SNP Discovery via Genomic Reduction, Barcoding, and 454-Pyrosequencing in Amaranth

The grain amaranths are important pseudo-cereals native to the New World. During the last decade they have garnered increased international attention for their nutritional quality, tolerance to abiotic stress, and importance as a symbol of indigenous cultures. We report the development of a novel genomic reduction protocol based on restriction-site conservation and multiplex identifiers (MID) barcodes to discover single nucleotide polymorphisms (SNPs) from a pooled amaranth library of four mapping parents. The incorporation of MID barcodes allowed for DNA sample pooling, sequence deconvolution, and the identifi cation of SNPs for specific mapping populations without additional genotyping. Approximately 1.3 million sequence-reads with an average read length of 440 bp were collected from a single 454-pyrosequencing run. Contigs specific to each of the four mapping populations were assembled. The assembled contigs had an average read-length of 464 bp, and an average read-depth of 16X. A total of 27,658 SNPs were observed across all populations. The average base coverage at all SNPs was 20X. Thirty-four of 35 (97%) predicted SNPs were verified via resequencing and the random genomic distribution of the SNPs generated using this approach was shown in Arabidopsis. The method does not require a priori sequence information and should be readily adaptable to other species.