Genomic Characterization of DArT Markers Based on High-Density Linkage Analysis and Physical Mapping to the Eucalyptus Genome

Abstract

Diversity Arrays Technology (DArT) provides a robust, high throughput, cost-effective method to query thousands of sequence polymorphisms in a single assay. Despite the extensive use of this genotyping platform for numerous plant species, little is known regarding the sequence attributes and genome-wide distribution of DArT markers. We investigated the genomic properties of the 7,680 DArT marker probes of a Eucalyptus array, by sequencing them, constructing a high density linkage map and carrying out detailed physical mapping analyses to the Eucalyptus grandis reference genome. A consensus linkage map with 2,274 DArT markers anchored to 210 microsatellites and a framework map, with improved support for ordering, displayed extensive collinearity with the genome sequence. Only 1.4 Mbp of the 75 Mbp of still unplaced scaffold sequence was captured by 45 linkage mapped but physically unaligned markers to the 11 main Eucalyptus pseudochromosomes, providing compelling evidence for the quality and completeness of the current Eucalyptus genome assembly. A highly significant correspondence was found between the locations of DArT markers and predicted gene models, while most of the 89 DArT probes unaligned to the genome correspond to sequences likely absent in E. grandis, consistent with the pan-genomic feature of this multi-Eucalyptus species DArT array. These comprehensive linkage-to-physical mapping analyses provide novel data regarding the genomic attributes of DArT markers in plant genomes in general and for Eucalyptus in particular. DArT markers preferentially target the gene space and display a largely homogeneous distribution across the genome, thereby providing superb coverage for mapping and genome-wide applications in breeding and diversity studies. Data reported on these ubiquitous properties of DArT markers will be particularly valuable to researchers working on less-studied crop species who already count on DArT genotyping arrays but for which no reference genome is yet available to allow such detailed characterization.

DArT Markers Effectively Target Gene Space in the Rye Genome

“…The preferential sampling of gene-rich regions is achieved through the use of methylation sensitive restriction enzymes in the genome complexity reduction stage, which is a crucial element of the DArT protocol. DArT genotyping panels had been established for a considerable number of species, including orphan/resource poor crops (Gupta et al, 2008; Petroli et al, 2012 for a reference list). To date sequence information was obtained for sets of selected DArT markers from a few genotyping panels.…”

“…To date sequence information was obtained for sets of selected DArT markers from a few genotyping panels. The species in question include Eucalyptus (Petroli et al, 2012), oats (Tinker et al, 2009), sugarcane (Aitken et al, 2014), wheat (Marone et al, 2012), apple (Schouten et al, 2012), and potato (Traini et al, 2013). This is the first report concerning rye DArT marker sequences.…”

“…Sequence information for DArT markers of interest can be thus easily obtained through sequencing of amplified inserts. As demonstrated by several studies, this sequence knowledge can in turn can provide basis for the attribution of functional meaning to those markers (Tinker et al, 2009; Marone et al, 2012; Petroli et al, 2012; Aitken et al, 2014), and for candidate gene identification.…”

Climate adaptation and ecological restoration in eucalypts

“…Our genomic studies have predominantly employed genome-wide scans using molecular markers such as DArtSeq (Sansaloni et al 2011;Petroli et al 2012). These use 'genomic representation' approaches that utilise restriction enzymes to produce DNA fragments, which are subsequently sequenced using next-generation platforms to produce thousands of markers from across the genome.…”

Genetic and Genomic Resources of Small Millets

“…The genome sequencing in finger millet is a distant reality at the moment because of the complex polyploid genome. However, the NGS-based genome sequencing techniques, such as restriction site associated DNA sequencing (RAD-seq), GBS, and Diversity Arrays Technology (DArT), which have potentially led to discovery of SNP markers and genetic analysis even in non-model crop plants (Wenzl et al, 2004;Davey et al, 2011;Etter et al, 2011;Elshire et al, 2011;Kilian et al, 2012;Petroli et al, 2012), may be adopted as alternative approaches. These techniques are very robust with the power of multiplexing large number of samples at less sample cost.…”