Abstract
BackgroundFlow cytometry facilitates sorting of single chromosomes and chromosome arms which can be used for targeted genome analysis. However, the recovery of microgram amounts of DNA needed for some assays requires sorting of millions of chromosomes which is laborious and time consuming. Yet, many genomic applications such as development of genetic maps or physical mapping do not require large DNA fragments. In such cases time-consuming de novo sorting can be minimized by utilizing whole-genome amplification.ResultsHere we report a protocol optimized in barley including amplification of DNA from only ten thousand chromosomes, which can be isolated in less than one hour. Flow-sorted chromosomes were treated with proteinase K and amplified using Phi29 multiple displacement amplification (MDA). Overnight amplification in a 20-microlitre reaction produced 3.7 – 5.7 micrograms DNA with a majority of products between 5 and 30 kb. To determine the purity of sorted fractions and potential amplification bias we used quantitative PCR for specific genes on each chromosome. To extend the analysis to a whole genome level we performed an oligonucleotide pool assay (OPA) for interrogation of 1524 loci, of which 1153 loci had known genetic map positions. Analysis of unamplified genomic DNA of barley cv. Akcent using this OPA resulted in 1426 markers with present calls. Comparison with three replicates of amplified genomic DNA revealed >99% concordance. DNA samples from amplified chromosome 1H and a fraction containing chromosomes 2H – 7H were examined. In addition to loci with known map positions, 349 loci with unknown map positions were included. Based on this analysis 40 new loci were mapped to 1H.ConclusionThe results indicate a significant potential of using this approach for physical mapping. Moreover, the study showed that multiple displacement amplification of flow-sorted chromosomes is highly efficient and representative which considerably expands the potential of chromosome flow sorting in plant genomics.
Highlights
Flow cytometry facilitates sorting of single chromosomes and chromosome arms which can be used for targeted genome analysis
Optimization of chromosome treatment for DNA amplification After DNA purification, 7 – 70 ng of chromosomal DNA were obtained from flow-sorted fractions, which were subsequently used for multiple displacement amplification
Genome wide survey of amplified barley DNA We previously developed an oligonucleotide pool assay (OPA) for interrogation of 1524 barley single nucleotide polymorphism (SNP) [36]
Summary
Flow cytometry facilitates sorting of single chromosomes and chromosome arms which can be used for targeted genome analysis. Many genomic applications such as development of genetic maps or physical mapping do not require large DNA fragments. In such cases time-consuming de novo sorting can be minimized by utilizing whole-genome amplification. The most attractive application has been the construction of chromosome- and chromosome arm-specific BAC libraries [17,18,19] Their availability greatly facilitates development of physical contig maps [20] and positional gene cloning [21] in species with complex genomes
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