Random Tagging GBS in Kiwifruit

Genotyping by sequencing (GBS) is a targeted approach developed to reduce the genome complexity using a restriction enzyme, and to discover genetic markers when a priori sequence information is unavailable. Commonly 64 bases around the target site are used for variant discovery. Sufficient coverage at each locus is essential to distinguish heterozygous from homozygous sites accurately. The number of GBS samples able to be pooled in one lane is limited since a balance is needed between the numbers of restriction sites and the read depth required at each site per sample for accurate single-nucleotide polymorphism calling. Loci bias was observed using a slight modification of the Elshire method: some restriction sites were represented in higher proportions while others were poorly represented or absent. This bias could be due to the quality of genomic DNA, endonuclease and ligase reaction efficiency, the distance between restriction sites, preferential amplification of small library restriction fragments, and bias towards cluster formation of small amplicons during the sequencing process. To overcome these issues, we developed a GBS method based on randomly tagging genomic DNA (rtGBS). By randomly landing on the genome, we can find restriction sites that are far apart, and not detected by the standard GBS (stdGBS) method. The study comprises two types of biological replicates: different plants and two independent DNA extractions per plant; and three types of technical replicates: four samples of each DNA extraction, standard versus random tagging GBS methods, and two independent library amplifications, each sequenced in separate lanes.