Preparation of Barley Pollen Mother Cells for Confocal and Super Resolution Microscopy

Recombination (crossover) drives the release of genetic diversity in plant breeding programs. However, in barley, recombination is skewed toward the telomeric ends of its seven chromosomes, restricting the re-assortment of about 30% of the genes located in the centromeric regions of its large 5.1 Gb genome. A better understanding of meiosis and recombination could provide ways of modulating crossover distribution and frequency in barley as well as in other grasses, including wheat. While most research on recombination has been carried out in the model plant Arabidopsis thaliana, recent studies in barley (Hordeum Vulgare) have provided new insights into the control of crossing over in large genome species. A major achievement in these studies has been the use of cytological procedures to follow meiotic events. This protocol provides detailed practical steps required to perform immunostaining of barley meiocytes (pollen mother cells) for confocal or structured illumination microscopy.</p

Isolation and fine mapping of Rps6: An intermediate host resistance gene in barley to wheat stripe rust

A plant may be considered a nonhost of a pathogen if all known genotypes of a plant species are resistant to all known isolates of a pathogen species. However, if a small number of genotypes are susceptible to some known isolates of a pathogen species this plant maybe considered an intermediate host. Barley (Hordeum vulgare) is an intermediate host for Puccinia striiformis f. sp. tritici (Pst), the causal agent of wheat stripe rust. We wanted to understand the genetic architecture underlying resistance to Pst and to determine whether any overlap exists with resistance to the host pathogen, Puccinia striiformis f. sp. hordei (Psh). We mapped Pst resistance to chromosome 7H and show that host and intermediate host resistance is genetically uncoupled. Therefore, we designate this resistance locus Rps6. We used phenotypic and genotypic selection on F2:3 families to isolate Rps6 and fine mapped the locus to a 0.1 cM region. Anchoring of the Rps6 locus to the barley physical map placed the region on two adjacent fingerprinted contigs. Efforts are now underway to sequence the minimal tiling path and to delimit the physical region harbouring Rps6. This will facilitate additional marker development and permit identification of candidate genes in the region

An investigation of causes of false positive single nucleotide polymorphisms using simulated reads from a small eukaryote genome

Background: Single Nucleotide Polymorphisms (SNPs) are widely used molecular markers, and their use has increased massively since the inception of Next Generation Sequencing (NGS) technologies, which allow detection of large numbers of SNPs at low cost. However, both NGS data and their analysis are error-prone, which can lead to the generation of false positive (FP) SNPs. We explored the relationship between FP SNPs and seven factors involved in mapping-based variant calling - quality of the reference sequence, read length, choice of mapper and variant caller, mapping stringency and filtering of SNPs by read mapping quality and read depth. This resulted in 576 possible factor level combinations. We used error- and variant-free simulated reads to ensure that every SNP found was indeed a false positive. Results: The variation in the number of FP SNPs generated ranged from 0 to 36,621 for the 120 million base pairs (Mbp) genome. All of the experimental factors tested had statistically significant effects on the number of FP SNPs generated and there was a considerable amount of interaction between the different factors. Using a fragmented reference sequence led to a dramatic increase in the number of FP SNPs generated, as did relaxed read mapping and a lack of SNP filtering. The choice of reference assembler, mapper and variant caller also significantly affected the outcome. The effect of read length was more complex and suggests a possible interaction between mapping specificity and the potential for contributing more false positives as read length increases. Conclusions: The choice of tools and parameters involved in variant calling can have a dramatic effect on the number of FP SNPs produced, with particularly poor combinations of software and/or parameter settings yielding tens of thousands in this experiment. Between-factor interactions make simple recommendations difficult for a SNP discovery pipeline but the quality of the reference sequence is clearly of paramount importance. Our findings are also a stark reminder that it can be unwise to use the relaxed mismatch settings provided as defaults by some read mappers when reads are being mapped to a relatively unfinished reference sequence from e.g. a non-model organism in its early stages of genomic exploration

Haplotype based genotyping-by-sequencing in oat genome research

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Identification of manganese efficiency candidate genes in winter barley (Hordeum vulgare) using genome wide association mapping

BACKGROUND: Manganese (Mn) has several essential functions in plants, including a role as cofactor in the oxygen evolving complex (OEC) of photosystem II (PSII). Manganese deficiency is a major plant nutritional disorder in winter cereals resulting in significant yield reductions and winter kill in more severe cases. Among the winter cereals, genotypes of winter barley are known to differ considerably in tolerance to Mn deficiency, but the genes controlling the Mn deficiency trait remains elusive. RESULTS: Experiments were conducted using 248 barley varieties, cultivated in six distinct environments prone to induce Mn deficiency. High-throughput phenotyping for Mn deficiency was performed by chlorophyll a (Chl a) fluorescence analysis to quantify the quantum yield efficiency of PSII. High-throughput phenotyping in combination with ICP-OES based multi-element analyses allowed detection of marker-trait associations by genome wide association (GWA) mapping. Several key candidate genes were identified, including PSII subunit proteins, germin like proteins and Mn superoxide dismutase. The putative roles of the encoded proteins in Mn dependent metabolic processes are discussed. CONCLUSIONS: Fifty-four candidate genes were identified by Chl a fluorescence phenotyping and association genetics. Tolerance of plants to Mn deficiency, which is referred to as Mn efficiency, appeared to be a complex trait involving many genes. Moreover, the trait appeared to be highly dependent on the environmental conditions in field. This study provides the basis for an improved understanding of the parameters influencing Mn efficiency and is valuable in future plant breeding aiming at producing new varieties with improved tolerance to cultivation in soil prone to induce Mn deficiency. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-016-3129-9) contains supplementary material, which is available to authorized users

Genotyping-by-sequencing on the ion torrent platform in barley

The characterization of genetic polymorphism is a crucial step in both genetic studies and breeding programs. Genotyping-by-sequencing (GBS) constitutes one of the most attractive approaches for this purpose, especially in a genome as large as that of barley. The genome sequencing project undertaken by the International Barley Sequencing Consortium (IBSC) has produced a structured reference genome for the cultivar Morex [1] that can serve as an excellent resource for the analysis of GBS data. The genome assembly for this species [2] is thought to adequately capture the gene-rich portion of the genome (~80% of the entire genome). In this chapter, we describe the entire GBS process, from library preparation to the analysis of read data to produce a high-quality catalog of single nucleotide polymorphism (SNP) markers using the barley reference genome