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Construction and Application for QTL Analysis of a Restriction Site Associated DNA (RAD) Linkage Map in Barley

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Construction and Application for QTL Analysis of a Restriction Site Associated DNA (RAD) Linkage Map in Barley

Yada Chutimanitsakun et al. BMC Genomics.

Abstract

Background: Linkage 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).

Results: Using 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.

Conclusions: We 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.

Figures

Figure 1
Figure 1
Segregation distortion on chromosome 2H linkage maps in the Oregon Wolfe Barley mapping population. The results of mapping with two different data sets are shown in A) the OWB-2383 map + 463 RAD loci, B) the 436 RAD and morphological marker loci and C) the OWB-2383 map. The X axis represents map distance in cM and the Y axis represent -log of the χ2 p-value for segregation distortion. A positive value means distortion in favor of OWB-D whereas a negative value means distortion in favor of OWB-R. Dashed lines represent significance thresholds at 0.05. Marker positions are represented as perpendicular lines to the X axis.
Figure 2
Figure 2
Macro-scale syntenic relationships between barley and Brachypodium revealed with sequenced RAD markers. RAD sequences anchored by linkage analysis are distributed across the seven Hordeum linkage groups. Alignments to orthologous sequence loci in Brachypodium are shown. Solid lines denote relationships supported by EST sequence comparison. Two dashed lines indicate sequence alignments that do not coincide with expected chromosomal relationships.

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