CHARACTERIZATION OF SOYBEAN CULTIVARS: RAPID HPLC PROFILING BASED ON PROTEIN MARKERS - Soybean and Wheat Crops: Growth, Fertilization, and Yield - page 69

C HARACTERIZATION OF S OYBEAN C ULTIVARS
The differentiation among the increasing number of soybean cultivars is not an easy task
since many of them are genetically very close. Traditional methodologies for the
identification of soybean cultivars are based on phenotypic characters [14, 43, 44]. Since
many different soybean cultivars are indistinguishable based on these characteristics, other
methodologies have raised as alternatives for cultivar characterization [45].
In addition to morphological markers, different types of DNA-based markers have also
been applied for studying genetic diversity in soybean: RFLP (Restriction Fragment Length
Polymorphism), AFLP (Amplified Fragment Length Polymorphism), RAPD (Random
Amplified Polymorphic DNAs), DAF (DNA Amplification Fingerprinting), and SSR (Simple
Sequence Repeats) [13, 46-54]. In general, the results of these studies revealed the huge
genetic diversity of soybean cultivars and the difficulty in their differentiation and only the
application of microsatellite markers such as SSR resulted to be more suitable for genotypic
identification [52-54]. Moreover, the application of these techniques requires complicated
procedures and specialized techniques and laboratory equipment being both time-consuming
and costly and, thus, not suitable for routine analysis [55].
The identification of soybean cultivars through the analysis of soybean proteins using
different electrophoretic techniques has also been widely employed. Most electrophoretic
systems employed either starch or polyacrylamide gels in which proteins were separated
based on their molecular weight and charge density [6, 7, 12, 14, 22, 33, 41, 45, 56]. In
addition to the low reproducibility, tediousness, and time cost of these methodologies, one-
dimensional electrophoresis, in general, did not enable the differentiation among soybean
cultivars. As example, Figure 1 shows the sodium dodecyl sulphate polyacrilamide gel
electrophoresis (SDS-PAGE) profile of total proteins and 11S and 7S fractions for seven
different soybean varieties not observing differences in profiles among cultivars [33]. The use
of isoelectrically focused gels and blotting were also tried in gel electrophoresis but in no case
successful results regarding cultivar differentiation were reported [57, 58].
The application of two-dimensional SDS-PAGE has resulted advantageous in the
selection of high quality soybean varieties [1, 3, 13, 25, 59]. The application of this technique
requires a prefractionation step to reduce the complexity of the sample and minimize protein
degradation due to the presence of proteases, and to remove interfering compounds such as
lipids, salts, nucleic acids, polyphenols, alkaloids, pigments, terpenes, organic acids, and
other compounds. The extraction with trichloroacetic acid/acetone is very useful for
minimizing protein degradation and removing interfering compounds while the introduction
of an immobilized pH gradient has improved the separation of proteins. Natarajan et al. [1,
13, 60] observed variability in glycinin polypeptides, β-conglycinin, and soybean seed
allergens (Gly m Bd 60K, Gly m Bd 30K, and Gly m Bd 28K) between wild and cultivated
soybean but they could not find significant differences within the same group of soybean
cultivars. Zarcadas et al. [3] observed differences in the proteome and subunit expression of
glycinin and β-conglycinin among two tofu and eleven null soybean genotypes (lacking a
subunit or part of a subunit of β-conglycinin). Nevertheless, they also reported [59] strong
similarities in the overall distribution pattern of glycinin, β-conglycinin, and total proteins
among other soybean cultivars. The application of image analysis to the spots using a