Construction and application for QTL analysis of a Restriction Site Associated DNA (RAD) linkage map in barley

Abstract

BackgroundLinkage maps are an integral resource for dissection of complex genetic traits in plant and animal species. Canonical map construction follows a well-established workflow: an initial discovery phase where genetic markers are mined from a small pool of individuals, followed by genotyping of selected mapping populations using sets of marker panels. A newly developed sequence-based marker technology, Restriction site Associated DNA (RAD), enables synchronous single nucleotide polymorphism (SNP) marker discovery and genotyping using massively parallel sequencing. The objective of this research was to assess the utility of RAD markers for linkage map construction, employing barley as a model system. Using the published high density EST-based SNP map in the Oregon Wolfe Barley (OWB) mapping population as a reference, we created a RAD map using a limited set of prior markers to establish linakge group identity, integrated the RAD and prior data, and used both maps for detection of quantitative trait loci (QTL).ResultsUsing the RAD protocol in tandem with the Illumina sequence by synthesis platform, a total of 530 SNP markers were identified from initial scans of the OWB parental inbred lines - the "dominant" and "recessive" marker stocks - and scored in a 93 member doubled haploid (DH) mapping population. RAD sequence data from the structured population was converted into allele genotypes from which a genetic map was constructed. The assembled RAD-only map consists of 445 markers with an average interval length of 5 cM, while an integrated map includes 463 RAD loci and 2383 prior markers. Sequenced RAD markers are distributed across all seven chromosomes, with polymorphic loci emanating from both coding and noncoding regions in the Hordeum genome. Total map lengths are comparable and the order of common markers is identical in both maps. The same large-effect QTL for reproductive fitness traits were detected with both maps and the majority of these QTL were coincident with a dwarfing gene (ZEO) and the VRS1 gene, which determines the two-row and six-row germplasm groups of barley.ConclusionsWe demonstrate how sequenced RAD markers can be leveraged to produce high quality linkage maps for detection of single gene loci and QTLs. By combining SNP discovery and genotyping into parallel sequencing events, RAD markers should be a useful molecular breeding tool for a range of crop species. Expected improvements in cost and throughput of second and third-generation sequencing technologies will enable more powerful applications of the sequenced RAD marker system, including improvements in de novo genome assembly, development of ultra-high density genetic maps and association mapping.

Genome sequence of the cultivated cotton Gossypium arboreum

“…Young leaves of parents and individuals from the F 2 population were collected for DNA extraction using the Plant Genomics DNA kit (Qiagen). Genomic DNA from this mapping population was digested with the restriction endonuclease EcoRI and processed into RAD libraries 38 . Every 20 cotton individuals were pooled into 1 sequencing library with nucleotide multiplex identifiers (4, 6 and 8 bp), and each sequencing library was barcoded.…”

High-resolution skim genotyping by sequencing reveals the distribution of crossovers and gene conversions in Cicer arietinum and Brassica napus

“…In eggplant, RADsequencing was able to call 10,000 SNPs, 1,600 InDels and 1,800 putative SSRs (Barchi et al, 2011). In Hordeum vulgare RAD sequencing was applied to identify 530 SNPs of which 445 were used to generate a genetic map (Chutimanitsakun et al, 2011 Recombination is the major source of genetic variation, through the shuffling of sets of genes to produce novel allelic combinations. Both crossover and noncrossover based recombination is observed in crop genomes and both are initiated by double strand breaks (DSBs) during meiosis.…”

Next-generation hybridization and introgression

“…RAD markers are short sequences of DNA adjacent to a restriction enzyme recognition site, in which SNPs are compared between individuals (full method in Miller et al, 2007b, and summarized in Figure 2c). This technique has been used in various organisms such as Drosophila (Miller et al, 2007b), Neurospora (Baird et al, 2008), the pitcher plant mosquito (Emerson et al, 2010), zebrafish (Miller et al, 2007a), the three spine stickleback (Miller et al, 2007b), trout (Hohenlohe et al, 2011) and barley (Chutimanitsakun et al, 2011). This approach sequences a subset of the genome to reduce costs and uses barcodes for individual samples to allow bulk sequencing.…”

“…These methods harvest NGS technologies in the most effective manner, using them for both polymorphic marker design and automated high-throughput genotyping (Miller et al, 2007b). RAD tag sequencing is one example that satisfies the target of sampling many individuals for a large number of loci, and the recent application of RAD tags in the complex genome of barley indicates this method can be successfully applied to repetitive genomes (Chutimanitsakun et al, 2011). Alternative approaches to genome-wide sampling allow important sequence variation to be amplified in a more targeted manner.…”