Reduced-Cost Genotyping by Resequencing in Peanut Breeding Programs Using Tecan Allegro Targeted Resequencing V2.

The identification of informative molecular markers is useful for linkage mapping and can benefit genome-wide association studies by providing fine-scale information about sequence variations. However, high-throughput genotyping approaches are not cost-effective for labs that require frequent use, such as breeding programs that need to perform genotyping on large populations with hundreds of individuals. The number of single nucleotide polymorphism markers generated by those approaches can be far more than needed for most breeding programs; instead, breeders focus on the use of at most hundreds of polymorphic molecular markers for analysis. To help make use of molecular markers a routine tool for breeding programs, we aim to develop a cost-effective genotyping system by using the Tecan Allegro Targeted Resequencing V2 kit. This provides a customized probe design, which indicates that all the DNA fragments synthesized are known targets. SNPs obtained from previous peanut next-generation sequencing data were pre-filtered and selected as targets. These SNP targets were polymorphic among different tetraploid accessions and were selected to be distinguishable from paralogs. A total of 5154 probes were designed to detect 2770 SNP targets and were tested on 48 accessions, which include some closely related sister lines from a breeding population. The results indicated that genotyping by a targeted resequencing approach reduced the cost from around USD 28 (SNP chip and GBS) to USD 18 per sample, while providing polymorphic markers with accurate SNP calls. With this cost-effective genotyping platform, pre-selected SNP markers can be used effectively and routinely for more breeding programs.