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

Jesse A Poland,Patrick J Brown,Mark E Sorrells,Jean-Luc Jannink,Tongming Yin

doi:10.1371/journal.pone.0032253

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.

Highlights

The development of molecular markers and genomic resources in barley and wheat has always been a formidable task due the massive, complex, and, in the case of wheat, polyploid genomes [1,2,3]
We developed a modified GBS approach using two enzymes and a Y-adapter to generate ‘‘uniform’’ GBS libraries where Adapter 1 and Adapter 2 are on opposite ends of every fragment
To utilize PstI as a rare-cutting restriction enzyme we first developed a set of GBS barcodes compatible with the PstI restriction site and overhang

Summary

Introduction

The development of molecular markers and genomic resources in barley and wheat has always been a formidable task due the massive, complex, and, in the case of wheat, polyploid genomes [1,2,3]. Genotyping-by-sequencing (GBS) was developed as a simple but robust approach for complexity reduction in large complex genomes [19] Both RAD sequencing and GBS target the genomic sequence flanking restriction enzyme sites to produce a reduced representation of the genome. When combined with Yadapters for the common restriction site, the use of two enzymes differs from the original GBS protocol in that amplified fragments in the two-enzyme libraries will all consist of the barcoded forward adapter and the common reverse adapter. This type of library construction greatly simplifies quantification of the library prior to sequencing. A form of this complexity reduction approach has been successfully applied in sequencing pools of BAC libraries for construction of physical maps [20]

Methods

Results

Conclusion