18S rRNA Gene & ITS Sequencing

Molecular Identification of Fungi

18S rRNA Gene

Similar to the bacterial 16S rRNA genes, the eukaryotic 18S rRNA gene has both conserved and variable regions. The 18S rRNA gene sequences or/and their associated transcribed spacers (internal transcribed spacer; ITS) combined are the currently employed biomarkers to classify fungi and eukaryotes. The ITS region is found to include the ITS1 which is located between 18S and 5.8S rDNA and ITS2 region which if found between 5.8S and 28S rDNA. It has been observed that the variability in ITS1 and ITS2 is greater than 18S rDNA gene variability, so it has an edge over 18S to identify fungi and lower eukaryotes at species and subspecies levels.

The 18S rRNA gene, similar to the 16S rRNA gene is a DNA sequence present in the eukaryotic genome that encodes the smaller subunit of the ribosomal RNA. The 18S rRNA gene also contains both conserved and variable regions. The conserved regions reveal the family relationship among species, whereas the variable regions reflect the disparities in sequence. As 18S rDNA is also found to be conserved in course of the evolution, it is considered as a potential biomarker for classification above the species level.

ITS Region and 26S rRNA gene

ITS (Internal Transcribed Spacer) refers to the spacer DNA region situated between the small-subunit rRNA and large-subunit rRNA genes in the chromosome. Owing to the relatively lower pressure in the natural selection during evolution, ITS can be employed for taxonomic classification and study relative abundance. Currently, it is mostly used in the classification of fungus.

At present, the 18S rRNA gene sequencing study is gaining importance for identification of fungi and algae. Generally, the ITS1/ITS4 primers are used for amplification of the ITS region which can be substituted with universal primers ITS2, ITS3, and ITS5. This ITS sequence can be compared with available public databases maintained by NCBI, EMBL, or databases DDBJ, and PDB to locate a statistically significant genetic neighbor. The ITS sequencing can also be combined with sequencing the 26S rRNA (D1/D2) region to improve the identification of fungi.