Jordi Comadran

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.

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.

The USDA barley core collection: genetic diversity, population structure, and potential for genome-wide association studies.

New sources of genetic diversity must be incorporated into plant breeding programs if they are to continue increasing grain yield and quality, and tolerance to abiotic and biotic stresses. Germplasm collections provide a source of genetic and phenotypic diversity, but characterization of these resources is required to increase their utility for breeding programs. We used a barley SNP iSelect platform with 7,842 SNPs to genotype 2,417 barley accessions sampled from the USDA National Small Grains Collection of 33,176 accessions. Most of the accessions in this core collection are categorized as landraces or cultivars/breeding lines and were obtained from more than 100 countries. Both STRUCTURE and principal component analysis identified five major subpopulations within the core collection, mainly differentiated by geographical origin and spike row number (an inflorescence architecture trait). Different patterns of linkage disequilibrium (LD) were found across the barley genome and many regions of high LD contained traits involved in domestication and breeding selection. The genotype data were used to define ‘mini-core’ sets of accessions capturing the majority of the allelic diversity present in the core collection. These ‘mini-core’ sets can be used for evaluating traits that are difficult or expensive to score. Genome-wide association studies (GWAS) of ‘hull cover’, ‘spike row number’, and ‘heading date’ demonstrate the utility of the core collection for locating genetic factors determining important phenotypes. The GWAS results were referenced to a new barley consensus map containing 5,665 SNPs. Our results demonstrate that GWAS and high-density SNP genotyping are effective tools for plant breeders interested in accessing genetic diversity in large germplasm collections.

Effects of ascertainment bias and marker number on estimations of barley diversity from high-throughput SNP genotype data

The capability of molecular markers to provide information of genetic structure is influenced by their number and the way they are chosen. This study evaluates the effects of single nucleotide polymorphism (SNP) number and selection strategy on estimates of germplasm diversity and population structure for different types of barley germplasm, namely cultivar and landrace. One hundred and sixty-nine barley landraces from Syria and Jordan and 171 European barley cultivars were genotyped with 1536 SNPs. Different subsets of 384 and 96 SNPs were selected from the 1536 set, based on their ability to detect diversity in landraces or cultivated barley in addition to corresponding randomly chosen subsets. All SNP sets except the landrace-optimised subsets underestimated the diversity present in the landrace germplasm, and all subsets of SNP gave similar estimates for cultivar germplasm. All marker subsets gave qualitatively similar estimates of the population structure in both germplasm sets, but the 96 SNP sets showed much lower data resolution values than the larger SNP sets. From these data we deduce that pre-selecting markers for their diversity in a germplasm set is very worthwhile in terms of the quality of data obtained. Second, we suggest that a properly chosen 384 SNP subset gives a good combination of power and economy for germplasm characterization, whereas the rather modest gain from using 1536 SNPs does not justify the increased cost and 96 markers give unacceptably low performance. Lastly, we propose a specific 384 SNP subset as a standard genotyping tool for middle-eastern landrace barley.

Genome-wide association mapping of agronomic and morphologic traits in highly structured populations of barley cultivars.

Genome-wide association study (GWAS) has become an obvious general approach for studying traits of agricultural importance in higher plants, especially crops. Here, we present a GWAS of 32 morphologic and 10 agronomic traits in a collection of 615 barley cultivars genotyped by genome-wide polymorphisms from a recently developed barley oligonucleotide pool assay. Strong population structure effect related to mixed sampling based on seasonal growth habit and ear row number is present in this barley collection. Comparison of seven statistical approaches in a genome-wide scan for significant associations with or without correction for confounding by population structure, revealed that in reducing false positive rates while maintaining statistical power, a mixed linear model solution outperforms genomic control, structured association, stepwise regression control and principal components adjustment. The present study reports significant associations for sixteen morphologic and nine agronomic traits and demonstrates the power and feasibility of applying GWAS to explore complex traits in highly structured plant samples.

Variation in the interaction between alleles of HvAPETALA2 and microRNA172 determines the density of grains on the barley inflorescence

Significance We show that the characteristic variation in the density of grains observed along the inflorescence (spike) of modern cultivated barley (Hordeum vulgare) is the consequence of a perturbed interaction between a microRNA, miR172, and its corresponding binding site in the mRNA of an APELATA2 (AP2)-like transcription factor, HvAP2. Our data indicate that variation in the miR172-driven turnover of HvAP2 regulates the length of a developmental window that is required for elongation of the internodes along the axis of the spike, and this variation results in the striking differences in the size and shape of the barley inflorescence. Within the cereal grasses, variation in inflorescence architecture results in a conspicuous morphological diversity that in crop species influences the yield of cereal grains. Although significant progress has been made in identifying some of the genes underlying this variation in maize and rice, in the temperate cereals, a group that includes wheat, barley, and rye, only the dosage-dependent and highly pleiotropic Q locus in hexaploid wheat has been molecularly characterized. Here we show that the characteristic variation in the density of grains along the inflorescence, or spike, of modern cultivated barley (Hordeum vulgare) is largely the consequence of a perturbed interaction between microRNA172 and its corresponding binding site in the mRNA of an APELATA2 (AP2)-like transcription factor, HvAP2. We used genome-wide association and biparental mapping to identify HvAP2. By comparing inflorescence development and HvAP2 transcript abundance in an extreme dense-spike mutant and its nearly isogenic WT line, we show that HvAP2 turnover driven by microRNA 172 regulates the length of a critical developmental window that is required for elongation of the inflorescence internodes. Our data indicate that this heterochronic change, an altered timing of developmental events caused by specific temporal variation in the efficiency of HvAP2 turnover, leads to the striking differences in the size and shape of the barley spike.

Genome-wide association mapping of frost tolerance in barley ( Hordeum vulgare L.)

BackgroundFrost tolerance is a key trait with economic and agronomic importance in barley because it is a major component of winter hardiness, and therefore limits the geographical distribution of the crop and the effective transfer of quality traits between spring and winter crop types. Three main frost tolerance QTL (Fr-H1, Fr-H2 and Fr-H3) have been identified from bi-parental genetic mapping but it can be argued that those mapping populations only capture a portion of the genetic diversity of the species. A genetically broad dataset consisting of 184 genotypes, representative of the barley gene pool cultivated in the Mediterranean basin over an extended time period, was genotyped with 1536 SNP markers. Frost tolerance phenotype scores were collected from two trial sites, Foradada (Spain) and Fiorenzuola (Italy) and combined with the genotypic data in genome wide association analyses (GWAS) using Eigenstrat and kinship approaches to account for population structure.ResultsGWAS analyses identified twelve and seven positive SNP associations at Foradada and Fiorenzuola, respectively, using Eigenstrat and six and four, respectively, using kinship. Linkage disequilibrium analyses of the significant SNP associations showed they are genetically independent. In the kinship analysis, two of the significant SNP associations were tightly linked to the Fr-H2 and HvBmy loci on chromosomes 5H and 4HL, respectively. The other significant kinship associations were located in genomic regions that have not previously been associated with cold stress.ConclusionsHaplotype analysis revealed that most of the significant SNP loci are fixed in the winter or facultative types, while they are freely segregating within the un-adapted spring barley genepool. Although there is a major interest in detecting new variation to improve frost tolerance of available winter and facultative types, from a GWAS perspective, working within the un-adapted spring germplasm pool is an attractive alternative strategy which would minimize statistical issues, simplify the interpretation of the data and identify phenology independent genetic determinants of frost tolerance.

Structural and Temporal Variation in Genetic Diversity of European Spring Two-Row Barley Cultivars and Association Mapping of Quantitative Traits

Two hundred sixteen barley (Hordeum vulgare L.) cultivars were selected to represent the diversity and history of European spring two‐row barley breeding and to search for alleles controlling agronomic traits by association genetics. The germplasm was genotyped with 7864 gene‐based single nucleotide polymorphism markers and corresponding field trial trait data relating to growth and straw strength were obtained at multiple European sites. Analysis of the marker data by statistical population genetics approaches revealed two important trends in the genetic diversity of European two‐row spring barley, namely, i) directional selection for approximately 14% of total genetic variation of the population in the last approximately 50 yr and ii) highly uneven genomic distribution of genetic diversity. Association analysis of the phenotypic and genotypic data identified multiple loci affecting the traits investigated, some of which co‐map with selected regions. Collectively, these data show that the genetic makeup of European two‐row spring barley is evolving under breeder selection, with signs of extinction of diversity in some genomic regions, suggesting that “breeding the best with the best” is leading towards fixation of some breeder targets. Nevertheless, modern germplasm also retains many regions of high diversity, suggesting that site‐specific genetic approaches for allele identification and crop improvement such as association genetics are likely to be successful.

Whole-genome association mapping in elite inbred crop varieties.

We have previously shown that linkage disequilibrium (LD) in the elite cultivated barley (Hordeum vulgare) gene pool extends, on average, for <1-5 cM. Based on this information, we have developed a platform for whole genome association studies that comprises a collection of elite lines that we have characterized at 3060 genome-wide single nucleotide polymorphism (SNP) marker loci. Interrogating this data set shows that significant population substructure is present within the elite gene pool and that diversity and LD vary considerably across each of the seven barley chromosomes. However, we also show that a subpopulation comprised of only the two-rowed spring germplasm is less structured and well suited to whole genome association studies without the need for extensive statistical intervention to account for structure. At the current marker density, the two-rowed spring population is suited for fine mapping simple traits that are located outside of the genetic centromeres with a resolution that is sufficient for candidate gene identification by exploiting conservation of synteny with fully sequenced model genomes and the emerging barley physical map.