An other example of DNA markers generated by polymerase chain reaction (PCR) technique was ISSR (Inter Simple Sequence Repeat) marker. In a Q & A session of the AGH635 lecture, one of the student asked the following question: “What was ISSR marker and how can one generate the marker?”
ISSR markers are similar to RAPD markers. The only different is the primer used to generate the marker. In RAPD marker, one use a single random nucleotide sequences as oligo-nucleotide primer to generate the RAPD markers. On the other hand, ISSR marker used a single “simple sequence repeat (SSR) sequences” as the oligo-nucleotide primer to generate the ISSR markers.
Simple sequence repeats (Figure 1.A. and 1.B.) were DNA repeats having di- or tri-nucleotides as the repeat unit, such as: AC and GT (di-nucleotides) or ACT and GCA (tri-nucleotides). Moreover, the basic unit was repeated only ten or so times. In the plant genome, however, it can also be found GCTA (tetra-nucleotides), GCATAC (penta-nucleotide), and other types as the repeat unit.
In the plant genome, di- or tri-nucleotide repeats occur more frequently. These simple repeat nucleotide (SSR) sequences are also exist and intersperse all over the genome (Fig. 2). As such, it will be possible to find two repeat sequences located adjacent of each other in the genome (Fig. 3). One can use such SSR sequences to generate ISSR marker (Fig. 4).
Let’s assume there was the following pattern of di-nucleotides SSR: 5′–X1(CT)nX2–3′ in the genome (X1 and X2 can be any nucleotide, respectively). Therefore, one might be able to find the following sequences some where in the genome:
- 5′–X1X2CTCTCTCTCTCTCT X3X4–3′ (i.e. [CT]7)
- 5′–X1X2CTCTCTCTCTCTCTCTCTCT X3X4–3′ (i.e. [CT]10)
- 5′–X1X2CTCTCTCTCT X3 X4–3′ (i.e. [CT]5)
- 5′–X1X2AGAGAGAGAGAGAG X3X4–3′ (i.e. [CT]7)
- 5′–X1X2AGAGAGAGAGAGAGAGAGAG X3X4–3′ (i.e. [CT]10)
- 5′–X1X2AGAGAGAGAG X3X4–3′ (i.e. [CT]5)
Based on the SSR sequences above, the repeat unit are CT or AG and the unit is repeated n times (i.e. n=7, 10, or 5 times). The sequences no. 1-3 are recognized either as (CT)7, (CT)10, or (CT)5 while those of no. 4-6 as (AG)7, (AG)10, or (AG)5, respectively.
If we take a look closer, the repeat motif of the first three SSR (SSR no.1-3) and the second ones (SSR no.4-6) are actually all the same, i.e. (CT)n repeats. However, the first three sets of SSR (SSR no.1-3) were in one orientation (5′–>3′, to the right) and the second sets (SSR no.4-6) were in the other (opposite) orientation than the first (3′<–5′, to the left). The above SSR example could also be viewed as repeats of (AG)n in different (opposite) orientations.
Lets look further at the examples above and focus on the nucleotide at the X3 & X4 positions. If we take a closer look at the either (CT)n or (AG)n SSR above, the nucleotide at the position of X3 and X4 could be either A/C/G/T. Therefore, there were a number of possibilities in the genome for the X3 and X4, such as: …(CT)nAX4…., …(CT)nCX4…., …(CT)nGX4…., and …(CT)nTX4…., or …(AG)nTX4…, …(AG)nGX4…, …(AG)nCX4…, and …(AG)nAX4…, and so on.
In the ISSR analysis, nucleotide positions of X3 and X4 are used to increase specificity of the ISSR primer and to generate locus specific ISSR markers. Without the addition of X3 and or X4 nucleotides in the ISSR primers, the primers will generate non-specific amplified PCR products. The design ISSR primers were usually consisted of . 11-21 bases of oligo nucleotides, such as: (CT)5G, (CT)5T, (CT)5A, or (CT)5C (the CT represent the repeat unit, the 5 is the number of repeats, and the “G”, “T”, “A”, or “C” at the 3’-end of the primer was used to specify which loci would be amplified [see Fig. 5]).Those cases illustrated in Figure 1 – Fig. 5 were the bases of the development of ISSR markers. Moreover, if ones explore further about the SSR sequences and the genomic DNA, one would find the following :
- The SSR sequences (Figure 1) could be found interspersed in different part of the plant genome and distributed throughout the genome as illustrated in Figure 2.
- The SSR sequences could be in the same orientation (i.e. reside in the same strand, such as position 1, 3, 5, and 7) or in the opposite direction (i.e. reside in different strands, such as position 1 & 2; 3 & 4; 3 & 6; 4 & 6; and 5 & 6) and either in inward or outward direction as illustrated in Fig. 3.
- The positions of two SSR sequences could be separated by a few hundreds or thousands bases apart as illustrated in Fig. 3.
- Two SSR sequences, that are in opposite direction and flanking DNA sequences of less than 3,000 bases, can be amplified using a single SSR related oligonucleotide primer. The PCR generated a certain size of PCR products that could be used as DNA marker, also known as ISSR marker (Fig 4).
- The ISSR markers most of the time are polymorphic among different individuals.
Finally, the SSR sequences, such as position 5 & 6, as illustrated in Fig. 3, were usually present in a number of locations in the genome. Therefore, if a single SSR associated sequences (i.e. [AC]5G) is used to PCR amplify target DNA using plant genomic DNA as template, then it should be possible to amplify a number of DNA fragments. In one PCR reaction – a number of ISSR markers can be generated using a single SSR associated sequences of oligonucleotide primer (Fig. 6). Therefore, similar to RAPD marker – ISSR marker is also known as the multi-locus markers.