An evaluation of genotyping by sequencing (GBS) to map the <em>Breviaristatum-e (ari-e)</em> locus in cultivated barley

ABSTRACT: We explored the use of genotyping by sequencing (GBS) on a recombinant inbred line population (GPMx) derived from a cross between the two-rowed barley cultivar ‘Golden Promise’ (ari-e.GP/Vrs1) and the six-rowed cultivar ‘Morex’ (Ari-e/vrs1) to map plant height. We identified three Quantitative Trait Loci (QTL), the first in a region encompassing the spike architecture gene Vrs1 on chromosome 2H, the second in an uncharacterised centromeric region on chromosome 3H, and the third in a region of chromosome 5H coinciding with the previously described dwarfing gene Breviaristatum-e (Ari-e). BACKGROUND: Barley cultivars in North-western Europe largely contain either of two dwarfing genes; Denso on chromosome 3H, a presumed ortholog of the rice green revolution gene OsSd1, or Breviaristatum-e (ari-e) on chromosome 5H. A recessive mutant allele of the latter gene, ari-e.GP, was introduced into cultivation via the cv. ‘Golden Promise’ that was a favourite of the Scottish malt whisky industry for many years and is still used in agriculture today. RESULTS: Using GBS mapping data and phenotypic measurements we show that ari-e.GP maps to a small genetic interval on chromosome 5H and that alternative alleles at a region encompassing Vrs1 on 2H along with a region on chromosome 3H also influence plant height. The location of Ari-e is supported by analysis of near-isogenic lines containing different ari-e alleles. We explored use of the GBS to populate the region with sequence contigs from the recently released physically and genetically integrated barley genome sequence assembly as a step towards Ari-e gene identification. CONCLUSIONS: GBS was an effective and relatively low-cost approach to rapidly construct a genetic map of the GPMx population that was suitable for genetic analysis of row type and height traits, allowing us to precisely position ari-e.GP on chromosome 5H. Mapping resolution was lower than we anticipated. We found the GBS data more complex to analyse than other data types but it did directly provide linked SNP markers for subsequent higher resolution genetic analysis

Quantitative trait loci associated with different polar metabolites in perennial ryegrass - providing scope for breeding towards increasing certain polar metabolites

peer-reviewedBackground Recent advances in the mapping of biochemical traits have been reported in Lolium perenne. Although the mapped traits, including individual sugars and fatty acids, contribute greatly towards ruminant productivity, organic acids and amino acids have been largely understudied despite their influence on the ruminal microbiome. Results In this study, we used a targeted gas-chromatography mass spectrometry (GC-MS) approach to profile the levels of 25 polar metabolites from different classes (sugars, amino acids, phenolic acids, organic acids and other nitrogen-containing compounds) present in a L. perenne F2 population consisting of 325 individuals. A quantitative trait (QTL) mapping approach was applied and successfully identified QTLs regulating seven of those polar metabolites (L-serine, L-leucine, glucose, fructose, myo-inositol, citric acid and 2, 3-hydroxypropanoic acid).Two QTL mapping approaches were carried out using SNP markers on about half of the population only and an imputation approach using SNP and DArT markers on the entire population. The imputation approach confirmed the four QTLs found in the SNP-only analysis and identified a further seven QTLs. Conclusions These results highlight the potential of utilising molecular assisted breeding in perennial ryegrass to modulate a range of biochemical quality traits with downstream effects in livestock productivity and ruminal digestion.This study was financed through a Research Stimulus Fund Grant by the Irish Department of Agriculture, Fisheries and Marine (RSF 06–346). AF, CH and DS acknowledge support from The Scottish Government’s Rural and Environment Science and Analytical Services Division

Identifying spring barley cultivars with differential response to tillage

Cultivars and some cultivar mixtures of spring barley were grown under inversion and non-inversion tillage conditions for three or four years and assessed for disease and yield in order to obtain genotypes that can be used to determine the mechanisms of cultivation adaptation. In general, the higher-yielding cultivars under inversion tillage conditions gave lower yields under non-inversion tillage, whereas low-yielding older cultivars showed relatively smaller reductions in yield under non-inversion tillage. A few cultivars showed preferential yield performance for either inversion or non-inversion tillage and this was irrespective of their overall yield performance. There was no pedigree or breeding programme link between these cultivars and no above-ground gross morphological trait observed was associated with tillage adaptation. Root hairs may contribute to inversion tillage adaptation as a root hair absence mutant was associated with non-inversion adaptation and it is likely that other root-associated traits are responsible also for tillage adaptation. There was no overall cultivar or tillage interaction with rhynchosporium symptoms but a differential tillage interaction may occur in individual years. We have identified clearly contrasting cultivars and tested their across-season robustness with respect to tillage treatment for further detailed mechanistic studies and identification of tillage adaptation traits

Development and Application of a Simple Plaque Assay for the Human Malaria Parasite Plasmodium falciparum.

Malaria is caused by an obligate intracellular protozoan parasite that replicates within and destroys erythrocytes. Asexual blood stages of the causative agent of the most virulent form of human malaria, Plasmodium falciparum, can be cultivated indefinitely in vitro in human erythrocytes, facilitating experimental analysis of parasite cell biology, biochemistry and genetics. However, efforts to improve understanding of the basic biology of this important pathogen and to develop urgently required new antimalarial drugs and vaccines, suffer from a paucity of basic research tools. This includes a simple means of quantifying the effects of drugs, antibodies and gene modifications on parasite fitness and replication rates. Here we describe the development and validation of an extremely simple, robust plaque assay that can be used to visualise parasite replication and resulting host erythrocyte destruction at the level of clonal parasite populations. We demonstrate applications of the plaque assay by using it for the phenotypic characterisation of two P. falciparum conditional mutants displaying reduced fitness in vitro

Phloem connectivity and transport are not involved in mature plant resistance (MPR) to Potato Virus Y in different potato cultivars, and MPR does not protect tubers from recombinant strains of the virus

This research was funded by UK Research and Innovation (BBSRC Grant # BB/L011840/1; Ecology and Epidemiology of Infectious Diseases), by the Scottish Government's Rural and Environment Science and Analytical Services (RESAS) Division, and by the James Hutton Institute.The aims of this study were: i) to investigate mature plant resistance (MPR) against four strains of Potato virus Y (PVYO, PVYN, PVYNTN and PVYN−Wi) in potato cultivars that differ in maturity (e.g. early or maincrop) at different developmental stages, and ii) to determine whether phloem translocation of photoassimilates at different stages including the source-sink transition influences MPR. The data showed that MPR was functional by the flowering stage in all cultivars, and that the host-pathogen interaction is highly complex, with all three variables (potato cultivar, virus strain and developmental stage of infection) having a significant effect on the outcome. However, virus strain was the most important factor, and MPR was less effective in protecting tubers from recombinant virus strains (PVYNTN and PVYN−Wi). Development of MPR was unrelated to foliar phloem connectivity, which was observed at all developmental stages, but a switch from symplastic to apoplastic phloem unloading early in tuber development may be involved in the prevention of tuber infections with PVYO. Recombinant virus strains were more infectious than parental strains and PVYNTN has a more effective silencing suppressor than PVYO, another factor that may contribute to the efficiency of MPR. The resistance conferred by MPR against PVYO or PVYN may be associated with or enhanced by the presence of the corresponding strain-specific HR resistance gene in the cultivar.Publisher PDFPeer reviewe

Identification, utilisation and mapping of novel transcriptome-based markers from blackcurrant (Ribes nigrum)

<p>Abstract</p> <p>Background</p> <p>Deep-level second generation sequencing (2GS) technologies are now being applied to non-model species as a viable and favourable alternative to Sanger sequencing. Large-scale SNP discovery was undertaken in blackcurrant (<it>Ribes nigrum </it>L.) using transcriptome-based 2GS 454 sequencing on the parental genotypes of a reference mapping population, to generate large numbers of novel markers for the construction of a high-density linkage map.</p> <p>Results</p> <p>Over 700,000 reads were produced, from which a total of 7,000 SNPs were found. A subset of polymorphic SNPs was selected to develop a 384-SNP OPA assay using the Illumina BeadXpress platform. Additionally, the data enabled identification of 3,000 novel EST-SSRs. The selected SNPs and SSRs were validated across diverse <it>Ribes </it>germplasm, including mapping populations and other selected <it>Ribes </it>species.</p> <p>SNP-based maps were developed from two blackcurrant mapping populations, incorporating 48% and 27% of assayed SNPs respectively. A relatively high proportion of visually monomorphic SNPs were investigated further by quantitative trait mapping of theta score outputs from BeadStudio analysis, and this enabled additional SNPs to be placed on the two maps.</p> <p>Conclusions</p> <p>The use of 2GS technology for the development of markers is superior to previously described methods, in both numbers of markers and biological informativeness of those markers. Whilst the numbers of reads and assembled contigs were comparable to similar sized studies of other non-model species, here a high proportion of novel genes were discovered across a wide range of putative function and localisation. The potential utility of markers developed using the 2GS approach in downstream breeding applications is discussed.</p

TetraploidSNPMap: Software for Linkage Analysis and QTL Mapping in Autotetraploid Populations Using SNP Dosage Data

An earlier software application of ours, TetraploidMap for Windows, enabled linkage analysis and quantitative trait locus interval mapping to be carried out in an experimental cross of an autotetraploid species, using both dominant markers such as amplified fragment length polymorphisms and codominant markers such as simple sequence repeats. The size was limited to 800 markers, and quantitative trait locus mapping was conducted for each parent separately due to the difficulties in obtaining a reliable consensus map for the 2 parents. Modern genotyping technologies now give rise to datasets of thousands of single nucleotide polymorphisms, and these can be scored in autotetraploid species as single nucleotide polymorphism dosages, distinguishing among the heterozygotes AAAB, AABB, and ABBB, rather than simply using the presence or absence of an allele. The dosage data is more informative about recombination and leads to higher density linkage maps. The current program, TetraploidSNPMap, makes full use of the dosage data, and has new facilities for displaying the clustering of single nucleotide polymorphisms, rapid ordering of large numbers of single nucleotide polymorphisms using a multidimensional scaling analysis, and phase calling. It also has new routines for quantitative trait locus mapping based on a hidden Markov model, which use the dosage data to model the effects of alleles from both parents simultaneously. A Windows-based interface facilitates data entry and exploration. It is distributed with a detailed user guide. TetraploidSNPMap is freely available from our GitHub repository

Plant exudates may stabilize or weaken soil depending on species, origin and time

We hypothesized that plant exudates could either gel or disperse soil depending on their chemical characteristics. Barley (Hordeum vulgare L. cv. Optic) and maize (Zea mays L.cv. Freya) root exudates were collected using an aerated hydroponic method and compared to chia (Salvia hispanica L.) seed exudate, a commonly used root exudate analogue. Sandy loam soil passed through a 500-μm mesh was treated with each exudate at a concentrationof 4.6 mg exudate g-1 dry soil. Two sets of soil samples were prepared, One set of treated soil samples was maintained at 4oC to suppress microbial processes. To characterize the effect of decomposition, the second set of samples was incubated at 16C for 2 weeks at – 30 kPa matric potential. Gas chromatography–mass spectrometry (GC–MS) analysis of the exudates found that barley had the largest organic acid content and chia the largest content of sugars (polysaccharide-derived or free), and maize was in between barley and chia. Yield stress of amended soil samples was measured by an oscillatory strain sweep test with a cone plate rheometer. When microbial decomposition was suppressed at 4oC, yield stress increased 20-fold for chia seed exudate and two-fold for maize root exudate compared to the control, whereas for barley root exudate it decreased to half. The yield stress after 2 weeks of incubation compared to soil with suppressed microbial decomposition increased by 85% for barley root exudate, but for chia and maize it decreased to by 87% and 54%, respectively. Barley root exudation might therefore disperse soil and this could facilitate nutrient release. The maize root and chia seed exudates gelled soil, which could create a more stable soil structure around roots or seeds

Identification of barley genetic regions influencing plant-microbe interactions and carbon cycling in soil

Purpose: Rhizodeposition shapes soil microbial communities that perform important processes such as soil C mineralization, but we have limited understanding of the plant genetic regions influencing soil microbes. Here, barley chromosome regions affecting soil microbial biomass-C (MBC), dissolved organic-C (DOC) and root biomass were characterised. Methods: A quantitative trait loci analysis approach was applied to identify barley chromosome regions affecting soil MBC, soil DOC and root biomass. This was done using barley Recombinant Chromosome Substitution Lines (RCSLs) developed with a wild accession (Caesarea 26-24) as a donor parent and an elite cultivar (Harrington) as recipient parent. Results: Significant differences in root-derived MBC and DOC and root biomass among these RCSLs were observed. Analysis of variance using single nucleotide polymorphisms genotype classes revealed 16 chromosome regions influencing root-derived MBC and DOC. Of these chromosome regions, five on chromosomes 2H, 3H and 7H were highly significant and two on chromosome 3H influenced both root-derived MBC and DOC. Potential candidate genes influencing root-derived MBC and DOC concentrations in soil were identified. Conclusion: The present findings provide new insights into the barley genetic influence on soil microbial communities. Further work to verify these barley chromosome regions and candidate genes could promote marker assisted selection and breeding of barley varieties that are able to more effectively shape soil microbes and soil processes via rhizodeposition, supporting sustainable crop production systems