Mapping-by-sequencing accelerates forward genetics in barley

Mascher et al. Genome Biology Mapping-by-sequencing accelerates forward genetics in barley Martin Mascher 0 Matthias Jost 0 Joel-Elias Kuon Axel Himmelbach Axel Afalg Sebastian Beier Uwe Scholz Andreas Graner Nils Stein 0 Equal contributors Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), OT Gatersleben , Corrensstrae 3, 06466 Stadt Seeland , Germany Mapping-by-sequencing has emerged as a powerful technique for genetic mapping in several plant and animal species. As this resequencing-based method requires a reference genome, its application to complex plant genomes with incomplete and fragmented sequence resources remains challenging. We perform exome sequencing of phenotypic bulks of a mapping population of barley segregating for a mutant phenotype that increases the rate of leaf initiation. Read depth analysis identifies a candidate gene, which is confirmed by the analysis of independent mutant alleles. Our method illustrates how the genomic resources of barley together with exome resequencing can underpin mapping-by-sequencing. - Background The recent profound transformation of molecular biology by next-generation sequencing (NGS) technologies [1] and the ready availability of reference genome sequences [2] has enriched the plant geneticists toolbox with what Schneeberger and Weigel named fast-forward genetics [3]. Combining classical bulked-segregant analysis [4] with aligning NGS read data to a reference genome has made gene cloning essentially a single-step computational procedure once a mapping population has been established [5]. Within a few days time, mapping intervals can be delineated in silico and mined for likely candidate genes, deprecating marker saturation, and physical mapping of the target interval. Since its original implementation as ShoreMap in an F2 population of Arabidopsis thaliana, mapping-by-sequencing has been extended to other population types such as isogenic backcross populations [6,7] as well as to other plant and animal species such as rice [8], maize [9], mouse, and zebrafish [10]. All successful attempts at mapping-by-sequencing in these species could take advantage of high-quality mapbased reference sequences. A reference genome embeds almost all genes of a species in a genomic context, a crucial prerequisite for mapping-by-sequencing, as sequencing of phenotypic bulks provides only allele frequencies at variant positions, but no genotypic data that could be used to construct a genetic map de novo to infer marker order. How this order can be derived in the absence of a reference genome and how rapid NGS-based gene isolation may be implemented in species for which only draft genome assemblies are available is not obvious. Galvao et al. [11] have proposed the collinear gene order in related species as a proxy for gene order in species without a reference genomes, but have also noted that this synteny-based approach may adversely affect mapping resolution. A novel bioinformatical procedure to find causal mutations by whole genome sequencing without using positional information has been applied to find causal variants in plant species with small genomes [12]. In addition to its importance for agriculture, barley (Hordeum vulgare L.) has been a model organism of genetics throughout the 20th century and boasts excellent resources for forward genetics. A large number of barley mutants had been created from the 1940s to the 1970s when mutation breeding programs flourished [13-16]. These mutant lines have been classified phenotypically and are nowadays maintained and distributed by seed banks. To further support the utilization of these resources in research and breeding, 881 original mutants have been backcrossed to cultivar (cv.) Bowman as a recurrent parent to obtain mutant alleles in a nearly isogenic background. Array-based genotyping of these introgression lines confirmed and broadly delimited introgression intervals [17]. This legacy of half a century of meticulous research has been recently complemented by several mutant populations [18,19] that were obtained in a systemic way via mutagenesis with ethyl methanesulfonate (EMS) to empower reverse genetics. In this regard, the mutants of barley have been instrumental in confirming candidate genes discovered through mapping in bi-parental populations [20] or association panels [21]. However, the full exploitation of the allelic diversity captured in these resources for basic research and crop improvement has been impeded by the lack of a reference genome sequence of barley. The major obstacles in assembling the barley genome are its sheer size (5 Gb) and its high content of repetitive DNA (80%), which pose a heavy sequencing load and put a challenge for current assembly algorithms [22]. Boosted by the enormous increase in sequencing throughput, extensive sequence datasets have accumulated recently and have been integrated with a genome-wide physical map and high-density genetic maps [23]. A large fraction of low-copy portion of the barley genome is now represented by contigs of a whole-genome shotgun assembly which are positioned with a resolution of approximately 3 cM [24]. Moreover, an exome capture assay designed on the basis of the annotated sequence assembly has made approximately 60 Mb of mRNA-coding sequence accessible to cost-efficient high-throughput resequencing [25]. To date, the complex sequence framework of barley has not been used as a backbone for mapping-bysequencing. Though the hopes are high, concerns remain that the fragmentary and incompletely ordered structure of the sequence assembly and the only partial representation of the gene complement may stall fastforward genetics. Leveraging the physically and genetically anchored sequence assembly, exome sequencing and the extensive mutant collections available to the barley research community, we put mapping-by-sequencing to the test in barley and were able to rapidly identify a gene underlying the many-noded dwarf (mnd) phenotype. Results mnd mutant phenotype The original mnd mutant was generated by X-ray mutagenesis at our institute in the 1950s [13]. The most conspicuous characteristic of mnd plants is their shortened plastochron, that is, a faster rate of leaf initiation. Mutants have on average two times more leaves than wildtype plants as a result of a faster emergence of leaves (Figure 1). Moreover, culm internode lengths are decreased in the mutant. Despite the larger number of internode (eight to nine in the mutant versus four to five in the wildtype), plant height is reduced by about one third under field conditions, but not in the greenhouse (Figure 1d). Apart from spacing, also the shape of leaves is altered in the mutant: leaves are narrower and more erect compared to the wildtype. Additional characteristics of mnd are an increased number of tillers (vegetative shoot branches arising from lateral meristems) and shorter spikes (Figure 1b; Additional file 1: Figure S1). Allele freq (...truncated)