Agriculture Reference
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differences, as with any other phenotypic marker. Genetic polymorphism can be detected when multiple
forms of an enzyme are observed.
After the enzyme bands are stained, running front (Rf) values can be calculated to aid in the identiica-
tion and reference of speciic bands. The Rf value is the distance a band migrates or travels in a gel divided
by the distance the tracker dye or reference protein travels in a gel during the same time period. Rf values
provide a way to identify or reference enzyme bands that are constant among different laboratories.
Figure 9.3. Isoelectric focusing (IEF) is used to differentiate between genotypes that are different in pH gradi-
ent and electrical charges (courtesy of ESTA Laboratories).
Isozyme analysis is a highly reproducible and robust method of variety testing, as well as a tool for
studying crop evolution, genetic erosion and genetic stability. One limitation of isozyme analysis is that
markers may be inluenced by environmental factors or developmental stage. More than 100 isozyme sys-
tems have been used, and in many cases enzyme loci have been mapped. Examples of the many species for
which isozyme analysis has been successful include barley (Fedak, 1974; Liu et al., 1999), corn (Orman
et al., 1991; Smith, 1988), lima bean (Maquet et al., 1997), onion (Rouamba et al., 2001) potato (Ortiz and
Huaman, 2001), rye (Persson and Von Bothmer, 2000), soybean (Stephens et al., 1998), sugar beet (Oleo et
al., 1992), and wheat (Cheniany et al., 2007; Langston et al., 1980; Suseelan et al., 1987).
storage Protein Analysis-Polyacrylamide Gel electrophoresis
Seed storage protein analysis by polyacrylamide gel electrophoresis has been used to characterize cultivated
varieties as well as study the diversity of wild species. Even with the recent dominance of DNA-marker
technology, cultivar testing by electrophoretic separation of seed storage proteins remains a widely used
method of genotype identiication. Although used for varietal identiication of a range of crops (Vaz et al.,
2004; Javaid et al., 2004; Jha and Ohri, 1996), the main use of this method has been for varietal evaluation
of cereals through proiling variations in seed storage protein.
Although crude protein extractions have been used for electrophoretic analysis, the banding patterns
of two protein groups in particular, both found in cereal endosperm, are usually targeted. These are prola-
mines, considered unique to cereal seeds, and glutelins. Prolamines are given different names in different
cereals, avenins in oats, zeins in corn, gliadins in wheat and hordein in barley.
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