Research Projects

ABSTRACT

European rivers are inhabited with widely distributed Thymallus thymallus, occurring in many evolutionary lineages bound to specific geographical areas. Among them there are the lineages populating the Adriatic and Sava drainage. Adriataic grayling (A. grayling) inhabits the Po and Adige drainages in Italy, and the Soča drainage in Slovenia. Although A. grayling is mostly regarded as T. thymallus, it was on the basis of morphological description already in 1848 described as separate species T. aeliani. The distinctness of A. grayling has been confirmed also by genetic analyses.

A. grayling in Slovenia is much endangered due to intense supportive stocking lasting till 2006, with artificially reared Sava grayling, which resulted in hybrid population and extinction of genetically pure individuals.

Since A. grayling and artificially reared Sava population originate from rather different environment, they are not only evolutionary but most likely also adaptively divergent. Crossing of adaptively divergent lineages can cause substitution of alleles that increase fitness in local environment, with introduced ones that do not possess this ability (out-breeding depression) leading to fitness reduction of the local population.

An attempt to repopulate A. grayling in Slovenia is based on artificial propagation of genetically most appropriate individuals captured in nature, which however normally do not exceed 40% genetic purity. They are sorted out using neutral microsatellite loci. As neutral markers do not provide any information on environmental selection, a question arises whether out-breeding depression has already affected A. grayling, and whether genitor individuals, selected using neutral markers, retain sufficient adaptiveness to natural environment and pass this trait to their progeny to be used for repopulation stocking. Recently, A. grayling population with more than 90% of indigenous genetic share was discovered in the upper Adige River in Italy. Their translocation into the Soča drainage might contribute to the elevation of the Adriatic genetic share in Slovenian population; however, it is questionable whether the populations are adaptively compatible. Artificial propagation of grayling fry is demanding yet very much alleviated if performed under controlled conditions in fish-farm; but this approach may cause domestication and consequently poor adaptation of the released progeny into the wild. A question is how large adaptive divergence between fish-farm and wild grayling population truly is.

To answer these questions, we will apply molecular approach for the detection of markers, which are affected by selection due to different environment (adaptive markers). We will carry this out via identification of SNP loci associated with the adaptive traits. Sufficient quantitative extent of SNP loci will be assured using high-throughput NGS technique (i.e., RADseq). Quantification of adaptive variation among compared groups will be assessed by identifying outlier loci that deviate from neutral loci by their high Fst value.

Adaptive differentiation will be studied also by assessing levels of DNA methylation across the compared groups. On the basis of this signal we will make inferences on differences in gene expression and on epigenetically dependent adaptation to different environment.

The results of the project will substantially help to update and improve strategies of the genetic management of A. grayling and in this way importantly contribute to more efficient repopulation of this species in Slovenia.

According to our knowledge, this project will represent the first conservation genomic study on wild organisms in Slovenia and will be, beside scientific novelty, a conceptual starting point for similar studies on other threatened species.

THE PHASES OF THE PROJECT AND THEIR REALIZATION

WP 1: Selection of populations and sample collection

WP2: RAD sequencing and library preparation

WP3: Bioinformatics analysis of SNPs identified with RAD sequencing

WP4: Individual and population genetic analyses based on RAD sequencing

WP5: List of identified neutral and outlier SNPs for grayling

WP6: DNA methylation assay

WP7: DNA methylation bioinformatics

WP8: Assessing the status of the Adriatic grayling based on genetic and epigenetic analyses

The time frame of the project:

The first year:

• Sample collection: we will arrange the collection of samples in Slovenia and abroad in collaboration with colleagues from Angling Club Tolmin, Angling Club Bled, Fisheries Research Institute Slovenia and A. Gandolfi (Italy),
• Isolation of high quality DNA,
• Start of genotyping with RAD sequencing (libraries preparation),
• Selection of genes for DNA methylation assay,
• Bisulphite conversion of isolated DNA,
• Design of bisulphite sequencing PCR assay,
• Preparation of scientific publications;

The second year:

• Continuation of genotyping with RAD sequencing,
• Bioinformatics data analysis of RAD sequencing (detection of outlier and neutral SNPs; Annotation; Blast search, GO ontology),
• Identification of neutral SNP markers and markers under selection (outlier SNPs),
• Sequencing of bisulphite-converted DNA,
• Identification of methylated and non-methylated sites in candidate genes,
• Preparation of scientific publications;

The third year:

• Identification of and markers under selection (outlier SNPs),
• Selection of SNP markers characteristic for the Adriatic grayling and useful in subsequent steps of conservation genomic and repopulation of grayling in the Soča basin,
• Population genetic analyzes based on the large number of SNP markers from RAD sequencing,
• Presentation of all newly discovered SNP markers potentially useful for population genetic analyzes of other populations of grayling,
• Analyses of group/population differentiation based on proportion of methylated sites in candidate genes,
• Comparison of group/population differentiation based on outlier SNPs and proportion of methylated sites
• Evaluation of the status of the Adriatic grayling in the Soča basin and in Italy, according to the information obtained during the project; evaluation of potential translocation of grayling from Italy to Slovenia; comparison with the published results of the classical population genetic analyzes,
• Preparation of scientific publications;

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