ABSTRACT: Genotyping platforms, as critical supports for genomics, genetics, and molecular breeding, have been well implemented at national institutions/universities in developed countries and multinational seed companies that possess high-throughput, automatic, large-scale, and shared facilities. In this study, we integrated an improved genotyping by target sequencing (GBTS) system with capture-in-solution (liquid chip) technology to develop a multiple single-nucleotide polymorphism (mSNP) approach in which mSNPs can be captured from a single amplicon. From one 40K maize mSNP panel, we developed three types of markers (40K mSNPs, 251K SNPs, and 690K haplotypes), and generated multiple panels with various marker densities (1K–40K mSNPs) by sequencing at different depths. Comparative genetic diversity analysis was performed with genic versus intergenic markers and di-allelic SNPs versus non-typical SNPs. Compared with the one-amplicon-one-SNP system, mSNPs and within-mSNP haplotypes are more powerful for genetic diversity detection, linkage disequilibrium decay analysis, and genome-wide association studies. The technologies, protocols, and application scenarios developed for maize in this study will serve as a model for the development of mSNP arrays and highly efficient GBTS systems in animals, plants, and microorganisms. This study reports the development of high-resolution multiple-SNP (mSNP) arrays, which can capture multiple SNPs from single amplicons, through integrating an improved genotyping by target sequencing approach with liquid chip technology. From a 40K maize mSNP panel, multiple panels with 1K–40K mSNPs (including up to 251K SNPs and 690 haplotypes) were generated by sequencing at different depths, then tested in various genetic analyses using a large number of maize inbred lines. The technical protocol can be widely used in other organisms.
