2.2. DNA Extraction, GBS Library Preparation and Sequencing

DNA was extracted using the Sherlock AX Isolation KIT (A&A Biotechnology, Gdynia, Poland) according to the provided protocol for both kinds of biological material. The calamus was taken as a source of DNA from plumage.

DNA was pooled in groups according to the breed, with ten individuals per pool. As a result, we obtained 34 pools with a final DNA concentration of 100 ng/µL (Table 1).

DNA pools prepared for the experiment.

Prepared DNA pools were digested overnight in 37 °C with the restriction enzyme PstI-HF (100,000 U/mL, New England Biolabs, Ipswich, MA, USA). Afterwards, products of digestion were ligated with 4.8 ng of adaptors using T4 DNA ligase (400 U/µL, New England Biolabs, Ipswich, MA, USA) in 60 min incubation in 22 °C. In our analysis, we used 48 indexed adapters designed with GBS Barcode Generator software as described in the previous study [8]. The ligation mixture was purified using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the standard protocol. Purified libraries were finally amplified in PCR reaction with universal primers (12.5 pmol/µL) and Taq Master Mix (New England Biolabs, Ipswich, MA, USA). After another round of purification, obtained libraries were qualitatively examined using Tape Station 2200 system (Agilent Technologies, Santa Clara, CA, USA) and quantified by Qubit DS DNA assay (Thermo Fisher Scientific, Waltham, MA, USA). The final step of the GBS libraries preparation was normalization to 10 nM concentration and frozen in −20 °C for further use.

The obtained libraries were sequenced in a single 50 bp run on the HiScanSQ system (Illumina, San Diego, CA, USA) with the use of TruSeq v3 chemistry and v3HiSeqflowcell.