Development of improved Eucalyptus genotypes involves the routine identification of breeding stock and superior clones. Currently, microsatellites and random amplified polymorphic DNA markers are the most widely used DNA-based techniques for fingerprinting of these trees. While these techniques have provided rapid and powerful fingerprinting assays, they are constrained by their reliance on gel or capillary electrophoresis, and therefore, relatively low throughput of fragment analysis.
Diversity Arrays Technology (DArT) can detect and type DNA variation at several hundred genomic loci in parallel without relying on sequence information. Here we show that it can be effectively applied to genetic mapping and diversity analyses of barley, a species with a 5,000-Mbp genome. We tested several complexity reduction methods and selected two that generated the most polymorphic genomic representations. Arrays containing individual fragments from these representations generated DArT fingerprints with a genotype call rate of 98.0% and a scoring reproducibility of at least 99.8%. The fingerprints grouped barley lines according to known genetic relationships. To validate the Mendelian behavior of DArT markers, we constructed a genetic map for a cross between cultivars Steptoe and Morex. Nearly all polymorphic array features could be incorporated into one of seven linkage groups (98.8%). The resulting map comprised ≈385 unique DArT markers and spanned 1,137 centimorgans. A comparison with the restriction fragment length polymorphism-based framework map indicated that the quality of the DArT map was equivalent, if not superior, to that of the framework map. These results highlight the potential of DArT as a generic technique for genome profiling in the context of molecular breeding and genomics.
Although 50 years have passed since the structure of DNA was deciphered (1), the study of DNA variation emerged as a field of scientific endeavor only in the last 25 years. Two groups of technologies were developing in parallel from the very beginning: DNA sequencing and molecular markers. DNA sequencing technology developed quickly from proof of concept (2, 3) to an automated process (4), enabling the field of genomics. Molecular marker technologies progressed rapidly as well. Based on the Southern blot technique (5), Botstein et al. (6) developed the restriction fragment length polymorphism (RFLP) technique as a method for creating genetic linkage maps.
Development of the PCR technique spawned two important molecular marker techniques: amplified fragment length polymorphism (AFLP) (7) and simple sequence repeats (8). Thousands of studies using molecular markers in plants, including hundreds in barley, have been published but are not referenced because of space limitations.
DNA sequencing and molecular marker technologies started to merge when the accumulated sequence data began to yield information on sequence variation among different accessions of the same species. It was soon noted that single-nucleotide polymorphism (SNP) is the most abundant marker type, promising nearly unlimited supply of markers (9). Many alternatives were developed for the SNP assay (primer extension, selective ligation) and the platform to type assays in high throughput (DNA chip, printed and self-assembling arrays, matrix-assisted laser desorption ionization/time-of-flight mass spectroscopy) (10–14).
For humans and a limited number of model organisms, the throughput of SNP assays has increased impressively, and assay costs have decreased correspondingly. Yet discovering sequence polymorphism in nonmodel species is difficult, which is particularly true for many crops with limited resources and often complex, polyploid genomes. We have developed Diversity Arrays Technology (DArT) to enable whole-genome profiling of such crops without the need of sequence information. DArT is based on microarray hybridizations that detect the presence versus absence of individual fragments in genomic representations as described by Jaccoud et al. (15).
For our initial proof-of-concept study, we selected a species with a simple genome (rice) and used AFLP-like complexity reduction methods to generate genomic representations (15). Here we apply a non-AFLP version of DArT to barley, a species with a complex genome nearly twice as large as the human genome and 13 times larger than that of rice (16). We show that DArT can be used to effectively create a medium-density genetic map, a result that points to its potential as a generic technique for high-throughput genome profiling of plants.