Luke Ramsay

Recent history of artificial outcrossing facilitates whole-genome association mapping in elite inbred crop varieties

Genomewide association studies depend on the extent of linkage disequilibrium (LD), the number and distribution of markers, and the underlying structure in populations under study. Outbreeding species generally exhibit limited LD, and consequently, a very large number of markers are required for effective whole-genome association genetic scans. In contrast, several of the worlds major food crops are self-fertilizing inbreeding species with narrow genetic bases and theoretically extensive LD. Together these are predicted to result in a combination of low resolution and a high frequency of spurious associations in LD-based studies. However, inbred elite plant varieties represent a unique human-induced pseudooutbreeding population that has been subjected to strong selection for advantageous alleles. By assaying 1,524 genomewide SNPs we demonstrate that, after accounting for population substructure, the level of LD exhibited in elite northwest European barley, a typical inbred cereal crop, can be effectively exploited to map traits by using whole-genome association scans with several hundred to thousands of biallelic SNPs.

Isolation, characterisation and mapping of simple sequence repeat loci in potato

Solanum tuberosum L. DNA sequences containing simple sequence repeat (SSR) motifs were extracted from the EMBL database, cDNA and selectively enriched small-insert DNA libraries. Enrichment was achieved using either triplex affinity capture or single-strand hybridisation selection. One hundred and twelve primer pairs which successfully amplified products of the correct size from potato DNA were ultimately designed and synthesised. Ninety-eight of these revealed length polymorphisms in a panel of four diploid and two tetraploid clones, in agreement with the high information content of this class of markers which has been found in other species. All of the markers were assigned a quality score of 1–5 based on their potential usefulness. Eighty-nine loci from 65 of the primer pairs were located on two genetic linkage maps of potato by segregation analysis of the amplified alleles. Fifty-two of the SSRs were clearly single locus. The maps were aligned using 23 SSR primer pairs and 13 RFLP loci mapped in both populations. The markers described constitute a class which should replace Restriction Fragment Length Polymorphisms (RFLP) as the markers of choice for future genetic studies in potato. The sequences of the primers, together with other information on these markers are provided.

Natural variation in a homolog of Antirrhinum CENTRORADIALIS contributed to spring growth habit and environmental adaptation in cultivated barley

As early farming spread from the Fertile Crescent in the Near East around 10,000 years before the present, domesticated crops encountered considerable ecological and environmental change. Spring-sown crops that flowered without the need for an extended period of cold to promote flowering and day length–insensitive crops able to exploit the longer, cooler days of higher latitudes emerged and became established. To investigate the genetic consequences of adaptation to these new environments, we identified signatures of divergent selection in the highly differentiated modern-day spring and winter barleys. In one genetically divergent region, we identify a natural variant of the barley homolog of Antirrhinum CENTRORADIALIS (HvCEN) as a contributor to successful environmental adaptation. The distribution of HvCEN alleles in a large collection of wild and landrace accessions indicates that this involved selection and enrichment of preexisting genetic variants rather than the acquisition of mutations after domestication.

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.

A chromosome conformation capture ordered sequence of the barley genome

Cereal grasses of the Triticeae tribe have been the major food source in temperate regions since the dawn of agriculture. Their large genomes are characterized by a high content of repetitive elements and large pericentromeric regions that are virtually devoid of meiotic recombination. Here we present a high-quality reference genome assembly for barley (Hordeum vulgare L.). We use chromosome conformation capture mapping to derive the linear order of sequences across the pericentromeric space and to investigate the spatial organization of chromatin in the nucleus at megabase resolution. The composition of genes and repetitive elements differs between distal and proximal regions. Gene family analyses reveal lineage-specific duplications of genes involved in the transport of nutrients to developing seeds and the mobilization of carbohydrates in grains. We demonstrate the importance of the barley reference sequence for breeding by inspecting the genomic partitioning of sequence variation in modern elite germplasm, highlighting regions vulnerable to genetic erosion.

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.

QTL for field resistance to late blight in potato are strongly correlated with maturity and vigour

Field resistance to Phytophthora infestans, the causal agent of foliage and tuber blight in cultivated potatoes, earliness (maturity) and vigour, were examined in a diploid segregating potato population grown in replicated trials over three consecutive growing seasons. A genetic linkage map of this population was constructed in parallel using PCR-based SSR, AFLP and CAPS markers. Analysis of the trait scores alongside the marker segregation data allowed the identification of regions of the genome which were significantly correlated with components of the respective characters. The most significant associations for all four traits were with marker alleles on potato linkage group V originating from the male (susceptible) parent. In the case of foliage resistance to late blight, the positions of the majority of the effects, which were located on eleven of the twelve potato linkage groups, have been detected in previous [16] and parallel studies [21]. The absence of Solanum demissum-derived R genes for hypersensitive response to late blight and the co-localisation of QTL for resistance, vigour and earliness suggest that developmental and/or physiological factors play a major role in determining the level of foliage resistance in this population. In contrast with previous findings, a negative correlation was found between foliage and tuber blight resistance.

A barley cultivation-associated polymorphism conveys resistance to powdery mildew

Barley (Hordeum vulgare) has played a pivotal role in Old World agriculture since its domestication about 10,000 yr ago. Barley plants carrying loss-of-function alleles (mlo) of the Mlo locus are resistant against all known isolates of the widespread powdery mildew fungus. The sole mlo resistance allele recovered so far from a natural habitat, mlo-11, was originally retrieved from Ethiopian landraces and nowadays controls mildew resistance in the majority of cultivated European spring barley elite varieties. Here we use haplotype analysis to show that the mlo-11 allele probably arose once after barley domestication. Resistance in mlo-11 plants is linked to a complex tandem repeat array inserted upstream of the wild-type gene. The repeat units consist of a truncated Mlo gene comprising 3.5 kilobases (kb) of 5′-regulatory sequence plus 1.1 kb of coding sequence. These generate aberrant transcripts that impair the accumulation of both Mlo wild-type transcript and protein. We exploited the meiotic instability of mlo-11 resistance and recovered susceptible revertants in which restoration of Mlo function was accompanied by excision of the repeat array. We infer cis-dependent perturbation of transcription machinery assembly by transcriptional interference in mlo-11 plants as a likely mechanism leading to disease resistance.

Mapping new EMBL-derived barley microsatellites and their use in differentiating German barley cultivars

Abstract By searching the EMBL DNA sequence database, we were able to develop 39 new, database-derived barley microsatellites. Eighteen of these EMBL microsatellites were mapped either to the interspecific barley map Lerche×BGRC41936 (L×41), the Igri×Franka map (I×F, Graner et al. 1991), or to both maps simultaneously. In addition, all 39 EMBL microsatellites were assigned to individual barley chromosomes by PCR screening of wheat barley addition lines. Both studies verified a random distribution of the microsatellites within the barley genome. Subsequently, 22 EMBL microsatellites were used to assess the genetic similarity among a set of 28, mainly German, barley cultivars and two wild form accessions. Spring and winter cultivars could be easily differentiated using the first coordinate of a principal coordinate analysis. Whereas the group of spring barley cultivars appeared rather homogeneous, winter barley cultivars could be divided into three subgroups. Two H. v. ssp. spontaneum accessions were included in the assessment of genetic similarity. They were placed among the winter barley cultivars. Based on the assessment of the 30 barley cultivars and accessions, the polymorphism information content (PIC) of each EMBL microsatellite has been calculated. The average PIC value among the EMBL microsatellites was equal to 0.38, which ascertains the value of these microsatellites as a genetic tool in barley genome research projects.

The emergence of whole genome association scans in barley

Barley geneticists are currently using association genetics to identify and fine map traits directly in elite plant breeding material. This has been made possible by the development of a highly parallel SNP assay platform that provides sufficient marker density for genome-wide scans and linkage disequilibrium-led gene identification. By leveraging the combined resources of the barley research and breeding sectors, marker-trait associations are being identified and a renewed interest has emerged in novel strategies for barley improvement. New database and visualization tools have been developed and statistical methods adapted from human genetics to account for complexities in the datasets. Exciting early results suggest that association genetics will assume a central role in establishing genotype-to-phenotype relationships.