Abstract
High-throughput genotyping arrays continue to be an attractive, cost-effective alternative to sequencing based approaches. We have developed a new 50k Illumina Infinium iSelect genotyping array for barley, a cereal crop species of major international importance. The majority of SNPs on the array have been extracted from variants called in exome capture data of a wide range of European barley germplasm. We used the recently published barley pseudomolecule assembly to map the exome capture data, which allowed us to generate markers with accurate physical positions and detailed gene annotation. Markers from an existing and widely used barley 9k Infinium iSelect array were carried over onto the 50k chip for backward compatibility. The array design featured 49,267 SNP markers that converted into 44,040 working assays, of which 43,461 were scorable in GenomeStudio. Of the working assays, 6,251 are from the 9k iSelect platform. We validated the SNPs by comparing the genotype calls from the new array to legacy datasets. Rates of agreement averaged 98.1 and 93.9% respectively for the legacy 9k iSelect SNP set (Comadran et al., 2012) and the exome capture SNPs. To test the utility of the 50k chip for genetic mapping, we genotyped a segregating population derived from a Golden Promise × Morex cross (Liu et al., 2014) and mapped over 14,000 SNPs to genetic positions which showed a near exact correspondence to their known physical positions. Manual adjustment of the cluster files used by the interpreting software for genotype scoring improved results substantially, but migration of cluster files between sites led to a deterioration of results, suggesting that local adjustment of cluster files is required on a site-per-site basis. Information relating to the markers on the chip is available online at https://ics.hutton.ac.uk/50k.
Highlights
Barley (Hordeum vulgare L.) is a cereal crop of major importance, ranked fourth grain crop in the world by the UN’s Food and Agriculture Organization in terms of production volume
The 9k Illumina Infinium iSelect Custom Genotyping BeadChip (Comadran et al, 2012) included 2,832 markers developed for the previous GoldenGate assays and 5,010 additional markers based on Single nucleotide polymorphisms (SNPs) marker discovery in Illumina RNAseq data from 10 UK elite cultivars
In this set we categorised each accession into different groups based on growth habit, row type, date of introduction and origin, where possible. From this we identified a set of 148 accessions representing most of the variation and allelic diversity (Figure 1 groups highlighted as: central European old cultivars; spring 6 row; spring 2 row; winter 2 row and winter 6 row). This was supplemented with a further 22 landraces sourced from the Germplasm Resources Unit (GRU) at the John Innes Institute and Science and Advice for Scottish Agriculture (SASA), for which no SNP data was available for comparison
Summary
Barley (Hordeum vulgare L.) is a cereal crop of major importance, ranked fourth grain crop in the world by the UN’s Food and Agriculture Organization in terms of production volume (http:// faostat.fao.org). Single nucleotide polymorphisms (SNPs) are today’s marker of choice due to their abundance, relative ease of discovery in Barley 50k iSelect SNP Array high-throughput sequencing data (Kumar et al, 2012) and costs per data point. High throughput genotyping in barley was first introduced in 2006 with the development of two Illumina GoldenGate assays (Fan et al, 2003) that featured 1,572 SNP markers each (Close et al, 2009). A new genotyping platform followed in 2009 that introduced larger numbers of markers based on SNP discovery in Generation Sequencing data. The 9k Illumina Infinium iSelect Custom Genotyping BeadChip (Comadran et al, 2012) included 2,832 markers developed for the previous GoldenGate assays and 5,010 additional markers based on SNP marker discovery in Illumina RNAseq data from 10 UK elite cultivars. The iSelect technology currently offers the potential of up to 700k custom targets (SNPs, indels, and CNVs) per array and 24 samples can be processed at a time per chip (https://www. illumina.com/content/dam/illumina-marketing/documents/ products/technotes/technote_iselect_design.pdf)
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