Q&A in AGH635 Course: In RAPD Marker Analysis – What is Called the Locus and What is the Allele?

The term locus (pl.: loci) is a common terminology associated with a location in the genome of an organism. In the Mendelian genetics, the locus controlling flower color clearly describes a location somewhere in the genome associated with gene(s) controlling flower color. Meanwhile, the term allele is associated with alternative types or genes reside in certain locus. In the above example, the alternative type or flower color in a flower color locus can be one causing a red flower color phenotype (i.e. M allele). Alternatively, the other allele for this locus is one causing a white color flower (i.e. m allele).

In a Q & A session of the AGH635 lecture, one of the student asked the following question: “Is the term loci and alleles also applicable when one discuss about RAPD (Randomly Amplified Polymorphic DNA) marker? What are the loci and what  are the allele in the RAPD marker analysis?”

Before posting the answer to that question, let me remind you what RAPD marker is. As everyone already know, RAPD markers are simply a PCR amplified DNA fragments generated using a random 10-mers oligonucleotide (a random sequences of 10 nucleotides) as primer and genomic DNA as template (see Figure 1).

Figure 4. Representative position of random sequences existed in the plat genome. A number of loci of the random sequences can be amplified using random primer to generate PCR products. The products can be used as markers (RAPD markers)

Figure 1. Representative position of random sequences existed in the plant genome. A number of loci of the random sequences can be amplified using random primer to generate PCR products. The products can be used as markers (RAPD markers)

In a single PCR reaction using certain random primer, there could be a number of different sizes of amplified DNA fragments. A certain size of the PCR amplified fragment using a single random-oligonucleotide primer is generated from certain location in the genome. Hence, different sizes of the PCR amplified fragments are generated from different locations in the genome (Figure 2 & Figure 3).

Figure 2. Three DNA fragments are amflified from three different location in the genome using a single random OPA9 oligo-nucleotide primer

Figure 2. Three DNA fragments are amplified from three different locations in the genome using a single random OPA9 oligo-nucleotide primer

Figure 3. Three different sizes of PCR amplified products, using a single random OPA9 oligonucleotide primer from 10 plant samples

Figure 3. Three different sizes of PCR amplified products (fragment #1, #2, and #3), using a single random OPA9 oligonucleotide primer from 10 plant samples

When one analyzes RAPD marker for a number of individuals in a population using a single random-oligonucleotide primer (i.e. OPA9) and one looks at certain size of PCR amplified product (i.e. Fragment #2), there will be two possibilities. For one individual, this amplified product might be present (sample #1, #3, #4, and #5) while for the others might be absent (i.e. sample #2, #6, #7, #8, #9, and #10).

Back to the questions – one of the AGH635 student’s asked, the locus in an RAPD marker analysis is usually named after the primer used and the amplified product sizes detected. In the Figure 3, PCR amplification of the plants using a single random oligonucleotide primer (i.e. OPA9) resulted in three different loci since there are three different sizes of amplified products. Hence, three loci are identified in this RAPD analysis.

In RAPD analysis – one PCR reaction using a single random oligonucleotide primer generates a number of marker loci. Because of that feature, RAPD marker is also known as multi-loci marker.

The rules in calling the RAPD marker locus are: “Name of random oligonucleotide primer” used to generate the RAPD marker and followed by “the sizes of the PCR amplified products.” If one use Figure 3 above and pay attention to only Fragment #1 (i.e. the size is ca. 1,100 base pair [bp]), then the name of that particular RAPD marker locus is OPA9-1,100. Similarly, one could define the loci for fragment #2 (ca. 1,000 bp) and fragment # 3 (ca. 950 bp) as OPA9-1,000 and OPA9-950, respectively.

If one looks at a particular locus (i.e. OPA9-1,100) and survey the amplified product across samples, then the PCR amplified product is either present or absent. If one use Figure 3 above and pay attention to only Fragment #1 (OPA9-1,100 locus), the amplified product is present [+] in almost all samples (9 samples) and absent [-] in only one sample (sample #9). For OPA9-1,000 locus, the PCR amplified product is only present [+] in four samples (sample #1, #3, #4, and #5) and absent [-] in the other samples. Meanwhile, for OPA-950 locus, the PCR product is present [+] in all samples.

In RAPD marker analysis, the present and absence of amplified product in a specific locus represent the alternative alleles for that locus. Therefore, there are only two possible alleles for each locus in the RAPD analysis, such as: present and absence of PCR amplified product.

To summarize, in RAPD marker analysis:

  • the locus is defined as the name of the primer, followed by the size of the amplified product (i.e. OPA9-1,100, whereas OPA9 is the name of random primer used to amplify the product and 1,100 is the size of the amplified product).
  • There are only two alternative alleles for each locus in RAPD marker. For OPA9-1.100 locus, the amplified PCR product is either present or absence. The present or absence of PCR amplified product is the alternative allele for a locus in RAPD marker analysis.

About PMB Lab: Prof. Sudarsono

This blog is dedicated as a communication media among alumni associated with PMB Lab, Dept. of Agronomy and Horticulture, Fac. of Agriculture, IPB, Bogor – Indonesia. It contains various information related to alumni activities, PMB Lab’s on going activities and other related matters.
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One Response to Q&A in AGH635 Course: In RAPD Marker Analysis – What is Called the Locus and What is the Allele?

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