Isoelectric Focusing of Esterases for Fine Fescue Identification1

There is a growing need for a technique to validate identities of commercial lots of seed, particularly at the cultivar level. A technique that would aid in identification of species and subspecies of a genus such as Festuca would also be helpful. The purpose of the present work was to develop such methods. Protein was extracted from ground and delipified seeds of cultivars or selections of known origin. Extracts were quantitatively added to polyacrylamide gel (with ampholite) in either rods or slabs and subjected to isoelectric focusing over a pH gradient of 4 to 9. After staining, up to 20 esterase bands. Isoelectric focusing on gel slabs provided a technique for simultaneous comparisons, both qualitative and quantitative, of band patterns of up to 18 entries side‐by‐side. Densitometric graphing aided in revealing consistent differences in relative density of specific bands, and made rapid identification of cultivars possible. Environmental stability of esterase band patterns characterizing cultivars was demonstrated using samples from the same seed fields for 3 years and also from different fields each year. Neither year of production nor location of production significantly altered the isozyme banding pattern that characterized each cultivar. Species and subspecies among the fine fescues were shown to encompass cultivars having similarities in esterase patterns that differed from those of other subspecies. Those illustrated include Festuca rubra subsp. rubra, F. rubra subsp. commutata, F. rubra subsp. trichophytla, F. tenuifolia F. ovina subsp. duriuscula = F. longifolia, and F. ovina subsp. glauca. We concluded that the esterase patterns developed were free from environmental influences and can be used satisfactorily for identification of fine fescue cultivars.