Characterisation of<Emphasis Type="Italic">fusarium graminearum</Emphasis> and<Emphasis Type="Italic">F. culmorum</Emphasis> isolates by mycotoxin production and aggressiveness to wheat

A total of 27Fusarium culmorum isolates from Germany and 41F. graminearum isolates from Kenya were investigated for aggressiveness and mycotoxin production on wheat ears. In addition, ergosterol content of the kernels from ears inoculated withF. graminearum was determined and theF. culmorum isolates were tested for mycotoxin productionin vitro. For both pathogens, isolates markedly differed in aggressiveness. 59% and 37% of theF. culmorum isolates produced NIV and DON, respectively,in vivo andin vitro. The DON-producing isolates also produced 3-acDONin vitro. The more aggressive isolates produced mainly DON while the less aggressive isolates produced mainly NIV. 12% and 85% of theF. graminearum isolates produced NIV and DON, respectively. The highly aggressive isolates produced higher amounts of DON, aggressiveness being highly correlated to DON content in the kernels. NIV-producing isolates were less aggressive. Ergosterol content of kernels was moderately correlated to aggressiveness but highly correlated to DON content. Disease severity was associated with kernel weight reduction.     

Geographic distribution of phylogenetic species of the <Emphasis Type="Italic">Fusarium graminearum</Emphasis> species complex and their 8-ketotrichothecene chemotypes on wheat spikes in Iran

Isolates of the Fusarium graminearum species complex (FGSC, n = 446) were collected from wheat spikes from northern and western regions of Iran with a history of Fusarium head blight (FHB) occurrences. The trichothecene mycotoxin genotypes/chemotypes, the associated phylogenetic species, and geographical distribution of these isolates were analyzed. Two phylogenetic species, Fusarium asiaticum and F. graminearum, were identified and were found to belong to sequence characterized amplified region (SCAR) groups V and I. Isolates from F. asiaticum species lineage 6 were within SCAR group V, whereas F. graminearum species lineage 7 were of SCAR group I. Of the 446 isolates assayed, 274 were F. asiaticum species predominantly of the nivalenol (NIV) genotype, while other isolates were either deoxynivalenol (DON) plus 3-acetyldeoxynivalenol (3-AcDON) or DON plus 15-acetyldeoxynivalenol (15-AcDON) genotype. Based on Tri7 gene sequences, a new subpopulation of 15-AcDON producers was observed among F. asiaticum strains in which 11-bp repeats were absent in the Tri7 sequences. The trichothecene chemotype was confirmed and quantified by high-performance liquid chromatography (HPLC) in 46 FGSC isolates. Isolates produced NIV (33.4–108.2 μg/g) and DON (64.7–473.6 μg/g) plus either 3-AcDON (51.4–142.4 μg/g) or 15-AcDON (24.1–99.3 μg/g). Among FGSC isolates, F. asiaticum produced the highest levels of trichothecenes. Using BIOCLIM based on the climate data of 20-year during 1994–2014, modelling geographical distribution of FGSC showed that F. asiaticum was restricted to warmer and humid areas with a median value of mean annual temperature of about 17.5 °C and annual rainfall of 658 mm, respectively (P < 0.05). In contrast, F. graminearum (only 15-AcDON producers) was restricted to cooler and drier areas, with a median value of the mean annual temperature of 14.4 °C and an annual rainfall of 384 mm, respectively (P < 0.05). Based on climate parameters at anthesis, the recorded distribution of F. graminearum and F. asiaticum was similar to that based on BIOCLIM parameters. Therefore, geographic differences on the wheat-growing areas in Iran have had a significant effect on distribution of FGSC and their trichothecene chemotypes.     

Cumulation of mycotoxins in maize cobs infected with<Emphasis Type="Italic">Fusarium gramihearum</Emphasis>

Maize cobs withFusarium ear rot were collected at 1986 season and five infected byFusarium graminearum were analyzed for presence of triohothecenes and zearalenone. Collected material was subsampled forFusarium damaged kernels and corresponding axial stems and healthy looking kernels. All investigated cobs contained deoxynivalenol (DON) (range 18.0–131.5 mg/kg) and zearalenone (ZEA) (range 0.38–2.17 mg/kg), in four cobs 15-acetyl-deoxynivalenol (15-AcDON) (range 5.2–6.2 mg/kg) was present and two cobs besides three all metabolites contained 3-acetyl-deoxynivalenol (3-AcD0N) (range 0.5–0.8 mg/kg).The average of individual toxins amount in axial stems: in mg/kg was equal to: DON — 110.36, ZEA — 4.57, 15-AcD0N — 16.66, and 3-AcD0N — 1.32.Fusarium damaged kernels contained in average the following amount (mg/kg) of: DON 77.00, ZEA 0.98, 15-AcD0N 3.78 and 3-AcD0N 0.06. Healthy looking kernels contained DON 1.96 mg/kg and ZEA 0.07 mg/kg only. Cooccurrence of 3-AcDON and 15-AcDON in two samples was an interesting finding. The amount of DON in total cob was highly correlated (r = 0.94) with percentage ofFusarium damaged kernels in given ear.     

An UDP-Glucosyltransferase Gene from Barley Confers Disease Resistance to <Emphasis Type="Italic">Fusarium</Emphasis> Head Blight

UDP-glucosyltransferases (UGTs) contribute to Fusarium head blight (FHB) resistance of wheat and barley by glycosylating the deoxynivalenol (DON), which is produced by Fusarium fungus. In this study, seven alleles of barley HvUGT14077 (GenBank No.GU170356.1) were cloned using RT-PCR. Among them, HvUGT-10W1, which was isolated from a FHB resistant barley variety 10W1, was significantly up-regulated in young spikes after F. graminearum (F.g) inoculation. HvUGT-10W1::GFP was subcellularly located in the plasma membrane and cytoplasm of the wheat protoplasts. In vitro antifungal activity assay showed that the HvUGT-10W1 protein exerted obvious inhibition against the growth of F.g. The silencing of the HvUGT-10W1 by virus-induced gene silencing (VIGS) resulted in compromised FHB resistance of 10W1, which was shown by the increased infected colonies on the leaves. These indicated that the barley HvUGT-10W1 may also contribute to F.g resistance in barley and provided a potential candidate gene to develop transgenic barley with enhanced FHB resistance.     

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.     

Comparison of DNA Microarray,Loop-Mediated Isothermal Amplification (LAMP) and Real-Time PCR with DNA Sequencing for Identification of <Emphasis Type="Italic">Fusarium</Emphasis> spp. Obtained from Patients with Hematologic Malignancies

The performance of three molecular biology techniques, i.e., DNA microarray, loop-mediated isothermal amplification (LAMP), and real-time PCR were compared with DNA sequencing for properly identification of 20 isolates of Fusarium spp. obtained from blood stream as etiologic agent of invasive infections in patients with hematologic malignancies. DNA microarray, LAMP and real-time PCR identified 16 (80%) out of 20 samples as Fusarium solani species complex (FSSC) and four (20%) as Fusarium spp. The agreement among the techniques was 100%. LAMP exhibited 100% specificity, while DNA microarray, LAMP and real-time PCR showed 100% sensitivity. The three techniques had 100% agreement with DNA sequencing. Sixteen isolates were identified as FSSC by sequencing, being five Fusarium keratoplasticum, nine Fusarium petroliphilum and two Fusarium solani. On the other hand, sequencing identified four isolates as Fusarium non-solani species complex (FNSSC), being three isolates as Fusarium napiforme and one isolate as Fusarium oxysporum. Finally, LAMP proved to be faster and more accessible than DNA microarray and real-time PCR, since it does not require a thermocycler. Therefore, LAMP signalizes as emerging and promising methodology to be used in routine identification of Fusarium spp. among cases of invasive fungal infections.     

Central Role of Salicylic Acid in Resistance of Wheat Against <Emphasis Type="Italic">Fusarium graminearum</Emphasis>

Head blight caused by Fusarium graminearum (F. graminearum) is one of the major threats to wheat and barley around the world. The importance of this disease is due to a reduction in both grain yield and quality in infected plants. Currently, there is limited knowledge about the physiological mechanisms involved in plant resistance against this pathogen. To reveal the physiological mechanisms underlying the resistance to F. graminearum, spikes of resistant (Sumai3) and susceptible (Falat) wheat cultivars were analyzed 4 days after inoculation, as the first symptoms of pathogen infection appeared. F. graminearum inoculation resulted in a greater induction level and activity of salicylic acid (SA), callose, phenolic compounds, peroxidase, phenylalanine ammonia lyase (PAL), and polyphenol oxidase in resistant versus susceptible cultivars. Soil drench application to spikes of SA, 24 h before inoculation with F. graminearum alleviated Fusarium head blight symptoms in both resistant and susceptible cultivars. SA treated plants showed a significant increment in hydrogen peroxide (H₂O₂) production, lipid peroxidation, SA, and callose content. SA-induced H₂O₂ level seems to be related to increased superoxide dismutase and decreased catalase activities. In addition, real-time quantitative PCR analysis showed that SA pretreatment induced expression of PAL genes in both infected and non-infected head tissues of the susceptible and resistant cultivars. Our data showed that soil drench application of SA activates antioxidant defense responses and may subsequently induce systemic acquired resistance, which may contribute to the resistance against F. graminearum. These results provide novel insights about the physiological and molecular role of SA in plant resistance against hemi-biotrophic pathogen infection.     

Molecular mapping of QTL for Fusarium head blight resistance introgressed into durum wheat

Key message

The major QTL for FHB resistance from hexaploid wheat line PI 277012 was successfully introgressed into durum wheat and minor FHB resistance QTL were detected in local durum wheat cultivars. A combination of these QTL will enhance FHB resistance of durum wheat.

Abstract

Fusarium head blight (FHB), caused by Fusarium graminearum, is a devastating disease of durum wheat. To combat the disease, great efforts have been devoted to introgress FHB resistance from its related tetraploid and hexaploid wheat species into adapted durum cultivars. However, most of the quantitative trait loci (QTL) for FHB resistance existing in the introgression lines are not well characterized or validated. In this study, we aimed to identify and map FHB resistance QTL in a population consisting of 205 recombinant inbred lines from the cross between Joppa (a durum wheat cultivar) and 10Ae564 (a durum wheat introgression line with FHB resistance derived from the hexaploid wheat line PI 277012). One QTL (Qfhb.ndwp-2A) from Joppa and two QTL (Qfhb.ndwp-5A and Qfhb.ndwp-7A) from 10Ae564 were identified through phenotyping of the mapping population for FHB severity and DON content in greenhouse and field and genotyping with 90K wheat Infinium iSelect SNP arrays. Qfhb.ndwp-2A explained 14, 15, and 9% of the phenotypic variation, respectively, for FHB severity in two greenhouse experiments and for mean DON content across the two greenhouse environments. Qfhb.ndwp-5A explained 19, 10, and 7% of phenotypic variation, respectively, for FHB severity in one greenhouse experiment, mean FHB severity across two field experiments, and mean DON content across the two greenhouse experiments. Qfhb.ndwp-7A was only detected for FHB severity in the two greenhouse experiments, explaining 9 and 11% of the phenotypic variation, respectively. This study confirms the existence of minor QTL in North Dakota durum cultivars and the successful transfer of the major QTL from PI 277012 into durum wheat.

     

FGsub: <Emphasis Type="Italic">Fusarium graminearum</Emphasis> protein subcellular localizations predicted from primary structures

Background

The fungal pathogen Fusarium graminearum (telomorph Gibberella zeae) is the causal agent of several destructive crop diseases, where a set of genes usually work in concert to cause diseases to crops. To function appropriately, the F. graminearum proteins inside one cell should be assigned to different compartments, i.e. subcellular localizations. Therefore, the subcellular localizations of F. graminearum proteins can provide insights into protein functions and pathogenic mechanisms of this destructive pathogen fungus. Unfortunately, there are no subcellular localization information for F. graminearum proteins available now. Computational approaches provide an alternative way to predicting F. graminearum protein subcellular localizations due to the expensive and time-consuming biological experiments in lab.

Results

In this paper, we developed a novel predictor, namely FGsub, to predict F. graminearum protein subcellular localizations from the primary structures. First, a non-redundant fungi data set with subcellular localization annotation is collected from UniProtKB database and used as training set, where the subcellular locations are classified into 10 groups. Subsequently, Support Vector Machine (SVM) is trained on the training set and used to predict F. graminearum protein subcellular localizations for those proteins that do not have significant sequence similarity to those in training set. The performance of SVMs on training set with 10-fold cross-validation demonstrates the efficiency and effectiveness of the proposed method. In addition, for F. graminearum proteins that have significant sequence similarity to those in training set, BLAST is utilized to transfer annotations of homologous proteins to uncharacterized F. graminearum proteins so that the F. graminearum proteins are annotated more comprehensively.

Conclusions

In this work, we present FGsub to predict F. graminearum protein subcellular localizations in a comprehensive manner. We make four fold contributions to this filed. First, we present a new algorithm to cope with imbalance problem that arises in protein subcellular localization prediction, which can solve imbalance problem and avoid false positive results. Second, we design an ensemble classifier which employs feature selection to further improve prediction accuracy. Third, we use BLAST to complement machine learning based methods, which enlarges our prediction coverage. Last and most important, we predict the subcellular localizations of 12786 F. graminearum proteins, which provide insights into protein functions and pathogenic mechanisms of this destructive pathogen fungus.

     

Induction of two cyclotide-like genes <Emphasis Type="Italic">Zmcyc1</Emphasis> and <Emphasis Type="Italic">Zmcyc5</Emphasis> by abiotic and biotic stresses in <Emphasis Type="Italic">Zea mays</Emphasis>

Cyclotides are small plant disulfide-rich and cyclic proteins with a diverse range of biological activities. Cyclotide-like genes show key sequence features of cyclotides and are present in the Poaceae. In this study the cDNA of the nine cyclotide-like genes were cloned and sequenced using 3′RACE from Zea mays. The gene expression of two of these genes (Zmcyc1 and Zmcyc5) were analyzed by real-time PCR in response to biotic (Fusarium graminearum, Ustilago maydis and Rhopalosiphum maydis) and abiotic (mechanical wounding, water deficit and salinity) stresses, as well as in response to salicylic acid and methyl jasmonate elicitors to mimic biotic stresses. All isolated genes showed significant similarity to other cyclotide-like genes and were classified in two separate clusters. Both Zmcyc1 and Zmcyc5 were expressed in all studied tissues with the highest expression in leaves and lowest expression in roots. Wounding, methyl jasmonate and salicylic acid significantly induced the expression of Zmcyc1 and Zmcyc5 genes, but the higher expression was observed for Zmcyc1 as compared with Zmcyc5. Expression levels of these two genes were also induced in inoculated leaves with F. graminearum, U. maydis and also in response to insect infestation. In addition, the 1000-base-pairs (bp) upstream of the promoter of Zmcyc1 and Zmcyc5 genes were identified and analyzed using the PlantCARE database and consequently a large number of similar biotic and abiotic cis-regulatory elements were identified for these two genes.     

Development of Defined Microbial Population Standards Using Fluorescence Activated Cell Sorting for the Absolute Quantification of <Emphasis Type="Italic">S. aureus</Emphasis> Using Real-Time PCR

In this article, four types of standards were assessed in a SYBR Green-based real-time PCR procedure for the quantification of Staphylococcus aureus (S. aureus) in DNA samples. The standards were purified S. aureus genomic DNA (type A), circular plasmid DNA containing a thermonuclease (nuc) gene fragment (type B), DNA extracted from defined populations of S. aureus cells generated by Fluorescence Activated Cell Sorting (FACS) technology with (type C) or without purification of DNA by boiling (type D). The optimal efficiency of 2.016 was obtained on Roche LightCycler^® 4.1. software for type C standards, whereas the lowest efficiency (1.682) corresponded to type D standards. Type C standards appeared to be more suitable for quantitative real-time PCR because of the use of defined populations for construction of standard curves. Overall, Fieller Confidence Interval algorithm may be improved for replicates having a low standard deviation in Cycle Threshold values such as found for type B and C standards. Stabilities of diluted PCR standards stored at ?20°C were compared after 0, 7, 14 and 30 days and were lower for type A or C standards compared with type B standards. However, FACS generated standards may be useful for bacterial quantification in real-time PCR assays once optimal storage and temperature conditions are defined.     

<Emphasis Type="Italic">HMG1A</Emphasis> and <Emphasis Type="Italic">PPARG</Emphasis> are differently expressed in the liver of fat and lean broilers

The expression of nine functional candidates for QT abdominal fat weight and relative abdominal fat content was investigated by real-time polymerase chain reaction (PCR) in the liver, adipose tissue, colon, muscle, pituitary gland and brain of broilers. The high mobility group AT-hook 1 (HMG1A) gene was up-regulated in liver with a ratio of means of 2.90 (P?≤?0.01) in the «fatty» group (relative abdominal fat content 3.5?±?0.18%, abdominal fat weight 35.4?±?6.09 g) relative to the «lean» group (relative abdominal fat content 1.9?±?0.56%, abdominal fat weight 19.2?±?5.06 g). Expression of this gene was highly correlated with the relative abdominal fat content (0.70, P?≤?0.01) and abdominal fat weight (0.70, P?≤?0.01). The peroxisome proliferator-activated receptor gamma (PPARG) gene was also up-regulated in the liver with a ratio of means of 3.34 (P?≤?0.01) in the «fatty» group relative to the «lean» group. Correlation of its expression was significant with both the relative abdominal fat content (0.55, P?≤?0.05) and the abdominal fat weight (0.57, P?≤?0.01). These data suggest that the HMG1A and PPARG genes were candidate genes for abdominal fat deposition in chickens. Searching of rSNPs in regulatory regions of the HMG1A and PPARG genes could provide a tool for gene-assisted selection.     

Trichothecene resistance in wheat: Development of molecular markers for PDR-type ABC transporter genes

Infection withFusarium graminearum andF. culmorum not only causes severe yield and quality losses, the most relevant concern is the contamination of cereal foods and feeds with trichothecenes (e.g. deoxynivalenol, DON). The ability to synthesize trichothecenes has been shown to be a virulence factor ofF. graminearum on wheat and, on the other hand, toxin resistance is most likely an important component of field resistance. Our hypothesis is that pleiotropic drug resistance mediated by PDR-type ABC transporter proteins (acting as membrane located drug efflux pumps) is a relevant mechanism of DON resistance not only in yeast but also in wheat. Goal of this project is the development of molecular markers for this gene family for use in marker-assisted plant breeding programs. The technical difficulties caused by the large size of the PDR-family are discussed.     

Feasibility of 3D UV-C treatment to reduce fungal growth and mycotoxin loads on maize and wheat kernels

Fungal disease of grain crops is a concern for the agricultural industry, resulting in economic losses. Aside from severe yield losses, mycotoxigenic fungi such as Penicillium and Fusarium can produce harmful mycotoxins, including deoxynivalenol (DON), zearalenone (ZEN), and ochratoxin A (OTA). This proof-of-concept study explored the feasibility and effects of ultraviolet (UV) C light at 253.7 nm to reduce fungal and mycotoxin loads on model surfaces as well as on maize and wheat kernels using benchtop 2D and 3D illumination strategies. Reduction of Penicillium verrucosum (98.6%) and Fusarium graminearum (88.8%) on agar was achieved using a UV-C dose of 100 mJ cm^?2. Naturally occurring fungal growth resembling P. verrucosum on maize was reduced by 79% after exposure to 5000 mJ cm^?2. Similarly, fungal growth resembling F. graminearum on maize was reduced by 60% with 1000 mJ cm^?2. On wheat, significant reduction of fungal growth was not observed. Maximal reduction of DON (97.3%), ZEN (75.4%), and OTA (91.2%) on filter paper was obtained using 15,000 mJ cm^?2. The overall reduction of DON (30%; 14%), ZEN (52%; 42%), and OTA (17%; 6%) on maize and wheat, respectively, was lower than on filter paper. Moisture and crude protein content as well as percent germination of maize kernels were not affected by UV-C treatment up to 5000 mJ cm^?2. This study has shown that 3D UV-C treatment is a feasible option for reducing Fusarium and Penicillium growth on maize kernels and, at higher doses, decreasing ZEN by ~?50%.     

Trichothecene mycotoxins in kernels and head fusariosis susceptibility in winter triticale

A single isolates ofFusarium graminearum Schwabe KF 366 andFusarium culmorum (W.G.Sm.) Sacc. KF 365 were used to infect 10 genotypes (9 lines and one cultivar) of winter triticale, 1 rye cultivar and 1 wheat cultivar, and amounts of mycotoxins in kernels were analysed at the same stage of development. One genotype of triticale CHD 353/79 and rye “Chodan” were found to be most resistant towards both species infection with lowest amount of mycotoxins (deoxynivalenol) content in kernels and also the lowest yield reduction. The most susceptible line CZR 142 cumulated in kernels about ten times higher amount of mycotoxins (up 53 mg DON/kg and 16 mg 3AcDON/kg, and 5 mg zearalenone/kg). GenerallyF, culmorum formed higher level of mycotoxins in kernels of infected heads thanF. graminearum. In kernels of more susceptible genotypes except deoxynivalenol, 3 acetyldeoxynivalenol and zearalenone also were present.     

<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains associated with the production of cachaça: identification and characterization by traditional and molecular methods (PCR,PFGE and mtDNA-RFLP)

A study of the genetic diversity of populations of Saccharomyces cerevisiae was conducted in ten different cachaça producers (alambiques) in the southern state of Minas Gerais, Brazil. A total of 106 isolates were identified by PCR using the primer SCREC114, specific to S. cerevisiae, by pulsed-field gel electrophoresis (PFGE) and by restriction fragment polymorphism of mitochondrial DNA analysis (RFLP-mtDNA). PCR showed a product of amplification to 61 isolates, enabling a rapid identification of S. cerevisiae in different alambiques. Nine different profiles were found by PFGE; all the yeasts identified as S. cerevisiae by PCR had profiles similar to that of the marker S. cerevisiae, highlighting the specificity of primer SCREC114. RFLP-mtDNA, using four different enzymes, enabled the grouping of strains of S. cerevisiae, with 80%–100% similarity. Some alambiques that had a higher frequency of S. cerevisiae characterized by PCR and PFGE, had a lower level of genetic diversity determined by RFLP-mtDNA, indicating the ability of these strains to lead the fermentative process.     

Genetic mapping and haplotype analysis of a locus for quantitative resistance to <Emphasis Type="Italic">Fusarium graminearum</Emphasis> in soybean accession PI 567516C

Key message

A major novel quantitative disease resistance locus, qRfg_Gm06, for Fusarium graminearum was genetically mapped to chromosome 6. Genomic-assisted haplotype analysis within this region identified three putative candidate genes.

Abstract

Fusarium graminearum causes seed, root rot, and seedling damping-off in soybean which contributes to reduced stands and yield. A cultivar Magellan and PI 567516C were identified with low and high levels of partial resistance to F. graminearum, respectively. Quantitative disease resistance loci (QDRL) were mapped with 241 F_7:8 recombinant inbred lines (RILs) derived from a cross of Magellan?×?PI 567516C. Phenotypic evaluation for resistance to F. graminearum used the rolled towel assay in a randomized incomplete block design. The genetic map was constructed from 927 polymorphic single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers. One major QDRL qRfg_Gm06 was detected and mapped to chromosome 6 with a LOD score of 20.3 explaining 40.2% of the total phenotypic variation. This QDRL was mapped to a?~400 kb genomic region of the Williams 82 reference genome. Genome mining of this region identified 14 putative candidate disease resistance genes. Haplotype analysis of this locus using whole genome re-sequencing (WGRS) of 106 diverse soybean lines narrowed the list to three genes. A SNP genotyping Kompetitive allele-specific PCR (KASP) assay was designed for one of the genes and was validated in a subset of the RILs and all 106 diverse lines.

     

The replication origin of <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis>

The putative replication origin of Azotobacter vinelandii was cloned as an autonomously replicating fragment after ligation to an antibiotic resistance cartridge. The resulting plasmids could be isolated and labelled by Southern hybridisation with the antibiotic resistance cartridge as probe and also visualised by electron microscopy. These plasmids integrated into the chromosome after a few generations, even in the recA mutant of A. vinelandii. The integrated copy of the plasmid was re-isolated from the chromosome and the DNA and its subfragments were cloned in the plasmid vector pBR322. A 200-bp DNA fragment was sufficient to allow the replication of pBR322 in an Escherichia coli polA strain. Electron microscopic analysis of this plasmid showed that replication initiated mostly within the A. vinelandii DNA fragment. The nucleotide sequence of the putative replication origin and its flanking regions was determined. In the sequence of the 200-bp fragment many of the distinctive features found in other replication origins are lacking. A greater variation from the consensus DnaA binding sequence was observed in A. vinelandii. Direct sequencing of the relevant genomic fragment was also carried after amplifying it from A. vinelandii chromosomal DNA by PCR. This confirmed that no rearrangements had taken place while the cloned fragment was resident in E. coli. It was shown by hybridisation that the 200-bp chromosomal origin fragment of A. vinelandii was present in three other field strains of Azotobacter spp.