Targeted enrichment by solution-based hybrid capture to identify genetic sequence variants in barley

Abstract

In barley and other cereal crops, phenological diversity drives adaptation to different cultivation areas. Improvement of barley yield and quality traits requires adaptation to specific production areas with introgression of favorable alleles dependent upon precise identification of the underlying genes. Combining targeted sequence capture systems with next-generation sequencing provides an efficient approach to explore target genetic regions at high resolution, and allows rapid discovery of thousands of genetic polymorphisms. Here, we apply a versatile target-capture method to detect genome-wide polymorphisms in 174 flowering time-related genes, chosen based on prior knowledge from barley, rice, and Arabidopsis thaliana . Sequences were generated across a phenologically diverse panel of 895 barley varieties, resulting a high mean depth coverage of ~25x allowing reliable discovery and calling of insertion-deletion (InDel) and single nucleotide polymorphisms (SNPs). Sequences of InDel and SNPs from the targeted enrichment were utilized to develop 67 Kompetitive Allele Specific PCR (KASP) markers for validation. This work provides researchers and breeders a comprehensive molecular toolkit for the selection of phenology-related traits in barley.

A global barley panel revealing genomic signatures of breeding in modern cultivars

“…Next, we performed high-resolution GWAS analyses of agronomically relevant traits, and detected novel and highly significant MTAs for all three traits not detected in our previous study [10, 11]. For example, we detected novel and highly significant MTAs for plant height, located within the phenology genes HORVU3Hr1G021140 (gigantea protein GI), and HORVU3Hr1G022170 (homeobox-leucine zipper protein ROC4).…”

“…To examine the origins and patterns of genetic diversity within the currently available barley cultivar gene pool, we assembled a global panel of 632 genotypes to represent barley genotypes from major global barley breeding programmes, including both historical cultivars and modern cultivars from 43 countries (S1 Fig, S1 File). The panel of 632 genotypes geographically diverse barley cultivars was genotyped using target capture [10, 11], low-coverage whole-genome sequencing (WGS), and genotyping-by-sequencing (GBS) by Diversity Arrays Technology (DArTseq). In total, 15,328 single-nucleotide polymorphisms (SNPs) and insertions and deletions (InDels) were detected via low-coverage WGS, 4,260 SNPs via target-enrichment sequencing, and 18,551 SNPs via DArTseq were distributed across 5,171 barley genes.…”

“…Genomic DNA was extracted from the leaves of a single barley plant per variety using the cetyl-trimethyl-ammonium bromide (CTAB) method as previously described [10, 11]. DNA quality was assessed on 1% agarose gels and quantified using a NanoDrop spectrophotometer (Thermo Scientific NanoDrop Products, Wilmington, Delaware USA).…”

“…To assess the genetic diversity of phenology and phenology-related genes in the global collection of barley landraces and cultivars, we designed a custom target-enrichment sequencing assay for loci implicated in the flowering pathway in barley and related plant species, as previously described [10, 11]. In short, the target-enrichment sequencing of genomic DNA regions was performed by solution-based hybrid capture using a synthetic library consisting of 13,588 RNA probes (MYbaits, MYcroarray®, Ann Arbour, MI, USA) following the manufacturer’s protocol (v.2.3.1).…”

“…Sequence files were post-run filtered and aligned to the latest release of the barley reference genome assembly [7] (IBSC v2) using Nuclear software v.3.6.16 (GYDLE Inc., Montreal, Canada). SNP variant discovery and genotype calling were performed using custom Perl scripts to produce a variant call format (VCF) v.4.2 genotype file based on the alignment files as previously described [10, 11]. Only SNPs with <10% missing values and a MAF >1% (4,260 SNPs) were used for subsequent analyses.…”