Advancements in next-generation sequencing technology have enabled whole genome re-sequencing in many species providing unprecedented discovery and characterization of molecular polymorphisms. There are limitations, however, to next-generation sequencing approaches for species with large complex genomes such as barley and wheat. Genotyping-by-sequencing (GBS) has been developed as a tool for association studies and genomics-assisted breeding in a range of species including those with complex genomes. GBS uses restriction enzymes for targeted complexity reduction followed by multiplex sequencing to produce high-quality polymorphism data at a relatively low per sample cost. Here we present a GBS approach for species that currently lack a reference genome sequence. We developed a novel two-enzyme GBS protocol and genotyped bi-parental barley and wheat populations to develop a genetically anchored reference map of identified SNPs and tags. We were able to map over 34,000 SNPs and 240,000 tags onto the Oregon Wolfe Barley reference map, and 20,000 SNPs and 367,000 tags on the Synthetic W9784 × Opata85 (SynOpDH) wheat reference map. To further evaluate GBS in wheat, we also constructed a de novo genetic map using only SNP markers from the GBS data. The GBS approach presented here provides a powerful method of developing high-density markers in species without a sequenced genome while providing valuable tools for anchoring and ordering physical maps and whole-genome shotgun sequence. Development of the sequenced reference genome(s) will in turn increase the utility of GBS data enabling physical mapping of genes and haplotype imputation of missing data. Finally, as a result of low per-sample costs, GBS will have broad application in genomics-assisted plant breeding programs.

/pdf/development-of-high-density-genetic-maps-for-barley-and-f8yjyw2gas.pdf

Development of High-Density Genetic Maps for Barley and Wheat Using a Novel Two-Enzyme Genotyping-by-Sequencing Approach

Rapid advances in “next-generation” DNA sequencing technology have brought the US$1000 human (Homo sapiens) genome within reach while providing the raw sequencing output for researchers to revolutionize the way populations are genotyped. To capitalize on these advancements, genotypingby-sequencing (GBS) has been developed as a rapid and robust approach for reduced-representation sequencing of multiplexed samples that combines genome-wide molecular marker discovery and genotyping. The fl exibility and low cost of GBS makes this an excellent tool for many applications and research questions in plant genetics and breeding. Here we address some of the new research opportunities that are becoming more feasible with GBS. Furthermore, we highlight areas in which GBS will become more powerful with the continued increase of sequencing output, development of reference genomes, and improvement of bioinformatics. The ultimate goal of plant biology scientists is to connect phenotype to genotype. In plant breeding, the genotype can then be used to predict phenotypes and select improved cultivars. Furthering our understanding of the connection between heritable genetic factors and the resulting phenotypes will enable genomics-assisted breeding to exist on the scale needed to increase global food supplies in the face of decreasing arable land and climate change. Next-Generation Genotyping

Genotyping-by-Sequencing for Plant Breeding and Genetics

Robbie Waugh and colleagues report that the EARLINESS PER SE (EPS2) locus is associated with spring growth habit and environmental adaptation in barley. Resequencing the barley homolog of CENTRORADIALIS, located within the EPS2 locus, in 216 spring and 207 winter barley accessions identified haplotypes at HvCEN that correspond with winter or spring growth habit. As early farming spread from the Fertile Crescent in the Near East around 10,000 years before the present1, 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 CENTRORADIALIS2 (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.

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

Despite advances in sequencing, the goal of obtaining a comprehensive view of genetic variation in populations is still far from reached. We sequenced 180 lines of A. thaliana from Sweden to obtain as complete a picture as possible of variation in a single region. Whereas simple polymorphisms in the unique portion of the genome are readily identified, other polymorphisms are not. The massive variation in genome size identified by flow cytometry seems largely to be due to 45S rDNA copy number variation, with lines from northern Sweden having particularly large numbers of copies. Strong selection is evident in the form of long-range linkage disequilibrium (LD), as well as in LD between nearby compensatory mutations. Many footprints of selective sweeps were found in lines from northern Sweden, and a massive global sweep was shown to have involved a 700-kb transposition.

Massive genomic variation and strong selection in Arabidopsis thaliana lines from Sweden

Genome-wide association studies (GWAS) based on linkage disequilibrium (LD) provide a promising tool for the detection and fine mapping of quantitative trait loci (QTL) underlying complex agronomic traits. In this study we explored the genetic basis of variation for the traits heading date, plant height, thousand grain weight, starch content and crude protein content in a diverse collection of 224 spring barleys of worldwide origin. The whole panel was genotyped with a customized oligonucleotide pool assay containing 1536 SNPs using Illumina's GoldenGate technology resulting in 957 successful SNPs covering all chromosomes. The morphological trait "row type" (two-rowed spike vs. six-rowed spike) was used to confirm the high level of selectivity and sensitivity of the approach. This study describes the detection of QTL for the above mentioned agronomic traits by GWAS. Population structure in the panel was investigated by various methods and six subgroups that are mainly based on their spike morphology and region of origin. We explored the patterns of linkage disequilibrium (LD) among the whole panel for all seven barley chromosomes. Average LD was observed to decay below a critical level (r2-value 0.2) within a map distance of 5-10 cM. Phenotypic variation within the panel was reasonably large for all the traits. The heritabilities calculated for each trait over multi-environment experiments ranged between 0.90-0.95. Different statistical models were tested to control spurious LD caused by population structure and to calculate the P-value of marker-trait associations. Using a mixed linear model with kinship for controlling spurious LD effects, we found a total of 171 significant marker trait associations, which delineate into 107 QTL regions. Across all traits these can be grouped into 57 novel QTL and 50 QTL that are congruent with previously mapped QTL positions. Our results demonstrate that the described diverse barley panel can be efficiently used for GWAS of various quantitative traits, provided that population structure is appropriately taken into account. The observed significant marker trait associations provide a refined insight into the genetic architecture of important agronomic traits in barley. However, individual QTL account only for a small portion of phenotypic variation, which may be due to insufficient marker coverage and/or the elimination of rare alleles prior to analysis. The fact that the combined SNP effects fall short of explaining the complete phenotypic variance may support the hypothesis that the expression of a quantitative trait is caused by a large number of very small effects that escape detection. Notwithstanding these limitations, the integration of GWAS with biparental linkage mapping and an ever increasing body of genomic sequence information will facilitate the systematic isolation of agronomically important genes and subsequent analysis of their allelic diversity.

/pdf/genome-wide-association-studies-for-agronomical-traits-in-a-3vwfogjrxx.pdf

Genome-wide association studies for agronomical traits in a world wide spring barley collection.

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.

https://www.pnas.org/content/pnas/107/50/21611.full.pdf

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

MYB transcription factors (TFs) represent one of the biggest TF families in plants, being involved in various specific plant processes, such as responses to biotic and abiotic stresses. The implication of MYB TFs in the tolerance mechanisms to abiotic stress is particularly interesting for crop breeding, since environmental conditions can negatively affect growth and productivity. Wheat is a worldwide-cultivated cereal, and is a major source of plant-based proteins in human food. In particular, durum wheat plays an important role in global food security improvement, since its adaptation to hot and dry conditions constitutes the base for the success of wheat breeding programs in future. In the present study, a genome-wide identification of R2R3-MYB TFs in durum wheat was performed. MYB profile search and phylogenetic analyses based on homology with Arabidopsis and rice MYB TFs led to the identification of 233 R2R3-TdMYB (Triticum durum MYB). Three Poaceae-specific MYB clusters were detected, one of which had never been described before. The expression of eight selected genes under different abiotic stress conditions, revealed that most of them responded especially to salt and drought stress. Finally, gene regulatory network analyses led to the identification of 41 gene targets for three TdR2R3-MYBs that represent novel candidates for functional analyses. This study provides a detailed description of durum wheat R2R3-MYB genes and contributes to a deeper understanding of the molecular response of durum wheat to unfavorable climate conditions.

/pdf/r2r3-mybs-in-durum-wheat-genome-wide-identification-poaceae-2zc7pw3m.pdf

R2R3-MYBs in Durum Wheat: Genome-Wide Identification, Poaceae-Specific Clusters, Expression, and Regulatory Dynamics Under Abiotic Stresses

The sweet cherry is an important fruit species that is widespread globally. In addition to the well-known traditional and modern varieties, a myriad of landraces is present in Europe, as well as in southern Italy. This study aims to evaluate the population structure, genetic relationships, and cases of duplicate samples in a collection of 143 accessions using GBS-derived SNP markers. The genetic material under investigation includes modern commercial varieties, ancient European and American varieties, landraces, and individuals retrieved from small orchards. Some of the known varieties were genetically analyzed here for the first time. In addition, several genotypes were collected from the Basilicata region (southern Italy), an area largely unexplored for sweet cherry genetic resources. The relationships among genotypes were assessed using four different methods: allele frequency and ancestry estimation, principal component analysis, Neighbor-Joining tree, and identity-by-state estimation. The analyses returned quite congruent results and highlighted the presence of four main genetic groups, namely: (i) American varieties, (ii) the ‘Germersdorfer-Ferrovia’ cluster, (iii) the ‘Burlat’ group, and (iv) the group of Italian landraces. The main drivers of clustering were ancestry, geographical distribution, and some important traits such as self-compatibility. The sweet cherries from Basilicata, herewith examined for the first time, were mostly distributed within the group of Italian landraces, being particularly linked to the autochthonous varieties of the Campania region. However, some genotypes were outside this group, thus suggesting the introduction of genetic material from other Italian regions or from European countries. The considerable amount of American and European modern varieties analyzed are genetically very closely related, suggesting a reduced genetic basis. In addition, we highlighted the discriminating ability of SNP markers to distinguish between an original variety and its mutant. Overall, our results may be useful in defining conservation strategies for sweet cherry germplasm and developing future breeding programs to enlarge the genetic basis of commercial varieties.

/pdf/sweet-cherry-diversity-and-relationships-in-modern-and-local-3ai21jkp.pdf

Sweet Cherry Diversity and Relationships in Modern and Local Varieties Based on SNP Markers

Durum wheat (Triticum turgidum subsp. durum (Desf.) Husn.) is an allotetraploid cereal crop of worldwide importance, given its use for making pasta, couscous, and bulgur. Under climate change scenarios, abiotic (e.g., high and low temperatures, salinity, drought) and biotic (mainly exemplified by fungal pathogens) stresses represent a significant limit for durum cultivation because they can severely affect yield and grain quality. The advent of next-generation sequencing technologies has brought a huge development in transcriptomic resources with many relevant datasets now available for durum wheat, at various anatomical levels, also focusing on phenological phases and environmental conditions. In this review, we cover all the transcriptomic resources generated on durum wheat to date and focus on the corresponding scientific insights gained into abiotic and biotic stress responses. We describe relevant databases, tools and approaches, including connections with other “omics” that could assist data integration for candidate gene discovery for bio-agronomical traits. The biological knowledge summarized here will ultimately help in accelerating durum wheat breeding.

/pdf/a-survey-of-the-transcriptomic-resources-in-durum-wheat-1h4zxstf.pdf

Diana L. Zuluaga

Papers

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

R2R3-MYBs in Durum Wheat: Genome-Wide Identification, Poaceae-Specific Clusters, Expression, and Regulatory Dynamics Under Abiotic Stresses

Sweet Cherry Diversity and Relationships in Modern and Local Varieties Based on SNP Markers

A Survey of the Transcriptomic Resources in Durum Wheat: Stress Responses, Data Integration and Exploitation