Development of High-Density Genetic Maps for Barley and Wheat Using a Novel Two-Enzyme Genotyping-by-Sequencing Approach

Abstract

Advancements in next-generation sequencing technology have enabled whole genome re-sequencing in many species providing unprecedented discovery and characterization of molecular polymorphisms. There are limitations, however, to next-generation sequencing approaches for species with large complex genomes such as barley and wheat. Genotyping-by-sequencing (GBS) has been developed as a tool for association studies and genomics-assisted breeding in a range of species including those with complex genomes. GBS uses restriction enzymes for targeted complexity reduction followed by multiplex sequencing to produce high-quality polymorphism data at a relatively low per sample cost. Here we present a GBS approach for species that currently lack a reference genome sequence. We developed a novel two-enzyme GBS protocol and genotyped bi-parental barley and wheat populations to develop a genetically anchored reference map of identified SNPs and tags. We were able to map over 34,000 SNPs and 240,000 tags onto the Oregon Wolfe Barley reference map, and 20,000 SNPs and 367,000 tags on the Synthetic W9784×Opata85 (SynOpDH) wheat reference map. To further evaluate GBS in wheat, we also constructed a de novo genetic map using only SNP markers from the GBS data. The GBS approach presented here provides a powerful method of developing high-density markers in species without a sequenced genome while providing valuable tools for anchoring and ordering physical maps and whole-genome shotgun sequence. Development of the sequenced reference genome(s) will in turn increase the utility of GBS data enabling physical mapping of genes and haplotype imputation of missing data. Finally, as a result of low per-sample costs, GBS will have broad application in genomics-assisted plant breeding programs.

Potential and limits to unravel the genetic architecture and predict the variation of Fusarium head blight resistance in European winter wheat (Triticum aestivum L.)

“…Marker systems that have been used for genome-wide fingerprinting in wheat include restriction fragment length polymorphism (Chao et al, 1989), amplified fragment length polymorphism (Barrett and Kidwell, 1998), simple sequence repeat (SSR) markers (Röder et al, 1998), diversity array technology (Akbari et al, 2006), singlenucleotide polymorphism (SNP) markers (Cavanagh et al, 2013), and most recently genotyping-by-sequencing approaches (Poland et al, 2012). When Heslot et al (2013) compared the accuracy of prediction of genomic selection based on genotyping-by-sequencing versus diversity array technology markers, they found advantages of genomic selection based on genotyping-by-sequencing data, which they interpreted to be mainly because of the higher number of markers.…”

Mapping resistance to the bird cherry-oat aphid and the greenbug in wheat using sequence-based genotyping

“…GBS in particular has proven to be very adequate for these tasks (Poland et al 2012;Saintenac et al 2013;Lu et al 2013). Here we constructed GBS libraries and employed QTL analyses in an F6 RIL population to map known and novel genomic regions associated to resistance to two aphid species in wheat.…”

“…DNA samples were pipetted in two 96-well plates of 72 and 70 samples each. Restriction, ligation and amplification processes were performed to construct the genomic libraries (Poland et al 2012;Saintenac et al 2013). Restriction of DNA was made with a restriction mix that consisted of 20µl of each sample, 3.0µl of NEB T4 DNA ligase buffer 3, 0.5µl of PstI (10 units), 1 µl of MseI (10 units) and 5.5µl of H 2 O. Ligation was performed by adding to the restriction digest (30µl) of each sample, 3µl of the unique barcode adapter, 8µl of the Y-adapter and 9µl of ligation NEB Master Mix (2 µl of T4 DNA ligase buffer, 0.5µl T4 DNA ligase and 6.5µl water).…”

“…Genotyping by sequencing (GBS) is one of the applications. In the development of GBS libraries the genome complexity is reduced by the use of methylation-sensitive restriction enzymes and in combination with multiplex sequencing it is possible to genotype entire populations efficiently (Elshire et al 2011;Poland et al 2012). This is particularly useful for species with large genomes such as wheat, where sequencing needs to target non-duplicated regions of the genome in order to produce high quality maps (Spindel et al 2013).…”

Breeding rootstocks for Prunus species: Advances in genetic and genomics of peach and cherry as a model

“…A number of methods to identify SNPs have been described (Ganal et al, 2009): by searching expressed sequence tag (EST) databases (Batley et al, 2003), amplicon re-sequencing (Choi et al, 2007), complete sequence of a genome (Velasco et al, 2007), and more recently, high throughput sequencing technology (Barbazuk et al, 2007). Advances in next-generation techniques have reduced the cost of DNA sequencing to the point where it is now feasible to perform genotypingby-sequencing (GBS) to analyze small-and large-sized genomes with high diversity and allow the identification of thousands of markers for a species (Elshire et al, 2011;Poland et al, 2012;Ward et al, 2013;Guajardo et al, 2015). Future applications of GBS in genetic improvement will allow breeders to perform genomic selection of new germplasm or species without having to first develop a molecular tool, which is needed with other types of molecular markers; it will also allow conservation biologists to determine population structure without needing previous knowledge of the genome or diversity of the species under study (Elshire et al, 2011).…”