Abstract

Whole-genome genotyping (WGG) stands as a pivotal element in genomic-assisted plant breeding. Nevertheless, sequencing-based approaches for WGG continue to be costly, primarily owing to the high expenses associated with library preparation and the laborious protocol. During prior development of foreground and background integrated genotyping by sequencing (FBI-seq), we discovered that any sequence-specific primer (SP) inherently possesses the capability to amplify a massive array of stable and reproducible non-specific PCR products across the genome. Here, we further improved FBI-seq by replacing the adapter ligated by Tn5 transposase with an arbitrary degenerate (AD) primer. The protocol for the enhanced FBI-seq unexpectedly mirrors a simplified thermal asymmetric interlaced (TAIL)-PCR, a technique that is widely used for isolation of flanking sequences. However, the improved TAIL-PCR maximizes the primer-template mismatched annealing capabilities of both SP and AD primers. In addition, leveraging of next-generation sequencing enhances the ability of this technique to assay tens of thousands of genome-wide loci for any species. This cost-effective, user-friendly, and powerful WGG tool, which we have named TAIL-PCR by sequencing (TAIL-peq), holds great potential for widespread application in breeding programs, thereby facilitating genome-assisted crop improvement.