Now that we have discussed in general terms the use of molecular markers and how they work, let’s next look more closely at different types of markers which you may choose to use in your plant breeding program. As mentioned earlier, most of the previously used molecular marker types have become obsolete now that SNPs have gained in popularity. SNPs have many advantages, including being high-throughput and low in cost. One older marker type, SSRs, are also still being used as of this writing, however, so are also included here. We will now briefly describe both of these marker types, starting first with SSRs.
Microsatellites, also called simple sequence repeats (SSRs), are tandemly arranged blocks of short nucleotide sequences, usually 1-10 nucleotides long (though more typically 2 or 3), repeated up to 50 times within the plant genome. The number of repeat units in the block can vary noticeably between individuals within a species. This variation can be targeted by PCR, by placing the primers either side of the block. This leads to highly reproducible, co-dominant, easily analyzed and polymorphic markers. As a result, SSRs represent one of the most widely used markers in MAB.
In Figure 7, the di-nucleotide motif “AG” is repeated 6 times in this example of a microsatellite. Primers would be designed from the sequence of the red flanking sequences. (See Powell et al. 1996)
Note that the differences seen (the polymorphisms) between organisms are due to the number of repeats, which leads to a difference in size of the amplified products (ie. The length of the DNA segment between the 2 primers), not a difference in the DNA sequence per se. This is illustrated in Figure 8-a below.
In this example, the amplified fragment from A is shorter than that from B, because the AG motif is repeated fewer times. As a result, the A amplicon runs faster through the gel than the B amplicon, and the polymorphism is recognized by the different positions of the bands (Figure 8-b).