Development of Single Chromosome Genomics in Plants

Dissecting nuclear genomes to separate chromosomes can reduce DNA sample complexity, validate genome, assemblies and assess structural genome variation. Flow cytometric sorting is suitable for the purification of large numbers of chromosomes. However, a chromosome of interest must be discriminated from other chromosomes based on karyotype. This is not possible in many species, where some chromosomes can only be sorted into groups of two or more chromosomes. An alternative option is to isolate a single copy of a chromosome and avoid contamination with other chromosomes. With this in mind, we have developed a protocol for the production of microgram quantities of DNA from single copies of flow-sorted chromosomes. Chromosome DNA sequence coverage may be increased by merging sequences obtained from several separate isolations. Our results indicate that single chromosome sequencing is suitable to identify genic sequences on individual chromosomes, develop chromosome-specific DNA markers, verify assignment of DNA sequence contigs to individual pseudomolecules, and validate whole genome assemblies. Moreover, single chromosome sequencing provides opportunities to analyze chromosome structural heterozygosity and determine haplotype phase in plants. By overcoming the need to discriminate individual chromosomes, our approach greatly expands the potential of chromosome genomics, and the method can be applied to any plant species from which chromosome samples suitable for flow cytometry can be prepared.