Malcolm Macaulay

Direct comparison of levels of genetic variation among barley accessions detected by RFLPs, AFLPs, SSRs and RAPDs

Abstract RFLPs, AFLPs, RAPDs and SSRs were used to determine the genetic relationships among 18 cultivated barley accessions and the results compared to pedigree relationships where these were available. All of the approaches were able to uniquely fingerprint each of the accessions. The four assays differed in the amount of polymorphism detected. For example, all 13 SSR primers were polymorphic, with an average of 5.7 alleles per primer set, while nearly 54% of the fragments generated using AFLPs were monomorphic. The highest diversity index was observed for AFLPs (0.937) and the lowest for RFLP (0.322). Principal co-ordinate analysis (PCoA) clearly separated the spring types from the winter types using RFLP and AFLP data with the two-row winter types forming an intermediate group. Only a small group of spring types clustered together using SSR data with the two-row and six-row winter varieties more widely dispersed. Direct comparisons between genetic similarity (GS) estimates revealed by each of the assays were measured by a number of approaches. Spearman rank correlation ranked over 70% of the pairwise comparisons between AFLPs and RFLPs in the same order. SSRs had the lowest values when compared to the other three assays. These results are discussed in terms of the choice of appropriate technology for different aspects of germplasm evaluation.

Genome-wide association mapping to candidate polymorphism resolution in the unsequenced barley genome

Although commonplace in human disease genetics, genome-wide association (GWA) studies have only relatively recently been applied to plants. Using 32 phenotypes in the inbreeding crop barley, we report GWA mapping of 15 morphological traits across ∼500 cultivars genotyped with 1,536 SNPs. In contrast to the majority of human GWA studies, we observe high levels of linkage disequilibrium within and between chromosomes. Despite this, GWA analysis readily detected common alleles of high penetrance. To investigate the potential of combining GWA mapping with comparative analysis to resolve traits to candidate polymorphism level in unsequenced genomes, we fine-mapped a selected phenotype (anthocyanin pigmentation) within a 140-kb interval containing three genes. Of these, resequencing the putative anthocyanin pathway gene HvbHLH1 identified a deletion resulting in a premature stop codon upstream of the basic helix-loop-helix domain, which was diagnostic for lack of anthocyanin in our association and biparental mapping populations. The methodology described here is transferable to species with limited genomic resources, providing a paradigm for reducing the threshold of map-based cloning in unsequenced crops.

INTERMEDIUM-C, a modifier of lateral spikelet fertility in barley, is an ortholog of the maize domestication gene TEOSINTE BRANCHED 1

The domestication of cereals has involved common changes in morphological features, such as seed size, seed retention and modification of vegetative and inflorescence architecture that ultimately contributed to an increase in harvested yield. In barley, this process has resulted in two different cultivated types, two-rowed and six-rowed forms, both derived from the wild two-rowed ancestor, with archaeo-botanical evidence indicating the origin of six-rowed barley early in the domestication of the species, some 8,600–8,000 years ago. Variation at SIX-ROWED SPIKE 1 (VRS1) is sufficient to control this phenotype. However, phenotypes imposed by VRS1 alleles are modified by alleles at the INTERMEDIUM-C (INT-C) locus. Here we show that INT-C is an ortholog of the maize domestication gene TEOSINTE BRANCHED 1 (TB1) and identify 17 coding mutations in barley TB1 correlated with lateral spikelet fertility phenotypes.

A representative, highly informative ’genotyping set’ of barley SSRs

Abstract We have developed a ’genotyping set’ of 48 SSR-based genetic markers for application in genetical studies of barley. The SSRs are a subset of a collection of approximately 600 SSRs available to the barley research community. They have been specifically chosen according to the following criteria: (1) they are single locus; (2) their product quality is good under standard assay conditions; (3) they are distributed across the barley genome; and (4) they exhibit reasonably high polymorphic information content (PIC) values in the cultivated barley gene-pool. To maximise genotyping throughput, one of each SSR primer pair was 5´ end-labelled with either fam, hex or tet fluorochromes to allow automated data capture after running the samples on a DNA sequencer. SSR product sizes were assembled from a reference set of 24 barley genotypes which allowed the construction of ’graphical genotypes’ of each of the individual lines. The graphical genotypes provide a convenient tool for interrogating genetic similarity in the individuals surveyed. The product sizes were compared to those obtained from end-labelling one of the primers with 33P and separating the products by denaturing PAGE followed by autoradiography. Although inconsistencies in size were common, they could generally be easily resolved. A reference manual for use of the ’genotyping set’ has been produced and is available as a PDF download file at http://www.scri.sari.ac.uk/ssr/pdf. These well-characterised barley SSRs, for the first time, provide a common set of robust PCR-based tools which can be used to integrate and compare information collected from fundamental and/or applied genetic studies on barley in different laboratories across the world.

Epigenetic Remodeling of Meiotic Crossover Frequency in Arabidopsis thaliana DNA Methyltransferase Mutants

Meiosis is a specialized eukaryotic cell division that generates haploid gametes required for sexual reproduction. During meiosis, homologous chromosomes pair and undergo reciprocal genetic exchange, termed crossover (CO). Meiotic CO frequency varies along the physical length of chromosomes and is determined by hierarchical mechanisms, including epigenetic organization, for example methylation of the DNA and histones. Here we investigate the role of DNA methylation in determining patterns of CO frequency along Arabidopsis thaliana chromosomes. In A. thaliana the pericentromeric regions are repetitive, densely DNA methylated, and suppressed for both RNA polymerase-II transcription and CO frequency. DNA hypomethylated methyltransferase1 (met1) mutants show transcriptional reactivation of repetitive sequences in the pericentromeres, which we demonstrate is coupled to extensive remodeling of CO frequency. We observe elevated centromere-proximal COs in met1, coincident with pericentromeric decreases and distal increases. Importantly, total numbers of CO events are similar between wild type and met1, suggesting a role for interference and homeostasis in CO remodeling. To understand recombination distributions at a finer scale we generated CO frequency maps close to the telomere of chromosome 3 in wild type and demonstrate an elevated recombination topology in met1. Using a pollen-typing strategy we have identified an intergenic nucleosome-free CO hotspot 3a, and we demonstrate that it undergoes increased recombination activity in met1. We hypothesize that modulation of 3a activity is caused by CO remodeling driven by elevated centromeric COs. These data demonstrate how regional epigenetic organization can pattern recombination frequency along eukaryotic chromosomes.

Variation in the leaf sodium content of the Hordeum vulgare (barley) cultivar Maythorpe and its derived mutant cv. Golden Promise

Tests for shoot and root sodium content were carried out on various barley cultivars (Hordeum vulgare) and experimental lines including wild barley (H. spontaneum) and derivatives. Lines were grown in hydroculture with and without the addition of salt (NaCl), and sodium concentrations in shoots and roots were determined. Variation in shoot sodium content was found between the various lines; in contrast, no significant differences were found between the lines tested for root sodium content. The most significant finding was the variation in shoot sodium content between the two cultivars Golden Promise and Maythorpe. Golden Promise is a direct γ-ray induced mutant of the cultivar Maythorpe and the reduced shoot sodium content of Golden Promise can be attributed to radiation treatment.

An exceptionally high nucleotide and haplotype diversity and a signature of positive selection for the eIF4E resistance gene in barley are revealed by allele mining and phylogenetic analyses of natural populations

In barley, the eukaryotic translation initiation factor 4E (eIF4E) gene situated on chromosome 3H is recognized as an important source of resistance to the bymoviruses Barley yellow mosaic virus and Barley mild mosaic virus. In modern barley cultivars, two recessive eIF4E alleles, rym4 and rym5, confer different isolate‐specific resistances. In this study, the sequence of eIF4E was analysed in 1090 barley landraces and noncurrent cultivars originating from 84 countries. An exceptionally high nucleotide diversity was evident in the coding sequence of eIF4E but not in either the adjacent MCT‐1 gene or the sequence‐related eIF(iso)4E gene situated on chromosome 1H. Surprisingly, all nucleotide polymorphisms detected in the coding sequence of eIF4E resulted in amino acid changes. A total of 47 eIF4E haplotypes were identified, and phylogenetic analysis using maximum likelihood provided evidence of strong positive selection acting on this barley gene. The majority of eIF4E haplotypes were found to be specific to distinct geographic regions. Furthermore, the eI4FE haplotype diversity (uh) was found to be considerably higher in East Asia, whereas SNP genotyping identified a comparatively low degree of genome‐wide genetic diversity in 16 of 17 tested accessions (each carrying a different eIF4E haplotype) from this same region. In addition, selection statistic calculations using coalescent simulations showed evidence of non‐neutral variation for eIF4E in several geographic regions, including East Asia, the region with a long history of the bymovirus‐induced yellow mosaic disease. Together these findings suggest that eIF4E may play a role in barley adaptation to local habitats.

Statistical analysis of a linkage experiment in barley involving quantitative trait loci for height and ear-emergence time and two genetic markers on chromosome 4.

SummaryThe quantitative traits height and ear-emergence date were analyzed in the F2 progeny of a cross between a tall winter barley cultivar (Gerbel) and a short spring barley cultivar (Heriot). The trait distributions were found to be related to the genotypes at two biochemical loci, β-amylase (Bmy1) and water-soluble protein (Wsp3), which are known to lie on the long arm of chromosome 4. Linkages between each trait and the markers were investigated using normal mixture models. The two parental phenotypes and the heterozygote phenotype of Bmy1 were distinguishable so the model could be used directly to estimate linkage between Bmy1 and a quantitative trait locus (QTL) for height (Height). The Gerbel homozygote and heterozygote phenotype of Wsp3 could not be distinguished and the model was adapted accordingly. The proportion of plants requiring vernalization was consistent with control by two independent genes acting epistatically, and a normal mixture model based on a two-gene hypothesis was fitted to the distribution of ear-emergence date to estimate linkage between the marker loci and a QTL for ear-emergence date (Vrn1). The parameters of each model were the recombination fraction between the marker locus and the QTL and the means and standard deviations associated with each QTL genotype; these were estimated by maximum likelihood. The fitted distributions correspond well to those observed and the order of the loci along the chromosome is inferred to be Height — Vrn1 — Bmy1 — Wsp3, with Wsp3 being the most distal.

Comparison of shoot dry weight, Na+ content and δ13C values of ari-e and other semi-dwarf barley mutants under salt-stress

Four breviaristatum (short awned and semi-dwarf) barley mutants; ari-e.1, ari-e.119, ari-e.156 and ari-e.228 were compared with other semi-dwarf mutants; Golden Promise, Alf, Pallas and Diamant along with their non-mutant parents; Bonus, Foma, Maythorpe, Bomi and Valticky, for response to salt stress. Plants were exposed to hydroponic salt treatments (NaCl at 25 and 175 mol m-3) for 4 weeks, after which response was measured in terms of shoot dry weight, sodium content and δ13C. In general ari-e mutants and Golden Promise had significantly lower Na+ contents than the other mutants. They also had significantly more negative δ13C values than the other lines in stressed (175 mol m-3 NaCl) conditions. There was a positive correlation (r = 0.71, p < 0.01) between shoot Na+ and δ13C values so that δ13C became less negative with increasing Na+ content. Shoot dry weights were compared to shoot Na+ and δ13C values. The ari-e and Golden Promise mutants showed less reduction in dry matter production in salt stress relative to the control treatment than all the other lines. The data suggest that ari-e mutants and Golden Promise are better adapted to salt stressed environments than the other lines examined. Tests for gibberellic acid sensitivity revealed that ari-e mutants and Golden Promise responded weakly to GA3, while other dwarf mutants Pallas, Diamant and Alf along with their parents Bonus, Foma, Maythorpe, Valticky and Bomi were highly sensitive. Our results support previous findings that ari-e mutants and the GPert mutant are allelic.

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.