An evaluation of genotyping by sequencing (GBS) to map the Breviaristatum-e (ari-e) locus in cultivated barley

Hui Liu 0 4 Micha Bayer 0 Arnis Druka 0 Joanne R Russell 0 Christine A Hackett 3 Jesse Poland 2 Luke Ramsay 0 Pete E Hedley 0 Robbie Waugh 0 1 0 Cell and Molecular Sciences, The James Hutton Institute, Invergowrie , Dundee, Scotland DD2 5DA , UK 1 Division of Plant Sciences, James Hutton Institute , Invergowrie , The University of Dundee. College of Life Sciences , Dundee, Scotland DD2 5DA , UK 2 Hard Winter Wheat Genetics Research Unit, USDA-ARS and Department of Agronomy, Kansas State University , 4011 Throckmorton, Manhattan, KS 66506 , USA 3 Biomathematics and Statistics Scotland (BioSS), Invergowrie , Dundee, Scotland DD2 5DA , UK 4 Biomedical Sciences Research Complex, University of St Andrews , North Haugh, St Andrews, Scotland KY16 9ST , UK We explored the use of genotyping by sequencing (GBS) on a recombinant inbred line population (GPMx) derived from a cross between the two-rowed barley cultivar 'Golden Promise' (ari-e.GP/Vrs1) and the six-rowed cultivar 'Morex' (Ari-e/vrs1) to map plant height. We identified three Quantitative Trait Loci (QTL), the first in a region encompassing the spike architecture gene Vrs1 on chromosome 2H, the second in an uncharacterised centromeric region on chromosome 3H, and the third in a region of chromosome 5H coinciding with the previously described dwarfing gene Breviaristatum-e (Ari-e). Background: Barley cultivars in North-western Europe largely contain either of two dwarfing genes; Denso on chromosome 3H, a presumed ortholog of the rice green revolution gene OsSd1, or Breviaristatum-e (ari-e) on chromosome 5H. A recessive mutant allele of the latter gene, ari-e.GP, was introduced into cultivation via the cv. 'Golden Promise' that was a favourite of the Scottish malt whisky industry for many years and is still used in agriculture today. Results: Using GBS mapping data and phenotypic measurements we show that ari-e.GP maps to a small genetic interval on chromosome 5H and that alternative alleles at a region encompassing Vrs1 on 2H along with a region on chromosome 3H also influence plant height. The location of Ari-e is supported by analysis of near-isogenic lines containing different ari-e alleles. We explored use of the GBS to populate the region with sequence contigs from the recently released physically and genetically integrated barley genome sequence assembly as a step towards Ari-e gene identification. Conclusions: GBS was an effective and relatively low-cost approach to rapidly construct a genetic map of the GPMx population that was suitable for genetic analysis of row type and height traits, allowing us to precisely position ari-e.GP on chromosome 5H. Mapping resolution was lower than we anticipated. We found the GBS data more complex to analyse than other data types but it did directly provide linked SNP markers for subsequent higher resolution genetic analysis. - Background Barley (Hordeum vulgare L.) is a diploid (2n = 14) economically important cereal crop and genetic model for small grain temperate cereals. Golden Promise (GP) is a two-rowed UK spring barley cultivar, and is currently the most responsive genotype for barley genetic transformation. Also, because of its unique properties, the malt extracted from GP is used to distil a number of signature Single Malt Scotch whiskies such as Macallan and Glengoyne. It is a primary induced gamma-ray mutant derivative of the barley cultivar Maythorpe, and is known to contain a mutation in Breviaristatum-e (Ari-e). This mutation in Ari-e in GP (ari-e.GP, also referred to in the literature as GP erectoides) causes a semidwarfing phenotype that has been used widely in barley cultivar development (especially in Scotland) to shorten straw length and reduce the severity of lodging. GP is also susceptible to several fungal pathogens, has short awns (as well as being dwarf ), reduced internode length and shows a measure of tolerance to salt [1]. Genetic analysis has previously located ari-e.GP to barley chromosome 5H as a quantitative trait locus (QTL) influencing plant height, and physiological studies have confirmed its relative insensitivity to the addition of exogenous gibberellic acid (GA3) [2]. The Ari-e gene has not yet been cloned although it was recently mapped as a height QTL using the tools of contemporary biometrical genetics in a complex three-way cross [3]. Over the past two decades, many molecular tools have been developed in barley to enable genetic research [4-9]. The primary focus has been the construction of molecular marker-based genetic linkage maps that can be leveraged for mapping genes of interest and subsequent marker assisted selection in breeding programs. These have been applied to discover, dissect and manipulate genes determining a range of simple and complex traits. Because of their value, accompanied by their increasing use in genetics and breeding, there has been a continual drive to both reduce marker costs and to avoid ascertainment issues [10] while at the same time enhancing flexibility and marker throughput per assay. It is therefore appropriate that new developments in marker technology are both explored and thoroughly evaluated against the current state of the art. Now that next generation sequencing (NGS) technology has been shown to be capable of discovering and genotyping thousands of markers across almost any genome of interest at low cost and in a single step, a current debate is whether sequence-based genotyping methods are ready to replace many of the established and widely used tools such as highly-multiplex Single Nucleotide Polymorphism (SNP) platforms [9]. Available sequence-based genotyping methods generally rely upon the use of restriction enzymes to produce a reduced representation of the non-repetitive (low copy) regions of the genome. Restriction site-associated genomic DNA (RAD) typing is such an approach and has been used in several species for the construction of linkage maps and application in QTL analyses [11]. In barley, a RAD linkage map was recently produced in a double haploid population and used for QTL analysis [12]. Elshire and colleagues [13] subsequently described a similar but more straightforward method of genotyping by sequencing (GBS) which works effectively in 96-well (or higher) plate assays. GBS was originally developed for high-resolution association studies in maize [14] and, like RAD, has been extended to a range of species with complex genomes. A two-enzyme GBS protocol has now been developed that produces a uniform library for sequencing and has been applied to both wheat and barley [15]. This GBS approach has been shown to be suited to genetic analysis of rapeseed, lupin, lettuce, switchgrass, soybean, and maize [16-20]. In this report, our biological objective was to identify at high resolution the genetic location of the ari-e.GP semi-dwarfing gene of cultivated barley. However, as a sequence assembly of the barley genome has just been published [21,22], we also wanted to use a seq (...truncated)