Adult brown trout were caught via electrofishing around the beginning of the spawning season from the River Aare and its tributaries Gürbe, Worble, Giesse, Kiese, and Rotache (Bern canton, Switzerland). They were kept in the Fischereistützpunkt Reutigen until eggs could be stripped from females. Adults were narcoticised when ready to spawn and gametes were stripped for block-wise full-factorial in vitro fertilisations (e.g. Marques da Cunha et al. 2019). Photographs were taken from all fish, standard length and wet weight were determined, and fin clips were taken for molecular analyses. All fish were then released back into the streams of origin. Samples of 24 freshly fertilized eggs per experimentally produced full-sib family were used for various laboratory experiments (e.g. Marques da Cunha et al. 2019). The remaining embryos were raised in the Fischereistützpunkt Reutigen and used for stocking the streams of parental origin and various nursery streamlets, following the routine of the stocking program of the Bern canton. These stocked fish were later sampled at various stages of their life cycle, and 13 microsatellite markers could be used to assign these fish to their parents.

Marques da Cunha L., Uppal A., Seddon E., Nusbaumer D., Vermeirssen E.L.M., Wedekind C. 2019. No additive genetic variance for tolerance to ethynylestradiol exposure in natural populations of brown trout (Salmo trutta). Evol. Appl. 12(5), 940-950. (doi:10.1111/eva.12767).

Standardized DNA extracts were sent to Ecogenics GmbH (Balgach, Switzerland) for microsatellite typing at 13 loci (i.e. Brun13, Brun25, BS131, MST-15, Mst543, MST-591, Ssa-197, Ssa-85, SSOSL438, Str12INRA, Str2INRA, Str58CNRS and T3-13) using in house protocols (see Supplementary Material of Palejowski et al. 2022 for full details). Amplification of microsatellite loci was conducted in three multiplex reactions. Multiplex one and two, each containing primers for the amplification of four microsatellites, was performed using 2X QIAGEN® Multi-plex PCR Master Mix (Qiagen GmbH, Hilden, Germany). Multiplex three included primers for the amplification of five microsatellite loci and was amplified using 2X HotStarTaq Mastermix (Qiagen GmbH, Hilden, Germany). Before thermal cycling, a prolonged denaturation step (95°C for 15 min) was included followed by 35 cycles with 94°C for 30 s, 58°C for 90 s, 72°C for 60 s and a final elongation step of 30 min at 72°C. Fragment analyses were performed on a 3730XL DNA Analyzer (Applied Biosystems, Foster City, CA, USA) with a GeneScan LIZ500 size standard (Applied Biosystems) and allele calling was performed using the GeneMarker V2.6.4 software (SoftGenetics LLC, State College, PA, USA).

Palejowski H., Bylemans J., Ammann V., Marques da Cunha L., Nusbaumer D., Castro I., Uppal A., Mobley K.B., Knörr S., Wedekind C. 2022 Sex-specific life history affected by stocking in juvenile brown trout. Front. Ecol. Evol. 10, 869925. (doi:10.3389/fevo.2022.869925).

These fish were used to experimentally produce embryos in a large number of full-factorial breeding blocks. Subsamples of these embryos were used in various types of laboratory experiments or released into different streamlets and sampled at various stages.