Single-Nucleotide Polymorphism Genotyping in Mapping Populations via Genomic Reduction and Next-Generation Sequencing: Proof of Concept

Abstract

We report the results of a proof-of-concept experiment that validates the use of multiplex identifier (MID) barcodes and next-generation sequencing (454-pyrosequencing and Illumina GAIIe) to simultaneously discover and genotype single nucleotide polymorphisms (SNPs) from mapping populations using pooled genomic reduction libraries. The genomic reduction library utilized here consisted of 60 individuals from an (L.) Heynh. mapping population. A total of 1720 and 504 SNPs were de novo identified and genotyped across the mapping population using only bioinformatic analyses of the progeny sequence fragments from the 454-pyrosequencing and Illumina datasets, respectively. The average base coverage at the SNPs was 4.5x for the 454-pyrosequencing dataset and 18.2x for the Illumina dataset. Cross validation of the genotypic scores between datasets showed 99.91% accuracy. Linkage mapping with the 454-pyrosequencing dataset produced five highly supported linkage groups that were collinear with the physical map ( = 0.981), further validating the accuracy of the genotyping method. The unadjusted cost per data point ranged from US$0.07 to $0.147, suggesting that the technique could be broadly used for large-scale genotyping and has particular value for plant species with limited monetary resources as it circumvents the need for post-SNP discovery genotype assay development.