A Sequence-Ready Physical Map of Barley Anchored Genetically by Two Million Single-Nucleotide Polymorphisms    

Abstract

Barley (Hordeum vulgare) is an important cereal crop and a model species for Triticeae genomics. To lay the foundation for hierarchical map-based sequencing, a genome-wide physical map of its large and complex 5.1 billion-bp genome was constructed by high-information content fingerprinting of almost 600,000 bacterial artificial chromosomes representing 14-fold haploid genome coverage. The resultant physical map comprises 9,265 contigs with a cumulative size of 4.9 Gb representing 96% of the physical length of the barley genome. The reliability of the map was verified through extensive genetic marker information and the analysis of topological networks of clone overlaps. A minimum tiling path of 66,772 minimally overlapping clones was defined that will serve as a template for hierarchical clone-by-clone map-based shotgun sequencing. We integrated whole-genome shotgun sequence data from the individuals of two mapping populations with published bacterial artificial chromosome survey sequence information to genetically anchor the physical map. This novel approach in combination with the comprehensive whole-genome shotgun sequence data sets allowed us to independently validate and improve a previously reported physical and genetic framework. The resources developed in this study will underpin fine-mapping and cloning of agronomically important genes and the assembly of a draft genome sequence.

A homolog of Blade-On-Petiole 1 and 2 (BOP1/2) controls internode length and homeotic changes of the barley inflorescence

“…Two additional features facilitated HvLax-a identification: the target gene was included in the target enrichment assay, and the mode of mutagenesis resulted in complete deletion of the functional gene. As the barley genome sequence was largely an unordered draft (Mayer et al, 2012;Ariyadasa et al, 2014), our initial genetic analysis revealed a large physical region of the chromosome represented by highly fragmented sequence information that required a substantial amount of further work to fully resolve. In the near future, the cloning of genes in the nonrecombining part of barley chromosomes will become much more efficient, given the impending release of a highly improved physical map-based reference sequence.…”

“…As the WGS contig bearing the candidate gene was not linked to the barley physical map, the candidate gene MLOC_61451.6 was used to PCR screen a barley BAC library, and four clones (HVVMRXALLeA0046E04, HVVMRXALLeA0122D17, HVVMRXALLeA0209O12, and HVVMRXALLeA0379H14), all belonging to the same BAC contig (FPC_2862) of the physical map (Ariyadasa et al, 2014), were identified. A minimum tiling path of overlapping nonredundant BAC clones, spanning the complete FPC_2862, was shotgun sequenced and de novo assembled, yielding 2.3 Mb of unique sequence (Supplemental Table S6).…”

Marker development using SLAF-seq and whole-genome shotgun strategy to fine-map the semi-dwarf gene ari-e in barley

“…The unexpected results might be due to the limited resolution of the current barley physical map while the non-coding region may play a key role in inducing semi-dwarfness. Owing to the large size of the barley genome (5.1 Gb) and the high repeat content (80 %), the current physical map comprises 9,265 contigs with a cumulative size of 4.9 Gb representing 96 % of the physical length of the barley genome [70]. Chromosomal assignments between the POPSEQ and IBSC maps agree in 97.6 % of the cases and discordant contig placements mostly occurred in the genetic centromere due to the severely reduced recombination frequency [55, 68].…”

“…Moreover, most of the map-based cloned barley genes have been located in the distal regions of the chromosome. Considering that ari-e occurred near the centromere of barley 5H [25], the repetitive nature of the barley genome [55, 70, 71] and limited resolution of the barley physical map, there may be other unanchored candidate genes for the ari-e semi-dwarf gene.…”

Identification of nuclear genes controlling chlorophyll synthesis in barley by RNA-seq

“…Availability of barley genome reference sequence [17] facilitates application of WGS-based approaches (such as RNA-seq) for investigating genetic networks responsible for phenotypic variation. RNA-seq approach has been already employed successfully to uncover the history of wild barley domestication and distribution [18], for studying the gene family of barley aquaporines [19] or creating SNP maps of barley [20]. …”