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, 9 (7), e102448
eCollection

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

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

Yung-Fen Huang et al. PLoS One.

Abstract

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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Number of GBS loci vs. sequencing depth.
Number of GBS SNP loci called in 53 OxT mapping progeny at increasing sequencing depth, filtered at four levels of completeness (25%, 50%, 75%, and 90%). Other filtering parameters were constant, with heterozygosity ≤10% and minor allele frequency ≥30%. A sequencing depth index of 1 represents the average read depth that would be achieved with 95 samples multiplexed in a standard Illumina sequencing run giving approximately 2×108 short reads. Thus, an index of 2 would be equivalent to twice this number of reads or half of this plexity.
Figure 2
Figure 2. Number of GBS loci vs. sample size.
Number of GBS loci called in samples of size N from a set of 360 diverse oat lines filtered at four levels of completeness (25%, 50%, 75%, and 90%) is shown. Other filtering parameters were constant, with heterozygosity ≥10% and MAF ≥5%.
Figure 3
Figure 3. Distribution of GBS loci across the oat genome.
Maps of each chromosome (delineated by blue lines and labeled on left) are divided into 5 cM bins with 0 cM starting at the top. Red bars show numbers of loci detected by two pipelines, green shows those detected only by the population-filtering pipeline, and violet shows those detected only by the UNEAK pipeline. Numerals inside boxes show total GBS loci by chromosome. A summary of placed GBS loci by pipeline and by sub-genome is shown.
Figure 4
Figure 4. Scatter plots of PC1 vs. PC2.
The k10 correction is shown: (A) coloured based on clustering from genotypic data, (B) coloured based on geographic origins.
Figure 5
Figure 5. LD decay plot.
r2 estimates were plotted against the average map distance (recombination frequency expressed in cM): (A) relationship fit using the mutation model (Hill-Weir), (B) relationship fit using the recombination-drift model (Sved). Population structure was estimated using the k10 correction.

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Publication types

Grant support

This work was supported by the Canadian Crop Genomics Initiative as part of Agriculture and Agri-Food Canada research grant 1885. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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