coincides with the morphological species boundaries. Neither was there any knowledge about the relation between geographical and climatic conditions today on the Galapagos and the occurrence of the two species. We characterized twelve accessions of S. galapagense, 22 of S. cheesmaniae, and as reference one of S. lycopersicum for whitefly resistance using no-choice experiments. Whitefly resistance was found in S. galapagense only and was associated with the presence of relatively high levels of acyl sugars and the presence of glandular trichomes of type I and IV.It is likely that a minimum level of acyl sugars and the presence of glandular trichomes type IV are needed to achieve an effective level of resistance. Genetic fingerprinting using 3316 polymorphic SNP markers did not show a clear differentiation between the two species endemic to the Galapagos. Acyl sugar accumulation as well as the climatic and geographical conditions at the collection sites of the accessions did not follow the morphological species boundaries. Altogether, our results suggest that S. galapagense and S. cheesmaniae might be considered as morphotypes rather than two species and that their co-existence is likely the result of selective pressure. Plants possess several resistance mechanisms acting at different time points during the interaction with herbivorous insect. Before any contact with the insects, plants emit an array of volatile organic compounds that can act as attractant or repellent of insects.Bemisia tabaci use a set of plant-derived cues in the process of host plant selection. It recognizes mainly monoterpenes (p-cymene, γ-terpinene and β-myrcene, α-phellandrene) and sesquiterpenes (7-epizingiberene and R-curcumene). Previously the line FCN93-6-2, which was derived from a cross between a susceptible tomato cultivar (Uco Plata INTA) and S. habrochaites (FCN3-5) was proved to be non-preferred by the greenhouse whitefly Trialeurodes vaporariorum. We identified chemical cues produced by FCN93-6-2 and S. habrochaites that can affect the preference of the whitefly B. tabaci as well as the potential chromosomal region(s) of S. habrochaites harbouring the genes involved in the preference. Two S. habrochaites accessions (CGN1.1561 and in FCN3-5) and the line FCN93-6-2 were non-preferred by B. tabaci when the whiteflies could get in direct contact with the plant and also when the whiteflies were offered olfactory cues only. The non-preference was independent of trichome type IV and of the presence of methyl-ketones but associated to the presence of monoterpenes in lower concentrations. Functional validation of the candidate metabolites and of the different introgressions is still needed. Once the insect has landed on a plant, another set of resistance mechanisms enter into action. We have described a 3.06 Mbp introgression on top of Chromosome 5 (OR-5) from the wild tomato species S. habrochaites (CGN1.1561). For the identification of OR-5, we went from the selection of specific F2 plants to the development of F2BC4S1 and F2BC4S2 families. This introgression was sufficient to reduce whitefly fecundity without an evident effect on whitefly survival. The identification of mechanisms exclusively affecting whitefly fecundity and independent of trichomes type IV opens new doors for resistance breeding to whiteflies that may be especially interesting in greenhouse cultivation combination with natural enemies of the whitefly. As an additional layer of defences, plants can perceive stress signals and respond to them in a specific way through induction of their immune system. This induction can also be triggered by exposing the plants to priming agents like hormones, some xenobiotic chemicals, like benzothiadiazole (BTH), β-aminobutyric acid (BABA), and sugars. Although the effect of priming agents was shown in laboratory and field studies, little is known about the effect of the genetic background of tomato on the extent of the priming, e.g. do genotypes varying in their level of resistance to insects and pathogens respond in the same way to a priming agent. We assessed the effect of selected priming agents on the effectiveness of natural defence in tomato. A set of no-choice and choice bioassays was conducted using tomato genotypes varying in their level of basal resistance to Bemisia tabaci and pathogens. We observed that whitefly survival and oviposition were not affected by the priming treatment in no-choice assays. Overall, in choice assays, fructose treated plants were more preferred by whiteflies than control plants. A genotype specific effect of priming was seen for the line FCN93-6-2. On this tomato line, JA and BABA applications decreased the number of whiteflies, e.g. making them less preferred. In this thesis, I have gone from the screening of wild relatives of tomatoes to in depth characterization of resistance mechanisms. I have identified resistance mechanisms targeting specific stages of the whitefly life cycle, thus providing new tools for breeding durable whitefly resistance in tomato.

Clavibacter michiganensis subsp. michiganensis (Cmm) is the pathogen causing bacterial canker in tomato. The disease was described for the first time in 1910 in Michigan, USA. Cmmis considered the most harmful bacteria threatening tomato. Disease transmission occurs via seed and symptoms become visible at least 20 days after infection. Due to its complex strategy and transmission, Cmm is under quarantine regulation in EU and other countries. There is no method to stop disease progress in plants after infection. Thus, disease management consists usually of chemical treatments as protection and by careful clean cultural practices. However, the use of resistant varieties is the most effective and environmentally friendly method. Unfortunately, there is no cultivar harboring effective resistance on the market although efforts to get resistant varieties already started in the 60s. Our aim in this thesis was to develop valuable genetic material for breeders in order to enable them to release resistant cultivars and provide comprehensive scientific knowledge for further detailed research about Cmm. Our scientific activity in this thesis started with the identification of new Cmm resistance sources and confirmation of existent ones. In Chapter 3 we have screened a collection of wild tomatoes for resistance to Cmm. We made use of Real Time TaqMan PCR for intensive phenotyping. Using wilting and bacterial concentration as parameters for evaluation of the sources, we have identified new sources and confirmed existent ones. We have decided to continue further with one new source, S. pimpinellifolium, and one existent source, S . arcanum. We continued our research in Chapter 4 with a genetic analysis of the new source coming from S. pimpinellifolium. A recombinant inbred line population between the resistant parent, S. pimpinellifolium, and the susceptible parent S. lycopersicum was evaluated in three different environments. Wilting, bacterial concentration, and stem discoloration were the scored parameters. Responses of resistance in different environments were determined and genomic regions responsible for different responses were mapped. In Chapter 5, we have continued our research by fine mapping of previously identified genomic regions and developing nearly isogenic lines containing those genomic regions. By doing fine mapping, we made use of old stock DNA and recently developed different types of SNP marker technology. Previously identified Quantitative Trait Loci(QTLs)could be more precisely delimited. During isogenic line development, embryo rescue was used in order to break the genetic barrier between our S. arcanum source and tomato. Marker assisted backcrossing was applied to obtain lines with a minimum of donor parent in a faster way. By using this method we gained two generations of backcrossing. In order to obtain comprehensive information about different Cmm isolates inTurkey, we have performed multi locus sequence analysis (MLST) analysis on a Cmm collection, which was collected in 20 years of time in different part of Turkey. In Chapter 6 a statistical analysis of this collection revealed that measurement of clonality of this collection was possible as well as it was possible to predict the virulence level of strainsusing a subset of housekeeping genes. All knowledge gained by our experiments and knowledge coming from literature about Cmm have led a Review paper, Chapter 2, by which comprehensive information about Cmm resistance sources, genetic analysis of these sources, detection methods of Cmm, infection strategies of Cmm and interaction with its host was discussed. In conclusion, two good Cmm resistance sources and several tools and methods are available for breeders. Genomic regions of these sources associated with resistance were determined. Wider knowledge about Cmm detection, Cmm infection and Cmm interaction with its host are available for further research.

Termites normally rely on gut symbionts to decompose organic matter but the Macrotermitinae domesticated Termitomyces fungi to produce their own food. This transition was accompanied by a shift in the composition of the gut microbiota, but the complementary roles of these bacteria in the symbiosis have remained enigmatic. We obtained high-quality annotated draft genomes of the termite Macrotermes natalensis, its Termitomyces symbiont, and gut metagenomes from workers, soldiers, and a queen. We show that members from 111 of the 128 known glycoside hydrolase families are represented in the symbiosis, that Termitomyces has the genomic capacity to handle complex carbohydrates, and that worker gut microbes primarily contribute enzymes for final digestion of oligosaccharides. This apparent division of labor is consistent with the Macrotermes gut microbes being most important during the second passage of comb material through the termite gut, after a first gut passage where the crude plant substrate is inoculated with Termitomyces asexual spores so that initial fungal growth and polysaccharide decomposition can proceed with high efficiency. Complex conversion of biomass in termite mounds thus appears to be mainly accomplished by complementary cooperation between a domesticated fungal monoculture and a specialized bacterial community. In sharp contrast, the gut microbiota of the queen had highly reduced plant decomposition potential, suggesting that mature reproductives digest fungal material provided by workers rather than plant substrate.

Nagaan van gezondheidsbevorderende werking van paddestoelen als middel om de consument te kunnen overtuigen van de extra voordelen van paddenstoel consumptie. Als eerste stap stelt PPO Paddenstoelen voor om, in een samenwerking met de vakgroep Humane Biologie van de Universiteit Maastricht, onderzoek te doen naar effecten van de consumptie van glucanen op het cholesterolgehalte.

Ca. 60 accessies van champignonlijnen uit de collectie van Plant Breeding Wageningen UR zijn geteeld en de vruchtlichamen genalayseerd op vetzuur derivaten en nucleotiden (o.a. mono sodium glutamaat (MSG)/umami smaak). Bij deze analyse is een “common control sample” meegenomen dat elders is geproduceerd (Food Analysis Laboratory Control Center, Biochemistry Department (0308), Virginia Tech). Voor onderzoek naar metabolieten in paddenstoelen is internationaal afgesproken dat deze standaard wordt meegenomen om analytische variaties te kunnen onderscheiden van echte verschillen in samenstelling bij analysen op verschillende laboratoria.

The present-day cultivars of button mushroom are too sensitive to mechanical damage. Consequently, mushrooms for the fresh market are still handpicked, which is a main cost factor for the Dutch mushroom industry. The main objective of this thesis is to use the phenotypic variation and segregation to study the genetic determinants explaining the differences in bruising sensitivity and generate breeding stocks that can be used to construct commercial varieties that are suitable for mechanical harvest. In addition, the genetic basis of other quality-related traits was investigated. For this, segregating populations were generated and used to study in more detail the life cycle of the bisporicvariety. The availability of the whole genome sequence allowsa more detailed study of recombination frequencies generating knowledge invaluable for further mushroom breeding.

Ca. 60 accessies van champignonlijnen uit de collectie van Plant Breeding Wageningen UR zijn geteeld en de vruchtlichamen genalayseerd op vetzuur derivaten en nucleotiden (o.a. mono sodium glutamaat (MSG)/umami smaak). Bij deze analyse is een “common control sample” meegenomen dat elders is geproduceerd (Food Analysis Laboratory Control Center, Biochemistry Department (0308), Virginia Tech). Voor onderzoek naar metabolieten in paddenstoelen is internationaal afgesproken dat deze standaard wordt meegenomen om analytische variaties te kunnen onderscheiden van echte verschillen in samenstelling bij analysen op verschillende laboratoria.

Selective lignin degrading fungi can be used as pre-treatment to make cellulose in plant cell walls accessible for rumen microbes. According to previous studies, Ceriporiopsis subvermispora and Lentinula edodes can increase the in vitro rumen degradability of lignocellulosic biomass in 7 to 8 weeks. To shorten this treatment three additives, urea (1 µg/g or 10 µg/g), manganese (15 µg/g or 150 µg/g) and linoleic acid (0.5 mM or 1 mM), were tested during C. subvermispora or L. edodes treatment of wheat straw and wood chips. After 2 and 4 weeks samples were taken to analyse the in vitro rumen degradability and fibre content. Two weeks of fungal treatment did not increase the in vitro rumen degradability of wheat straw or wood chips and additives did not have an effect on this. Addition of manganese (both concentrations) to C. subvermispora on wood chips did result in more lignin degradation. Linoleic acid (1 mM) and manganese (15 µg/g) enhanced lignin degradation of wood chips by L. edodes after 2 weeks of treatment. Fungal treatment for four weeks increased the in vitro rumen degradability. However additives did not enhance this. Urea and manganese did result in higher lignin degradation after 4 weeks of treatment of wheat straw with C. subvermispora. The cellulose content was not changed during the fungal treatment, without or with either additive.

The present-day cultivars of button mushroom are too sensitive to mechanical damage. Consequently, mushrooms for the fresh market are still handpicked, which is a main cost factor for the Dutch mushroom industry. The main objective of this thesis is to use the phenotypic variation and segregation to study the genetic determinants explaining the differences in bruising sensitivity and generate breeding stocks that can be used to construct commercial varieties that are suitable for mechanical harvest. In addition, the genetic basis of other quality-related traits was investigated. For this, segregating populations were generated and used to study in more detail the life cycle of the bisporicvariety. The availability of the whole genome sequence allowsa more detailed study of recombination frequencies generating knowledge invaluable for further mushroom breeding.

This paper describes the progress that has been made since the draft genome sequence of potato has been obtained and the analyses that need to be done to make further progress. Although sequencing has become less expensive and read lengths have increased, making optimal use of the information obtained is still difficult, certainly in the tetraploid potato crop. Major challenges in potato genomics are standardized genome assembly and haplotype analysis. Sequencing methods need to be improved further to achieve precision breeding. With the current new generation sequencing technology, the focus in potato breeding will shift from phenotype improvement to genotype improvement. In this respect, it is essential to realize that different alleles of the same gene can lead to different phenotypes depending on the genetic background and that there is significant epistatic interaction between different alleles. Genome-wide association studies will gain statistical power when binary single nucleotide polymorphism (SNP) data can be replaced with multi-allelic haplotype data. Binary SNP can be distributed across the many different alleles per locus or may be haplotype-specific, and potentially tag specific alleles which clearly differ in their contribution to a certain trait value. Assembling reads from the same linkage phase proved to allow constructing sufficiently long haplotype tracts to ensure their uniqueness. Combining large phenotyping data sets with modern approaches to sequencing and haplotype analysis and proper software will allow the efficiency of potato breeding to increase.

Nagaan van gezondheidsbevorderende werking van paddestoelen als middel om de consument te kunnen overtuigen van de extra voordelen van paddenstoel consumptie. Als eerste stap stelt PPO Paddenstoelen voor om, in een samenwerking met de vakgroep Humane Biologie van de Universiteit Maastricht, onderzoek te doen naar effecten van de consumptie van glucanen op het cholesterolgehalte.

Tomato (Solanum lycopersicum) is affected by a wide range of biotic stresses, of which Bemisia tabaci is one of the most important.Bemisia tabaci affects tomato directly through phloem sap feeding, and indirectly through its ability to be the vector of a large number of viruses. Different methods are available for whitefly control, and although several biological control agents are used against whiteflies in greenhouse cultivation, chemical control still is an essential component in open field tomato production. Breeding for host plant resistance is considered as one of the most promising methods in insect pest control in crop plants, and especially it is a promising alternative in whitefly control. Resistance to whiteflies was found in several wild relatives of tomato like Solanum peruvianum, S. pennellii, S. habrochaites, S. lycopersicum var. cerasiforme, S. pimpinellifolium andS. galapagense. In spite of previous breeding efforts, whiteflies are still a problem in tomato cultivation. The aim of my research was to identify and understand resistance mechanisms targeting specific stages of the whitefly life cycle in order to provide breeders with tools for developing whitefly resistant varieties. I assessed the natural variation and whitefly resistance in Solanum galapagense and S. cheesmaniae, two wild tomato species endemic to the Galapagos Islands. Previously, Solanum galapagense and S. cheesmaniae were classified as two species based on a morphological species concept, but with molecular markers no clear separation could be made. So far, only a limited number of accessions/populations of S. galapagense and S. cheesmaniae have been evaluated for insect resistance and therefore it was unknown if the insect resistance coincides with the morphological species boundaries. Neither was there any knowledge about the relation between geographical and climatic conditions today on the Galapagos and the occurrence of the two species. We characterized twelve accessions of S. galapagense, 22 of S. cheesmaniae, and as reference one of S. lycopersicum for whitefly resistance using no-choice experiments. Whitefly resistance was found in S. galapagense only and was associated with the presence of relatively high levels of acyl sugars and the presence of glandular trichomes of type I and IV.It is likely that a minimum level of acyl sugars and the presence of glandular trichomes type IV are needed to achieve an effective level of resistance. Genetic fingerprinting using 3316 polymorphic SNP markers did not show a clear differentiation between the two species endemic to the Galapagos. Acyl sugar accumulation as well as the climatic and geographical conditions at the collection sites of the accessions did not follow the morphological species boundaries. Altogether, our results suggest that S. galapagense and S. cheesmaniae might be considered as morphotypes rather than two species and that their co-existence is likely the result of selective pressure. Plants possess several resistance mechanisms acting at different time points during the interaction with herbivorous insect. Before any contact with the insects, plants emit an array of volatile organic compounds that can act as attractant or repellent of insects.Bemisia tabaci use a set of plant-derived cues in the process of host plant selection. It recognizes mainly monoterpenes (p-cymene, γ-terpinene and β-myrcene, α-phellandrene) and sesquiterpenes (7-epizingiberene and R-curcumene). Previously the line FCN93-6-2, which was derived from a cross between a susceptible tomato cultivar (Uco Plata INTA) and S. habrochaites (FCN3-5) was proved to be non-preferred by the greenhouse whitefly Trialeurodes vaporariorum. We identified chemical cues produced by FCN93-6-2 and S. habrochaites that can affect the preference of the whitefly B. tabaci as well as the potential chromosomal region(s) of S. habrochaites harbouring the genes involved in the preference. Two S. habrochaites accessions (CGN1.1561 and in FCN3-5) and the line FCN93-6-2 were non-preferred by B. tabaci when the whiteflies could get in direct contact with the plant and also when the whiteflies were offered olfactory cues only. The non-preference was independent of trichome type IV and of the presence of methyl-ketones but associated to the presence of monoterpenes in lower concentrations. Functional validation of the candidate metabolites and of the different introgressions is still needed. Once the insect has landed on a plant, another set of resistance mechanisms enter into action. We have described a 3.06 Mbp introgression on top of Chromosome 5 (OR-5) from the wild tomato species S. habrochaites (CGN1.1561). For the identification of OR-5, we went from the selection of specific F2 plants to the development of F2BC4S1 and F2BC4S2 families. This introgression was sufficient to reduce whitefly fecundity without an evident effect on whitefly survival. The identification of mechanisms exclusively affecting whitefly fecundity and independent of trichomes type IV opens new doors for resistance breeding to whiteflies that may be especially interesting in greenhouse cultivation combination with natural enemies of the whitefly. As an additional layer of defences, plants can perceive stress signals and respond to them in a specific way through induction of their immune system. This induction can also be triggered by exposing the plants to priming agents like hormones, some xenobiotic chemicals, like benzothiadiazole (BTH), β-aminobutyric acid (BABA), and sugars. Although the effect of priming agents was shown in laboratory and field studies, little is known about the effect of the genetic background of tomato on the extent of the priming, e.g. do genotypes varying in their level of resistance to insects and pathogens respond in the same way to a priming agent. We assessed the effect of selected priming agents on the effectiveness of natural defence in tomato. A set of no-choice and choice bioassays was conducted using tomato genotypes varying in their level of basal resistance to Bemisia tabaci and pathogens. We observed that whitefly survival and oviposition were not affected by the priming treatment in no-choice assays. Overall, in choice assays, fructose treated plants were more preferred by whiteflies than control plants. A genotype specific effect of priming was seen for the line FCN93-6-2. On this tomato line, JA and BABA applications decreased the number of whiteflies, e.g. making them less preferred. In this thesis, I have gone from the screening of wild relatives of tomatoes to in depth characterization of resistance mechanisms. I have identified resistance mechanisms targeting specific stages of the whitefly life cycle, thus providing new tools for breeding durable whitefly resistance in tomato.

Apple scab, caused by the fungal pathogen Venturia inaequalis, is one of the most devastating diseases for the apple growing industry in temperate zones with humid springs and summers. Breeding programs around the world have identified several sources of resistance, of which the Rvi6 (Vf) gene from Malus floribunda 821 has been the most widely used. The appearance of Rvi6-virulent strains of V. inaequalis in several European countries have underlined the necessity of pyramiding different effective resistance genes for durably resistant cultivars. Here we report the mapping of the new apple scab resistance gene Rvi18 (V25) from the selection 1980-015-025 of the apple breeding program at Wageningen University and Research Centre, The Netherlands. This gene was fine mapped on the proximal part of LG11 to a region of 34 Kb in the apple genome sequence of ‘Golden Delicious’, using 894 progeny plants, and SSR, DArT, AFLP, and SNP markers. One gene on the ‘Golden Delicious’ reference genome was identified as the potential susceptibility allele of the resistance gene. Moreover, an SSR marker has been developed of which one of its amplicons sizes is highly specific for Rvi18, thus facilitating the directed pyramiding of resistance genes through marker assisted breeding.

Potato (Solanum tuberosum L.) requires abundant nitrogen (N) to perform well and has low nitrogen use efficiency (NUE). We assessed phenotypic variation among 189 potato cultivars for NUE and the association between NUE and ecophysiological variables describing canopy development (CDv), under high and low N input. In 2009 and 2010, 189 cultivars were grown with N supply (soil N + fertiliser N) of 75 or 180 kg N/ha at Bant, the Netherlands. CDv was assessed weekly as the percentage of soil covered by green potato leaves (%SC). Data were analysed using a model that described CDv as a function of thermal time, based on the Beta function and estimates of cardinal temperatures. Nitrogen significantly affected model-derived, biologically relevant, curve-fit parameters for each cultivar. The t 1 (i.e., thermal time required to reach maximum soil cover (Vx)) was higher at low than at high N. Other parameters were higher at high than at low N, especially Vx and the period over which it was maintained. Nitrogen also affected tuber dry matter yield, tuber size and weight distributions, N content and N uptake but not tuber dry matter percentage. The total area under the %SC curve was highly correlated with yield in both years. Cultivars performing well under high N also performed well under low N. There was large variation in NUE component traits among cultivars; maturity type partially explained this variation. Variables of the CDv model captured this variation, N effects on light interception and its correlation with yield.

Late blight caused by the plant pathogenic oomycete Phytophthora infestans is known as one of the most destructive potato diseases. Plant breeders tend to employ NB-LRR-based resistance for introducing genetically controlled late blight resistance in their breeding lines. However, P. infestans is able to rapidly escape this type of resistance, and hence, NB-LRR-based resistance in potato cultivars is often not durable. Previously, we identified a novel type of Phytophthora resistance in Arabidopsis. This resistance is mediated by the cell surface receptor LecRK-I.9, which belongs to the family of L-type lectin receptor kinases. In this study, we report that expression of the Arabidopsis LecRK-I.9 gene in potato and Nicotiana benthamiana results in significantly enhanced late blight resistance. Transcriptional profiling showed strong reduction in salicylic acid (SA)-mediated defence gene expression in LecRK-I.9 transgenic potato lines (TPLs). In contrast, transcripts of two protease inhibitor genes accumulated to extreme high levels, suggesting that LecRK-I.9-mediated late blight resistance is relying on a defence response that includes activation of protease inhibitors. These results demonstrate that the functionality of LecRK-I.9 in Phytophthora resistance is maintained after interfamily transfer to potato and N. benthamiana and suggest that this novel type of LecRK-based resistance can be exploited in breeding strategies to improve durable late blight resistance in Solanaceous crops.

Detailed karyotypes of Lilium longiflorum and L. rubellum were constructed on the basis of chromosome arm lengths, C-banding, AgNO3staining, and PI-DAPI banding, together with fluorescence in situ hybridisation (FISH) with the 5S and 45S rDNA sequences as probes. The C-banding patterns that were obtained with the standard BSG technique revealed only few minor bands on heterologous positions of the L. longiflorum and L. rubellum chromosomes. FISH of the 5S and 45S rDNA probes on L. longiflorum metaphase complements showed overlapping signals at proximal positions of the short arms of chromosomes 4 and 7, a single 5S rDNA signal on the secondary constriction of chromosome 3, and one 45S rDNA signal adjacent to the 5S rDNA signal on the subdistal part of the long arm of chromosome 3. In L. rubellum, we observed co-localisation of the 5S and 45S rDNA sequences on the short arm of chromosomes 2 and 4 and on the long arms of chromosomes 2 and 3, and two adjacent bands on chromosome 12. Silver staining (Ag-NOR) of the nucleoli and NORs in L. longiflorum and L. rubellum yielded a highly variable number of signals in interphase nuclei and only a few faint silver deposits on the NORs of mitotic metaphase chromosomes. In preparations stained with PI and DAPI, we observed both red- and blue-fluorescing bands at different positions on the L. longiflorum and L. rubellum chromosomes. The red-fluorescing or so-called reverse PI-DAPI bands always coincided with rDNA sites, whereas the blue-fluorescing DAPI bands corresponded to C-bands. Based on these techniques, we could identify most of chromosomes of the L. longiflorum and L. rubellum karyotypes.Key words: fluorescence in situ hybridisation, FISH, 5S rDNA, 45S rDNA, C-banding, reverse PI-DAPI banding.

Rhizoctonia solani Kühn is a serious plant pathogenic fungus, causing various types of damage to sugar beet (Beta vulgaris L.). In Europe, the disease is spreading and becoming a threat for the growing of this crop. Plant resistance seems to be the most practical and economical way to control the disease. Experiments were carried out to optimise a greenhouse procedure to screen plants of sugar beet for resistance to R. solani. In the first experiment, two susceptible accessions were evaluated for root and leaf symptoms, after being grown in seven different soil mixtures and inoculated with R. solani. The fungus infected all plants. It was concluded that leaf symptoms were not reliable for the rating of disease severity. Statistically significant differences between the soil mixtures were observed, and there were no significant differences between the two accessions. The two soil mixtures, showing the most severe disease symptoms, were selected for a second experiment, including both resistant and susceptible accessions. As in the first experiment, root symptoms were recorded using a 1¿7 scale, and a significant expression of resistance was observed. The average severity of the disease in the greenhouse experiment generally was comparable with the infection in field experiments, and the ranking of the accessions was the same in the two types of experiments. It was concluded that evaluation procedures in the greenhouse could be used as a rapid assay to screen sugar beet plants for resistance to R. solani

This paper describes the development of a reliable transformation protocol for onion and shallot (Allium cepa L.) which can be used year-round. It is based on Agrobacterium tumefaciens as a vector, with three-week old callus, induced from mature zygotic embryos, as target tissue. For the development of the protocol a large number of parameters were studied. The expression of the uidA gene coding for -glucuronidase was used as an indicator in the optimization of the protocol. Subspecies (onion and shallot) and cultivar were important factors for a successful transformation: shallot was better than onion and for shallot cv. Kuning the best results were obtained. Also, it was found that constantly reducing the size of the calli during subculturing and selection by chopping, thus enhancing exposure to the selective agent hygromycin, improved the selection efficiency significantly. Furthermore, callus induction medium and co-cultivation period showed a significant effect on successful stable transformation. The usage of different Agrobacterium strains, callus ages, callus sources and osmotic treatments during co-cultivation did not influence transformation efficiency. The highest transformation frequency (1.95%), was obtained with shallot cv. Kuning. A total of 11 independent transformed callus lines derived from zygotic embryos were produced: seven lines from shallot and four lines from onion. Large differences in plantlet production were observed among these lines. The best line produced over 90 plantlets. Via PCR the presence of the uidA and hpt (hygromycin phosphotransferase) genes could be demonstrated in these putative transformed plants. Southern hybridization showed that most lines originated from one transformation event. However, in one line plants were obtained indicating the occurrence and rescue of at least three independent transformation events. This suggested that T-DNA integration occurred in different cells within the callus. Most transgenic plants only had one copy of T-DNA integrated into their genomes. FISH performed on 12 plants from two different lines representing two integration events showed that original T-DNA integration had taken place on the distal end of chromosomes 1 or 5. A total of 83 transgenic plants were transferred to the greenhouse and these plants appeared to be diploid and normal in morphology

Genomic DNA blot hybridization is traditionally used to demonstrate that, via genetic transformation, foreign genes are integrated into host genomes. However, in large genome species, such as Allium cepa L., the use of genomic DNA blot hybridization is pushed towards its limits, because a considerable quantity of DNA is needed to obtain enough genome copies for a clear hybridization pattern. Furthermore, genomic DNA blot hybridization is a time-consuming method. Adaptor ligation PCR (AL-PCR) of genomic DNA flanking T-DNA borders does not have these drawbacks and seems to be an adequate alternative to genomic DNA blot hybridization. Using AL-PCR we proved that T-DNA was integrated into the A. cepa genome of three transgenic lines transformed with Agrobacterium tumefaciens EHA105 (pCAMBIA 1301). The AL-PCR patterns obtained were specific and reproducible for a given transgenic line. The results showed that T-DNA integration took place and gave insight in the number of T-DNA copies present. Comparison of AL-PCR and previously obtained genomic DNA blot hybridization results pointed towards complex T-DNA integration patterns in some of the transgenic plants. After cloning and sequencing the AL-PCR products, the junctions between plant genomic DNA and the T-DNA insert could be analysed in great detail. For example it was shown that upon T-DNA integration a 66bp genomic sequence was deleted, and no filler DNA was inserted. Primers located within the left and right flanking genomic DNA in transgenic shallot plants were used to recover the target site of T-DNA integration

Studies on in vitro storage of enset under slow-growth conditions were carried out to develop an efficient protocol for conservation of the genetic diversity of the crop. The response to different growth retardation treatments was examined using three enset clones collected from southwestern Ethiopia. In vitro cultures could be effectively maintained for 6 months at 15 °C and 18 °C on MS medium supplemented with 10 M BAP, in the presence of mannitol at concentrations of 0, 1 or 2% as a growth retardant. Shoots were subsequently recovered and multiplied on MS medium supplemented with 10 and 20 M BAP at 25 °C and rooted shoots were successfully transferred to the greenhouse. Incubation at the lower temperature (15 °C) and the presence of mannitol in the culture medium had a significantly positive effect on maintenance, measured by the number of recovered shoots after storage.

Plant Breeding is the art of selecting and discarding genetic material to achieve crop improvement. Favourable alleles resulting in quality improvement or disease resistance must be added, while unfavourable alleles must be removed. The source for novel alleles can be other varieties, landraces or crop wild relatives. The identification of allelic variation is referred to as allele mining. Before allelic variation can be used for breeding purposes several steps need to be taken. First of all an inventory is needed of the available genetic resources. Phenotypic screens are needed to uncover potential expected and even unanticipated alleles. Next, using genetic and molecular tools, the alleles responsible for the identified traits must be traced and distinguished in order to be introgressed into new varieties. In this review we focus on the identification of novel disease resistance traits in the agronomically important genus Solanum. The fact that R genes are present in multigene clusters within the genome, which often include many paralogs necessitates thorough discussion on the distinction between alleles and paralogs. Often such a distinction cannot easily be made. An overview is given of how natural resources can be tapped, e.g. how germplasm can be most efficiently screened. Techniques are presented by which alleles and paralogs can be distinguished in functional and/or genetic screens, including also a specific tagging of alleles and paralogs. Several examples are given in which allele and paralog mining was successfully applied. Also examples are presented as to how allele mining supported our understanding about the evolution of R gene clusters. Finally an outlook is provided how the research field of allele mining might develop in the near future.

Restoration of formerly nutrient-poor and species-rich grasslands generally leads to an increase in species diversity. However, species without a persistent seed bank and with poor dispersal ability often do not re-establish spontaneously. Here, reintroduction is an option. If existing populations are comparable in their genetic composition, any population will do. This is not the case if populations have local adaptations. Unfortunately, whether populations are adapted locally is not easily determined, in contrast to assessing differentiation using neutral genetic markers. We used AFLP to study genetic diversity of Cirsium dissectum and Succisa pratensis within and among several Junco-Molinion plant communities in the Netherlands (up to 200 km apart) that were potential source populations, and followed the reintroduction using seeds from these populations. Also, vegetative growth phase characteristics of three populations of C. dissectum were analyzed under controlled conditions. Most of the genetic variation in these cross-fertilizing species was found within populations. Small but significant genetic differences in band frequencies were found among populations (Fst 0.100 - 0.135). The first generation of reintroduced plants contained less polymorphic bands than the source populations. The genetic differences caused by reintroduction using a limited number of seeds (founder effects) were significant in all except one case (Fst 0.012 - 0.101 between source and corresponding reintroduced population), but the magnitude was smaller than the source population differentiation. In assignment tests, reintroduced populations resembled their source population more than any other population, but all populations contained sizeable proportions of plants that were assigned to most similar plants from other populations, indicating that the populations are only marginally distinct. Calculations show that reintroduction from more than one source population introduces significantly more polymorphic bands into the new population, capitalizing on the existence of band frequency differences among populations.

Door de natuurontwikkeling op rivieroevers zijn er nieuwe kansen voor ooibossen en daarmee voor de zwarte populier gecreeerd. Langs de Rijn en de Waal is het verloop van de herkolonisatie onderzocht

The beet armyworm (Spodoptera exigua Hübner) is the most important pest in tropical Allium cultivations. All shallot ( Allium cepa L. group Aggregatum) cultivars are susceptible to this pest. Therefore accessions from three wild Allium species, namely A. galanthum Kar. et Kir., A. fistulosum L. and A. roylei Stearn, next to A. cepa L. were used to screen for resistance. First of all, a reliable bio-assay had to be developed. To this end transparent plastic cages with in total 5 plants of one accession per cage were placed on perlite in a heated greenhouse. Five 3-day old larvae were inoculated on each plant. Eight days after inoculation the number of surviving larvae per cage and the mean fresh weight per larva was determined. The lowest larval survival (36€was found on A. roylei. This was not, however, significantly different from other Allium accessions. Significant differences were found in the fresh weight per larva fed on different Allium accessions. The larvae survived on A. roylei had a very low fresh weight (10.3 mg per larva), while those on an accession of A. fistulosum had the highest fresh weight (45.1 mg per larva). The larval fresh weight on A. roylei was lower than all the other accessions except from the tropical shallot cultivar Bawang Bali. To check whether or not a toxic compound was involved in the resistance present in A. roylei, ten accessions from four Allium species were screened. Five 3-day old larvae were inoculated on regularly replaced leaf material of each accession of Allium species. No significant differences were found in mean fresh weight per larva and mean survival of larvae among different accessions. There were also no significant differences in pupal weight and developmental time. All larvae became pupae 10 days after inoculation. The data indicate that there is no toxic compound present in A. roylei. These results are underlined by the observation in the greenhouse bio-assay that A. roylei plants were equally damaged by the beet armyworm compared to other Allium species. The results obtained so far therefore suggest that introduction of resistance to S. exigua via the exploitation of variation for resistance to the beet armyworm in A. roylei is unclear and that genetic engineering using Cry sequences could provide a way forward.

The effect of several media components on the germination percentage of ovules in intraspecific T. gesneriana L. crosses was studied by using two embryo rescue techniques, viz. ovary-slice culture followed by ovule culture and direct ovule culture. The addition of 9% sucrose to medium for ovary-slice culture, started at 3 or at 5 weeks after pollination (WAP), significantly improved the germination percentage as compared to 5% sucrose. The germination percentage did not differ between both sucrose concentrations (3% and 5%) used in ovule culture started 4 weeks later with ovules excised from the ovary-slices (at 9 WAP). Similar germination percentages were obtained with media containing the full or half of the concentrations of micronutrients and macronutrients of the MS-medium during ovary-slice culture and ovule culture. For direct ovule culture, started at 4, at 6, and at 8 WAP, the germination percentages did not differ between ovules cultured on media with 3%, 6% or 9% sucrose. The addition of the cytokinin BAP (0.01 or 0.1 mg l-1) had no effect on the germination percentage. The use of liquid-shaken culture resulted in germination percentages which were similar to those on agar-solidified media. Analysis of the carbohydrate concentration of the media revealed that, in both media for ovary-slice culture and for ovule culture, ultimately all sucrose is converted into glucose and fructose. The total concentration of carbohydrates decreased with 19%–48% in the media for ovary-slice culture, whereas the total concentration of carbohydrates did not decrease remarkably in media for ovule culture.

Interspecific hybrids between Lilium longiflorum (L, 2n = 2x = 24) and Lilium rubellum (R, 2n = 2x = 24) were produced with the aim of transferring desirable horticultural traits from L. rubellum to L. longiflorum. All F1 hybrids (LR, 2n = 2x = 24) and BC1 individuals (LLR, 2n = 3x = 36) were phenotypically uniform for plant height, flowering time, leaf shape and flower colour. The BC1 plants were, in spite of their triploid nature, fertile and could be used as a female parent in backcrossmgs with autotetraploid L. longiflorum (LLLL, Zn = 4x = 48). Twelve BC2 individuals were obtained and three of them were selected for further chromosome analysis. As L. longiflorum and L. rubellum chromosomes were indistinguishable in the hybrids, genomic in-situ hybridization (GISH) was applied to establish the parentage of the chromosomes of the F1 hybrids and the BC1 and BC2 progenies. GISH confirmed the LLRR constitution of the doubled amphimonoploid (allodiploid), and the LLR constitution of all BC1 plants. The three selected BC2 plants were, as expected, aneuploid, containing three complete sets of L. longiflorum chromosomes and six, seven or eight L. rubellum chromosomes, respectively. However, L/R translocation or recombinant chromosomes could not be demonstrated in the mitotic metaphase complements of the F1, BC1 and BC2 plants. In spite of the high frequencies of homoeologous recombination in the F1 hybrids (LR) pollen was found to be sterile in all cases. At metaphase I of the pollen mother cells of the BC1 plants, genome painting did not reveal any cases of homoeologous pairing and recombination between L and R chromosomes. This lack of exchange between homoeologous chromosome segments indicates complete preferential pairing of the L and R chromosomes in the F1 (amphidiploid) and BC1 plants. It seems that the preferential pairing in the F1 and BC1 hybrids hinder the introgression of the chromosome segments or species-specific genes into the recipient for breeding purposes.

Heterogeneity of the internal transcribed spacer ITS1 of the rDNA within individuals ofTulipa gesneriana L.,T. kaufmanniana Regel, and their interspecific hybrids was analyzed by PCRRFLP, using the polymorphic restriction enzymesRsaI andHinfI, and by nucleotide sequence analysis. In most cases, the sum of the sizes of the restriction fragments was higher than the entire length of the undigested ITS fragment, indicating heterogeneity at the restriction sites within an individual. Differences in band intensities within the restriction patterns indicate the occurrence of variation in copy number of these different ITS1 variants within individuals. Automated sequencing without a visual inspection often failed to detect existing heterogeneity within sequences, resulting in a discrepancy between the sequencing and restriction analysis results. By visual interpretation of the sequences, the restriction patterns could mostly be predicted well. Fluorescence in situ hybridization (FISH) experiments in fourTulipa species revealed the occurrence of several rDNA spots. The number of rDNA loci varied from seven inT. gesneriana ‘Christmas Marvel’ to ten inT. australis Link. This might explain the occurrence of heterogeneity in ITS sequences inTulipa, as homogenization of variants has to take place over different loci.

Two complete sets of Allium fistulosum L.– A. cepa monosomic addition lines (2n=2x+1=17) together with an AFLP linkage map based on a cross between A. cepa and A. roylei Stearn were used to re-evaluate the eight A. cepa linkage groups identified in the mapping study. The linkage groups could be assigned to individual, physical chromosomes. The low level of molecular homology between A. cepa and A. fistulosum enabled the identification of 186 amplified fragment length polymorphisms (AFLP™ markers) present in A. cepa and not in A. fistulosum with ten different primer combinations. With the monosomic addition lines the distribution of the markers over the eight chromosomes of A. cepa could be determined. Of these 186 AFLP markers 51 were absent in A. roylei and consequently used as markers in the mapping study (A. cepa ×A. roylei cross). Therefore, these 51 AFLP markers could be used to assign the eight A. cepa linkage groups identified in the mapping study to physical chromosomes. Seven isozyme and three CAPS markers were also included. Two of the linkage groups had to be split because they included two sets of markers corresponding to different chromosomes. A total of 20 (approx. 10%) of the A. cepa-specific AFLP markers were amplified in more than one type of the monosomic addition lines, suggesting unlinked duplications. The co-dominant isozyme and CAPS markers were used to identify the correspondence of linkage groupsoriginating from A. cepa or from A. roylei.

Eight microsatellite markers were developed for the lepidopteran species Tortrix viridana using an enrichment protocol. The loci were highly variable with number of alleles ranging from four to 38. Six of the eight loci were in Hardy¿Weinberg equilibrium. The other two were linked to the Z-chromosome. Values of observed heterozygosity ranged for the autosomal loci from 0.510 to 0.957. All loci will be useful to study dispersal and the autosomal loci, as well for phylogeographical studies.

This paper reviews current approaches to validation of analytical chemical methods. It identifies some shortcomings of existing validation schemes such as insufficient coverage of variability in space or time, and mismatches between validation criteria and intended use of the method, e.g. the use in regulatory control. A general statistical modelling approach for combining different aspects of validation is recommended, and illustrated with an example. This type of modelling, based on components of variation, is advocated as the basis for the development of new statistically underpinned validation schemes which integrate current validation and quality assurance activities.

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.Key words: STMS, SSR, repeats, genetic diversity.

Lycopersicon peruvianum LA2157 originates from 1650 m above sea level and harbours several beneficial traits for cultivated tomatoes such as cold tolerance, nematode resistance and resistance to bacterial canker (Clavibacter michiganensis ssp. michiganensis). In order to identify quantitative trait loci (QTLs) for bacterial canker resistance, a QTL mapping approach was carried out in an F2 population derived from the interspecific F1 between Lycopersicon esculentum cv Solentos and L. peruvianum LA2157. Three QTLs for resistance mapped to chromosomes 5, 7 and 9 respectively. The resistance loci were additive and co-dominant with the QTL on chromosome 7 explaining the largest part of the variation for resistance in the F2 population. The combination of this QTL with either of the other two QTLs conferred a resistance similar to the level in the resistant parent L. peruvianum. Some RFLP markers flanking this QTL on chromosome 7 were converted into SCAR markers allowing efficient marker-assisted selection of plants with high resistance to bacterial canker.