Marker Assisted Selection

With the advent of marker-assisted selection (MAS), a new breeding tool is available to make more accurate and useful selections in breeding populations. MAS allows heritable traits to be linked to the DNA that is responsible for controlling that trait. These stretches of DNA or QTLs (Quantitative Trait Loci) can be detected through molecular biological means.

For detection, we use the method of Polymerase Chain Reaction (PCR) to amplify stretches of DNA that are linked to heritable traits such as yield or disease resistance (SDS and SCN). This method is useful because the DNA that we amplify is different (polymorphic) between cultivars. It is the difference that we use to determine whether the plant has the desired trait or not.

The process in which the differential DNA sites (or primer sites) are discovered comes from genetic mapping techniques, i.e. AFLP, RFLP, RAPD, microsattelites. An example; Forrest is resistant to SDS and SCN, while Essex is susceptible. These cultivars were crossed and then we had the resultant population of ExFs. The Forrest and Essex parents were screened with many different sets of primers (DNA markers) to see if we could achieve differential amplification. If a difference was seen then the polymorphic marker was tested in the resultant population from the Essex x Forrest cross. If the DNA molecular marker score (genotype data) matched with the phenotypic data (Yield, SDS, or SCN score from the field or greenhouse) the marker is considered as an informative marker. Therefore, it can be used in a marker assisted selection breeding program.

To learn how MAS works, basic molecular biology principles need to be understood. The methods we employ are DNA extraction, PCR amplification, DNA separation with electrophoresis, and DNA visualization.

In order to examine the DNA from a plant it first must be extracted out of the cell. To begin the extraction the cell’s integrity must be disrupted or lysed. Then the DNA will be separated away from the rest of the cellular debris, i.e. proteins, fats, polysaccharides, phenols, etc. Various methods for this are available, ranging from the complex to the simple. With a marker assisted selection breeding program the simpler methods are necessary since they are time and cost effective.

PCR is an effective method for generating large quantities of a specific DNA sequence from a small amount of starting DNA. The amplification which can result in a million-fold increase of specific DNA sequences occurs in a three step reaction. A typical PCR reaction involves repeating the same basic temperature cycling. The first step is denaturation of double stranded DNA to single stranded at high temperature (94° C). The DNA is then allowed to cool to around 48° C which is when short synthetic primers will anneal. The last step is the synthesis of the DNA by a thermostable DNA polymerase enzyme (72° C). The cycle is then repeated many times, which will allow amplification from one copy to millions of copies in three to five hours using a PCR thermocycler machine.

The amplification of specific regions of DNA is accomplished through the specificity of the short synthetic primers. This technique is useful for a MAS breeding program because the results are reliable. The same plant will give the same PCR fragments in repeated amplification reactions. The different alleles from different plants will have different amplification products (length or fluorescence).

In order to separate out the different lengths of PCR amplified DNA, the sample is placed into a gel matrix and then an electric current is applied. The DNA has a negative charge and will move towards the positive electrode. The smaller fragments will move through the matrix faster than the larger fragments. This will allow for the differences in size to be visualized. With primers fluorescently labeled, the amplification of one allele or the other will increase the corresponding fluorescence (different allele specific primers have different fluorescent labels).

The PCR amplified products are not visible with the naked eye, we therefor employ several methods to detect the amplified DNA (radioactivity, fluorescence, or ethidium bromide). The PCR products are then recorded on film or computer disk, scored for genotype, and subjected to data analysis.