Charlene P. Wight

Using Genotyping-By-Sequencing (GBS) for Genomic Discovery in Cultivated Oat

Advances in next-generation sequencing offer high-throughput and cost-effective genotyping alternatives, including genotyping-by-sequencing (GBS). Results have shown that this methodology is efficient for genotyping a variety of species, including those with complex genomes. To assess the utility of GBS in cultivated hexaploid oat (Avena sativa L.), seven bi-parental mapping populations and diverse inbred lines from breeding programs around the world were studied. We examined technical factors that influence GBS SNP calls, established a workflow that combines two bioinformatics pipelines for GBS SNP calling, and provided a nomenclature for oat GBS loci. The high-throughput GBS system enabled us to place 45,117 loci on an oat consensus map, thus establishing a positional reference for further genomic studies. Using the diversity lines, we estimated that a minimum density of one marker per 2 to 2.8 cM would be required for genome-wide association studies (GWAS), and GBS markers met this density requirement in most chromosome regions. We also demonstrated the utility of GBS in additional diagnostic applications related to oat breeding. We conclude that GBS is a powerful and useful approach, which will have many additional applications in oat breeding and genomic studies.

A molecular linkage map with associated QTLs from a hulless × covered spring oat population

In spring-type oat (Avena sativa L.), quantitative trait loci (QTLs) detected in adapted populations may have the greatest potential for improving germplasm via marker-assisted selection. An F6 recombinant inbred (RI) population was developed from a cross between two Canadian spring oat varieties: ‘Terra’, a hulless line, and ‘Marion’, an elite covered-seeded line. A molecular linkage map was generated using 430 AFLP, RFLP, RAPD, SCAR, and phenotypic markers scored on 101 RI lines. This map was refined by selecting a robust set of 124 framework markers that mapped to 35 linkage groups and contained 35 unlinked loci. One hundred one lines grown in up to 13 field environments in Canada and the United States between 1992 and 1997 were evaluated for 16 agronomic, kernel, and chemical composition traits. QTLs were localized using three detection methods with an experiment-wide error rate of approximately 0.05 for each trait. In total, 34 main-effect QTLs affecting the following traits were identified: heading date, plant height, lodging, visual score, grain yield, kernel weight, milling yield, test weight, thin and plump kernels, groat β-glucan concentration, oil concentration, and protein. Several of these correspond to QTLs in homologous or homoeologous regions reported in other oat QTL studies. Twenty-four QTL-by-environment interactions and three epistatic interactions were also detected. The locus controlling the covered/hulless character (N1) affected most of the traits measured in this study. Additive QTL models with N1 as a covariate were superior to models based on separate covered and hulless sub-populations. This approach is recommended for other populations segregating for major genes. Marker-trait associations identified in this study have considerable potential for use in marker-assisted selection strategies to improve traits within spring oat breeding programs.

SNP Discovery and Chromosome Anchoring Provide the First Physically-Anchored Hexaploid Oat Map and Reveal Synteny with Model Species

A physically anchored consensus map is foundational to modern genomics research; however, construction of such a map in oat (Avena sativa L., 2n = 6x = 42) has been hindered by the size and complexity of the genome, the scarcity of robust molecular markers, and the lack of aneuploid stocks. Resources developed in this study include a modified SNP discovery method for complex genomes, a diverse set of oat SNP markers, and a novel chromosome-deficient SNP anchoring strategy. These resources were applied to build the first complete, physically-anchored consensus map of hexaploid oat. Approximately 11,000 high-confidence in silico SNPs were discovered based on nine million inter-varietal sequence reads of genomic and cDNA origin. GoldenGate genotyping of 3,072 SNP assays yielded 1,311 robust markers, of which 985 were mapped in 390 recombinant-inbred lines from six bi-parental mapping populations ranging in size from 49 to 97 progeny. The consensus map included 985 SNPs and 68 previously-published markers, resolving 21 linkage groups with a total map distance of 1,838.8 cM. Consensus linkage groups were assigned to 21 chromosomes using SNP deletion analysis of chromosome-deficient monosomic hybrid stocks. Alignments with sequenced genomes of rice and Brachypodium provide evidence for extensive conservation of genomic regions, and renewed encouragement for orthology-based genomic discovery in this important hexaploid species. These results also provide a framework for high-resolution genetic analysis in oat, and a model for marker development and map construction in other species with complex genomes and limited resources.

Identification of molecular markers for aluminium tolerance in diploid oat through comparative mapping and QTL analysis

The degree of aluminium tolerance varies widely across cereal species, with oats (Avena spp.) being among the most tolerant. The objective of this study was to identify molecular markers linked to aluminium tolerance in the diploid oat A. strigosa. Restriction fragment length polymorphism markers were tested in regions where comparative mapping indicated the potential for orthologous quantitative trait loci (QTL) for aluminium tolerance in other grass species. Amplified fragment length polymorphism (AFLP) and sequence-characterized amplified region (SCAR) markers were used to provide additional coverage of the genome. Four QTL were identified. The largest QTL explained 39% of the variation and is possibly orthologous to the major gene found in the Triticeae as well as Alm1 in maize and a minor gene in rice. A second QTL may be orthologous to the Alm2 gene in maize. Two other QTL were associated with anonymous markers. Together, these QTL accounted for 55% of the variation. A SCAR marker linked to the major QTL identified in this study could be used to introgress the aluminium tolerance trait from A. strigosa into cultivated oat germplasm.

A consensus map in cultivated hexaploid oat reveals conserved grass synteny with substantial subgenome rearrangement

We constructed a hexaploid oat consensus map from 12 populations representing 19 parents. The map represents the most common physical chromosome arrangements in oat. Deviations from the consensus map may indicate physical rearrangements. Large chromosomal translocations vary among different varieties. There is regional synteny with rice but considerable subgenome rearrangement.

High-density marker profiling confirms ancestral genomes of Avena species and identifies D-genome chromosomes of hexaploid oat

Key messageGenome analysis of 27 oat species identifies ancestral groups, delineates the D genome, and identifies ancestral origin of 21 mapped chromosomes in hexaploid oat.AbstractWe investigated genomic relationships among 27 species of the genus Avena using high-density genetic markers revealed by genotyping-by-sequencing (GBS). Two methods of GBS analysis were used: one based on tag-level haplotypes that were previously mapped in cultivated hexaploid oat (A. sativa), and one intended to sample and enumerate tag-level haplotypes originating from all species under investigation. Qualitatively, both methods gave similar predictions regarding the clustering of species and shared ancestral genomes. Furthermore, results were consistent with previous phylogenies of the genus obtained with conventional approaches, supporting the robustness of whole genome GBS analysis. Evidence is presented to justify the final and definitive classification of the tetraploids A. insularis, A. maroccana (=A. magna), and A. murphyi as containing D-plus-C genomes, and not A-plus-C genomes, as is most often specified in past literature. Through electronic painting of the 21 chromosome representations in the hexaploid oat consensus map, we show how the relative frequency of matches between mapped hexaploid-derived haplotypes and AC (DC)-genome tetraploids vs. A- and C-genome diploids can accurately reveal the genome origin of all hexaploid chromosomes, including the approximate positions of inter-genome translocations. Evidence is provided that supports the continued classification of a diverged B genome in AB tetraploids, and it is confirmed that no extant A-genome diploids, including A. canariensis, are similar enough to the D genome of tetraploid and hexaploid oat to warrant consideration as a D-genome diploid.

Population Genomics Related to Adaptation in Elite Oat Germplasm

An oat association‐mapping panel contributed by active breeding programs worldwide. Characterized population structure and found subdivisions related to adaptation Characterized genome‐wide and chromosome‐specific linkage disequilibrium Performed association‐mapping and post hoc modeling of heading date Found several consistently associated QTL

Quantitative Trait Loci Affecting Oil Content, Oil Composition, and Other Agronomically Important Traits in Oat

Groat oil content and composition are important determinants of oat (Avena sativa L.) quality. We investigated these traits in a population of 146 recombinant inbred lines from a cross between ‘Dal’ (high oil) and ‘Exeter’ (low oil). A linkage map consisting of 475 Diversity Arrays Technology (DArT) markers spanning 1271.8 cM across 40 linkage groups was constructed. Quantitative trait locus (QTL) analysis for groat oil content and composition was conducted using grain samples grown at Aberdeen, ID, in 1997. Quantitative trait locus analysis for multiple agronomic traits was also conducted using data collected from hill plots and field plots in Ottawa, ON, in 2010. Using simple and composite interval mapping methods, QTLs for oil content, palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), and linolenic acid (18:3) were identified. Two of the loci associated with oil content were associated with all of the fatty acids examined in this study, and most oil‐related QTL showed similar patterns of effect on the fatty acid profile. These results suggest the presence of pleiotropic effects on oil‐related traits through influences at specific nodes of the oil synthesis pathway. In addition, 12 QTL‐associated markers (likely representing nine unique regions) were associated with plant height, heading date, lodging, and protein content.

Haplotype-based genotyping-by-sequencing in oat genome research

Summary In a de novo genotyping‐by‐sequencing (GBS) analysis of short, 64‐base tag‐level haplotypes in 4657 accessions of cultivated oat, we discovered 164741 tag‐level (TL) genetic variants containing 241224 SNPs. From this, the marker density of an oat consensus map was increased by the addition of more than 70000 loci. The mapped TL genotypes of a 635‐line diversity panel were used to infer chromosome‐level (CL) haplotype maps. These maps revealed differences in the number and size of haplotype blocks, as well as differences in haplotype diversity between chromosomes and subsets of the diversity panel. We then explored potential benefits of SNP vs. TL vs. CL GBS variants for mapping, high‐resolution genome analysis and genomic selection in oats. A combined genome‐wide association study (GWAS) of heading date from multiple locations using both TL haplotypes and individual SNP markers identified 184 significant associations. A comparative GWAS using TL haplotypes, CL haplotype blocks and their combinations demonstrated the superiority of using TL haplotype markers. Using a principal component‐based genome‐wide scan, genomic regions containing signatures of selection were identified. These regions may contain genes that are responsible for the local adaptation of oats to Northern American conditions. Genomic selection for heading date using TL haplotypes or SNP markers gave comparable and promising prediction accuracies of up to r = 0.74. Genomic selection carried out in an independent calibration and test population for heading date gave promising prediction accuracies that ranged between r = 0.42 and 0.67. In conclusion, TL haplotype GBS‐derived markers facilitate genome analysis and genomic selection in oat.

Genomic relationships among sixteen Avena species based on (ACT)6 trinucleotide repeat FISH

Knowledge of the locations of repeat elements could be very important in the assembly of genome sequences and their assignment to physical chromosomes. Genomic and species relationships among 16 species were investigated using fluorescence in situ hybridization (FISH) with the Am1 and (ACT)6 probes. The Am1 oligonucleotide probe was particularly enriched in the C genomes, whereas the (ACT)6 trinucleotide repeat probe showed a diverse distribution of hybridization patterns in the A, AB, C, AC, and ACD genomes but might not be present in the B and D genomes. The hybridization pattern of Avena sativa was very similar to that of A. insularis, indicating that this species most likely originated from A. insularis as a tetraploid ancestor. Although the two FISH probes failed to identify relationships of more species, this proof-of-concept approach opens the way to the use of FISH probes in assigning other signature elements from genomic sequence to physical chromosomes.