Molecular Phylogenetics of the Wild Perennial Relatives of Soybean (Glycine Subgenus Glycine): Intron-Containing Nuclear Genes

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9614984 Doyle The wild perennial species of Glycine subgenus Glycine (family Leguminosae) are the closest known relatives to the soybean, Glycine max, and its wild progenitor, Glycine soja, in Asia. The subgenus comprises 16 species native to Australia, two of which are widespread in the Pacific region. They occur in a variety of habitats including very dry areas of central Australia. Particular species are of potential economic interest as sources of drought and disease resistance, and consequently have been used in breeding programs with the cultivated soybean both in the U.S. and Australia. Relationships among the species of subgenus Glycine have been studied using many approaches, and groupings ("genome groups") have been hypothesized primarily on the basis of artificial hybridizations. Prof. Jeff Doyle's studies some years ago using chloroplast DNA (cpDNA) provided the first rigorous phylogenetic hypotheses for the subgenus. Although results were in substantial agreement with earlier biosystematic studies, some areas of disagreement were noted with the genome groupings. Within the genome groups, however, cpDNA data either failed to resolve relationships, or produced hypotheses that were incongruent with traditional taxonomic boundaries. The chloroplast evidence could be giving the "right" answers, in which case taxonomic boundaries should be reassessed and realigned; alternatively, the genealogical pattern of cpDNA data may not be tracking the historical pattern of speciation in the subgenus, for any of several possible reasons. Testing these alternatives using molecular (genetic) data requires the use of DNA sequences capable of showing historical independence from the chloroplast genome. The most obvious choices are nuclear genes; however, apart from the nuclear ribosomal gene locus, nuclear gene sequences appropriate for the low levels of divergence expected for closely related species within a genus have not been available. This is a general problem in molecular systematics that is not unique to Glycine. Non-coding regions of nuclear genes theoretically should provide variation suitable for phylogenetic reconstruction at this taxonomic level. Of the various types of non-coding regions, intervening sequences (introns) afford technical advantages because they are flanked by coding regions (exons) whose higher degree of sequence conservatism facilitates the design of oligonucleotide primers for amplification by the polymerase chain reaction (PCR). In preliminary work, Prof. Doyle has shown that introns of one such locus, histone H3-D, provide useful phylogenetic data in Glycine; and he and his Australian colleague, Dr. Brown, plan to utilize DNA sequences from this genetic locus to test a wide range of chloroplast and non-molecular hypotheses in the subgenus. These include relationships among and within the diploid genome groups as well as the origins and diversification of at least one of the three polyploid complexes in the subgenus. The basic methodology includes amplification by PCR, DNA sequencing of PCR products either directly or after molecular cloning, and phylogenetic analysis.