Between each copy of the 18S-5.8S-28S ribosomal DNA sequence within an intra-chromosomal repeat unit are non-transcribed spacers (cf ITS, which are transcribed and excised from the RNA transcript later). The sequence of these segments is entirely free of selective constraint (ITS must continue to fold in such a way that nucleases can cut the transcript at the appropriate points). For this reason, the non-transcribed spacers (NTS), otherwise known as intergenic spacers (IGS) are sensitive markers of evolutionary change, tracking drift more rapidly than the ITS. As with ITS they are thought to be under the constraint of concerted evolution, and so rapid change can only follow disruption of panmixis in a population.

The Intergenic Spacer has been examined in G. salaris and G. thymalli by Cunningham's group and by the Oslo group. In their first study, Collins & Cunningham (2000) amplified a range of fragments from the IGS. This makes it impossible to be sure how long the IGS is; the longest fragment is 2.62 Kb but it is not clear how the fragments relate to each other, and it is possible that IGS of different lengths exist within the cassette.

Collins & Cunningham's (2000) primers for IGS:

5'-GCTGATTTAATGAGCC-3'

5'-AGGTTAGTTTTACCCTACT-3'

Amplification conditions:

10 mins 96°C then addition of Taq polymerase.

then:

94°C 1 min

50°C 1 min

72°C 2.5 mins

for 35 cycles.

then 5 mins at 72°C to complete extension.

Sterud et al.'s primers for IGS locus:

5'-CTGGCTATAATCACGTAAGACTGC-3'

5'-AAGATACTCATTTGACTCGGTGTG-3'

These primers do not amplify the whole IGS locus, only the repeat region.

Amplification conditions: Initial denaturing step 5 mins at 96°C before addition of Taq polymerase.

Then:

96°C 1 min

55°C 1 min

72°C 2 min

for 35 cycles

then 10 mins at 72°C to complete extension.

References

Sterud, E., Mo, T.A., Collins, C.M. & Cunningham, C.O. (2002).The use of host specificity, pathogenicity, and molecular markers to differentiate between Gyrodactylus salaris Malmberg, 1957 and G. thymalli Zitnan, 1960 (Monogenea: Gyrodactylidae). Parasitology 124 203-213.