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.