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 d iploid genomes become duplicated, homoeologous loci in allopolyploids. Alth ough homoeologous loci have been identified using classical genetic and lin kage mapping approaches, there are few examples of the isolation and molecu lar characterization of homoeologous loci from allopolyploids. Here we desc ribe two methods for the isolation of orthologous duplicated loci and demon strate the feasibility of the techniques using allotetraploid cotton. The m ethods utilize restriction-digested, size-fractionated genomic DNA as a tem plate for either plasmid cloning or PCR amplification. This fractionation p rocedure, when combined with Southern hybridization and mapping information , can separate homoeologues from each other and from more divergent cross-h ybridizing sequences (paralogues). Each homoeologue can be then be isolated from a pool of size-fractionated genomic DNA that is enriched for target l oci. While these methods were specifically designed for isolating homoeolog ous loci from allotetraploids, they should be applicable to a broad spectru m of diploid and polyploid plants and be useful for studying both ancient a nd recent gene duplications. In addition, the procedures enrich for ortholo gous sequences, which can be used for phylogenetic analysis. Thus, a genera l approach is detailed for the isolation of nuclear genes for phylogeny rec onstruction.