Simple methods for isolating homeologous loci from allopolyploid genomes

During allopolyploid formation, divergent sets of chromosomes are combined in a common nucleus. Accordingly, loci that are single-copy in progenitor diploid genomes become duplicated, homoeologous loci in allopolyploids. Although homoeologous loci have been identified using classical genetic and linkage mapping approaches, there are few examples of the isolation and molecular characterization of homoeologous loci from allopolyploids. Here we describe two methods for the isolation of orthologous duplicated loci and demonstrate the feasibility of the techniques using allotetraploid cotton. The methods utilize restriction-digested, size-fractionated genomic DNA as a template for either plasmid cloning or PCR amplification. This fractionation procedure, when combined with Southern hybridization and mapping information, can separate homoeologues from each other and from more divergent cross-hybridizing sequences (paralogues). Each homoeologue can be then be isolated from a pool of size-fractionated genomic DNA that is enriched for target loci. While these methods were specifically designed for isolating homoeologous loci from allotetraploids, they should be applicable to a broad spectrum of diploid and polyploid plants and be useful for studying both ancient and recent gene duplications. In addition, the procedures enrich for orthologous sequences, which can be used for phylogenetic analysis. Thus, a general approach is detailed for the isolation of nuclear genes for phylogeny reconstruction.