Targeted Next Generation Amplicon Sequencing of Diverse Winter Wheat Vernalization and Photoperiod Genes as a Method for Variant Discovery

Winter wheat exhibits wide variation for flowering time in response to cold treatment and day length. Association mapping and QTL analyses have implicated the wheat VRN1 and PPD1 loci as important determinants for timing developmental phases.  Although much progress has been made in cloning and characterizing variation at the major vernalization and photoperiod loci, evaluation of the allelic variation available to wheat breeders is needed.  To identify novel gene variants that may contribute to flowering time variation, we undertook a project to sequence the VRN1 and PPD1 loci from diverse winter wheat lines and to characterize critical variants that may have a functional or regulatory role. Advances in next generation sequencing (NGS) has reduced the cost of DNA sequencing allowing for the development of novel methods to address many biological questions. NGS platforms provide a much higher sequencing throughput than Sanger sequencing which can facilitate the discovery of novel gene variants. Here, we present a protocol to construct gene specific targeted amplicon libraries of winter wheat vernalization and photoperiod genes for sequencing using the Illumina MiSeq platform and bioinformatic workflow for downstream analysis. This method was a cost effective approach to screen for novel gene variants in winter wheat and can be applied to a wide range of organism