Clean vector technology for marker-free transgenic ornamentals

Marker-free transgenic crops confer several advantages over transgenic crops equipped with e.g. genes coding for antibiotic resistance. Firstly, the European Union encourages notifiers for introductions of GMOs in the environment to ¿avoid or minimize the inclusion of superfluous transgenes or sequences¿ and it promotes the use of clean vector systems. Secondly, the number of selection genes allowing the preferential growth of transformed cells and tissues is limited and often a gene transfer protocol for a specific crop or even a cultivar depends on the use of one specific selectable marker gene. Hence, stacking of genes within the same transgenic line is difficult once a selectable marker gene has been introduced. If these marker genes can be removed, the subsequent introduction of the next gene-of-interest is greatly facilitated. At Plant Research International a system has been developed for specific elimination of any introduced DNA/gene sequences using site-specific recombination combined with selection for successful removal using a negative selection system. Completely marker-free transgenic plants have been obtained using a model vector, both in an efficient transformation system (strawberry) as well as in a non-efficient transformation system (apple). Frequencies were more than adequate. Presently. A versatile vector set providing a choice of several selectable markers and carrying a multiple cloning site for receiving cassettes of the gene-of-interest is available for application in, amongst others, ornamental crop

Clean vector technology for marker-free transgenic fruit crops

Marker-free transgenic crops confer several advantages over transgenic crops equipped with selection genes coding e.g. for antibiotic resistance. Firstly, the European Union has prepared a guidance document for risk assessment of GM-crops to be introduced in the environment (E.U. Joint Working Group on Novel Foods and GMO’s, 2003). In this document based on compliance to consumer demands the EU encourages to “avoid or minimise the inclusion of superfluous transgenes or sequences”. EU thus promotes the use of clean vector systems. Secondly, the number of selection genes allowing the preferential growth of transformed cells and tissues is limited. Often a gene transfer protocol for a specific crop or even a cultivar depends on the use of one specific selectable marker gene. Hence, stacking of genes within the same transgenic line is difficult once a selectable marker gene has been introduced. If these marker genes can be removed, the subsequent introduction of the next gene-of-interest is greatly facilitated. At Plant Research International a system has been developed for specific elimination of any introduced DNA/gene sequences using site-specific recombination combined with selection for successful removal using a negative selection system. Completely marker-free transgenic plants have been obtained using a model vector, both in an efficient transformation system (strawberry) as well as in a non-efficient transformation system (apple). Frequencies were more than adequate. Presently a versatile vector set providing a choice of several selectable markers and carrying a multiple cloning site for receiving cassettes of the gene-of-interest is available for application in, amongst others, fruit crops

Three Combined Quantitative Trait Loci from Nonhost Lactuca saligna Are Sufficient to Provide Complete Resistance of Lettuce Against Bremia lactucae

The nonhost resistance of wild lettuce (Lactuca saligna) to downy mildew (Bremia lactucae) is based on at least 15 quantitative trait loci (QTL), each effective at one or more plant developmental stages. We used QTL pyramiding (stacking) to determine how many of these QTL from L. saligna are sufficient to impart complete resistance towards B. lactucae to cultivated lettuce, L. sativa. The alleles of four of the most promising QTL, rbq4, rbq5, rbq6+11, and rbq7 are effective at both the young and adult plant stages. Lines with these four QTL in all possible combinations were generated by crossing the respective backcross inbred lines (BIL). Using the 11 resulting lines (combiBIL), we determined that combinations of three QTL, rbq4, rbq5, and rbq6+11, led to increased levels of resistance; however, one QTL, rbq7, did not add to the resistance level when combined with the other QTL. One line, tripleBIL268, which contains the three QTL rbq4, rbq5, and rbq6+11, was completely resistant to B. lactucae at the young plant stage. This suggests that these three QTL are sufficient to confer the complete resistance of the nonhost L. saligna and any additional QTL in L. saligna are redundant. Histological analysis of B. lactucae infection in L. saligna, the BIL, and the combiBIL 48 h after inoculation revealed different microscopical phenotypes of resistance. The QTL differed with respect to the stage of the infection process with which they interfere

Isolation and characterisation of a strawberry fruit-specific promotor

In order to achieve specific expression of transgenes in strawberry fruits, the availability of tissue- (receptacle) specific promoter sequences is desired. For this reason, 5'-upstream sequences of the strawberry expansin gene FaExp2, which is expressed in a fruit-specific manner, have been isolated. To characterise the promoter activity of the isolated sequences, fragments of 0.7 kb (0.7pFaExp2)and 1.6 kb (1.6pFaExp2) have been fused to the ß-glucuronidase reporter gene (gus). In transgenic strawberry plants transformed with either 0.7pFaExp2-gus or1.6pFaExp2-gus, a fruit-specific expression pattern was observed for both promoter constructs. However, quantitative RT-PCR revealed that gus expression levels driven by the 1.6pFaExp2 promoter fragment were much more higher. In addition to the expression in fruits, both promoter fragments also seemed to direct gene expression in the achenes and to some extent in epidermal and subepidermal tissues of petioles and stems of flowers and fruits. It is concluded that both promoter sequences are suitable for directing transgene expression in strawberry fruits in a specific wa

Fine mapping quantitative resistances to downy mildew in lettuce revealed multiple sub-QTLs with plant stage dependent effects reducing or even promoting the infection

Previous studies on the genetic dissection of the complete resistance of wild lettuce, Lactuca saligna, to downy mildew revealed 15 introgression regions that conferred plant stage dependent quantitative resistances (QTLs). Three backcross inbred lines (BILs), carrying an individual 30–50 cM long introgression segment from L. saligna in a cultivated lettuce, L. sativa, background, reduced infection by 60–70 % at young plant stage and by 30–50 % at adult plant stage in field situations. We studied these three quantitative resistances in order to narrow down their mapping interval and determine their number of loci, either single or multiple. We performed recombinant screenings and developed near isogenic lines (NILs) with smaller overlapping L. saligna introgressions (substitution mapping). In segregating introgression line populations, recombination was suppressed up to 17-fold compared to the original L. saligna × L. sativaF2 population. Recombination suppression depended on the chromosome region and was stronger suppressed at the smallest introgression lengths. Disease evaluation of the NILs revealed that the resistance of all three BILs was not explained by a single locus but by multiple sub-QTLs. The 17 L. saligna-derived sub-QTLs had a smaller and plant stage dependent resistance effect, some segments reducing; others even promoting downy mildew infection. Implications for lettuce breeding are outlined

Effects of stacked quantitative resistances to downy mildew in lettuce do not simply add up

Key message In a stacking study of eight resistance QTLs in lettuce against downy mildew, only three out of ten double combinations showed an increased resistance effect under field conditions. Abstract Complete race nonspecific resistance to lettuce downy mildew, as observed for the nonhost wild lettuce species Lactuca saligna, is desired in lettuce cultivation. Genetic dissection of L. saligna’s complete resistance has revealed several quantitative loci (QTL) for resistance with field infection reductions of 30–50 %. To test the effect of stacking these QTL, we analyzed interactions between homozygous L. saligna CGN05271 chromosome segments introgressed into the genetic background of L. sativa cv. Olof. Eight different backcross inbred lines (BILs) with single introgressions of 30–70 cM and selected predominately for quantitative resistance in field situations were intercrossed. Ten developed homozygous lines with stacked introgression segments (double combinations) were evaluated for resistance in the field. Seven double combinations showed a similar infection as the individual most resistant parental BIL, revealing epistatic interactions with ‘less-than-additive’ effects. Three double combinations showed an increased resistance level compared to their parental BILs and their interactions were additive, ‘less-than-additive’ epistatic and ‘more-than-additive’ epistatic, respectively. The additive interaction reduced field infection by 73 %. The double combination with a ‘morethan-additive’ epistatic effect, derived from a combination between a susceptible and a resistant BIL with 0 and 30 % infection reduction, respectively, showed an average field infection reduction of 52 %. For the latter line, an attempt to genetically dissect its underlying epistatic loci by substitution mapping did not result in smaller mapping intervals as none of the 22 substitution lines reached a similar high resistance level. Implications for breeding and the inheritance of L. saligna’s complete resistance are discussed

QTL mapping of thrips resistance in pepper

Thrips is one of the most damaging pests in pepper (Capsicum). Resistance to thrips was identified in Capsicum annuum. This study was aimed at the elucidation of the genetic background of thrips resistance in Capsicum through QTL mapping. The QTL analysis was carried out for Frankliniella occidentalis resistance in an F2 population consisting of 196 plants derived from an interspecific cross between the highly resistant C. annuum AC 1979 as female parent and the highly susceptible C. chinense 4661 as male parent. Fifty-seven SSR, 109 AFLP, and 5 SNP markers were used to construct a genetic map with a total length of 1636 cM. Damage caused by larvae and the survival of first and second instar larval stages observed in a no-choice test were used as parameters of resistance. Interval mapping detected one QTL for each of these parameters, all co-localizing near the same marker on chromosome 6. Use of this marker as co-factor in a multiple-QTL mapping analysis failed to uncover any additional QTLs. This QTL explained about 50 % of the genetic variation, and the resistance allele of this QTL was inherited from the resistant parent. Thrips resistance was not linked to trichome density

Metabolomics and molecular marker analysis to explore pepper (Capsicum sp.) biodiversity

An overview of the metabolic diversity in ripe fruits of a collection of 32 diverse pepper (Capsicum sp.) accessions was obtained by measuring the composition of both semi-polar and volatile metabolites in fruit pericarp, using untargeted LC–MS and headspace GC–MS platforms, respectively. Accessions represented C. annuum, C. chinense, C. frutescens and C. baccatum species, which were selected based on variation in morphological characters, pungency and geographic origin. Genotypic analysis using AFLP markers confirmed the phylogenetic clustering of accessions according to Capsicum species and separated C. baccatum from the C. annuum–C. chinense–C. frutescens complex. Species-specific clustering was also observed when accessions were grouped based on their semi-polar metabolite profiles. In total 88 semi-polar metabolites could be putatively identified. A large proportion of these metabolites represented conjugates of the main pepper flavonoids (quercetin, apigenin and luteolin) decorated with different sugar groups at different positions along the aglycone. In addition, a large group of acyclic diterpenoid glycosides, called capsianosides, was found to be highly abundant in all C. annuum genotypes. In contrast to the variation in semi-polar metabolites, the variation in volatiles corresponded well to the differences in pungency between the accessions. This was particularly true for branched fatty acid esters present in pungent accessions, which may reflect the activity through the acyl branch of the metabolic pathway leading to capsaicinoids. In addition, large genetic variation was observed for many well-established pepper aroma compounds. These profiling data can be used in breeding programs aimed at improving metabolite-based quality traits such as flavour and health-related metabolites in pepper fruits