Rapid Molecular Identification of Tetraselmis Using Enzymatic Digestion of the 18S rDNA Gene

Abstract

Tetraselmis is a genus of quadriflagellated single-celled green algae belonging to the Phylum Chlorophyta, commonly used in aquaculture with very promising biotechnological potential. The varied morphological characteristics, in some cases, have led to confusion in taxonomic identification. To solve this problem, new techniques based on molecular markers and restriction enzymes can ensure the identification of microalgae without sequencing. This study aimed to compare in silico modeling with an experimental restriction pattern based on the 18S rDNA gene for the identification of a microalgae strain. The strain grew in a culture medium, based on organic fertilizer. Theoretical analyses allowed the design of three primers based on the alignment of eight sequences obtained from NCBI, applying the Geneious Prime® 2019 and V1.3 and Oligo Calculator version 3.2. programs. The in silico restriction patterns was obtained with the NEBcutter v2.0 program. Experimental analyses began with the extraction of the DNA using the TENS protocol, then PCR amplification using PM-016F/PM-016R and PM-001F/PM-016R primers of 18S rDNA and finally the product was digested with BbvCI and Eco53kI; BstUI, RsaI and MspI enzymes. The DNA concentration extraction reached 3200 ng µl-1 and a purity of 2.0. The PCR amplified two products: 950 bp and 1400 bp, which brought us closer to identifying the microalgae. The in silico modeling and experimental restriction patterns showed similar fragments. In this way, the efficient response of restriction enzymes was demonstrated by confirming that the PM013 strain corresponds to the Tetraselmis genus. This method can be considered as a fast and safe alternative to identify wild microalgae in a basic molecular biology laboratory.