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During allopolyploid formation two 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 many 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 strictly 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, effectively separates homoeologues from each other and from more divergent cross-hybridizing sequences (paralogues). Each homoeologue is thereby confidently isolated from diflferent pools of size-fractionated genomic DNA. While these methods were specifically designed for isolating orthologous duplicated 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 yield strictly orthologous sequences, which are required for phylogenetic analysis. Thus, a general approach is detailed for the isolation of nuclear genes for phylogeny reconstruction.

Molecular evolution at homoeologous loci in allotetraploid cotton (Malvaceae: Gossypium hirsutum L)