Marker Process Using CAPS
CAPS markers can be used to conduct marker-assisted selection in plant
breeding programs. Marker-assisted selection is useful for several reasons as
follows:
1. Many agronomically important traits are challenging for traditional
phenotypic selection.
2. Many of these traits are difficult to measure or have low heritability.
3. Many traits are also expressed late in the growing season or inconsistently
between environments.
4. They may also be affected by allelic dominance, making it hard to distinguish
heterozygous breeding lines from homozygous dominant individuals.
Marker-assisted selection overcomes many of these obstacles.
Here is a breakdown of the general steps involved in MAS using a PCR test
(not possible with online tools):
1. DNA extraction: Marker-assisted selection begins with DNA extraction. DNA
extraction starts by grinding leaves or certain parts of seeds. The ground
tissue is suspended in an extraction buffer, and after this, there are several
rounds of centrifugation, alcohol-based rinses, and RNA treatments to
remove any residual RNA in the sample. The final product is high-quality
genomic
Alt text: DNA extraction
1 | Page
2. Conduct PCR: The next step is to conduct polymerase chain reaction,
or PCR. This process duplicates a specific part of the genome that is
interesting for the trait being studied. The following steps are being
followed:
Alt text: PCR test
Step 1: The genomic DNA is heated to separate the strands. This step is
called denaturation.
Step 2: After this, the temperature is reduced, which allows short
sequences of DNA, called primers, to bind to the specific region being
studied.
Step 3: The temperature is raised to 72 degrees, allowing an enzyme
called Taq polymerase to construct new DNA based on the original
templates. The DNA in the region of interest has now been duplicated.
This three-step process repeats between 30 and 40 times, exponentially
increasing the number of copies of the sequence of interest. The final
product of PCR is billions of copies of this specific sequence, which is
enough for other applications.
2 | Page
3. Cleaved Amplified Polymorphic
Sequences, or CAPS markers: It
works by specifically cutting one
allele of the sequence that was
duplicated in PCR. A restriction
enzyme, which cuts DNA of a
highly specific sequence, is
introduced to the samples. Some
samples with one variant will be
cut, while samples with the other Alt text: Amplified PCR curve
variant remain intact.
If the original DNA sequence amplified by PCR was, for example, 300 base
pairs in length, the resulting cut sequences could be, for example, 100 and
200 base pairs in length. Samples with a different allele will not be cut and
will remain at 300 base pairs in length. The DNA samples can then be run
on a gel and their lengths can be visualised.
4. Gel electrophoresis: Cut DNA fragments move further along the gel are
readily recognisable as they have travelled a different distance. They also
typically have multiple bands rather than a single band.
Alt text: DNA separation
3 | Page
5. Identify heterozygous or homozygous: Marker-assisted selection makes
it possible to determine whether individuals are heterozygous or
homozygous. With CAPS markers heterozygous individuals can have
fragments of all three sizes. They have fragments of 300 base pairs, 200
base pairs, and 100 base pairs.
Alt text: Heterozygous and homozygous
Marker-assisted selection is a powerful tool that can increase the
accuracy and intensity of selection, and its role in plant breeding is likely
to continue to grow in coming decades.
4 | Page