PCR-based markers diagnostic for spring and winter seasonal growth habit in barley

Toward the ultimate goal of replacing field-based evaluation of seasonal growth habit, we describe the design and validation of a multiplex polymerase chain reaction assay diagnostic for allelic status at the barley (Hordeum vulgare ssp. vulgare L.) vernalization locus, VRN-H1 By assaying for the presence of all known insertion–deletion polymorphisms thought to be responsible for the difference between spring and winter alleles, this assay directly tests for the presence of functional polymorphism at VRN-H1 Four of the nine previously recognized VRN-H1 haplotypes (including both winter alleles) give unique profiles using this assay. The remaining five spring haplotypes share a single profile, indicative of function-altering deletions spanning, or adjacent to, the putative “vernalization critical” region of intron 1. When used in conjunction with a previously published PCR-based assay diagnostic for alleles at VRN-H2, it was possible to predict growth habit in all the 100 contemporary UK spring and winter lines analyzed in this study. This assay is likely to find application in instances when seasonal growth habit needs to be determined without the time and cost of phenotypic assessment and during marker-assisted selection using conventional and multicross population analysis.

Association mapping of partitioning loci in barley

Background: Association mapping, initially developed in human disease genetics, is now being applied to plant species. The model species Arabidopsis provided some of the first examples of association mapping in plants, identifying previously cloned flowering time genes, despite high population sub-structure. More recently, association genetics has been applied to barley, where breeding activity has resulted in a high degree of population sub-structure. A major genotypic division within barley is that between winter- and spring-sown varieties, which differ in their requirement for vernalization to promote subsequent flowering. To date, all attempts to validate association genetics in barley by identifying major flowering time loci that control vernalization requirement (VRN-H1 and VRN-H2) have failed. Here, we validate the use of association genetics in barley by identifying VRN-H1 and VRN-H2, despite their prominent role in determining population sub-structure. Results: By taking barley as a typical inbreeding crop, and seasonal growth habit as a major partitioning phenotype, we develop an association mapping approach which successfully identifies VRN-H1 and VRN-H2, the underlying loci largely responsible for this agronomic division. We find a combination of Structured Association followed by Genomic Control to correct for population structure and inflation of the test statistic, resolved significant associations only with VRN-H1 and the VRN-H2 candidate genes, as well as two genes closely linked to VRN-H1 (HvCSFs1 and HvPHYC). Conclusion: We show that, after employing appropriate statistical methods to correct for population sub-structure, the genome-wide partitioning effect of allelic status at VRN-H1 and VRN-H2 does not result in the high levels of spurious association expected to occur in highly structured samples. Furthermore, we demonstrate that both VRN-H1 and the candidate VRN-H2 genes can be identified using association mapping. Discrimination between intragenic VRN-H1 markers was achieved, indicating that candidate causative polymorphisms may be discerned and prioritised within a larger set of positive associations. This proof of concept study demonstrates the feasibility of association mapping in barley, even within highly structured populations. A major advantage of this method is that it does not require large numbers of genome-wide markers, and is therefore suitable for fine mapping and candidate gene evaluation, especially in species for which large numbers of genetic markers are either unavailable or too costly.

Molecular barley breeding

Breeding progress in barley yield in the UK is being sustained at a rate in the order of 1% per annum against a background of declining seed sales. Commercial barley breeders are largely concentrating upon the elite local gene pool but with genotypic evidence suggesting that there is still considerable variation between current recommended cultivars, even those produced as half-sibs by the same breeder. Marker Assisted Selection (MAS) protocols could be substituted for conventional selection for a number of major-gene targets but, in the majority of cases, conventional selection is more resource efficient. Results from current QTL mapping studies have not yet identified sufficiently robust and validated targets for UK barley breeders to adopt MAS to assist in the selection of complex traits such as yield and malting quality. Results from multiple population mapping amongst the elite gene pool being utilised by breeders and from association studies of elite germplasm tested as part of the UK recommended list trial process do, however, show some promise.

Haplotype analysis of vernalization loci in European barley germplasm reveals novel VRN-H1 alleles and a predominant winter VRN-H1/VRN-H2 multi-locus haplotype

In barley, variation in the requirement for vernalization (an extended period of low temperature before flowering can occur) is determined by the VRN-H1, -H2 and -H3 loci. In European cultivated germplasm, most variation in vernalization requirement is accounted for by alleles at VRN-H1 and VRN-H2 only, but the range of allelic variation is largely unexplored. Here we characterise VRN-H1 and VRN-H2 haplotypes in 429 varieties representing a large portion of the acreage sown to barley in Western Europe over the last 60 years. Analysis of genotype, intron I sequencing data and growth habit tests identified three novel VRN-H1 alleles and determined the most frequent VRN-H1 intron I rearrangements. Combined analysis of VRN-H1 and VRN-H2 alleles resulted in the classification of seventeen VRN-H1/VRN-H2 multi-locus haplotypes, three of which account for 79% of varieties. The molecular markers employed here represent powerful diagnostic tools for prediction of growth habit and assessment of varietal purity. These markers will also allow development of germplasm to test the behaviour of individual alleles with the aim of understanding the relationship between allelic variation and adaptation to specific agri-environments.

Integration of retrotransposons-based markers in a linkage map of barley

A deeper understanding of random markers is important if they are to be employed for a range of objectives. The sequence specific amplified polymorphism (S-SAP) technique is a powerful genetic analysis tool which exploits the high copy number of retrotransposon long terminal repeats (LTRs) in the plant genome. The distribution and inheritance of S-SAP bands in the barley genome was studied using the Steptoe × Morex (S × M) double haploid (DH) population. Six S-SAP primer combinations generated 98 polymorphic bands, and map positions were assigned to all but one band. Eight putative co-dominant loci were detected, representing 16 of the mapped markers. Thus at least 81 of the mapped S-SAP loci were dominant. The markers were distributed along all of the seven chromosomes and a tendency to cluster was observed. The distribution of S-SAP markers over the barley genome concurred with the knowledge of the high copy number of retrotransposons in plants. This experiment has demonstrated the potential for the S-SAP technique to be applied in a range of analyses such as genetic fingerprinting, marker assisted breeding, biodiversity assessment and phylogenetic analyses.

Testing for unit roots in bounded time series

Many key economic and financial series are bounded either by construction or through policy controls. Conventional unit root tests are potentially unreliable in the presence of bounds, since they tend to over-reject the null hypothesis of a unit root, even asymptotically. So far, very little work has been undertaken to develop unit root tests which can be applied to bounded time series. In this paper we address this gap in the literature by proposing unit root tests which are valid in the presence of bounds. We present new augmented Dickey–Fuller type tests as well as new versions of the modified ‘M’ tests developed by Ng and Perron [Ng, S., Perron, P., 2001. LAG length selection and the construction of unit root tests with good size and power. Econometrica 69, 1519–1554] and demonstrate how these tests, combined with a simulation-based method to retrieve the relevant critical values, make it possible to control size asymptotically. A Monte Carlo study suggests that the proposed tests perform well in finite samples. Moreover, the tests outperform the Phillips–Perron type tests originally proposed in Cavaliere [Cavaliere, G., 2005. Limited time series with a unit root. Econometric Theory 21, 907–945]. An illustrative application to U.S. interest rate data is provided

Analysis of DNA polymorphism in ancient barley herbarium material: validation of the KASP SNP genotyping platform

The availability of crop specimens archived in herbaria and old seed collections represent valuable resources for the analysis of plant genetic diversity and crop domestication. The ability to extract ancient DNA (aDNA) from such samples has recently allowed molecular genetic investigations to be undertaken in ancient materials. While analyses of aDNA initially focused on the use of markers which occur in multiple copies such as the internal transcribed spacer region (ITS) within ribosomal DNA and those requiring amplification of short DNA regions of variable length such as simple sequence repeats (SSRs), emphasis is now moving towards the genotyping of single nucleotide polymorphisms (SNPs), traditionally undertaken in aDNA by Sanger sequencing. Here, using a panel of barley aDNA samples previously surveyed by Sanger sequencing for putative causative SNPs within the flowering-time gene PPD-H1, we assess the utility of the Kompetitive Allele Specific PCR (KASP) genotyping platform for aDNA analysis. We find KASP to out-perform Sanger sequencing in the genotyping of aDNA samples (78% versus 61% success, respectively), as well as being robust to contamination. The small template size (≥46 bp) and one-step, closed-tube amplification/genotyping process make this platform ideally suited to the genotypic analysis of aDNA, a process which is often hampered by template DNA degradation and sample cross-contamination. Such attributes, as well as its flexibility of use and relatively low cost, make KASP particularly relevant to the genetic analysis of aDNA samples. Furthermore, KASP provides a common platform for the genotyping and analysis of corresponding SNPs in ancient, landrace and modern plant materials. The extended haplotype analysis of PPD-H1 undertaken here (allelic variation at which is thought to be important for the spread of domestication and local adaptation) provides further resolution to the previously identified geographic cline of flowering-time allele distribution, illustrating how KASP can be used to aid genetic analyses of aDNA from plant species. We further demonstrate the utility of KASP by genotyping ten additional genetic markers diagnostic for morphological traits in barley, shedding light on the phenotypic traits, alleles and allele combinations present in these unviable ancient specimens, as well as their geographic distributions.

An optimized method for the extraction of bacterial mRNA from plant roots infected with Escherichia coli O157:H7

Analysis of microbial gene expression during host colonization provides valuable information on the nature of interaction, beneficial or pathogenic, and the adaptive processes involved. Isolation of bacterial mRNA for in planta analysis can be challenging where host nucleic acid may dominate the preparation, or inhibitory compounds affect downstream analysis, e.g., quantitative reverse transcriptase PCR (qPCR), microarray, or RNA-seq. The goal of this work was to optimize the isolation of bacterial mRNA of food-borne pathogens from living plants. Reported methods for recovery of phytopathogen-infected plant material, using hot phenol extraction and high concentration of bacterial inoculation or large amounts of infected tissues, were found to be inappropriate for plant roots inoculated with Escherichia coli O157:H7. The bacterial RNA yields were too low and increased plant material resulted in a dominance of plant RNA in the sample. To improve the yield of bacterial RNA and reduce the number of plants required, an optimized method was developed which combines bead beating with directed bacterial lysis using SDS and lysozyme. Inhibitory plant compounds, such as phenolics and polysaccharides, were counteracted with the addition of high-molecular-weight polyethylene glycol and hexadecyltrimethyl ammonium bromide. The new method increased the total yield of bacterial mRNA substantially and allowed assessment of gene expression by qPCR. This method can be applied to other bacterial species associated with plant roots, and also in the wider context of food safety.

Matching roots to their environment

Background Plants form the base of the terrestrial food chain and provide medicines, fuel, fibre and industrial materials to humans. Vascular land plants rely on their roots to acquire the water and mineral elements necessary for their survival in nature or their yield and nutritional quality in agriculture. Major biogeochemical fluxes of all elements occur through plant roots, and the roots of agricultural crops have a significant role to play in soil sustainability, carbon sequestration, reducing emissions of greenhouse gasses, and in preventing the eutrophication of water bodies associated with the application of mineral fertilisers. ● Scope This article provides the context for a Special Issue of Annals of Botany on ‘Matching Roots to Their Environment’. It first examines how land plants and their roots evolved, describes how the ecology of roots and their rhizospheres contributes to the acquisition of soil resources, and discusses the influence of plant roots on biogeochemical cycles. It then describes the role of roots in overcoming the constraints to crop production imposed by hostile or infertile soils, illustrates root phenotypes that improve the acquisition of mineral elements and water, and discusses high-throughput methods to screen for these traits in the laboratory, glasshouse and field. Finally, it considers whether knowledge of adaptations improving the acquisition of resources in natural environments can be used to develop root systems for sustainable agriculture in the future.

Root elongation rate is correlated with the length of the bare root apex of maize and lupin roots despite contrasting responses of root growth to compact and dry soils

Background We investigated interacting effects of matric potential and soil strength on root elongation of maize and lupin, and relations between root elongation rates and the length of bare (hairless) root apex. Methods Root elongation rates and the length of bare root apexwere determined formaize and lupin seedlings in sandy loam soil of various matric potentials (−0.01 to −1.6 MPa) and bulk densities (0.9 to 1.5 Mg m−3). Results Root elongation rates slowed with both decreasing matric potential and increasing penetrometer resistance. Root elongation of maize slowed to 10 % of the unimpeded rate when penetrometer resistance increased to 2 MPa, whereas lupin elongated at about 40 % of the unimpeded rate. Maize root elongation rate was more sensitive to changes in matric potential in loosely packed soil (penetrometer resistances <1 MPa) than lupin. Despite these differing responses, root elongation rate of both species was linearly correlated with length of the bare root apex (r2 0.69 to 0.97). Conclusion Maize root elongation was more sensitive to changes in matric potential and mechanical impedance than lupin. Robust linear relationships between elongation rate and length of bare apex suggest good potential for estimating root elongation rates for excavated roots.

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.

Interactions between the powdery mildew effector BEC1054 and barley proteins identify candidate host targets

There are over 500 candidate secreted effector proteins (CSEPs) or Blumeria effector candidates (BECs) specific to the barley powdery mildew pathogen Blumeria graminis f.sp. hordei. The CSEP/BEC proteins are expressed and predicted to be secreted by biotrophic feeding structures called haustoria. Eight BECs are required for the formation of functional haustoria. These include the RNase-like effector BEC1054 (synonym CSEP0064). In order to identify host proteins targeted by BEC1054, recombinant BEC1054 was expressed in E. coli, solubilized, and used in pull-down assays from barley protein extracts. Many putative interactors were identified by LC-MS/MS after subtraction of unspecific binders in negative controls. Therefore, a directed yeast-2-hybrid assay, developed to measure the effectiveness of the interactions in yeast, was used to validate putative interactors. We conclude that BEC1054 may target several host proteins, including a glutathione-S-transferase, a malate dehydrogenase, and a pathogen-related-5 protein isoform, indicating a possible role for BEC1054 in compromising well-known key players of defense and response to pathogens. In addition, BEC1054 interacts with an elongation factor 1 gamma. This study already suggests that BEC1054 plays a central role in barley powdery mildew virulence by acting at several levels.

Biofilm on the apical region of roots in primary teeth with vital and necrotic pulps with or without radiographically evident apical pathosis

Aim To evaluate, by scanning electron microscopy (SEM), the presence of biofilms on the external surfaces of the apical third of roots of human primary teeth with vital or necrotic pulps with and without radiographically evident periradicular pathosis. Methodology Eighteen teeth were selected: group I - normal pulp (n = 5), group II - pulp necrosis without radiographic evidence of periapical pathosis (n = 7) and group III - pulp necrosis with well-defined radiographic periapical pathosis (n = 6). After extraction, the teeth were washed with saline and immersed in 0.03 g mL(-1) trypsin solution for 20 min. The teeth were then washed in sodium cacodilate buffer and stored in receptacles containing modified Karnovsky solution. The teeth were sectioned, dehydrated in an ethanol series, critical-point dried with CO(2), sputter coated with gold and the external root surface in the apical third examined by SEM. Results In the teeth of groups I and II, the apical root surfaces were covered by collagen fibres, with no evidence of bacteria (100%). In the teeth of group III, the root apices had no collagen fibres but revealed resorptive areas containing microorganisms (cocci, bacilli, filaments and spirochetes) in all cases (100%). Conclusion Microorganisms organized as biofilms on the external root surface (extraradicular infection) were detected in primary teeth with pulp necrosis and radiographically visible periapical pathosis.

Abscisic acid and auxin accumulation in Catasetum fimbriatum roots growing in vitro with high sucrose and mannitol content

Endogenous contents of indolyl-3-acetic acid (IAA) and abscisic acid (ABA) were quantified in excised roots of Catasetum fimbriatum (Orchidaceae) cultured in vitro on solidified Vacin and Went medium with 1, 2, 4, 6, 8 and 10 % sucrose, as well as 2 % sucrose plus mannitol. Maximum root growth was observed in media with 4 % sucrose and 2 % sucrose plus 2.2 % mannitol, suggesting that a moderate water or osmotic stress promotes orchid root growth. Contents of both ABA and IAA increased in parallel to increasing sucrose concentration and a correlation between root elongation and the ABA/IAA ratio was observed. Incubating isolated C. fimbriatum roots with radiolabeled tryptophan, we showed an accumulation of IAA and its conjugates.