SNP-markers in Allium species to facilitate introgression breeding in onion.

BACKGROUND: Within onion, Allium cepa L., the availability of disease resistance is limited. The identification of sources of resistance in related species, such as Allium roylei and Allium fistulosum, was a first step towards the improvement of onion cultivars by breeding. SNP markers linked to resistance and polymorphic between these related species and onion cultivars are a valuable tool to efficiently introgress disease resistance genes. In this paper we describe the identification and validation of SNP markers valuable for onion breeding. RESULTS: Transcriptome sequencing resulted in 192 million RNA seq reads from the interspecific F1 hybrid between A. roylei and A. fistulosum (RF) and nine onion cultivars. After assembly, reliable SNPs were discovered in about 36 % of the contigs. For genotyping of the interspecific three-way cross population, derived from a cross between an onion cultivar and the RF (CCxRF), 1100 SNPs that are polymorphic in RF and monomorphic in the onion cultivars (RF SNPs) were selected for the development of KASP assays. A molecular linkage map based on 667 RF-SNP markers was constructed for CCxRF. In addition, KASP assays were developed for 1600 onion-SNPs (SNPs polymorphic among onion cultivars). A second linkage map was constructed for an F2 of onion x A. roylei (F2(CxR)) that consisted of 182 onion-SNPs and 119 RF-SNPs, and 76 previously mapped markers. Markers co-segregating in both the F2(CxR) and the CCxRF population were used to assign the linkage groups of RF to onion chromosomes. To validate usefulness of these SNP markers, QTL mapping was applied in the CCxRF population that segregates for resistance to Botrytis squamosa and resulted in a QTL for resistance on chromosome 6 of A. roylei. CONCLUSIONS: Our research has more than doubled the publicly available marker sequences of expressed onion genes and two onion-related species. It resulted in a detailed genetic map for the interspecific CCxRF population. This is the first paper that reports the detection of a QTL for resistance to B. squamosa in A. roylei.

Elicitin recognition confers enhanced resistance to Phytophthora infestans in potato.

Potato late blight, caused by the destructive Irish famine pathogen Phytophthora infestans, is a major threat to global food security(1,2). All late blight resistance genes identified to date belong to the coiled-coil, nucleotide-binding, leucine-rich repeat class of intracellular immune receptors(3). However, virulent races of the pathogen quickly evolved to evade recognition by these cytoplasmic immune receptors(4). Here we demonstrate that the receptor-like protein ELR (elicitin response) from the wild potato Solanum microdontum mediates extracellular recognition of the elicitin domain, a molecular pattern that is conserved in Phytophthora species. ELR associates with the immune co-receptor BAK1/SERK3 and mediates broad-spectrum recognition of elicitin proteins from several Phytophthora species, including four diverse elicitins from P. infestans. Transfer of ELR into cultivated potato resulted in enhanced resistance to P. infestans. Pyramiding cell surface pattern recognition receptors with intracellular immune receptors could maximize the potential of generating a broader and potentially more durable resistance to this devastating plant pathogen.

Accumulation of human EGF in nectar of transformed plants of Nicotiana langsdorffii x N. sanderae and transfer to honey by bees.

Honey has been used successfully in wound healing for thousands of years. The peptide hormone human epidermal growth factor (hEGF) is also known to have a beneficial effect in various wound healing processes via mechanisms that differ from those for honey. In this study, we show that hEGF can be incorporated into honey via nectar. Plants of Nicotiana langsdorffii x N. sanderae were transformed with the gene for hEGF, equipped with a nectary-targeted promoter and a signal sequence for secretion to nectar. These plants accumulated hEGF in the nectar. The maximum hEGF concentration recorded with ELISA in these plants is 2.5 ng·ml⁻¹. There is a significant linear relationship (P<0.001) between hEGF concentration and induction of hEGF-receptor phosphorylation. Since the flower morphology of these plants did not allow production of honey from their nectar, we used feeding solutions, spiked with synthetic hEGF, to study transfer of this peptide into honey through bee activity. Transfer of hEGF from a feeding solution to honey by bees occurred with retention of the hEGF concentration and the capacity to induce hEGF-receptor phosphorylation. These observations indicate that plants can function as a production platform for honey containing biologically active peptides, which may enhance wound healing and other biological processes.

Genetic analysis of the interaction between Allium species and arbuscular mycorrhizal fungi.

The response of Allium cepa, A. roylei, A. fistulosum, and the hybrid A. fistulosum × A. roylei to the arbuscular mycorrhizal fungus (AMF) Glomus intraradices was studied. The genetic basis for response to AMF was analyzed in a tri-hybrid A. cepa × (A. roylei × A. fistulosum) population. Plant response to mycorrhizal symbiosis was expressed as relative mycorrhizal responsiveness (R') and absolute responsiveness (R). In addition, the average performance (AP) of genotypes under mycorrhizal and non-mycorrhizal conditions was determined. Experiments were executed in 2 years, and comprised clonally propagated plants of each genotype grown in sterile soil, inoculated with G. intraradices or non-inoculated. Results were significantly correlated between both years. Biomass of non-mycorrhizal and mycorrhizal plants was significantly positively correlated. R' was negatively correlated with biomass of non-mycorrhizal plants and hence unsuitable as a breeding criterion. R and AP were positively correlated with biomass of mycorrhizal and non-mycorrhizal plants. QTLs contributing to mycorrhizal response were located on a linkage map of the A. roylei × A. fistulosum parental genotype. Two QTLs from A. roylei were detected on chromosomes 2 and 3 for R, AP, and biomass of mycorrhizal plants. A QTL from A. fistulosum was detected on linkage group 9 for AP (but not R), biomass of mycorrhizal and non-mycorrhizal plants, and the number of stem-borne roots. Co-segregating QTLs for plant biomass, R and AP indicate that selection for plant biomass also selects for enhanced R and AP. Moreover, our findings suggest that modern onion breeding did not select against the response to AMF, as was suggested before for other cultivated species. Positive correlation between high number of roots, biomass and large response to AMF in close relatives of onion opens prospects to combine these traits for the development of more robust onion cultivars.

Can the QTL for late blight resistance on potato chromosome 5 be attributed to foliage maturity type?

We investigated the association between late blight resistance and foliage maturity type in potato by means of molecular markers. Two QTLs were detected for foliage resistance against Phytophthora infestans (on chromosomes 3 and 5) and one for foliage maturity type (on chromosome 5). The QTL for resistance to late blight and the QTL for foliage maturity type on chromosome 5 appeared to be mapped on indistinguishable positions. We were interested whether this genetic linkage was due to closely linked but different genes, or due to one (or more) gene(s) with pleiotropic effects. We therefore developed an approach to detect QTLs, in which resistance to late blight was adjusted for foliage maturity type. This analysis revealed the same two QTLs for resistance against P. infestans, but the effect of the locus on chromosome 5 was reduced to only half the original effect. This is a strong indication that the two indistinguishable QTLs for foliage maturity type and for late blight resistance on chromosome 5 may actually be one gene with a pleiotropic effect on both traits. However, there was still a significant effect on resistance against P. infestans on the locus on chromosome 5 after adjusting for foliage maturity type. Therefore we cannot rule out the presence of two closely linked QTLs on chromosome 5: one with a pleiotropic effect on both late blight resistance and foliage maturity type, and another with merely an effect on resistance. In addition, the two QTLs for resistance to late blight showed an important epistatic interaction, suggesting that QTLs for resistance affect each other's expression.

Leaf position prevails over plant age and leaf age in reflecting resistance to late blight in potato.

ABSTRACT The effects of plant age, leaf age, and leaf position on race-nonspecific resistance against Phytophthora infestans were investigated in a series of field and controlled environment experiments with five different potato (Solanum tuberosum) cultivars. Leaf position proved to be the most significant factor; apical leaves were far more resistant to late blight than basal leaves. Plant age and leaf age had only minor effects; therefore, the resistance of a specific leaf remained about the same during its entire lifetime. The gradual increase in late blight resistance from basal leaves to apical leaves appeared to be a general effect, irrespective of cultivar, growing conditions, or resistance test. Therefore, it is important to consider leaf position in tests for late blight resistance, because contrasts in resistance may be ascribed erroneously to differences between genotypes or treatments, whereas they are actually caused by differences in leaf position.

Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarrays.

Fruit flavor is a result of a complex mixture of numerous compounds. The formation of these compounds is closely correlated with the metabolic changes occurring during fruit maturation. Here, we describe the use of DNA microarrays and appropriate statistical analyses to dissect a complex developmental process. In doing so, we have identified a novel strawberry alcohol acyltransferase (SAAT) gene that plays a crucial role in flavor biogenesis in ripening fruit. Volatile esters are quantitatively and qualitatively the most important compounds providing fruity odors. Biochemical evidence for involvement of the SAAT gene in formation of fruity esters is provided by characterizing the recombinant protein expressed in Escherichia coli. The SAAT enzyme showed maximum activity with aliphatic medium-chain alcohols, whose corresponding esters are major components of strawberry volatiles. The enzyme was capable of utilizing short- and medium-chain, branched, and aromatic acyl-CoA molecules as cosubstrates. The results suggest that the formation of volatile esters in fruit is subject to the availability of acyl-CoA molecules and alcohol substrates and is dictated by the temporal expression pattern of the SAAT gene(s) and substrate specificity of the SAAT enzyme(s).

Transgenic caraway, Carum carvi L.: a model species for metabolic engineering.

Regeneration in caraway was obtained via two different routes. Hypocotyls showed delayed shoot formation after a callus phase and at relatively low frequencies. In contrast, high-frequency, direct regeneration occurred when cotyledonary node explants were used. Transient expression of ß-glucuronidase was monitored after inoculation of both explant types with Agrobacterium tumefaciens AGL0(pMOG410). Gene transfer was more efficient when using cotyledonary node explants. This explant type also proved to be the best for stable transformation resulting in transgenic plants. Several parameters determining regeneration and transformation efficiency were tested. The percentage of explants giving one to numerous transgenic plants could be as high as 13%. This system for the rapid production of many transgenic caraway plants opens up possibilities for studying metabolic engineering with this crop.

Assessing non-specificity of resistance in wheat to head blight caused by inoculation with European strains of Fusarium culmorum, F. graminearum and F. nivale using a multiplicative model for interaction.

To determine whether resistance to Fusarium head blight in winter wheat is horizontal and non-species specific, 25 genotypes from five European countries were tested at six locations across Europe in the years 1990, 1991, and 1992. The five genotypes from each country had to cover the range from resistant to susceptible. The locations involved were Wageningen, Vienna, Rennes, Hohenheim, Oberer Lindenhof, and Szeged. In total, 17 local strains of Fusarium culmorum, F. graminearum, and F. nivale were used for experimental inoculation. One strain, F. culmorum IPO 39-01, was used at all locations. Best linear unbiased predictions (BLUPs) for the head blight ratings of the genotypes were formed within each particular location for each combination of year and strain. The BLUPs over all locations were collected in a genotype-by environment table in which the genotypic dimension consisted of the 25 genotypes, while the environmental dimension was made up of 59 year-by-strain-by-location combinations. A multiplicative model was fitted to the genotype by-environment interaction in this table. The inverses of the variances of the genotype-by-environment BLUPs were used as weights. Interactions between genotypes and environments were written as sums of products between genotypic scores and environmental scores. After correction for year-by-location influence very little variation in environmental scores could be ascribed to differences between strains. This provided the basis for the conclusion that the resistance to Fusarium head blight in winter wheat was of the horizontal and non-species specific type. There was no indication for any geographical pattern in virulence genes. Any reasonable aggressive strain, a F. culmorum strain for the cool climates and a F. graminearum strain for the warmer humid areas, should be satisfactory for screening purposes.