Every species of organism examined by researchers has revealed immense genetic variation or polymorphism (many forms), an indication of the presence of different genotypes (genetic makeup) in the population. It is however, impossible to infer the genotypes of plants simply by observing their visible characteristics or phenotypes. In many plant science laboratories, researchers employ electrophoresis to determine the mode of reproduction of plant species, to detect genetic variation within and between plant populations, and to identify plant genotypes. Also, researchers establish genetic relatedness in plants, that is, establish the most probable paternal parent or pollen donor within and outside a study site that sired seeds collected from a known maternal plant. To accomplish these tasks, researchers rely upon protein and DNA markers generated by gel electrophoresis.
Protein markers known as allozymes have been used extensively in a number of genetic analyses. Allozymes are electrophoretically distinct forms of an enzyme produced by different alleles (alternate forms of a gene). (An enzyme is a protein that speeds up the rate of a chemical reaction in an organism, without being consumed in the process.) Allozymes catalyze the same chemical reactions but have slightly different sequences of amino acids, the building blocks of proteins.
To analyze allozymes, researchers extract enzymes from plants and separate them on starch or polyacrylamide gels. Gels made up of potato starch are most commonly used because of their low cost and ease of use. After electrophoresis, the gel is submerged in a solution containing a dye and a substrate appropriate for the enzyme studied. The enzyme reacts with the substrate to produce a colored band on the gel. If the gel yields one colored discrete band in a lane, then that particular plant contains just one form of the enzyme, therefore the genotype of the plant must be homozygous (having two identical alleles of a gene). If the gel yields two colored bands, then the plant contains two forms of the enzyme and is therefore heterozygous (having two different alleles of a gene). Allozyme electrophoresis enables researchers to learn about the mode of reproduction of plants. For example, plant populations with high numbers of heterozygotes indicate a high level of cross-pollination, that is, the transfer of pollen (plant male gamete) by wind, insects, birds, bats, or other animals from one flowering plant to another. High numbers of homozygotes within a plant population indicate a high level of self-pollination, the transfer of pollen within a flower or between flowers of the same plant.
In many plant laboratories today, protein markers have been superseded by DNA markers, which are fragments of DNA that are distinguished from one another because of the differences in their base sequences. To generate DNA markers, DNA is extracted from plants and cut into fragments with special enzymes known as restriction enzymes. The fragments are then separated by electrophoresis on an agarose gel and analyzed.
Some of the widely used DNA markers in many laboratories are different lengths of DNA fragments, known as restriction fragment polymorphisms (RFLPs) or short sequences of DNA bases that are repeated many times in tandem (head to tail), called variable number of tandem repeats (VNTR), a type of RFLP. RFLP analysis distinguishes between heterozygous and homozygous genotypes. Heterozygotes yield two fragments on a gel, while homozygous genotypes yield a single fragment.
Another DNA marker, known as random amplified polymorphic DNAs (RAPDs), have become extremely popular DNA markers with plant scientists. They are DNA fragments amplified by a technique known as polymerase chain reaction (PCR) using a short primer consisting of ten nucleotides chosen randomly and then separated by size by agarose gel electrophoresis. Polymorphisms are revealed when a DNA fragment is amplified in one plant and fails to amplify in another. On a gel, the researcher checks for the presence or absence of the marker.
Many studies involving parentage of seeds and seed pods have been done using DNAmarkers. The DNAprofiles of seeds or seed pods are compared to maternal and paternal plants. If seeds were produced by cross-pollination, then half of the DNA bands of the seeds would be found in the maternal plant and the other half from the paternal plant.
Oluwatoyin O. Osunsanya
See also: Autoradiography; Chromatography; DNA in plants; Nucleic acids; Proteins and amino acids.
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