Abstract
We adapted and tested a Rapture assay as an enhancement of genotyping-by-sequencing (GBS) in oat (Avena sativa). This assay was based on an additional bait-based capture of specific DNA fragments representing approximately 10,000 loci within the enzyme-based complexity reduction provided by GBS. By increasing the specificity of GBS to include only those fragments that provided effective polymorphic markers, it was possible to achieve deeper sequence coverage of target markers, while simultaneously sequencing a greater number of samples on a single unit of next-generation sequencing. The Rapture assay consistently out-performed the GBS assay when filtering markers at 80% completeness or greater, even though the total number of reads per sample was only 25% that of GBS. The reduced sequencing cost per sample for Rapture more than compensated for the increased cost of the capture reaction. Thus, Rapture generated a more repeatable set of marker data at a cost per sample that was approximately 40% less than GBS. Additional advantages of Rapture included accurate identification of heterozygotes, and the possibility to increase the depth or length of sequence reads with less impact on the cost per sample. We tested Rapture for genomic selection and diversity analysis and concluded that it is an effective alternative to GBS or other SNP assays. We recommend the use of Rapture in oat and the development of similar assays in other crops with large complex genomes.
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Acknowledgements
The authors gratefully acknowledge professional assistance from Matthew Hayes, Brad De Haan, Denis Green, Kali Stewart, Julie Chapados, Kasia Dadej, and Wayne McCormick, as well as useful discussions and input from Charlene Wight, Alireza Nakhforoosh, and Shiaoman Chao. This work was funded as part of the ‘Oat Project’ through the Agriculture and Agri-Food Canada AgriScience Program, with matching industry support from the Canadian Field Crop Research Alliance (CFCRA).
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WAB, AI and NAT performed data analysis and wrote the manuscript; BB suggested the Rapture technique and advised on its development; AI and BB performed laboratory analyses; WY and JMF provided biological materials and advised on data interpretation. All authors edited and approved the final manuscript.
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Communicated by Hong-Qing Ling.
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122_2019_3496_MOESM2_ESM.xlsx
Key for de-multiplexing raw sequence files (available at http://www.ncbi.nlm.nih.gov/bioproject/590643) (XLSX 80 kb)
122_2019_3496_MOESM3_ESM.xlsx
Example of a Haplotag GBS passport showing tag-level haplotypes and tag counts for GBS versus Rapture assays performed on the same set of accessions (XLSX 38 kb)
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Bekele, W.A., Itaya, A., Boyle, B. et al. A targeted genotyping-by-sequencing tool (Rapture) for genomics-assisted breeding in oat. Theor Appl Genet 133, 653–664 (2020). https://doi.org/10.1007/s00122-019-03496-w
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DOI: https://doi.org/10.1007/s00122-019-03496-w