Representative DNA Amplification from Single Plant Chromosomes

Genetic information of eukaryotes is divided into distinct subunits called chromosomes. This property can be exploited to reduce nuclear genome complexity and facilitate its analysis, mapping and sequencing. Current flow sorters may be used to isolate single chromosomes and nuclei into appropriate vessels. The minute amounts of DNA thus obtained, typically picograms DNA or less are too small for downstream analyses. Here we have optimized a protocol for amplification of DNA from single copies of plant chromosomes using multiple displacement amplification. The protocol utilizes high fidelity polymerase Phi29 to generate fragments exceeding 10 kb and produce microgram amounts of DNA. Although the chromosome sequence coverage is uneven, rendering the amplified DNA unsuitable for studies on CNV and repeat assembly, about 50% of gene loci are covered and their near complete sequences can be reconstructed. We also show that pooling technical replicates greatly improves chromosome coverage and gene representation. The amount and quality of DNA produced makes the method suitable for a variety of applications. The fact that only one particle is used as a template opens new opportunities for the application of chromosome genomics, such as the analysis of meiotic recombination, haplotyping and allele phasing, as well as the analysis of genomic heterogeneity at single cell level. Furthermore, this method enables obtaining chromosome-specific DNA in species in which separate chromosomes cannot be discriminated and purified. This work has been supported by the Czech Science Foundation (award P501/12/G090) and by the National Program of Sustainability I (award LO1204).