GSM2978605: 92: white earlobe; Gallus gallus; OTHER
Genomic DNA was isolated from whole blood using the NRBC Blood DNA Kit (Omega Bio-Tek, Norcross, GA, USA) following the manufacturer's instruction. In this study, the red jungle fowl genome (Gallus_Gallus 4.0, ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCA_000002315.2_Gallus_gallus-4.0) was chosen as the reference genome to perform enzyme digestion prediction with in-house related software. The fragments predictably digested by the enzyme that was selected for genome digestion should be in line with below rules, including a low proportion of that locating repetitive sequences, a uniform distribution across the genome to the greatest extent, a preferable identity between the fragment length and the experiment system and the digested fragments number predicted meeting the expectation (i.e., 100,000 SLAF). We herein selected restriction enzyme HaeIII to digest QYP DNA samples, predictably giving rise to 113,202 SLAF, with the length ranging from 364-444 bp. After digested by HaeIII, the genomic DNA fragments (SLAF) from each sample was added with A-tailing at 3' end, further ligated with Dual-index adaptor, and then amplified by PCR. After that, SLAF from each sample was purified and then mixed for further target fragment selection via gel electrophoresis and extraction, thereby obtaining the SLAF library for high-sequencing. Ultimately, SLAF libraries from each sample were sequenced on Illumina HiSeq 2500 platform, according to Illumina instructions (operated by Biomarker Biological Technology Co., Ltd., Beijing, China). We herein developed a total of 622,443 SLAF markers, including 424,810 polymorphic ones, and their corresponding average sequencing depth reached 10.03 ×.