Use of genetic markers for the detection of off-types for DUS phenotypic traits in the inbreeding crop, barley

Detection of crop off-types is of interest for multiple uses, including the assessment of uniformity for new plant variety applications during distinctness, uniformity and stability (DUS) testing for the awarding of plant breeders’ rights (PBR). Here, we investigate whether genetic markers, in this case Kompetitive Allele-Specific PCR (KASP), can be used for the identification off-types for phenotypes assessed for DUS in the inbreeding cereal crop, barley (Hordeum vulgare). To demonstrate proof of principle, KASP markers diagnostic for phenotypic expression of nine DUS phenotypes, and DNA from two barley varieties (‘Pelican’ and ‘Felicie’) carrying contrasting alleles at each marker were used. We found that for the majority of markers, it was possible to robustly call alleles down to template DNA concentrations of 2 ng, but not ≤ 0.2 ng. When used in mixtures of DNA consisting of ‘Felicie’ DNA spiked with different concentrations of ‘Pelican’ DNA, robust allele calling was possible in DNA mixtures down to 18 ng:2 ng. Collectively, this demonstrates that where diagnostic markers are available, molecular identification of a single off-type for a given DUS trait within a bulk of ten individuals should be possible. We validated this assumption, with all of the diagnostic genetic markers investigated found to robustly detect DUS off-types at a frequency of 10% in DNA extracted from tissue collected from pools of 10 individuals. Ultimately, this work demonstrates that, where diagnostic polymorphisms are known for DUS traits, KASP markers should be able to robustly detect off-types or cross-contamination within DNA samples from a diploid inbred species down to 10%. While just two varieties that contrasted for the eight DUS targeted were investigated in this study, as the markers used are diagnostic for their relevant phenotype (or a proportion of the variation observed for that phenotype), in theory the approach should be valid for any variety studied— although the introduction of novel alleles via spontaneous mutation or more exotic germplasm pools may mean that marker sets would need to be periodically added to or updated. However, we nevertheless demonstrate the principle that, for a subset of DUS traits, molecular markers can now be robustly used as a tool towards determining all three components of the DUS testing process in barley. These results are relevant for the assessment of varietal uniformity by crop breeders, crop testing authorities and germplasm maintenance, as well as highlighting the potential use of bulk samples rather than individual plant samples for assessment of distinctness by molecular methods

CWITools: A Python3 Data Analysis Pipeline for the Cosmic Web Imager Instruments

The Palomar Cosmic Web Imager (PCWI) and Keck Cosmic Web Imager (KCWI) are integral-field spectrographs on the Hale 5m telescope at Palomar Observatory and the Keck-2 10m telescope at W. M. Keck Observatory, respectively. In recent years, these instruments have been increasingly used to conduct survey work; in particular focused on the circumgalactic and intergalactic media at high redshift. Extracting faint signals from three-dimensional IFU data is a complex task which can become prohibitively difficult for large samples without the proper tools. We present CWITools, a package written in Python3 for the analysis of PCWI and KCWI data. CWITools is designed to provide a pipeline between the output of the standard instrument data reduction pipelines and scientific products such as surface brightness maps, spectra, velocity maps, as well as a wide array of associated models and measurements. While the package is designed specifically for PCWI and KCWI data, the package is open source and can be adapted to accommodate any three-dimensional integral field spectroscopy data. Here, we describe this pipeline, the methodology behind individual steps and provide example applications

A framework for gene mapping in wheat demonstrated using the Yr7 yellow rust resistance gene

We used three approaches to map the yellow rust resistance gene Yr7 and identify associated SNPs in wheat. First, we used a traditional QTL mapping approach using a double haploid (DH) population and mapped Yr7 to a low-recombination region of chromosome 2B. To fine map the QTL, we then used an association mapping panel. Both populations were SNP array genotyped allowing alignment of QTL and genome-wide association scans based on common segregating SNPs. Analysis of the association panel spanning the QTL interval, narrowed the interval down to a single haplotype block. Finally, we used mapping-by-sequencing of resistant and susceptible DH bulks to identify a candidate gene in the interval showing high homology to a previously suggested Yr7 candidate and to populate the Yr7 interval with a higher density of polymorphisms. We highlight the power of combining mapping-by-sequencing, delivering a complete list of gene-based segregating polymorphisms in the interval with the high recombination, low LD precision of the association mapping panel. Our mapping-by-sequencing methodology is applicable to any trait and our results validate the approach in wheat, where with a near complete reference genome sequence, we are able to define a small interval containing the causative gene

Seed storage proteins of faba bean (Vicia faba L): current status and prospects for genetic improvement

Faba bean (Vicia faba L.) is one of the foremost candidate crops for simultaneously increasing both sustainability and global supply of plant protein. On a dry matter basis, its seeds contain about 29% protein of which more than 80% consists of globulin storage proteins (vicilin and legumin). However, to achieve optimum utilization of this crop for human and animal nutrition, both protein content and quality have to be improved. Though initial investigations on the heritability of these traits indicated the possibility for genetic improvement, little has been achieved so far, partly due to the lack of genetic information coupled with the complex relationship between protein content and grain yield. This review reports on the current knowledge on Vicia faba seed storage proteins, their structure, composition, and genetic control, and highlights key areas for further improvement of the content and composition of Vicia faba seed storage proteins on the basis of recent advances in Vicia faba genome knowledge and genetic tools

Genetic variation at flowering time loci in wild and cultivated barley

The worldwide spread of barley cultivation required adaptation to agricultural environments far distant from those found in its centre of domestication. An important component of this adaptation is the timing of flowering, achieved predominantly in response to day length and temperature. Here, we use a collection of cultivars, landraces and wild barley accessions to investigate the origins and distribution of allelic diversity at four major flowering time loci, mutations at which have been under selection during the spread of barley cultivation into Europe. Our findings suggest that while mutant alleles at the PPD-H1 and PPD-H2 photoperiod loci occurred pre-domestication, the mutant vernalization non-responsive alleles utilized in landraces and cultivars at the VRN-H1 and VRN-H2 loci occurred post-domestication. The transition from wild to cultivated barley is associated with a doubling in the number of observed multi-locus flowering-time haplotypes, suggesting that the resulting phenotypic variation has aided adaptation to cultivation in the diverse ecogeographic locations encountered. Despite the importance of early-flowering alleles during the domestication of barley in Europe, we show that novel VRN alleles associated with early flowering in wild barley have been lost in domesticates, highlighting the potential of wild germplasm as a source of novel allelic variation for agronomic traits

The FLASHES Survey I: Integral Field Spectroscopy of the CGM around 48 z=2.3−3.1z=2.3-3.1 QSOs

We present the pilot study component of the Fluorescent Lyman-Alpha Structures in High-z Environments (FLASHES) Survey; the largest integral-field spectroscopy survey to date of the circumgalactic medium at $z=2.3-3.1$. We observed 48 quasar fields between 2015 and 2018 with the Palomar Cosmic Web Imager (Matuszewski et al. 2010). Extended HI Lyman-$\mathrm{\alpha}$ emission is discovered around 42/48 of the observed quasars, ranging in projected, flux-weighted radius from 21-71 proper kiloparsecs (pkpc), with 26 nebulae exceeding $100\mathrm{~pkpc}$ in effective diameter. The circularly averaged surface brightness radial profile peaks at a maximum of $\mathrm{1\times 10^{-17}~erg~s^{-1}~cm^{-2}~arcsec^{-2}}$ ($2\times10^{-15}~\mathrm{erg~s^{-1}~cm^{-2}~arcsec^{-2}}$ adjusted for cosmological dimming) and luminosities range from $1.9\times10^{43}~\mathrm{erg~s^{-1}}$ to $-14.1\times10^{43}~\mathrm{erg~s^{-1}}$. The emission appears to have a highly eccentric morphology and a maximum covering factor of $50\%$ ($60\%$ for giant nebulae). On average, the nebular spectra are red-shifted with respect to both the systemic redshift and Ly$\alpha$ peak of the quasar spectrum. The integrated spectra of the nebulae mostly have single or double-peaked line shapes with global dispersions ranging from $167~\mathrm{km~s^{-1}}$ to $690~\mathrm{km~s^{-1}}$, though the individual (Gaussian) components of lines with complex shapes mostly appear to have dispersions $\leq 400$ $\mathrm{km~s^{-1}}$, and the flux-weighted velocity centroids of the lines vary by thousands of $\mathrm{km~s^{-1}}$ with respect to the systemic QSO redshifts. Finally, the root-mean-square velocities of the nebulae are found to be consistent with gravitational motions expected in dark matter halos of mass $\mathrm{M_h \simeq10^{12.5} M_\odot}$. We compare these results to existing surveys at both higher and lower redshift

Elevated temperature drives a shift from selfing to outcrossing in the insect pollinated legume, faba bean (Vicia faba)

Climate change can threaten the reproductive success of plants, both directly, through physiological damage during increasingly extreme weather events, and indirectly, through disruption of plant–pollinator interactions. To explore how plant–pollinator interactions are modified by extreme weather, we exposed faba bean (Vicia faba) plants to elevated temperature for 5 d during flowering, simulating a heatwave. We then moved the plants to flight cages with either bumblebees or no pollinators, or to two field sites, where plants were enclosed in mesh bags or pollinated by wild insect communities. We used a morphological marker to quantify pollen movement between experimental plants. There was a substantial increase in the level of outcrossing by insect pollinators following heat stress. Proportion outcrossed seed increased from 17 % at control temperature to 33 % following heat stress in the flight cages, and from 31 % to 80 % at one field site, but not at the other (33 % to 32 %). Abiotic stress can dramatically shift the relative contributions of cross- and self-pollination to reproduction in an insect pollinated plant. The resulting increases in gene flow have broad implications for genetic diversity and functioning of ecosystems, and may increase resilience by accelerating the selection of more stress-tolerant genotypes

Molecular and phenotypic characterization of the alternative seasonal growth habit and flowering time in barley (Hordeum vulgare ssp. vulgare L.)

Barley can be classified into three major agronomic types, based on its seasonal growth habit (SGH): spring, winter and alternative. Winter varieties require exposure to vernalization to promote subsequent flowering and are autumn-sown. Spring varieties proceed to flowering in the absence of vernalization and are sown in the spring. The ‘alternative’ (also known as ‘facultative’) SGH is only loosely defined and can be sown in autumn or spring. Here, we investigate the molecular genetic basis of alternative barley. Analysis of the major barley vernalization (VRN-H1, VRN-H2) and photoperiod (PPD-H1, PPD-H2) response genes in a collection of 386 varieties found alternative SGH to be characterized by specific allelic combinations. Spring varieties possessed spring loci at one or both of the vernalization response loci, combined with long-day non-responsive ppd-H1 alleles and wild-type alleles at the short-day photoperiod response locus, PPD-H2. Winter varieties possessed winter alleles at both vernalization loci, in combination with the mutant ppd-H2 allele conferring delayed flowering under short-day photoperiods. In contrast, all alternative varieties investigated possessed a single spring allele (either at VRN-H1 or at VRN-H2) combined with mutant ppd-H2 alleles. This allelic combination is found only in alternative types and is diagnostic for alternative SGH in the collection studied. Analysis of flowering time under controlled environment found alternative varieties flowered later than spring control lines, with the difference most pronounced under short-day photoperiods. This work provides genetic characterization of the alternative SGH phenotype, allowing precise manipulation of SGH and flowering time within breeding programmes, and provides the molecular tools for classification of all three SGH categories within national variety registration processes

Evaluation of Claviceps purpurea isolates on wheat reveals complex virulence and host susceptibility relationships

Ergot of cereals, caused by Claviceps purpurea, results in yield loss and downgrading of infested grain because of toxic alkaloids in the sclerotia. Resistant wheat genotypes are known, but their effectiveness against different C. purpurea isolates over geographic regions has not been studied. The objective of this study was to examine the pathogenic variability among isolates of C. purpurea on wheat lines differing in resistance. Under controlled environmental conditions, 41 single spore isolates of C. purpurea were obtained from Canadian and UK collections and inoculated onto a set of wheat genotypes composed of durum wheat lines ‘Melita’, ‘Kyle’ and 9260B-173A, and hexaploid spring wheat lines ‘Cadillac’, ‘Vista’, ‘Kenya Farmer’, ‘Lee’ and HY630. Honeydew production and weight of sclerotia produced per spike were assessed. There were significant differences among the wheat genotypes for overall reactions to the pathogen isolates, and among pathogen isolates for geographic origin and host origin. Twenty virulence phenotypes were identified using the honeydew production data, and 23 virulence phenotypes identified using the sclerotial weight data from the 41 isolates. The existence of different virulence phenotypes indicates that variability in virulence exists in populations of C. purpurea, and knowledge of virulence phenotypes is necessary to effectively breed for resistant commercial lines