DISTINCTION BETWEEN MULTIPLE ISOLATES OF CHLORELLA VULGARIS (CHLOROPHYTA, TREBOUXIOPHYCEAE) AND TESTING FOR CONSPECIFICITY USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM AND ITS RDNA SEQUENCES¹

Abstract

Multiple strains of individual algal species are available from public culture collections, often with the same isolate being maintained in parallel at a number of collections under different culture regimes. To unravel genomic variation and to identify unique genotypes among such multiple strains, two approaches were used on a sample of 29 strains of Chlorella vulgaris Beijerinck, an alga of great value for applied research, from five culture collections. With the exception of two strains, internal transcribed spacer rDNA sequence data substantiated conspecificity of the studied strains and only minor sequence differences with the authentic ''Beijerinck isolate'' were observed. Amplified fragment length polymorphism (AFLP) detected considerable genomic variation when rDNA sequences were identical. Band detection and the construction of a binary matrix from AFLP patterns for phylogenetic analyses were fully automated, but comparison of similar patterns still required manual refinement. The AFLPs distinguished 11 unique genotypes and provided robust support for the presence of five cryptic species. This finding advocates the need to carefully record which strain has been used in any experiment or in applied research, because genomic variation may also correspond to differences in physiological/biochemical properties. No genomic differences could be detected between duplicate strains of the same isolate that were maintained by continuous subculturing over many decades or within those stored at ultralow temperatures.

Quality analysis of commercial Chlorella products used as dietary supplement in human nutrition

“…Most manufacturers have their own favourite production strains, whereas the species' origin and strain identification often remain disputable. According to Müller et al (2005), more than 200 citations on C. vulgaris were obtained in scientific publications, but less than 20 provided exact strain designations. Most published studies referred to the genus Chlorella and not to a particular species or even strain.…”

The phylogenetic position and phenotypic changes of a Chlorella-like alga during 5-year microcosm culture

“…However, the present results may not necessarily indicate that the rDNA sequences, which are present in multiple copies in the genome, are all exactly the same in the derived clones and identical to those of the ancestral strain. The use of AFLP methods has been shown to reveal considerable genomic variation in Chlorella-like algae when their rDNA sequences are similar (Müller et al 2005). Hence, in a future study of the microcosm strains, it may be worthwhile to use an AFLP approach to give supplementary information on the microcosm strain and its derived algal clones and possible variation at an intraspecific level.…”

Biological Constraints on the Production of Microalgal-Based Biofuels

“…Traditionally, algal cultures have been maintained by serial transfer, and although this has successfully maintained some taxa apparently unchanged for many decades (Müller et al, 2005 ;Day et al, 2010 ) , it cannot guarantee the longterm genotypic security of microbial strains. The only applicable alternative is to employ cryopreservation (storage at ultra-cold temperatures, i.e., <−130 °C) (Day and Stacey, 2008 ) .…”