Abstract
Next Generation Sequencing technologies significantly advance the development of molecular markers for molecular breeding. Dedicated NGS data-analysis procedures must be developed for de novo reference assembly and SNP discovery in crop species without a reference genome sequence. In outcrossing fodder crops, the high degree of polymorphism hampers de novo assembly, contig clustering, read mapping, and SNP discovery. Using selected candidate genes as case studies, we illustrate the reconstruction of a reference transcript sequence from RNA-seq data from multiple genotypes, we validate de novo transcript assembly by Sanger sequencing, and analyse how read mapping and SNP discovery parameters determine sensitivity and specificity during SNP discovery. Thus, we propose a general strategy to construct a non-redundant reference transcriptome for crops without a sequenced genome, using predicted proteins from a closely related model species as a guidance for clustering and annotation. This reference transcriptome is required for candidate gene discovery and exome-wide identification of polymorphisms.
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Acknowledgements
This research was supported by the Agency for the promotion of Innovation by Science and Technology (IWT) Flanders (project LO-080510) and by the Research Foundation Flanders (FWO Grant K208710N). RNA-seq data was generated in collaboration with Torben Asp, Jakob Hedegaard, and Christian Bendixen at Aarhus University, Denmark. We would like to thank Sabine van Glabeke and Nancy Mergan for excellent technical support.
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Ruttink, T., Sterck, L., Vermeulen, E., Rohde, A., Roldán-Ruiz, I. (2013). Development of an SNP Identification Pipeline for Highly Heterozygous Crops. In: Barth, S., Milbourne, D. (eds) Breeding strategies for sustainable forage and turf grass improvement. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-4555-1_16
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DOI: https://doi.org/10.1007/978-94-007-4555-1_16
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