Mycelial biomass and concentration of loline alkaloids driven by complex population structure in Epichloë uncinata and meadow fescue (Schedonorus pratensis).

Many efforts have been made to select and isolate naturally occurring animal-friendly Epichloë strains for later reinfection into elite cultivars. Often this process involves large-scale screening of Epichloë-infected wild grass populations where strains are characterized and alkaloids measured. Here, we describe for the first time the use of genotyping-by-sequencing (GBS) on a collection of 217 Epichloë-infected grasses (7 S. arundinaceum, 4 L. perenne, and 206 S. pratensis). This genotyping strategy is cheaper than complete genome sequencing, is suitable for a large number of individuals, and, when applied to endophyte-infected grasses, conveniently genotypes both organisms. In total, 6273 single nucleotide polymorphisms (SNPs) in the endophyte data set and 38 323 SNPs in the host data set were obtained. Our findings reveal a composite structure with three distinct endophyte clusters unrelated to the three main S. pratensis gene pools that have most likely spread from different glacial refugia in Eurasia. All three gene pools can establish symbiosis with E. uncinata. A comparison of the endophyte clusters with microsatellite-based fingerprinting of the same samples allows a quick test to discriminate between these clusters using two simple sequence repeats (SSRs). Concentrations of loline alkaloids and mycelial biomass are correlated and differ significantly among the plant and endophyte subpopulations; one endophyte strain has higher levels of lolines than others, and one specific host genotype is particularly suitable to host E. uncinata. These findings pave the way for targeted artificial inoculations of specific host-endophyte combinations to boost loline production in the symbiota and for genome association studies with the aim of isolating genes involved in the compatibility between meadow fescue and E. uncinata.

Integrating a newly developed BAC-based physical mapping resource for Lolium perenne with a genome-wide association study across a L. perenne European ecotype collection identifies genomic contexts associated with agriculturally important traits.

BACKGROUND AND AIMS: Lolium perenne (perennial ryegrass) is the most widely cultivated forage and amenity grass species in temperate areas worldwide and there is a need to understand the genetic architectures of key agricultural traits and crop characteristics that deliver wider environmental services. Our aim was to identify genomic regions associated with agriculturally important traits by integrating a bacterial artificial chromosome (BAC)-based physical map with a genome-wide association study (GWAS). METHODS: BAC-based physical maps for L. perenne were constructed from ~212 000 high-information-content fingerprints using Fingerprint Contig and Linear Topology Contig software. BAC clones were associated with both BAC-end sequences and a partial minimum tiling path sequence. A panel of 716 L. perenne diploid genotypes from 90 European accessions was assessed in the field over 2 years, and genotyped using a Lolium Infinium SNP array. The GWAS was carried out using a linear mixed model implemented in TASSEL, and extended genomic regions associated with significant markers were identified through integration with the physical map. KEY RESULTS: Between ~3600 and 7500 physical map contigs were derived, depending on the software and probability thresholds used, and integrated with ~35 k sequenced BAC clones to develop a resource predicted to span the majority of the L. perenne genome. From the GWAS, eight different loci were significantly associated with heading date, plant width, plant biomass and water-soluble carbohydrate accumulation, seven of which could be associated with physical map contigs. This allowed the identification of a number of candidate genes. CONCLUSIONS: Combining the physical mapping resource with the GWAS has allowed us to extend the search for candidate genes across larger regions of the L. perenne genome and identified a number of interesting gene model annotations. These physical maps will aid in validating future sequence-based assemblies of the L. perenne genome.

Clade classification of monolignol biosynthesis gene family members reveals target genes to decrease lignin in Lolium perenne.

In monocots, lignin content has a strong impact on the digestibility of the cell wall fraction. Engineering lignin biosynthesis requires a profound knowledge of the role of paralogues in the multigene families that constitute the monolignol biosynthesis pathway. We applied a bioinformatics approach for genome-wide identification of candidate genes in Lolium perenne that are likely to be involved in the biosynthesis of monolignols. More specifically, we performed functional subtyping of phylogenetic clades in four multigene families: 4CL, COMT, CAD and CCR. Essential residues were considered for functional clade delineation within these families. This classification was complemented with previously published experimental evidence on gene expression, gene function and enzymatic activity in closely related crops and model species. This allowed us to assign functions to novel identified L. perenne genes, and to assess functional redundancy among paralogues. We found that two 4CL paralogues, two COMT paralogues, three CCR paralogues and one CAD gene are prime targets for genetic studies to engineer developmentally regulated lignin in this species. Based on the delineation of sequence conservation between paralogues and a first analysis of allelic diversity, we discuss possibilities to further study the roles of these paralogues in lignin biosynthesis, including expression analysis, reverse genetics and forward genetics, such as association mapping. We propose criteria to prioritise paralogues within multigene families and certain SNPs within these genes for developing genotyping assays or increasing power in association mapping studies. Although L. perenne was the target of the analyses presented here, this functional subtyping of phylogenetic clades represents a valuable tool for studies investigating monolignol biosynthesis genes in other monocot species.

Analysis of PSP toxins in Norwegian mussels by a post-column derivatization HPLC method.

The aim of this study was to investigate the agreement between the AOAC mouse bioassay and an HPLC method for determining Paralytic Shellfish Poisoning (PSP) toxicity in blue mussels from the Norwegian coast. The AOAC mouse bioassay has traditionally been used for determining the toxin levels. Recently, an HPLC method for determining PSP toxins has been implemented and run in parallel with the bioassay. Four hundred mussel extracts from the last six years were analysed with both methods. A highly significant linear correlation between the methods was achieved (r = 0.84). However, the relation between the two methods was best described by a second-degree polynom. Totally, this model explained 87% of the variation in the HPLC method. By agreement analysis, it was possible to establish cut-off levels for the HPLC method related to the AOAC mouse bioassay with kappa values >0.80 for toxicity levels below 500 MU/100 g. The HPLC method could substitute the mouse bioassay in determining PSP toxicity in Norwegian mussels. Additionally, about 70 mussel samples were also analysed by the MIST Alert trade mark test kit. The kit did not give any false negative results compared with the regulatory limit, but 30% of the samples below the cut point were also determined as positive.

Determination of extractable organic chlorine and bromine by probe injection dual-microplasma atomic emission spectrometry.

A probe injection dual-microplasma spectrometer is evaluated as a low-cost alternative for the determination of extractable organic chlorine and bromine (EOCl and EOBr). The system consists of two 350 kHz plasmas sustained in the same stream of helium and a probe for sample application in the interplasma region. The sample was applied with a microsyringe into a small cup on the sample probe. Subsequently, the extraction solvent was gently evaporated, and the sample cup was pushed into the interplasma region. The first plasma was in direct contact with the sample probe and served to rapidly vaporize the sample material. The vaporized sample was then transferred to the second plasma, where atomic emission was measured for the determination of EOCl and EOBr. For both Cl and Br, 120 pg detection limits and 1000:1 halogen-to-carbon selectivities were obtained, and responses were linear over 3 orders of magnitude.

Elemental analysis of bovine liver by inductively coupled plasma atomic emission spectrometry by using a simple dissolution procedure.

A simple digestion with nitric acid followed by filtration of the undigested lipids was found to be suitable for the decomposition of bovine liver, prior to multielement determination by inductively coupled plasma atomic emission spectrometry. Using reference materials, accurate results were obtained for cadmium, copper, iron, manganese, molybdenum and zinc.

Continuing reject-repeat film analysis program.

A continuing image quality assurance and control program has been employed in the Department for over 9 years. One of the tools used in assessing quality has been reject-repeat analyses performed 9 times. The reject rate dropped from 15% in 1980-1981 to 8.4% in 1982. After moving to a new department with new film processing systems and, in part, new diagnostic equipment, the rejection rate increased to 13.2%. New and renewed procedures such as increased control and adjustment of technical equipment (in particular the automatic daylight film processing systems), information and education, decreased the rejection rates to 9.2 and 6.6% on 2 analysis occasions in 1987, and to 6.4% in 1988. Image quality assurance and control have to be continuous activities if they are to yield favourable and economically justifiable results.

Elimination of electrostatic discharges in automatic daylight film processor systems. A simple, cost-effective solution. Technical note.

Automatic daylight film processors may produce heavy electrostatic discharges on radiographic films when using other brands of film than those produced by the processor manufacturer. This is a disadvantage, as it can eliminate competition when purchasing film. By 1. earthing the processor feeder rollers, 2. coating them with aluminium paint, and 3. isolating the wheels with a conventional insulating spray for electric cables, the artifacts vanish, and various brands of film can be used.