Q&A in AGH635 Course: “What Have Caused Allele Diversities in ISSR Marker?”

In a Q & A session of the AGH635 lecture, one of the student asked me the following question: “What have caused allele diversity in ISSR marker?” The answer to this question was actually similar to one of RAPD marker that has previously been posted.

First, let’s recall what was the locus and what was allele in ISSR marker. In ISSR marker, just like in RAPD, one define the locus as “the primer used to generate ISSR and the size of the generated amplified product.” Let’s assume an ISSR primer was used to amplify genomic DNA of cacao, and there were three amplified products (550, 700, and 1100 bp, respectively). In this case, the primer was able to generate ISSR markers at three different loci, namely: (1) ISSR-550, (2) ISSR-700, and (3) ISSR-1100, respectively. As it has been discussed before, for each of the ISSR locus, there would be two possible allele, either (1) the presence or (2) the absence – of amplified product for that particular locus.

Let’s get back to the previous question, “What have caused allele diversities in ISSR marker?” Just like in RAPD marker, in ISSR marker there were also at least five ways that could cause ISSR allele variability, such as: (1) mutation at the annealing site of forward primer, (2) mutation at the annealing site of reverse primer, (3) mutation at the annealing site of both forward and reverse primers, (4) insertion in between annealing sites of the forward and the reverse primers, and (5) deletion in between annealing sites of the forward and reverse primer.

    • As it was illustrated in Figure 1. there was a point mutation – a single nucleotide substitution (changes from C to A or from G to T) at the 3′ end of the template DNA corresponding to the forward primer annealing site. Because of the mutation, forward primer can not be elongated and resulted in negative PCR amplification (ISSR marker [-]). Similarly, any other mutations at the forward primer annealing site preventing the forward primer to anneal at it or preventing elongation of the primer by DNA polymerase would result in negative PCR amplification (ISSR marker [-]).
Figure 1. Mutation in annealing site of the forward primer resulted in (-) PCR product

Figure 1. Mutation in annealing site of the forward primer resulted in (-) PCR product

    • As it was illustrated in Figure 2. there was a point mutation – a single nucleotide substitution (changes from C to G or from G to C) at the 3′ end of the template DNA corresponding to the reverse primer annealing site. Because of the mutation, reverse primer could not be elongated and resulted in negative PCR amplification (ISSR marker [-]). Similarly, any other mutations at the reverse primer annealing site preventing the reverse primer to anneal at it or preventing elongation of the primer by DNA polymerase would result in negative PCR amplification (ISSR marker [-]).
Figure 2. Mutation in annealing site of the reverse primer resulted in (-) PCR product

Figure 2. Mutation in annealing site of the reverse primer resulted in (-) PCR product

    • As it was illustrated in Figure 3. there were two point mutations – two nucleotide substitutions at the 3′ end of the template DNA corresponding to both the forward and reverse primer annealing sites. Because of the mutations, both forward and reverse primers can not be elongated and resulted in negative PCR amplification (ISSR marker [-]). Similarly, any other mutations at both the forward and reverse primer annealing sites preventing both the forward and reverse primer to anneal at them or preventing elongation of the primers by DNA polymerase would result in negative PCR amplification (ISSR marker [-]).

Figure 3. Mutation in annealing sites of both the forward and reverse primers resulted in (-) PCR product

  • As it was illustrated in Figure 4. there was an insertion mutation in between the forward and reverse primer annealing sites. Because of the insertion mutation, the PCR amplified product would be larger than the original one. Hence, at position of the original size, there was no amplified product(PCR [-]). On the other hand, there was a new size of PCR amplified product from the same locus. However, the new size of amplified product would be regarded as new locus because of the size difference.
Figure 4. Insertion mutation between the forward and reverse primer annealing sites, in addition to changing the marker (+) into marker (-), it also created a new ISSR marker locus at the size larger than the original site

Figure 4. Insertion mutation between the forward and reverse primer annealing sites, in addition to changing the marker (+) into marker (-), it also created a new ISSR marker locus at the size larger than the original site

  • As it was illustrated in Figure 5. there was a deletion mutation in between the forward and reverse primer annealing sites. Because of the deletion mutation, the PCR amplified product would be smaller than the original one. Hence, at position of the original size, there was no amplified product (PCR [-]). On the other hand, there was a new-smaller size of PCR amplified product from the same locus. However, the new siae of amplified product would be regarded as new locus because of the size difference.
Figure 5. Deletion mutation between the forward and reverse primer annealing sites, in addition to changing the marker (+) into marker (-), it also created a new ISSR marker locus at the size smaller than the original one

Figure 5. Deletion mutation between the forward and reverse primer annealing sites, in addition to changing the marker (+) into marker (-), it also created a new ISSR marker locus at the size smaller than the original one

There might be many other ways that can cause allele diversity in ISSR marker. However, five cases listed in this posting were most probably the major causes of the allele variation in ISSR marker.

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: “What Have Caused Allele Diversities in ISSR Marker?”

  1. Reblogged this on Plant Molecular Biology Lab, IPB, Bogor and commented:

    AGH635-2015: Part 3 of Lecture 3. ISSR Marker

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