BackgroundLinkage maps are an integral resource for dissection of complex genetic traits in plant and animal species. Canonical map construction follows a well-established workflow: an initial discovery phase where genetic markers are mined from a small pool of individuals, followed by genotyping of selected mapping populations using sets of marker panels. A newly developed sequence-based marker technology, Restriction site Associated DNA (RAD), enables synchronous single nucleotide polymorphism (SNP) marker discovery and genotyping using massively parallel sequencing. The objective of this research was to assess the utility of RAD markers for linkage map construction, employing barley as a model system. Using the published high density EST-based SNP map in the Oregon Wolfe Barley (OWB) mapping population as a reference, we created a RAD map using a limited set of prior markers to establish linakge group identity, integrated the RAD and prior data, and used both maps for detection of quantitative trait loci (QTL).ResultsUsing the RAD protocol in tandem with the Illumina sequence by synthesis platform, a total of 530 SNP markers were identified from initial scans of the OWB parental inbred lines - the "dominant" and "recessive" marker stocks - and scored in a 93 member doubled haploid (DH) mapping population. RAD sequence data from the structured population was converted into allele genotypes from which a genetic map was constructed. The assembled RAD-only map consists of 445 markers with an average interval length of 5 cM, while an integrated map includes 463 RAD loci and 2383 prior markers. Sequenced RAD markers are distributed across all seven chromosomes, with polymorphic loci emanating from both coding and noncoding regions in the Hordeum genome. Total map lengths are comparable and the order of common markers is identical in both maps. The same large-effect QTL for reproductive fitness traits were detected with both maps and the majority of these QTL were coincident with a dwarfing gene (ZEO) and the VRS1 gene, which determines the two-row and six-row germplasm groups of barley.ConclusionsWe demonstrate how sequenced RAD markers can be leveraged to produce high quality linkage maps for detection of single gene loci and QTLs. By combining SNP discovery and genotyping into parallel sequencing events, RAD markers should be a useful molecular breeding tool for a range of crop species. Expected improvements in cost and throughput of second and third-generation sequencing technologies will enable more powerful applications of the sequenced RAD marker system, including improvements in de novo genome assembly, development of ultra-high density genetic maps and association mapping.

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.

Recent advances in high-throughput genotyping have made it easier to combine information from different mapping populations into consensus genetic maps, which provide increased marker density and genome coverage compared to individual maps. Previously, a single nucleotide polymorphism (SNP)-based genotyping platform was developed and used to genotype 373 individuals in four barley (Hordeum vulgare L.) mapping populations. This led to a 2943 SNP consensus genetic map with 975 unique positions. In this work, we add data from six additional populations and more individuals from one of the original populations to develop an improved consensus map from 1133 individuals. A stringent and systematic analysis of each of the 10 populations was performed to achieve uniformity. This involved reexamination of the four populations included in the previous map. As a consequence, we present a robust consensus genetic map that contains 2994 SNP loci mapped to 1163 unique positions. The map spans 1137.3 cM with an average density of one marker bin per 0.99 cM. A novel application of the genotyping platform for gene detection allowed the assignment of 2930 genes to fl ow-sorted chromosomes or arms, confi rmed the position of 2545 SNP-mapped loci, added chromosome or arm allocations to an additional 370 SNP loci, and delineated pericentromeric regions for chromosomes 2H to 7H. Marker order has been improved and map resolution has been increased by almost 20%. These increased precision outcomes enable more optimized SNP selection for markerassisted breeding and support association genetic analysis and map-based cloning. It will also improve the anchoring of DNA sequence scaffolds and the barley physical map to the genetic map.

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Barley (Hordeum vulgare L.) is an economically important model plant for genetics research. Barley is currently served by an increasingly comprehensive set of tools for genetic analysis that have recently been augmented by high‐density genetic linkage maps built with gene‐based single nucleotide polymorphisms (SNPs). These SNP‐based maps need to be aligned with earlier generation maps, which were used for quantitative trait locus (QTL) detection, by integrating multiple types of markers into a single map. A 2383 locus linkage map was developed using the Oregon Wolfe Barley (OWB) Mapping Population to allow such alignments. The map is based on 1472 SNP, 722 DArT, and 189 prior markers which include morphological, simple sequence repeat (SSR), Restriction Fragment Length Polymorphism (RFLP), and sequence tagged site (STS) loci. This new OWB map forms, therefore, a useful bridge between high‐density SNP‐only maps and prior QTL reports. The application of this bridge concept is shown using malting‐quality QTLs from multiple mapping populations, as reported in the literature. This is the first step toward developing a Barley QTL Community Curation workbook for all types of QTLs and maps, on the GrainGenes website. The OWB‐related resources are available at OWB Data and GrainGenes Tools (OWB‐DGGT) (http://wheat.pw.usda.gov/ggpages/maps/OWB/).

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BackgroundConsiderations in applying association mapping (AM) to plant breeding are population structure and size: not accounting for structure and/or using small populations can lead to elevated false-positive rates. The principal determinants of population structure in cultivated barley are growth habit and inflorescence type. Both are under complex genetic control: growth habit is controlled by the epistatic interactions of several genes. For inflorescence type, multiple loss-of-function alleles in one gene lead to the same phenotype. We used these two traits as models for assessing the effectiveness of AM. This research was initiated using the CAP Core germplasm array (n = 102) assembled at the start of the Barley Coordinated Agricultural Project (CAP). This array was genotyped with 4,608 SNPs and we re-sequenced genes involved in morphology, growth and development. Larger arrays of breeding germplasm were subsequently genotyped and phenotyped under the auspices of the CAP project. This provided sets of 247 accessions phenotyped for growth habit and 2,473 accessions phenotyped for inflorescence type. Each of the larger populations was genotyped with 3,072 SNPs derived from the original set of 4,608.ResultsSignificant associations with SNPs located in the vicinity of the loci involved in growth habit and inflorescence type were found in the CAP Core. Differentiation of true and spurious associations was not possible without a priori knowledge of the candidate genes, based on re-sequencing. The re-sequencing data were used to define allele types of the determinant genes based on functional polymorphisms. In a second round of association mapping, these synthetic markers based on allele types gave the most significant associations. When the synthetic markers were used as anchor points for analysis of interactions, we detected other known-function genes and candidate loci involved in the control of growth habit and inflorescence type. We then conducted association analyses - with SNP data only - in the larger germplasm arrays. For both vernalization sensitivity and inflorescence type, the most significant associations in the larger data sets were found with SNPs coincident with the synthetic markers used in the CAP Core and with SNPs detected via interaction analysis in the CAP Core.ConclusionsSmall and highly structured collections of germplasm, such as the CAP Core, are cost-effectively phenotyped and genotyped with high-throughput markers. They are also useful for characterizing allelic diversity at loci in germplasm of interest. Our results suggest that discovery-oriented exercises in AM in such small arrays may generate a large number of false-positives. However, if haplotypes in candidate genes are available, they may be used as anchors in an analysis of interactions to identify other candidate regions harboring genes determining target traits. Using larger germplasm arrays, genome regions where the principal genes determining vernalization sensitivity and row type are located were identified.

Snap bean (Phaseolus vulgaris L.) breeding programs are tasked with developing cultivars that meet the standards of the vegetable processing industry and ultimately that of the consumer, all the while matching or exceeding the field performance of existing cultivars. While traditional breeding methods have had a long history of meeting these requirements, genetic marker technology, combined with the knowledge of important quantitative trait loci (QTL), can accelerate breeding efforts. In contrast to dry bean, snap bean immature pods and seeds are consumed as a vegetable. Several pod traits are important in snap bean including: reduced pod wall fiber, absence of pod suture strings, and thickened, succulent pod walls. In addition, snap bean pods are selected for round pod cross section, and pods tend to be longer with cylindrical seed shape. Seed color is an important trait in snap bean, especially those used for processing, as processors prefer white-seeded cultivars. The objective of this study was to investigate the genetic control of traits important to snap bean producers and processors. RR6950, a small seeded brown indeterminate type IIIA dry bean accession, was crossed to the Oregon State University (OSU) breeding line OSU5446, a type I Blue Lake four-sieve breeding line to produce the RR138 F4:6 recombinant inbred (RI) mapping population. We evaluated the RR138 RI population for processing and morphological traits, especially those affecting pods. The RR138 population was genotyped with the BARCBean6K_3 Beadchip, and single nucleotide polymorphisms (SNPs) were used to assemble a linkage map, and identify QTL for pod traits. The linkage map produced from this study contained 1689 SNPs across 1196cM. The map was populated with an average of one SNP per 1.4 cM, spanning 11 linkage groups. Seed and flower color genes B and P were located on Pv02 and Pv07, respectively. A QTL for string:pod length (PL) ratio was found on Pv02 controlling 32% of total genetic variation. QTL for a suite of important processing traits including pod wall fiber, pod height, pod width, and pod wall thickness were found clustering on Pv04 and controlled 21%, 26%, 18%, and 16% of genetic variation for each of these respective traits. A QTL for PL was found on Pv09 controlling 5% of genetic variation.