Parthenocarpic fruit development in tomato.

Parthenocarpic fruit development is a very attractive trait for growers and consumers. In tomato, three main sources of facultative parthenocarpy, pat, pat-2, pat-3/pat-4, are known to have potential applications in agriculture. The parthenocarpic fruit development in these lines is triggered by a deregulation of the hormonal balance in some specific tissues. Auxins and gibberellins are considered as the key elements in parthenocarpic fruit development of those lines. An increased level of these hormones in the ovary can substitute for pollination and trigger fruit development. This has opened up genetic engineering approaches for parthenocarpy that have given promising results, both in quality and quantity of seedless fruit production.

Lactuca saligna, a non-host for lettuce downy mildew ( Bremia lactucae), harbors a new race-specific Dm gene and three QTLs for resistance.

Lactuca sativa (lettuce) is susceptible to Bremia lactucae (downy mildew). In cultivated and wild Lactuca species, Dm genes have been identified that confer race-specific resistance. However, these genes were soon rendered ineffective by adaptation of the pathogen. Lactuca saligna (wild lettuce) is resistant to all downy mildew races and can be considered as a non-host. Therefore, L. saligna might be an alternative source for a more-durable resistance to downy mildew in lettuce. In order to analyze this resistance, we have developed an F(2) population based on a resistant L. saligna x susceptible L. sativa cross. This F(2) population was fingerprinted with AFLP markers and tested for resistance to two Bremia races NL14 and NL16. The F(2) population showed a wide and continuous range of resistance levels from completely resistant to completely susceptible. By comparison of disease tests, we observed a quantitative resistance against both Bremia races as well as a race-specific resistance to Bremia race NL16 and not to NL14. QTL mapping revealed a qualitative gene ( R39) involved in the race-specific resistance and three QTLs ( RBQ1, RBQ2 and RBQ3) involved in the quantitative resistance. The qualitative gene R39 is a dominant gene that gives nearly complete resistance to race NL16 in L. saligna CGN 5271 and therefore it showed features similar to Dm genes. The three QTLs explained 51% of the quantitative resistance against NL14, which indicated that probably only the major QTLs have been detected in this F(2) population. The perspectives for breeding for durable resistance are discussed.

Underestimation of language issues in frequently used accident investigation methods: a new taxonomy problem found in Dutch accident data.

A wide variety of methods exist in the field of accident investigation. The challenge to find the cause for each and every accident has perpetuated a complicated and fundamental debate. In spite of the different paradigms, the many branch specific investigation methods, decades of accumulated and documented accident investigation experience, the diversity in taxonomy of causal factors, and an increasing depth of general systematic analysis tools, there are still causal factors missing out. A recent study identifies language issues as an underestimated danger. Dutch Labour Inspectorate records identify procedures and communication as an important causal factor area for major accidents. Accident investigation method taxonomies that are frequently used in The Netherlands, were investigated on the content related to language issues. Language issues are found to be either present less than proportional with observed accident rates or not present at all in frequently applied accident investigation classification systems. Hence a new taxonomy problem was found in Dutch accident data. Language issues need more attention in accident investigation methods.

Efficient QTL detection for nonhost resistance in wild lettuce: backcross inbred lines versus F(2) population.

In plants, several population types [F(2), recombinant inbred lines, backcross inbred lines (BILs), etc.] are used for quantitative trait locus (QTL) analyses. However, dissection of the trait of interest and subsequent confirmation by introgression of QTLs for breeding purposes has not been as successful as that predicted from theoretical calculations. More practical knowledge of different QTL mapping approaches is needed. In this recent study, we describe the detection and mapping of quantitative resistances to downy mildew in a set of 29 BILs of cultivated lettuce (L. sativa) containing genome segments introgressed from wild lettuce (L. saligna). Introgression regions that are associated with quantitative resistance are considered to harbor a QTL. Furthermore, we compare this with results from an already existing F(2) population derived from the same parents. We identified six QTLs in our BIL approach compared to only three in the F(2) approach, while there were two QTLs in common. We performed a simulation study based on our actual data to help us interpret them. This revealed that two newly detected QTLs in the BILs had gone unnoticed in the F(2), due to a combination of recessiveness of the trait and skewed segregation, causing a deficit of the wild species alleles. This study clearly illustrates the added value of extended genetic studies on two different population types (BILs and F(2)) to dissect complex genetic traits.

A high-density, integrated genetic linkage map of lettuce (Lactuca spp.).

An integrated map for lettuce comprising of 2,744 markers was developed from seven intra- and inter-specific mapping populations. A total of 560 markers that segregated in two or more populations were used to align the individual maps. 2,073 AFLP, 152 RFLP, 130 SSR, and 360 RAPD as well as 29 other markers were assigned to nine chromosomal linkage groups that spanned a total of 1,505 cM and ranged from 136 to 238 cM. The maximum interval between markers in the integrated map is 43 cM and the mean interval is 0.7 cM. The majority of markers segregated close to Mendelian expectations in the intra-specific crosses. In the two L. saligna x L. sativa inter-specific crosses, a total of 155 and 116 markers in 13 regions exhibited significant segregation distortion. Data visualization tools were developed to curate, display and query the data. The integrated map provides a framework for mapping ESTs in one core mapping population relative to phenotypes that segregate in other populations. It also provides large numbers of markers for marker assisted selection, candidate gene identification, and studies of genome evolution in the Compositae.

QTL analysis of cadmium and zinc accumulation in the heavy metal hyperaccumulator Thlaspi caerulescens.

Thlaspi caerulescens (Tc; 2n = 14) is a natural Zn, Cd and Ni hyperaccumulator species belonging to the Brassicaceae family. It shares 88% DNA identity in the coding regions with Arabidopsis thaliana (At) (Rigola et al. 2006). Although the physiology of heavy metal (hyper)accumulation has been intensively studied, the molecular genetics are still largely unexplored. We address this topic by constructing a genetic map based on AFLP markers and expressed sequence tags (ESTs). To establish a genetic map, an F(2) population of 129 individuals was generated from a cross between a plant from a Pb/Cd/Zn-contaminated site near La Calamine, Belgium, and a plant from a comparable site near Ganges (GA), France. These two accessions show different degrees of Zn and, particularly, Cd accumulation. We analyzed 181 AFLP markers (of which 4 co-dominant) and 13 co-dominant EST sequences-based markers and mapped them to seven linkage groups (LGs), presumably corresponding to the seven chromosomes of T. caerulescens. The total length of the genetic map is 496 cM with an average density of one marker every 2.5 cM. This map was used for Quantitative Trait Locus (QTL) mapping in the F(2). For Zn as well as Cd concentration in root we mapped two QTLs. Three QTLs and one QTL were mapped for Zn and Cd concentration in shoot, respectively. These QTLs explain 23.8-60.4% of the total variance of the traits measured. We found only one common locus (LG6) for Zn and Cd (concentration in root) and one common locus for shoot and root concentrations of Zn (LG1) and of Cd (LG3). For all QTLs, the GA allele increased the trait value except for two QTLs for Zn accumulation in shoot (LG1 and LG4) and one for Zn concentration in root (LG1).

The Tomato Sequencing Project, the first cornerstone of the International Solanaceae Project (SOL).

The genome of tomato (Solanum lycopersicum) is being sequenced by an international consortium of 10 countries (Korea, China, the United Kingdom, India, The Netherlands, France, Japan, Spain, Italy and the United States) as part of a larger initiative called the 'International Solanaceae Genome Project (SOL): Systems Approach to Diversity and Adaptation'. The goal of this grassroots initiative, launched in November 2003, is to establish a network of information, resources and scientists to ultimately tackle two of the most significant questions in plant biology and agriculture: (1) How can a common set of genes/proteins give rise to a wide range of morphologically and ecologically distinct organisms that occupy our planet? (2) How can a deeper understanding of the genetic basis of plant diversity be harnessed to better meet the needs of society in an environmentally friendly and sustainable manner? The Solanaceae and closely related species such as coffee, which are included in the scope of the SOL project, are ideally suited to address both of these questions. The first step of the SOL project is to use an ordered BAC approach to generate a high quality sequence for the euchromatic portions of the tomato as a reference for the Solanaceae. Due to the high level of macro and micro-synteny in the Solanaceae the BAC-by-BAC tomato sequence will form the framework for shotgun sequencing of other species. The starting point for sequencing the genome is BACs anchored to the genetic map by overgo hybridization and AFLP technology. The overgos are derived from approximately 1500 markers from the tomato high density F2-2000 genetic map (http://sgn.cornell.edu/). These seed BACs will be used as anchors from which to radiate the tiling path using BAC end sequence data. Annotation will be performed according to SOL project guidelines. All the information generated under the SOL umbrella will be made available in a comprehensive website. The information will be interlinked with the ultimate goal that the comparative biology of the Solanaceae-and beyond-achieves a context that will facilitate a systems biology approach.

Development of AFLP markers in barley.

To investigate the application of amplified fragment length polymorphism (AFLP) markers in barley, 96 primer combinations were used to generate AFLP patterns with two barley lines, L94 and Vada. With seven primer combinations, only a few intense bands were obtained, probably derived from repeated sequences. With the majority of the remaining 89 primer combinations, on average about 120 amplification products were generated, and the polymorphism rate between the two lines was generally over 18%. Based on the number of amplified products and the polymorphism rate, the 48 best primer combinations were selected and tested on 16 barley lines, again including L94 and Vada. Using a subset of 24 primer combinations 2188 clearly visible bands within the range from 80 to 510 bp were generated; 55% of these showed some degree of polymorphism among the 16 lines. L94 versus Vada showed the highest polymorphism rate (29%) and Proctor versus Nudinka yielded the lowest (12%). The polymorphism rates per primer combination showed little dependence on the barley lines used. Hence the most efficient and informative primer combinations identified for a given pair of lines turned out to be highly efficient when applied to others. Generally, more than 100 common markers (possibly locus specific) among populations or crosses were easily identified by comparing 48 AFLP profiles of the parent lines. The existence of such a large number of markers common to populations will facilitate the merging of molecular marker data and other genetic data into one integrated genetic map of barley.

The development of lettuce backcross inbred lines (BILs) for exploitation of the Lactuca saligna (wild lettuce) germplasm.

Backcross inbred lines (BILs) were developed in which chromosome segments of Lactuca saligna (wild lettuce) were introgressed into L. sativa (lettuce). These lines were developed by four to five backcrosses and one generation of selfing. The first three generations of backcrossing were random. Marker-assisted selection began in the BC(4) generation and continued until the final set of BILs was reached. A set of 28 lines was selected that together contained 96% of the L. saligna genome. Of these lines, 20 had a single homozygous introgression (BILs), four had two homozygous introgressions (doubleBILs) and four lines had a heterozygous single introgression (preBILs). Segregation ratios in backcross generations were compared to distorted segregation ratios in an F(2) population, and the results indicated that most of the distorted segregations can be explained by genetic effects on pollen- or egg-cell fitness. By means of BIL association mapping we were able to map 12 morphological traits and hundreds of additional amplified fragment length polymorphic (AFLP) markers. The total AFLP map now comprises 757 markers. This set of BILs is very useful for future genetic studies.

Comparison and integration of four barley genetic maps.

Barley (Hordeum vulgare L.) is one of the most extensively studied food crops in recent molecular research. More than 1000 molecular markers have been located on the barley genome by using five independent populations. For the present study, four segregation data sets, 'Proctor' x 'Nudinka', 'Igri' x 'Franka', 'Steptoe' x 'Morex', and 'Harrington' x TR306, were downloaded from the publicly available GrainGenes databank. Since 22% of the markers are common to at least two of the independent data sets, we were able to establish an integrated map using the computer package JOINMAP v2.0. The integrated map contains 898 markers, covers 1060 cM, and removes many large gaps present in the individual maps. Comparison of the integrated map with the individual maps revealed that the overall linear order of markers is in good agreement and that the integrated map is consistent with the component maps. No significant reordering of markers was found. This conservative property of the barley genome makes the integrated map reliable and successful. Except for chromosome 7 (5H), marker clustering was observed in the centromeric regions, probably owing to the centromeric suppression of recombination. Based on this integrated map, geneticists and breeders can choose their favourite markers in any region of interest of the barley genome. Key words : Hordeum vulgare, RFLP, integrated map.

Use of locus-specific AFLP markers to construct a high-density molecular map in barley.

By using 25 primer combinations, 563 AFLP markers segregating in a recombinant inbred population (103 lines, F9) derived from L94/Vada were generated. The 38 AFLP markers in common to the existing AFLP/RFLP combined Proctor/Nudinka map, one STS marker, and four phenotypic markers with known map positions, were used to assign present AFLP linkage groups to barley chromosomes. The constructed high-density molecular map contains 561 AFLP markers, three morphological markers, one disease resistance gene and one STS marker, and covers a 1062-cM genetic distance, corresponding to an average of one marker per 1.9 cM. However, extremely uneven distributions of AFLP markers and strong clustering of markers around the centromere were identified in the present AFLP map. Around the centromeric region, 289 markers cover a genetic distance of 155 cM, corresponding to one marker per 0.5 cM; on the distal parts, 906 cM were covered by 277 markers, corresponding to one marker per 3.3 cM. Three gaps larger than 20 cM still exist on chromosomes 1, 3 and 5. A skeletal map with a uniform distribution of markers can be extracted from the high-density map, and can be applied to detect and map loci underlying quantitative traits. However, the application of this map is restricted to barley species since hardly any marker in common to a closely related Triticum species could be identified.

Resistance to powdery mildew (Oidium lycopersicum) in Lycopersicon hirsutum is controlled by an incompletely-dominant gene Ol-1 on chromosome 6.

The inheritance of resistance to powdery mildew (Oidium lycopersicum) in Lycopersicon hirsutum was investigated by disease tests in segregating populations obtained by hybridising tomato (L. esculentum) cv Moneymaker with the wild relative L. hirsutum G1.1560. One incompletely dominant gene Ol-1 was found to largely control resistance to the disease. To map Ol-1, DNA pools from seven resistant and ten susceptible F2 plants were analyzed for random amplified polymorphic DNA (RAPD). With 32 primers tested, one RAPD, primed with the sequence 5'-GACGTGGTGA-3', was observed between the susceptible and the resistant bulks, which cosegregated with resistance in the F2 population of L. esculentum × L. hirsutum G1.1560. This RAPD was mapped on chromosome 6 by using an F2 (L. esculentum × L. pennellii) already mapped for 49 RFLPs. RFLP analysis of the F2 from L. esculentum cv Moneymaker × L. hirsutum G1.1560 demonstrated that Ol-1 maps near the Aps-1 region on chromosome 6, in the vicinity of the resistance genes to Meloidogyne spp. (Mi) and to Cladosporium fulvum (Cf-2/Cf-5).

Artifacts are responsible for the translational activity of polyribosome preparations isolated from alfalfa mosaic virus-infected tobacco leaves.

We observed that polyribosome preparations isolated from alfalfa mosaic virus (A1MV)-infected tobacco leaves were contaminated with virion-derived material which could not be removed completely by sucrose gradient centrifugation or by magnesium ion precipitation. Upon incubation of polyribosome preparations with S 100 extracts from reticulocyte lysates, viral-encoded proteins were produced. Aurintricarboxylic acid (ATA), an inhibitor of initiation of translation, was used to inhibit de novo translation of the RNAS contaminating the polyribosome preparations. ATA concentrations, which did not inhibit peptide chain elongation on in vitro-produced polyribosomes, completely inhibited the translational activity of the tobacco polyribosomes. Hence the protein synthetic capacity of the tobacco polyribosome preparations is due to de novo translation of virion-derived material by vacant ribosomes present in the complementing S 100 extract. Efforts to activate the tobacco polyribosomes remain unsuccessful.

Dispersion of the Cf-4 disease resistance gene in Lycopersicon germplasm.

In the past, numerous Cf genes have been reported in tomato (Lycopersicon esculentum Mill.) that confer resistance against leaf mould (Cladosporium fulvum Cke.). We are interested in genetic variation at Cf loci. Therefore, previously uncharacterized Cf genes were further analysed. Recognition of the AVR4 elicitor, DNA gel blot analysis, PCR analysis and sequencing of part of the Cf-4 locus showed that a large proportion of the accessions tested harboured the Cf-4 resistance gene. We concluded that despite differences in nomenclature, all these accessions harbour the same Cf-4 locus, probably introgressed from the same donor. The origin of the Cf-4 locus and the reasons for discrepancies with earlier reports are discussed.

GATA- and GACA-repeats are not evenly distributed throughout the tomato genome.

This paper describes the distribution of highly polymorphic GATA- and GACA-containing DNA regions in tomato. To study the distribution of these polymorphic regions, a mapping experiment was done. The segregation of 32 GATA- and GACA-containing loci was analyzed in a F2 population from a cross between Lycopersicon esculentum and L. pennellii. From these loci, 28 could be mapped to 8 of the 12 tomato chromosomes. Both the GATA- and GACA-containing loci seem to cluster in the same chromosomal regions. To our knowledge, this is the first report on mapping of GATA- and GACA-containing loci in plants.

Genomic regions determining resistance to leaf stripe (Pyrenophora graminea) in barley.

Leaf stripe is a seed-borne disease of barley (Hordeum vulgare) caused by Pyrenophora graminea. Little is known about the genetics of resistance to this pathogen. In the present work, QTL analysis was applied on two recombinant inbred line (RIL) populations derived from two- and six-rowed barley genotypes with different levels of partial resistance to barley leaf stripe. Quantitative trait loci for partial resistance were identified using the composite interval mapping (CIM) method of PLABQTL software, using the putative QTL markers as cofactors. In the L94 x 'Vada' mapping population, one QTL for resistance was detected on chromosome 2H; the same location as the leaf-stripe resistance gene Rdg1 mapped earlier in 'Alf', where it confers complete resistance to the pathogen. An additional minor-effect QTL was identified by further analyses in this segregating population on chromosome 7H. In L94 x C123, two QTLs for resistance were mapped, one each on chromosomes 7H and 2H.

Mapping strategy for resistance genes in tomato based on RFLPs between cultivars: Cf9 (resistance to Cladosporium fulvum) on chromosome 1.

The contribution of introgressed regions derived from wild species to the genetic variation within the species of Lycopersicon esculentum was investigated by comparing the RFLP patterns of 2 introgression-free, obsolete cultivars ('Moneymaker' and 'Premier') and a modern cultivar ('Sonatine') that carries at least 5 introgressed resistance genes. In this analysis 195 mapped nuclear markers were used in combination with 6 restriction enzymes. Among the 1170 probe-enzyme combinations tested, only 3 showed a polymorphism between the 2 introgression-free cultivars. On the other hand 24 probe-enzyme combinations were found to exhibit polymorphisms between 'Moneymaker' and 'Sonatine'. These represented ten polymorphic loci distributed among 5 linkage groups on chromosomes 1, 3, 4, 6, and 9.On the assumption that most of the polymorphic loci corresponded to introgressed chromosome segments of wild species carrying resistance genes, linkages between these loci and the component resistance genes were examined by RFLP analysis of pairs of near-isogenic lines differing only for one particular resistance gene, and a variety of commercial cultivars having different resistance gene compositions. Two of the polymorphic linkage groups could thus be ascribed to resistance genes whose map positions were already known: Cf2 on chromosome 6 and Tm2a on chromosome 9, whereas another marker, TG301 on chromosome 1, could be assigned to the Cladosporium fulvum resistance gene Cf9 with a hitherto disputable map position. By linkage analysis of a segregating F2 population the genetic distance between the Cf9 gene and the marker TG301 was estimated at 5.5 ± 2.3 cM.

Tomato chromosome 1: high resolution genetic and physical mapping of the short arm in an interspecific Lycopersicon esculentum x L. peruvianum cross.

A detailed map of part of the short arm of chromosome 1 proximal to the Cf-4/Cf-9 gene cluster was generated by using an F2 population of 314 plants obtained from the cross between the remotely related species Lycopersicon esculentum and L. peruvianum. Six markers that cosegregate in an L. esculentum x L. pennellii F2 population showed high recombination frequencies in the present interspecific population, spanning an interval of approximately 13 cM. Physical distances between RFLP markers were estimated by pulsed field gel electrophoresis of high-molecular-weight DNA and by identifying YACs that recognized more than one RFLP marker. In this region 1 cM corresponded to 55-110 kb. In comparsion with the value of 730 kb per cM averaged over the entire genome, this reflects the remarkably high recombination frequencies in this region in the hybrid L. esculentum x L. peruvianum progeny population. The present data underline the fact that recombination is not a process that occurs randomly over the entire genome, but can vary dramatically in intensity between chromosomal regions and among populations.

Ribosomes are stalled during in vitro translation of alfalfa mosaic virus RNA 1.

In the presence of plant tRNAs the full-length translation product of alfalfa mosaic virus RNA 1 is produced in rabbit reticulocytes only at low mRNA concentration. At higher mRNA concentration translation is restricted to the 5' half of RNA 1. At high mRNA concentration the full-length product can be formed when additional plant tRNA and glutamine are supplied to the translation mixture. In contrast, in the presence of yeast or calf liver tRNA the translation pattern of alfalfa mosaic virus RNA 1 always results in the synthesis of the full-length product. Pulse-chase experiments in the presence of plant tRNAs show that the ribosomes pause at several positions in the 5' half of RNA 1. The pausing time is different at the different 'halting places'. Protein synthesis is resumed upon addition of glutamine, even when the addition is delayed for more than 3 h after the start of protein synthesis. Only one tRNA species, purified from wheat germ or tobacco, could promote full-length translation of RNA 1. This tRNA can be charged with glutamine. Analysis of the position of glutamine codons on RNA 1 shows a correlation between the positions of the CAA codons and the halting places of the ribosomes. The CAA codon (for any other codon) on its own cannot be responsible for the pausing of the ribosomes, since a variety of RNAs, known to contain all sense codons, are translated efficiently in rabbit reticulocyte lysates in the presence of plant tRNAs. Apparently other elements can restrict decoding of normal codons during protein chain elongation.

A genetic linkage map of the diplosporous chromosomal region in Taraxacum officinale (common dandelion; Asteraceae).

In this study, we mapped the diplosporous chromosomal region in Taraxacum officinale, by using amplified fragment length polymorphism technology (AFLP) in 73 plants from a segregating population. Taraxacum serves as a model system to investigate the genetics, ecology, and evolution of apomixis. The genus includes sexual diploid as well as apomictic polyploid, mostly triploid, plants. Apomictic Taraxacum is diplosporous, parthenogenetic, and has autonomous endosperm formation. Previous studies have indicated that these three apomixis elements are controlled by more than one locus in Taraxacum and that diplospory inherits as a dominant, monogenic trait ( Ddd; DIP). A bulked segregant analysis provided 34 AFLP markers that were linked to DIP and were, together with two microsatellite markers, used for mapping the trait. The map length was 18.6 cM and markers were found on both sides of DIP, corresponding to 5.9 and 12.7 cM, respectively. None of the markers completely co-segregated with DIP. Eight markers were selected for PCR-based marker development, of which two were successfully converted. In contrast to all other mapping studies of apomeiosis to date, our results showed no evidence for suppression of recombination around the DIP locus in Taraxacum. No obvious evidence for sequence divergence between the DIP and non- DIP homologous loci was found, and no hemizygosity at the DIP locus was detected. These results may indicate that apomixis is relatively recent in Taraxacum.