James Cockram

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.

Association mapping of partitioning loci in barley

BackgroundAssociation 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.ResultsBy 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).ConclusionWe 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.

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.

Application of genomics-assisted breeding for generation of climate resilient crops: progress and prospects

Climate change affects agricultural productivity worldwide. Increased prices of food commodities are the initial indication of drastic edible yield loss, which is expected to increase further due to global warming. This situation has compelled plant scientists to develop climate change-resilient crops, which can withstand broad-spectrum stresses such as drought, heat, cold, salinity, flood, submergence and pests, thus helping to deliver increased productivity. Genomics appears to be a promising tool for deciphering the stress responsiveness of crop species with adaptation traits or in wild relatives toward identifying underlying genes, alleles or quantitative trait loci. Molecular breeding approaches have proven helpful in enhancing the stress adaptation of crop plants, and recent advances in high-throughput sequencing and phenotyping platforms have transformed molecular breeding to genomics-assisted breeding (GAB). In view of this, the present review elaborates the progress and prospects of GAB for improving climate change resilience in crops, which is likely to play an ever increasing role in the effort to ensure global food security.

Global agricultural intensification during climate change: a role for genomics

Summary Agriculture is now facing the ‘perfect storm’ of climate change, increasing costs of fertilizer and rising food demands from a larger and wealthier human population. These factors point to a global food deficit unless the efficiency and resilience of crop production is increased. The intensification of agriculture has focused on improving production under optimized conditions, with significant agronomic inputs. Furthermore, the intensive cultivation of a limited number of crops has drastically narrowed the number of plant species humans rely on. A new agricultural paradigm is required, reducing dependence on high inputs and increasing crop diversity, yield stability and environmental resilience. Genomics offers unprecedented opportunities to increase crop yield, quality and stability of production through advanced breeding strategies, enhancing the resilience of major crops to climate variability, and increasing the productivity and range of minor crops to diversify the food supply. Here we review the state of the art of genomic‐assisted breeding for the most important staples that feed the world, and how to use and adapt such genomic tools to accelerate development of both major and minor crops with desired traits that enhance adaptation to, or mitigate the effects of climate change.

Genome Dynamics Explain the Evolution of Flowering Time CCT Domain Gene Families in the Poaceae

Numerous CCT domain genes are known to control flowering in plants. They belong to the CONSTANS-like (COL) and PREUDORESPONSE REGULATOR (PRR) gene families, which in addition to a CCT domain possess B-box or response-regulator domains, respectively. Ghd7 is the most recently identified COL gene to have a proven role in the control of flowering time in the Poaceae. However, as it lacks B-box domains, its inclusion within the COL gene family, technically, is incorrect. Here, we show Ghd7 belongs to a larger family of previously uncharacterized Poaceae genes which possess just a single CCT domain, termed here CCT MOTIF FAMILY (CMF) genes. We molecularly describe the CMF (and related COL and PRR) gene families in four sequenced Poaceae species, as well as in the draft genome assembly of barley (Hordeum vulgare). Genetic mapping of the ten barley CMF genes identified, as well as twelve previously unmapped HvCOL and HvPRR genes, finds the majority map to colinear positions relative to their Poaceae orthologues. Combined inter-/intra-species comparative and phylogenetic analysis of CMF, COL and PRR gene families indicates they evolved prior to the monocot/dicot divergence ∼200 mya, with Poaceae CMF evolution described as the interplay between whole genome duplication in the ancestral cereal, and subsequent clade-specific mutation, deletion and duplication events. Given the proven role of CMF genes in the modulation of cereals flowering, the molecular, phylogenetic and comparative analysis of the Poaceae CMF, COL and PRR gene families presented here provides the foundation from which functional investigation can be undertaken.

Evolutionary history of barley cultivation in Europe revealed by genetic analysis of extant landraces

BackgroundUnderstanding the evolution of cultivated barley is important for two reasons. First, the evolutionary relationships between different landraces might provide information on the spread and subsequent development of barley cultivation, including the adaptation of the crop to new environments and its response to human selection. Second, evolutionary information would enable landraces with similar traits but different genetic backgrounds to be identified, providing alternative strategies for the introduction of these traits into modern germplasm.ResultsThe evolutionary relationships between 651 barley landraces were inferred from the genotypes for 24 microsatellites. The landraces could be divided into nine populations, each with a different geographical distribution. Comparisons with ear row number, caryopsis structure, seasonal growth habit and flowering time revealed a degree of association between population structure and phenotype, and analysis of climate variables indicated that the landraces are adapted, at least to some extent, to their environment. Human selection and/or environmental adaptation may therefore have played a role in the origin and/or maintenance of one or more of the barley landrace populations. There was also evidence that at least some of the population structure derived from geographical partitioning set up during the initial spread of barley cultivation into Europe, or reflected the later introduction of novel varieties. In particular, three closely-related populations were made up almost entirely of plants with the daylength nonresponsive version of the photoperiod response gene PPD-H1, conferring adaptation to the long annual growth season of northern Europe. These three populations probably originated in the eastern Fertile Crescent and entered Europe after the initial spread of agriculture.ConclusionsThe discovery of population structure, combined with knowledge of associated phenotypes and environmental adaptations, enables a rational approach to identification of landraces that might be used as sources of germplasm for breeding programs. The population structure also enables hypotheses concerning the prehistoric spread and development of agriculture to be addressed.

The role of double-stranded break repair in the creation of phenotypic diversity at cereal VRN1 loci.

Nonhomologous repair of double-stranded breaks, although fundamental to the maintenance of genomic integrity in all eukaryotes, has received little attention as to its evolutionary consequences in the generation and selection of phenotypic diversity. Here we document the role of illegitimate recombination in the creation of novel alleles in VRN1 orthologs selected to confer adaptation to annual cropping systems in barley and wheat.

Molecular, phylogenetic and comparative genomic analysis of the cytokinin oxidase/dehydrogenase gene family in the Poaceae

The genomes of cereals such as wheat (Triticum aestivum) and barley (Hordeum vulgare) are large and therefore problematic for the map-based cloning of agronomicaly important traits. However, comparative approaches within the Poaceae permit transfer of molecular knowledge between species, despite their divergence from a common ancestor sixty million years ago. The finding that null variants of the rice gene cytokinin oxidase/dehydrogenase 2 (OsCKX2) result in large yield increases provides an opportunity to explore whether similar gains could be achieved in other Poaceae members. Here, phylogenetic, molecular and comparative analyses of CKX families in the sequenced grass species rice, brachypodium, sorghum, maize and foxtail millet, as well as members identified from the transcriptomes/genomes of wheat and barley, are presented. Phylogenetic analyses define four Poaceae CKX clades. Comparative analyses showed that CKX phylogenetic groupings can largely be explained by a combination of local gene duplication, and the whole-genome duplication event that predates their speciation. Full-length OsCKX2 homologues in barley (HvCKX2.1, HvCKX2.2) and wheat (TaCKX2.3, TaCKX2.4, TaCKX2.5) are characterized, with comparative analysis at the DNA, protein and genetic/physical map levels suggesting that true CKX2 orthologs have been identified. Furthermore, our analysis shows CKX2 genes in barley and wheat have undergone a Triticeae-specific gene-duplication event. Finally, by identifying ten of the eleven CKX genes predicted to be present in barley by comparative analyses, we show that next-generation sequencing approaches can efficiently determine the gene space of large-genome crops. Together, this work provides the foundation for future functional investigation of CKX family members within the Poaceae.

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.