A high-resolution map of the <Emphasis Type="Italic">Grp1</Emphasis> locus on chromosome V of potato harbouring broad-spectrum resistance to the cyst nematode species <Emphasis Type="Italic">Globodera pallida</Emphasis> and <Emphasis Type="Italic">Globodera rostochiensis</Emphasis>

The Grp1 locus confers broad-spectrum resistance to the potato cyst nematode species Globodera pallida and Globodera rostochiensis and is located in the GP21-GP179 interval on the short arm of chromosome V of potato. A high-resolution map has been developed using the diploid mapping population RHAM026, comprising 1,536 genotypes. The flanking markers GP21 and GP179 have been used to screen the 1,536 genotypes for recombination events. Interval mapping of the resistances to G. pallida Pa2 and G. rostochiensis Ro5 resulted in two nearly identical LOD graphs with the highest LOD score just north of marker TG432. Detailed analysis of the 44 recombinant genotypes showed that G. pallida and G. rostochiensis resistance could not be separated and map to the same location between marker SPUD838 and TG432. It is suggested that the quantitative resistance to both nematode species at the Grp1 locus is mediated by one or more tightly linked R genes that might belong to the NBS-LRR class. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. A. Finkers-Tomczak and S. Danan contributed equally to this research.     

Somatic hybrids between transgenic <Emphasis Type="Italic">Solanum tuberosum</Emphasis> potato plants and transplastome <Emphasis Type="Italic">Solanum rickii</Emphasis> plants

Experiments on fusion of mesophyllic protoplasts of Solanum tuberosum (Lugovskoi and Slavyanka cultivars) possessing the nptII gene in the nuclear DNA with transplastome Solanum rickii plants (which possess the aadA gene) that we have derived previously, are performed. Hybrid plants with the genes aadA and nptII, the chloroplasts of S. rickii and S. tuberosum, and a hybrid nuclear genome were obtained in a selection medium containing the antibiotics kanamycin, spectomycin, and streptomycin. The result is confirmed by results of PCR analyses.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Echinacea pallida</Emphasis> from leaf explants

Summary A method has been developed for the induction of adventitious shoots from leaf tissue of Echinacea pallida with subsequent whole-plant regeneration. Proliferating callus and shoot cultures were derived from leaf tissue explants placed on Murashige and Skoog medium supplemented with 6-benzylaminopurine and naphthaleneacetic acid combinations. The optimum shoot regeneration frequency (63%) and number of shoots per explant (2.3 shoots per explant) was achieved using media supplemented with 26.6 μM 6-benzylaminopurine and 0.11 μM naphthaleneacetic acid. Rooting of regenerated shoot explants was successful on Murashige and Skoog medium, both with and without the addition of indole-3-butyric acid. All plantlets survived acclimatization, producing phenotypically normal plants in the greenhouse. This study demonstrates that leaf tissue of E. pallida is competent for adventitious shoot regeneration and establishes a useful method for the micropropagation of this important medicinal plant.     

Mapping of loci from <Emphasis Type="Italic">Solanum lycopersicoides</Emphasis> conferring resistance or susceptibility to <Emphasis Type="Italic">Botrytis cinerea</Emphasis> in tomato

Cultivated tomato (Solanum lycopersicum, syn. Lycopersicon esculentum) is susceptible to the necrotrophic ascomycete and causal agent of gray mold, Botrytis cinerea. Resistance to this fungal pathogen is elevated in wild relatives of tomato, including Solanum lycopersicoides. An introgression line population (IL) containing chromosomal segments of S. lycopersicoides within the background of tomato cv. VF36 was used to screen the genome for foliar resistance and susceptibility to B. cinerea. Based on this screen, putative quantitative trait loci (QTL) were identified, five for resistance and two for susceptibility. Four resistance QTL decreased infection frequency while the fifth reduced lesion diameter. One susceptibility QTL increased infection frequency whereas the other increased lesion diameter. Overlapping chromosomal segments provided strong evidence for partial resistance on chromosomes 1 and 9 and for elevated susceptibility on chromosome 11. Segregation analysis confirmed the major resistance QTL on the long arm of chromosome 1 and susceptibility on chromosome 11. Linkage of partial resistance to chromosome 9 could not be confirmed. The usefulness of these data for resistance breeding and for map-based cloning of foliar resistance to B. cinerea is discussed.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Functional analysis of <Emphasis Type="Italic">Xa3/Xa26</Emphasis> family members in rice resistance to <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis>

Plant disease resistant (R) genes are frequently clustered in the genome. The diversity of members in a complex R-gene family may provide variation in resistance specificity. Rice Xa3/Xa26, conferring resistance to Xanthomonas oryzae pv. oryzae (Xoo) encodes a leucine-rich repeat (LRR) receptor kinase-type protein and belongs to a multigene family, consisting of Xa3/Xa26, MRKa, MRKc and MRKd in rice cultivar Minghui 63. MRKa and MRKc are intact genes, while MRKd is a pseudogene. Complementary analyses showed that MRKa and MRKc could not mediate resistance to Xoo when regulated by their native promoters, but MRKa not MRKc conferred partial resistance to Xoo when regulated by a strong constitutive promoter. Plants carrying truncated XA3/XA26, which lacked the kinase domain, were compromised in their resistance to Xoo. However, the kinase domain of MRKa could partially restore the function of the truncated XA3/XA26 in resistance. MRKa and MRKc showed similar expression pattern as Xa3/Xa26, which expressed only in the vascular systems of different tissues. The expressional characteristic of MRKa and MRKc perfectly fits the function of genes conferring resistance to Xoo, a vascular pathogen. These results suggest that although MRKa and MRKc cannot mediate bacterial blight resistance nowadays, they may be once effective genes for Xoo resistance. Their expressional characteristic and sequence similarity to Xa3/Xa26 will provide templates for generating novel recognition specificity to face the evolution of Xoo. In addition, both LRR and kinase domains encoded by Xa3/Xa26 and MRKa are the functional determinants and MRKa-mediated resistance is dosage-dependent. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Application of DNA markers linked to the potato <Emphasis Type="Italic">H1</Emphasis> gene conferring resistance to pathotype Ro1 of <Emphasis Type="Italic">Globodera rostochiensis</Emphasis>

Ninety-one potato genotypes (cultivars and breeding lines) selected as resistant or susceptible to pathotype Ro1 of Globodera rostochiensis were screened for the presence of two PCR markers, 0.14 and 0.76 kb in length. Both PCR markers were linked with the H1 gene, located at the distal end of the long arm of chromosome V, and were present in 88 to 100% of the resistant cultivars and breeding lines. The 0.76 kb PCR marker was detected in all resistant genotypes and in approximately 86% of susceptible breeding lines as well as in all susceptible cultivars. The 0.14 kb marker was detected in 88% of resistant breeding lines and in 94% of resistant cultivars. Most of the susceptible genotypes tested (91% of cultivars, but only 50% of breeding lines) did not show the presence of the 0.14 kb marker. We conclude that the 0.14 kb H1 marker is likely to be useful for the proper selection of potato genotypes resistant to the Ro1 pathotype of G. rostochiensis.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Functional characterization of the pollen-specific <Emphasis Type="Italic">SBgLR</Emphasis> promoter from potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.)

SBgLR (Solanum tuberosum genomic lysine-rich) gene was isolated from a potato genomic library using SB401 (S. berthaultii 401) cDNA as probe. RT-PCR analysis of SBgLR gene expression profile and microscopic analysis of green fluorescent protein (GFP) expression in tobacco plants transformed with SBgLR promoter-GFP reporters indicate that SBgLR is a pollen-specific gene. A series of 5′deletions of SBgLR promoter were fused to the β-glucuronidase (GUS) gene and stably introduced into tobacco plants. Histochemical and quantitative assays of GUS expression in transgenic plants allowed us to localize an enhancer of SBgLR promoter to the region −345 to −269 relative to the translation start site. This 76 bp (−345 to −269) fragment enhanced GUS expression in leaves, stems and roots when fused to −90/+6 CaMV 35S minimal promoter. Deletion analysis showed that a cis-element, which can repress gene expression in root hairs, was located in the region −345 to −311. Further study indicated that the −269 to −9 region was sufficient to confer pollen-specific expression of GFP when fused to CaMV 35S enhancer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Authors Zhihong Lang and Peng Zhou contributed equally to this work.     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

Electrofusion of protoplasts from <Emphasis Type="Italic">Solanum tuberosum</Emphasis>, <Emphasis Type="Italic">S. bulbocastanum</Emphasis> and <Emphasis Type="Italic">S. pinnatisectum</Emphasis>

This paper discusses a number of experiments performed, involving the fusion by an electric field of mesophyll protoplasts from Solanum tuberosum cv. Bintje, S. tuberosum dihaploid clones 243, 299 and the wild tuberous disease-resistant species S. bulbocastanum and S. pinnatisectum. Three fusion experiments (S. bulbocastanum + S. tuberosum dihaploid 243, S. pinnatisectum + S. tuberosum cv. Bintje and S. pinnatisectum + S. tuberosum dihaploid 299) yielded 542 calli, the 52 ones of which produced shoots. Obtained regenerants were estimated by the flow-cytometry (FC) and RAPD analysis to determine hybrid plants.The utilisation of the FC as a useful method for detecting somatic hybrids is also discussed in this paper. The combination S. bulbocastanum + S. tuberosum dihaploid 243 led to the creation of eight somatic hybrids, the combination S. pinnatisectum + S. tuberosum cv. Bintje yielded four somatic hybrids and the combination S. pinnatisectum + S. tuberosum dihaploid 299 resulted in no hybrid regenerants. Morphology in vitro, growth vigour and production of tuber-like structures were evaluated in hybrid plants. Plants were transferred in vivo for further estimation (acclimatization, habitus evaluation and tuberization ability).     

Gene transfer into <Emphasis Type="Italic">Solanum tuberosum</Emphasis> via <Emphasis Type="Italic">Rhizobium</Emphasis> spp.

Agrobacterium tumefaciens-mediated transformation (ATMT) is the preferred technique for gene transfer into crops. A major disadvantage of the technology remains the complexity of the patent landscape that surrounds ATMT which restricts its use for commercial applications. An alternative system has been described (Broothaerts et al. in Nature 433:629-633, 2005) detailing the propensity of three rhizobia to transform the model crop Arabidopsis thaliana, the non-food crop Nicotiana tabacum and, at a very low frequency, the monocotyledonous crop Oryza sativa. In this report we describe for the first time the genetic transformation of Solanum tuberosum using the non-Agrobacterium species Sinorhizobium meliloti, Rhizobium sp. NGR234 and Mesorhizobium loti. This was achieved by combining an optimal bacterium and host co-cultivation period with a low antibiotic regime during the callus and shoot induction stages. Using this optimized protocol the transformation frequency (calculated as % of shoots equipped with root systems with the ability to grow in rooting media supplemented with 25 μg/ml hygromycin) of the rhizobia strains was calculated at 4.72, 5.85 and 1.86% for S. meliloti, R. sp. NGR234 and M. loti respectively, compared to 47.6% for the A. tumefaciens control. Stable transgene integration and expression was confirmed via southern hybridisation, quantitative PCR analysis and histochemical screening of both leaf and/or tuber tissue. In light of the rapid advances in potato genomics, combined with the sequencing of the potato genome, the ability of alternative bacteria species to genetically transform this major food crop will provide a novel resource to the Solanaceae community as it continues to develop potato as both a food and non-food crop.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Mapping <Emphasis Type="Italic">Ol-4</Emphasis>, a gene conferring resistance to <Emphasis Type="Italic">Oidium neolycopersici</Emphasis> and originating from <Emphasis Type="Italic">Lycopersicon peruvianum</Emphasis> LA2172, requires multi-allelic,single-locus markers

Lycopersicon peruvianum LA2172 is completely resistant to Oidium neolycopersici, the causal agent of tomato powdery mildew. Despite the large genetic distance between the cultivated tomato and L. peruvianum, fertile F₁ hybrids of L. esculentum cv. Moneymaker × L. peruvianum LA2172 were produced, and a pseudo-F₂ population was generated by mating F₁ half-sibs. The disease tests on the pseudo-F₂ population and two BC₁ families showed that the resistance in LA2172 is governed by one dominant gene, designated as Ol-4. In the pseudo-F₂ population, distorted segregation was observed, and multi-allelic, single-locus markers were used to display different marker-allele configurations per locus. Parameters for both distortion and linkage between genetic loci were determined by maximum likelihood estimation, and the necessity of using multi-allelic, single-locus markers was illustrated. Finally, a genetic linkage map of chromosome 6 around the Ol-4 locus was constructed by using the pseudo-F₂ population.     

<Emphasis Type="Italic">qRfg3</Emphasis>, a novel quantitative resistance locus against <Emphasis Type="Italic">Gibberella</Emphasis> stalk rot in maize

Key message

A quantitative trait locus  qRfg3 imparts recessive resistance to maize Gibberella stalk rot. qRfg3 has been mapped into a 350-kb interval and could reduce the disease severity index by ~26.6%.

Abstract

Gibberella stalk rot, caused by the fungal pathogen Fusarium graminearum, severely affects maize yield and grain quality worldwide. To identify more resistance quantitative trait loci (QTLs) against this disease, we analyzed a recombinant inbred line (RIL) population derived from a cross between resistant H127R and susceptible C7-2 inbred lines. Within this population, maize resistance to Gibberella stalk rot had high broad-sense heritability. A major QTL, qRfg3, on chromosome 3 was consistently detected across three field trials, accounting for 10.7–19.4% of the total phenotypic variation. Using a progeny-based sequential fine-mapping strategy, we narrowed qRfg3 down to an interval of ~350 kb. We further demonstrated that qRfg3 is a recessive resistance locus to Gibberella stalk rot that reduced the disease severity index by ~26.6%. Both the gene location and recessive genetic mode distinguish qRfg3 from other stalk rot resistance loci. Hence, qRfg3 is valuable as a complement to existing resistance QTLs to improve maize resistance to Gibberella stalk rot.

     

Over-expressing a UDP-glucosyltransferase gene (<Emphasis Type="Italic">Ta-UGT</Emphasis><Subscript><Emphasis Type="Italic">3</Emphasis></Subscript>) enhances <Emphasis Type="Italic">Fusarium</Emphasis> Head Blight resistance of wheat

Wheat Fusarium Head Blight (FHB), mainly caused by Fusarium graminearum (F.g), is a destructive fungal disease worldwide. FHB can not only cause considerable reduction in yield, but more seriously, can contaminate grain by trichothecene toxins released by the fungus. Here, we report new insights into the function and underlying mechanisms of a UDP-glycosyltransferase gene, Ta-UGT ₃ , that is involved in FHB resistance in wheat. In our previous study, Ta-UGT ₃ was found to enhance host tolerance against deoxynivalenol (DON) in Arabidopsis. In this study, four transgenic lines over-expressing Ta-UGT ₃ in a FHB highly susceptible wheat variety, Alondra’s, were obtained and characterized. 3 years of assays using single floret inoculation with F.g indicated that all four transgenic lines exhibited significantly enhanced type II resistance to FHB and less DON accumulation in the grains compared to the untransformed control. Histological observation using GFP labelled F.g was in agreement with the above test results since over-expression of Ta-UGT ₃ dramatically inhibited expansion of F.g. To explore the putative mechanism of resistance mediated by Ta-UGT ₃ , microarray analysis, qRT-PCR and hormone measurements were performed. Microarray analysis showed that DON up-regulated genes, such as TaNPR1, in the susceptible control, and down-regulated genes in F.g inoculated transgenic lines, while qRT-PCR showed that some defence related genes were up-regulated in F.g inoculated transgenic lines. Ta-UGT ₃ over-expression also changed the contents of the endogenous hormones SA and JA in the spikes. These data suggest that Ta-UGT ₃ positively regulates the defence responses to F.g, perhaps by regulating defence-related and DON-induced downstream genes.