Restriction Fragment Length Polymorphisms
Torbert R. Rocheford
Corn breeders have traditionally developed improved varieties by selecting plants with desirable phenotypes (visual appearance). A plant's phenotype, however, is determined not only by its genotype (genetic composition) but also by the environment in which it is grown.
Because effects of the environment may mask effects of the plant's genotype, the phenotype may provide an inaccurate measure of a plant's actual genetic composition or potential. Consequently, to identify genetically superior plants, very elaborate, expensive plant-breeding techniques are used. By providing direct information about the genetic material of plants, restriction fragment length polymorphism (RFLP) technology may help overcome some of the problems associated with traditional methods of identifying genetically superior plants.
DNA consists of nucleotide bases that comprise genes (coding regions) and the regions between genes (noncoding regions). Variation in the DNA sequence composition of individual plants can be detected by restriction endonucleases. These enzymes recognize specific short (four- or six-) nucleotide base sequences of DNA and cleave the DNA strands at these points. Using electrophoresis, an electrical current is produced across the length of an agarose gel. Because DNA is negatively charged, DNA fragments migrate to the positively charged end at a rate proportional to their relative size.
The fractionated DNA is transferred to nylon membranes by a process called Southern blotting. Previously cloned pieces of DNA from regions of the plant genome under study are labeled with radioactive or nonradioactive labeling methods. The labeled pieces of DNA are hybridized or annealed to the fractionated DNA on the membrane. The membrane is then exposed to X-ray film to detect the regions where the cloned DNA hybridized to the fractionated DNA. The patterns on the film will reveal restriction enzyme-generated hybridization fragments of different lengths (polymorphisms), thus the basis for the term "Restriction Fragment Length Polymorphisms" (RFLPs). Also known as molecular markers, RFLPs mark different chromosomal regions.
Through evaluation of genetic progenies segregating for RFLP markers and agronomic traits of interest, RFLP markers can be associated or linked with genes controlling important traits. For plant breeding, DNA can be isolated from experimental plants and their RFLP patterns determined with the DNA clones that are linked to important traits. Plants with the desirable RFLP pattern are identified and selected for further breeding, regardless of the environment in which the plants were grown.
In the DNA sequence analsysis here, each band represents a different allele of the same gene encoding maise seed protein.
Torbert R. Rocheford, assistant professor of corn breeding, Department of Agronomy
