Razvoj molekularnih orodij za ohranitev jadranskega lipana (Thymallus aeliani)

V evropskih rekah živi sladkovodna vrsta lipan Thymallus thymallus, ki se pojavlja v več

evolucijskih linijah, vezanih na različna geografska območja. Sem sodita tudi liniji, ki

naseljujeta jadranski in savski rečni sistem. Jadranski lipan (j. lipan) živi v porečju Pada in

Adiže v Italiji in v porečju Soče v Sloveniji. Čeprav danes j. lipana večinoma uvrščajo v vrsto

T. thymallus, je bil na osnovi morfološkega opisa že l. 1848 poimenovan kot samostojna

vrsta T. aeliani. Različnost j. lipana so potrdile tudi sodobne genetske raziskave.

J. lipan je v Sloveniji zelo ogrožen zaradi intenzivnega podpornega vlaganja z umetno

vzrejenim savskim lipanom, ki je trajalo do l. 2006, kar je povzročilo nastanek hibridne

populacije in izginotje čistih primerkov j. lipana v soškem porečju.

Ker j. lipan in ribogojniška savska populacija izhajata iz zelo različnega okolja, med njima

zelo verjetno poleg evolucijske obstaja tudi adaptivna divergenca. Križanje adaptivno

divergentnih linij lahko privede do zamenjave alelov, ki vzdržujejo prilagojenost na lokalno

okolje, z alohtonimi, ki te lastnosti nimajo (depresija zaradi »out-breedinga«), in do znižanja

»fitnessa« lokalne populacije.

Poskus repopulacije j. lipana v Sloveniji temelji na vzreji genetsko najprimernejših osebkov

iz narave, ki pa ne presegajo 40% genetske čistosti. Njihova identifikacija poteka z

nevtralnimi mikrosatelitnimi markerji, ki pa ne dajejo informacije o selekcijskem pritisku

okolja, zato obstaja vprašanje, ali je pri j. lipanu prišlo do depresije zaradi »out-breedinga«,

in tudi, ali plemenski lipani, odbrani z nevtralnimi markerji, ohranijo adaptivnost na naravno

okolje in le-to v zadostni meri prenesejo na svoje potomce, ki so namenjeni

repopulacijskemu vlaganju. V porečju Adiže so nedavno odkrili populacije j. lipana z več kot

90% avtohtonim genetskim deležem. Translokacija le-teh v soško porečje bi lahko

pripomogla k dvigu jadranskega genetskega deleža slovenske populacije, vendar obstaja

vprašanje, ali sta populaciji adaptivno kompatibilni. Vzreja mladic lipana je težavna a precej

olajšana, če temelji na plemenski jati, ki se jo vzdržuje v kontroliranih ribogojniških

razmerah, vendar pri tem obstaja nevarnost njene domestikacije in posledično slabe

prilagojenosti njenega potomstva na naravne razmere. Zastavlja se vprašanje, kako

obsežna je v resnici adaptivna divergenca med ribogojniško in divjo lipansko populacijo.

Da bi odgovorili na ta vprašanja, bomo uporabili molekularno orodje za odkrivanje

markerjev, na katerih se odraža vpliv selekcije zaradi različnega okolja (adaptivni markerji),

kar bomo izvajali preko identifikacije SNP-lokusov na kodirajočih regijah genoma. Zadosten

kvantitativni obseg SNP-lokusov bomo zagotovili z RAD sekvenciranjem. Kvantifikacijo

adaptivne diferenciacije med primerjanimi skupinami bomo ugotavljali preko identifikacije

odstopajočih SNP lokusov glede na njihovo visoko Fst vrednost (t.i. »outlier lokusi«).

Adaptivno diferenciacijo bomo na osnovi kandidatnih genov ugotavljali tudi preko različne

stopnje metilacije DNA primerjanih skupin in preko tega signala sklepali na njihove razlike v

stopnji genske ekspresije in posredno na epigenetsko pogojeno prilagajanje na specifično

okolje.

Rezultati projekta bodo bistveno pripomogli pri odločitvah genetskega upravljanja z

jadranskim lipanom in na ta način pomembno doprinesli k učinkovitejši repopulaciji te

ogrožene vrste v Sloveniji.

Po našem védenju bo v Sloveniji to prva raziskava, ki bo temeljila na varstveni genomiki

prostoživečih organizmov, in bo poleg raziskovalne novosti predstavljala tudi izhodišče za

nadaljnje podobne študije na drugih ogroženih vrstah.

 

PROJECT ABSTRACT

European rivers are inhabited with widely distributed freshwater fish grayling Thymallus

thymallus, occurring in many evolutionary lineages bound to specific geographical areas.

Among them there are the lineages populating the Adriatic and Sava drainage. Adriatic

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 recognized 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 (outbreeding

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.