Tunable Genotyping-By-Sequencing (tGBS®) Enables Reliable Genotyping of Heterozygous Loci

Abstract

Most Genotyping-by-Sequencing (GBS) strategies suffer from high rates of missing data and high error rates, particularly at heterozygous sites. Tunable genotyping-by-sequencing (tGBS®), a novel genome reduction method, consists of the ligation of single-strand oligos to restriction enzyme fragments. DNA barcodes are added during PCR amplification; additional (selective) nucleotides included at the 3'-end of the PCR primers result in more genome reduction as compared to conventional GBS methods. By adjusting the number of selective bases different numbers of genomic sites can be targeted for sequencing. Because this genome reduction strategy concentrates sequencing reads on fewer sites, SNP calls are based on more reads than conventional GBS, resulting in higher SNP calling accuracy (>97-99%) even for heterozygous sites and less missing data per marker. tGBS genotyping is expected to be particularly useful for genomic selection, which requires the ability to genotype populations of individuals that are heterozygous at many loci.

Parallels between natural selection in the cold‐adapted crop‐wild relative Tripsacum dactyloides and artificial selection in temperate adapted maize

“…In total, 47 tropical and subtropical lines (TSS) and 46 temperate lines (NSS and SS) were selected from the Hapmap3 dataset (Bukowski et al ., ), based on having a probability value of membership into either of these groups greater than 0.8 (Table S8; Olukolu et al ., ). Based on the recombination rates measured using high‐density genetic maps in maize (Ott et al ., ), XP‐CLR was run with the following parameters: sliding window size of 0.05 cM; a fixed number of SNPs per window of 100; downweighting of SNPs in high LD ( r 2 > 0.75) and a set of grid points as the putative selected allele positions were placed with a spacing of 1 kb across the whole genome. Each gene was assigned the maximum XP‐CLR values found within the region 5 kb upstream and downstream of the gene's annotation transcription start and transcription stop site.…”

Increased Power and Accuracy of Causal Locus Identification in Time Series Genome-wide Association in Sorghum

“…DNA was extracted from seedling-stage plants grown in the Beadle Center Greenhouse complex at University of Nebraska-Lincoln using the same seed employed for phenotyping. Sequencing libraries generated using a modified tGBS (tuneable-Genotyping-By-Sequencing) protocol (Ott et al, 2017) were constructed and paired-end (151-bp read length) sequenced in eight lanes of an Illumina HiSeqX instrument. This produced an average of 4 million paired-end reads per sample.…”

Largely unlinked gene sets targeted by selection for domestication syndrome phenotypes in maize and sorghum

“…Genetic maps for both species were sourced from public datasets. For maize, a genetic map was employed that included 10,085 markers genotyped in a set of 232 RILs from the maize IBM population using tGBS 44 while for sorghum a genetic map was employed which was constructed from a set of 3,418 markers genotyped in a set of 244 RILs from a grain sorghum x sweet sorghum cross using resequencing 45 .…”

“…Recombination rates in maize and sorghum were measured using high density genetic maps in maize 44 and sorghum 45 . Genetic maps were transfered to the more recent versions of the maize and sorghum genomes used in this analysis (B73 RefGen v3 and v3.1 respectively).…”