Analysis of DNA sequence polymorphism at the cMWG699 locus reveals phylogenetic relationships and allopolyploidy within Hordeum murinum subspecies.

Hordeum murinum L. is one of the most widely distributed species in the genus Hordeum. This species is composed of three subspecies with three ploidy levels, namely subsp. glaucum (2x=14), subsp. murinum (4x=28) and subsp. leporinum (4x=28, 6x=42). These three subspecies are morphologically similar and are frequently referred to as the 'murinum complex'. Although many cytological studies suggest that the murinum complex is allopolyploid, one inter-specific hybridization study suggested that it is autopolyploid. The goals of the present study are to identify nucleotide variation in the cMWG699 locus in the polyploid genomes of the murinum complex, to conduct molecular phylogenetic analysis of this locus, and to clarify the allo- versus auto-polyploidy status of the murinum complex. For this purpose, PCR-RFLP analysis was conducted with HhaI and SspI restriction enzymes on 80 H. murinum accessions. Single enzyme digestion data revealed polymorphism between diploid and polyploids, and double-digestion revealed polymorphism between tetra- and hexaploids. The nucleotide sequences of clones clearly show that polyploid murinum species are allopolyploid. In addition, DNA sequence analysis indicated that one donor of the tetraploid was subsp. glaucum (2x), as has been suggested previously by cytological studies. The other diploid donors were not identified, but at least one group of sequences common to 4x and 6x genomes (namely clonetype B) was highly diverged from 2x subsp. glaucum. The two tetraploid subspecies, 4x subsp. murinum and 4x subsp. leporinum, had identical DNA sequences, suggesting that these two subspecies are not differentiated at the cMWG699 locus.