Gene expression during anthesis and senescence in Iris flowers.

We investigated changes in gene expression in Iris hollandica flowers by microarray technology. Flag tepals were sampled daily, from three days prior to flower opening to the onset of visible senescence symptoms. Gene expression profiles were compared with biochemical data including lipid and protein degradation and DNA coiling, and with morphological data. Plasmodesmata of mesophyll cells closed about two days before flower opening, while in the epidermis they closed concomitant with opening. Similarly, the onset of visible senescence in the epidermis cells occurred about two days later than in the mesophyll. About 1400 PCR-amplified clones, derived from a subtractive cDNA library enriched for tepal-specific genes, were spotted and about 240 clones, including 200 that were expressed most differentially, were sequenced. The expression patterns showed three main clusters. One exhibited high expression during tepal growth (cluster A). These genes were putatively associated with pigmentation, cell wall synthesis and metabolism of lipids and proteins. The second cluster (B) was highly expressed during flower opening. The third cluster (C) related to the final stages of senescence, with genes putatively involved in signal transduction, and the remobilization of phospholipids, proteins, and cell wall compounds. Throughout the sampling period, numerous plant defence genes were highly expressed. We identified an ion channel protein putatively involved in senescence, and some putative regulators of transcription and translation, including a MADS-domain factor.

Tomato chromosome 6: effect of alien chromosomal segments on recombinant frequencies.

Variation in recombinant frequencies at two adjacent intervals on chromosome 6 of tomato (Lycopersicon esculentum Mill.) has been studied in seven lines that differ in the amount and origin of introgressed segments from wild species. These lines were all crossed to a genotype homozygous recessive for the markers tl, yv, and c, which define the centromere spanning region tl-yv and the long arm region yv-c. Recombinants were identified in large F2, populations consisting of over 30 000 plants in total. Application of molecular markers provided additional information on the distribution of crossover events within the centromere-containing interval tl-yv. A decrease in recombination at the marked intervals correlated with the presence of an alien segment. Suppression of recombination was up to sixfold in the centromere spanning interval tl-yv depending on the source and size of the introgression, and was restricted to the alien segments with no strong effect on the neighbouring intervals. Key words : recombinant frequency, Lycopersicon esculentum, morphological markers, introgressions, centromere.

Tomato chromosome 6: a high resolution map of the long arm and construction of a composite integrated marker-order map.

Integration of molecular and classical genetic maps is an essential requirement for marker-assisted breeding, quantitative trait locus mapping and map-based cloning. With respects to tomato, such maps are only available for the top part of chromosome 1, for chromosome 3 and for the short arm and the centromere proximal part of the long arm of chromosome 6. Employing an L. esculentum line carrying an L. hirsutum introgression we constructed an integrated linkage map for the telomere proximal part of the long arm of tomato chromosome 6, thereby completing the integrated map published previously. With an average map distance of only 0.6 cM the map provides detailed information on the relative position of molecular markers and several traits of economical importance, such as the fruit color marker B. Furthermore, two additional crosses using lines containing L. pennellii introgressions were performed to address the question as to how the recombination frequency in a marked interval on the long arm of chromosome 6 is affected by introgressed segments from different origins. It is concluded that recombination is not merely affected by the local level of homology but also by surrounding sequences. Combination of all the linkage data generated in various crosses described in this and other studies enabled the construction of the first integrated map of an entire tomato chromosome. This map carries 42 loci and shows the position of 15 classical genes relative to 59 molecular markers.

Integration of the classical and molecular linkage maps of tomato chromosome 6.

In the past, a classical map of the tomato genome has been established that is based on linkage data from intraspecific Lycopersicon esculentum crosses. In addition, a high density molecular linkage map has recently been constructed using a L. esculentum x L. pennellii cross. As the respective maps only partially match, they provide limited information about the relative positions of classical and molecular markers. In this paper we describe the construction of an integrated linkage map of tomato chromosome 6 that shows the position of cDNA-, genomic DNA- and RAPD markers relative to 10 classical markers. Integration was achieved by using a L. esculentum line containing an introgressed chromosome 6 from L. pennellii in crosses to a variety of L. esculentum marker lines. In addition, an improved version of the classical linkage map is presented that is based on a combined analysis of new linkage data for 16 morphological markers and literature data. Unlike the classical map currently in use, the revised map reveals clustering of markers into three major groups around the yv, m-2 and c loci, respectively. Although crossing-over rates are clearly different when comparing intraspecific L. esculentum crosses with L. esculentum x L. pennellii crosses, the clusters of morphological markers on the classical map coincide with clusters of genomic- and cDNA-markers on the molecular map constructed by Tanksley and coworkers.

Insect-resistant chrysanthemum calluses by introduction of a Bacillus thuringiensis crystal protein gene.

A 3'-end truncated crystal protein gene, derived from Bacillus thuringiensis (Bt) subsp. aizawai 7.21, encoding the toxic fragment of the insecticidal protein cryIA(b), was constructed. The gene was inserted into a transformation vector, also carrying the neomycin phosphotransferase II (nptII) gene and the beta-glucuronidase (gus) gene, and introduced in the oncogenic Agrobacterium tumefaciens strain A281, harbouring the Ti-plasmid pTiBO542. The recombinant Agrobacterium strain was used to transform leaf explants of chrysanthemum (Dendranthema grandiflora) cultivar Parliament. The resulting tumours were kanamycin-resistant, exhibited beta-glucuronidase activity and produced agropine and mannopine. In most tumours, all simultaneously transferred genes were expressed, owing to selection for the presence of both T-DNAs, but no correlation was found between the level of expression of the various genes. A bioassay was developed, in which larvae were fed with tumorous chrysanthemum tissue, in order to detect the effect of the transferred toxin gene on larval development. Using this bioassay with second instar larvae of Heliothis virescens (tobacco budworm), 17 tumour lines were tested. Several of these lines proved to be strongly inhibitory to larval growth. These results indicate that Bt-based insect resistance might be used as a tool in reducing the amount of pesticides used in chrysanthemum culture.

The root-knot nematode resistance gene (Mi) in tomato: construction of a molecular linkage map and identification of dominant cDNA markers in resistant genotypes.

A dominant allele at the Mi locus on chromosome 6 of tomato (Lycopersicon esculentum Mill) confers resistance to three species of root-knot nematodes (Meloidogyne). The resistance, which is associated with a localized necrotic response, was originally introduced into tomato from the wild species Lycopersicon peruvianum. As a step towards the molecular cloning of Mi, we have identified closely linked DNA markers from both cDNA and genomic DNA libraries as restriction fragment length polymorphisms (RFLPs). DNA from tomato populations segregating for nematode resistance was analyzed to generate a high-resolution genetic map of this region. Additional information on gene order was obtained by comparing the size of the introgressed L. peruvianum chromosomal segment within a collection of nematode-resistant tomato lines. Among the four cDNA markers that are tightly linked to Mi, three are dominant, i.e. L. peruvianum-specific. One cDNA marker corresponds to a gene family comprising 20-30 members, one of which is diagnostic for all nematode-resistant genotypes tested. The presence of non-homologous sequences around the Mi gene may contribute to the suppression of recombination in this region of the genome in crosses heterozygous for Mi. The potential of 'walking' from closely linked markers to Mi is discussed.

Genetic transformation of Chrysanthemum using wild type Agrobacterium strains; strain and cultivar specificity.

To develop an Agrobacterium mediated transformation protocol for chrysanthemum we studied the transformation efficiency of commonly used A.tumefaciens strains on 14 genotypes by comparing tumour size and frequency. One genotype was analyzed in detail using 14 strains of both A.tumefaciens and A.rhizogenes. Only a few genotype/strain combinations resulted in significant tumour formation. Especially 0-type strains were highly efficient. An 0-type strain was used to transfer genes for neomycine phosphotransferase (NPT II) and ß-glucuronidase (GUS) to a susceptible cultivar. Transfer of the GUS gene was confirmed by using the Polymerase Chain Reaction (PCR).