Development of high-resolution multiple-SNP arrays for genetic analyses and molecular breeding through genotyping by target sequencing and liquid chip

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.

Keywords: GBTS; LD; genotyping by target sequencing; linkage disequilibrium; mSNPs; multiple single-nucleotide polymorphisms; multiplexing PCR; sequence capture in-solution (liquid chip).

Figure 1

Genotyping data generated for multiple mSNP panels. Missing rates and the sequencing quantities required to develop multiple mSNP panels with different numbers of target loci (1K to 40K) and their corresponding SNPs, calculated for both mSNP and SNP markers.

Figure 2

SNP markers developed from different genomic regions. Numbers and types (transitions versus transversions) of the 251K SNPs occurring in genomic regions (CDS, intronic, intergenic, introns, and 5′ and 3′ UTRs).

Figure 3

Distribution of frequencies for insertions and deletions. The frequency was calculated for each 1-Mb interval across the maize genome. From outside to inside: chromosomes and their centromeres, insertion, deletion. Units on the circumference are megabases with centromeres indicated by blue bars.

Figure 4

Marker characterization. Minor allele frequency (MAF) (A), polymorphic information content (PIC) (B), gene diversity (C), and observed heterozygosity (D) for 40K high-PIC SNPs and 251K SNPs as revealed in 867 maize inbred lines.

Figure 5

Evaluation of markers by linkage disequilibrium (LD) analysis. Average LD decay by marker panels for the combined germplasm sample (n=867) (A; top), maize germplasm groups (temperate, tropical, and sweet) (B (middle) for 40K high-PIC SNPs; C(bottom) for 251K SNPs), and also for markers from different genomic regions (intergenic SNPs, genic SNPs and total)

Figure 6

Evaluation of markers by genome-wide association study (GWAS). Genome-wide association scans for cob color as an example to test the power of different marker types in gene mapping using 867 maize inbred lines. Manhattan plots (upper) and corresponding QQ plots (lower) are provided for 40K random SNPs, 40K high-PIC SNPs, 251K SNPs, and 690K haplotypes.

Figure 7

Technical procedure for improvement of genotyping by target sequencing (GBTS). A chart for mSNP development through GBTS with highlighted technical improvements and optimization.