Workpackage 1: Genetic characterisation of rotifer clones will enable the creation of mixtures of known rotifer clones needed in the other WPs

To establish a collection of characterised rotifer clones, suitable and standardised protocols to collect, preserve and transport the rotifer samples (cultures or resting eggs) are needed. The different clones will be maintained at the infrastructure of partner 1 and will be available for use in the other WPs.
The genetic characterisation can be split in two. First, the genetic characterisation of individual clones will be done by using three markers: part of the mitochondrial 16S rDNA gene, part of the genomic HSP60 gene and specific microsatellites for B. plicatilis and B. rotundiformis. Both the 16S rDNA marker (Garey et al., 1998) and the HSP60 marker (Cochrane et al., 1994) will be combined with the SSCP technique (Single Strand Conformation Polymorphism) (Orita et al., 1989, Lessa and Appelbaum, 1993). Until now 7 microsatellites and 2 minisatellites have been described for B. plicatilis and B. rotundiformis (Gómez et al., 1998; Boehm et al., 2000). Among these, the most useful ones have to be selected: the alleles of the selected microsatellite markers have to be distinct enough among a set of clones (e.g. technically this means no overlap due to stuttering).
Secondly, the characterisation of mixtures of clones (laboratory scale, WP2-5) will be done by using DGGE and by using the selected microsatellites. Microsatellites have already been used to determine the genotype of an individual. They have not been used to follow a culture containing a mixture of genotypes. But, if the alleles are distinct enough among a set of clones it should be possible to use microsatellites as a tool to describe mixtures of clones and their evolution during culture (WP2-5). In fact, applying microsatellites in this way is very similar to multiplexing various microsatellites, which is a common technique nowadays. Yet the application of microsatellites in the way suggested still needs to be validated, as also the application of the DGGE technique.
In order to validate both techniques as a tool to describe mixtures of Brachionus clones, artificial mixtures, with known ratios of clones, will be made. This way the detection limit for a certain clone in the background of a mixture can be determined. In addition the repeatability and reproducibility will be verified. Whenever unknown mixtures (commercial/hatchery scale) have to be characterised, fifty individuals will be isolated and separately characterised with the microsatellites to unravel the complexity of diversity present in the sample.
This double approach provides the possibility to compare the results produced in both ways. The characterisation of a mixture through the characterisation of individuals is the classic approach but is labour intensive. The other approach might yield less detailed information (depending on the outcome of this project) but is definitely less labour intensive and less expensive. It might become a cost-effective tool to monitor commercial Brachionus cultures.