Genome dna extraction kit

Blood was collected and stored at −20°C. Genomic DNA was isolated by using a Genome DNA Extraction Kit (TIANGEN, Beijing, China) according to the manufacturer's instruction. Primer pairs used for amplifying exon regions of MyoD1 were designed from reference sequences of the pigeon MyoD1 gene in GenBank (accession no: 102096590) by using Primer Premier 5.0 software (Premier Biosoft International, Palo Alto, CA). The primers were synthesized by TSINGKE Biotech Co., Ltd. (Hangzhou, China) and are listed in Table 1.
The PCR amplification was performed in reaction mixtures with a total reaction volume of 50 µL, containing 25 µL 2× Taq PCR MasterMix (including Mg²⁺, dNTP, and Taq DNA polymerase), 2 µL of each primer, 2 µL genomic DNA, and double-distilled water. The cycling protocol was 5 min at 94°C, 30 cycles of 94°C for 30 s, 40 s at the annealing temperature (Table 1), and extension at 72°C for 1 min, with a final extension at 72°C for 10 min. The PCR products were verified by 1.5% agarose gel electrophoresis and sequenced by Hangzhou Qingkezixi Biotech Co., Ltd (Hangzhou, China). To identify SNPs, sequences were analyzed with the DNAMAN software (DNAMAN Application 4.0.1.1, Lynnon BioSoft, San Ramon, CA, USA).

Sixty domestic pigeons (Columba livia) were used for the detection of SNPs in DRD2 gene and the analysis of the relationship between the SNPs and the reproduction traits. All birds (Taishen king pigeon) were obtained from Xingliang commercial pigeon farm (Wenzhou, China), hatched on the same day, male-female matched artificially based on pedigree data, and reared in a stair-step cage under the same nutritional and environmental conditions.
Four reproduction traits were measured according to The Poultry Production Performance Terms and Measurement Statistics Method (NY/T823-2004). These traits include egg production (total number of eggs; EP), total number of fertile eggs (FE), fertility rate (the ration of FE to EP; FR), and total hatching number (HN) within 500 days of age. Venous blood samples were collected from all sixty birds by veni-puncture and stored at −20°C. The genomic DNA was extracted using Genome DNA Extraction Kit (Tiangen, Beijing, China) according to the manufacturer's instructions. The quality of DNA was checked by both native DNA electrophoresis on 1.0% agarose gel and the ultraviolet spectrophotometer.