Two hypervariable regions have been identified in the alfalfa (Medicago sativa L) chloroplast genome. These regions initially were identified through restriction enzyme analysis and therefore are identified as the HindIII site and the HaeII site, the enzymes first used to discover their presence. The HindIII region was discovered as a hypervariable region within the Medicago sativa species (Schabel, 1996). The HaeII region was originally identified as hypervariable between Medicago species (Johnson and Palmer, 1989). After their discovery, through cloning and sequencing, PCR primers flanking the hypervariable restriction sites were designed, and now are used routinely to assay the size of fragment(s) represented in individual plants.

Variability at the HinDIII site is assayed by the amplification of a fragment about 800bp in length, while the HaeII site amplification yields a fragment of about 1100bp. Rounded to the nearest 50 bp, the HindIII fragment ranged from 200 to 1150bp, and the HaeII fragment ranged from 550 to 3950bp, with the frequencies shown in Table 1.

Comparisons of the polymorphic fragment distributions between PI lines from the Medicago sativa core collection yielded estimates of genetic distances between the PI lines. Principal component analysis of the distance data was used to develop a representation of the realtionships of the of PI lines and countries of origin. In general, the HindIII and HaeII regions yielded similar results. Specific size polymorphisms tended to be associated with geographic regions from which the PI's were collected. However, the distribution of polymorphic fragments in PI's collected from outside of the region of alfalfa origin, such as the US and Canada, tended to be much more heterogeneous. As a result, these PI lines often appeared to be more-or-less equally related to PI's from several geographic regions.