Characterization of cytoplasmic diversity in soybean (Glycine max L. Merr) using mitochondrial markers

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Virginia Tech

Soybean, Glycine max L. Merr, is used worldwide as an important source of protein and oil for a wide spectrum of edible feed and industrial purposes. Modem cultivars are derived from relatively few plant introductions (PIs) which severely limits diversity in soybean germplasm. The United States Department of Agriculture (USDA) maintains the soybean germplasm collection. Mitochondrial DNA sequences have been used as markers of diversity at the cytoplasmic level.

This project included three objectives. The first was a classification of the 208 varieties of the USDA's 'old domestic collection' of soybean varieties with two mitochondrial restriction fragment length polymorphisms (RFLP) markers. Molecular techniques were used to search for additional sources of cytoplasmic diversity available to soybean breeders. The two polymorphic markers were, a 2.3 kb HindIII fragment isolated from 'Williams 82' mitochondrial DNA, and a portion of the mitochondrial atp6 gene. These markers were used to distinguish cytoplasmic groups based on hybridization analysis of HindIII-digested soybean DNA Four major groups were observed with the 2.3 kb marker in the old domestic collection, and several minor subgroups were also detected.

The second objective included subcloning and sequencing the 0.9 kb and 1.7 kb HindIII-PstI clones flanking the 2.3 kb HindIII fragment from 'Williams 82' DNA The total 4.9 kb PstI sequence from 'Williams 82' mitochondrial DNA was used to search a sequence database for any homology to known mitochondrial sequences.

The third objective compared restriction maps of the four cytoplasmic types in the regions containing homology to the 4.9 kb PstI fragment. DNAs from the four cytoplasmic types were digested with five enzymes and four specific clones (0.9 kb PstI-HindIII, 0.8 kb HindIII-XbaI, 1.5 kb Xbal-HindIII, 1.7 kb HindIII-PstI) were used as hybridization probes in Southern analysis to examine RFLP patterns and construct comparative restriction maps of the four cytoplasmic types of DNA.