All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of Yangzhou University. Procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (Yangzhou University, China, 2012) and the Standards for the Administration of Experimental Practices (Jiangsu, China, 2008). The two goose breeds used in this study, Zhedong goose and Yangzhou goose, were raised in the breeding farm of Jiangsu Lihua Animal Husbandry Co., Ltd., Changzhou, China, according to the farm’s standard practice. One hundred female geese of each breed were selected randomly for the study. Geese were exposed to natural light and ambient temperature throughout this study and released to an open area during the day, when they were fed ad libitum with rice grain and, when possible, green grass.
Tissue sample collection
Geese were sacrificed by anesthetizing them with sodium pentobarbital. To investigate DBH expression patterns in different tissues, various tissues were removed, immediately frozen in liquid nitrogen, and stored at −80 °C for RNA isolation. These tissues were heart, liver, glandular stomach, lung, spleen, kidney, intestinum tenue, intestinum crissum, cerebrum, cerebellum, muscle, infundibulum of the oviduct, pituitary, hypothalamus, and the stroma of the ovary. A group of Zhedong geese were sacrificed in the pre-laying stage, when they were 120 days old. Three groups of 380-days-old Zhedong geese(5 geese/group) were selected: a laying group with an egg in the oviduct (ovulation, the release of an ovum from a ruptured follicle), a laying group without an egg in the oviduct (oviposition, the laying of the egg), and a brooding group(The goose sits in the nest and the distance between pubic bones is less than two finger widths). Another 5 laying Yangzhou geese with an egg in the oviduct (ovulation) was also selected for comparison with the Zhedong breed.
Zhedong goose DBH cDNA cloning and sequencing
Total RNA was extracted from collected tissue samples using TRIzol reagent according to the manufacturer’s instruction (TaKaRa, China) and re-suspended in RNase-free water. The concentration and purity were determined with a NanoDrop Spectrophotometer (NanoDrop, USA). After purification, 2 μg of total RNA was reverse transcribed using M-MLV reverse transcriptase (Promega, USA) according to the manufacturer’s protocol. Primers were designed according the unigene (Xu et al., ; Additional file 1) and reverse transcription PCR (RT-PCR) was performed using ovarian cDNA from geese. The PCR product was purified, cloned into the pMD19-T vector (TaKaRa, China), and subjected to sequence analysis. The 5′- and 3′-ends of DBH were amplified via rapid amplification of cDNA ends (RACE) using the 5´-RACE System for Rapid Amplification of cDNA Ends (Invitrogen, USA) and the 3′-Full RACE Kit (TaKaRa, China), respectively. RACE primers (Additional file 1) were designed using the partial DBH nucleotide sequence obtained from RT-PCR. Touchdown and nested PCRs were performed according to the manufacturer’s instructions. Amplicons were then cloned into a plasmid vector for nucleotide sequencing by Sangon Biotech (Shanghai, China).
The Zhedong goose cDNA and deduced DBH amino acid sequences were analyzed using DNAStar (version 7.1). Homology analyses were carried out using Clustal W (http://www.ebi.ac.uk/Tools/msa/). Conserved domains in the protein were identified by the conserved domain database (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). A rooted neighbor-joining tree was constructed to determine the phylogenetic relationship using MEGA 6.0 software with 1000 bootstrap replicates to establish the confidence level of each node.
DBH expression patterns in Zhedong goose and Yangzhou goose
To study expression of the cDNA encoding goose DBH (gDBH), we performed real-time quantitative PCR (qPCR) on total RNA isolated from the tissues. Assays were conducted in 20-μL reaction mixes using the SYBR Premix Ex Taq™ (TaKaRa, China) and performed on an ABI two-step RT-PCR system (Applied Biosystems 7500, USA) with diluted first-strand cDNA. The glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) served as an internal reference gene. Quantitative qPCR programs for DBH and GAPDH were: one cycle of 95 °C for 5 min, 40 cycles of 95 °C for 10 s, 60 °C for 34 s of data collection, and one cycle for the melting curve analysis. All cDNA synthesis reactions were carried out using 100 ng of total RNA per reaction and assayed in three to four technical replicates for each set of biological samples. The same methods were used to determine the DBH mRNA expression profile during the reproductive cycle. For the DBH mRNA expression profile of the pre-laying Zhedong geese, the chest muscle tissue served as a calibrator. For the differential expression analysis during the reproductive cycle, the oviduct tissue from pre-laying Zhedong geese served as a calibrator. To compare the expression patterns between the Zhedong geese and Yangzhou geese, the mean ΔCt value of the hypothalamus tissue of Zhedong geese within each group was used as the calibrator. Relative expression of mRNA was calculated using the 2-ΔΔCt method .
Identification of SNP on DBH in Zhedong goose and Yangzhou goose
According to the goose genome sequences(scaffold224136, scaffold224137), nine pairs of primers (shown in Additional file 1) were synthesized to identify the polymorphisms. PCR products were amplified from the DNA of Yangzhou geese and Zhedong geese, and sequenced directly by the GenScript Co., Ltd. (Nanjing, China). The obtained sequences were aligned by AlignIR(V2.0) software to screen the potential single nucleotide polymorphisms (SNPs) in the coding region.
Data analysis was performed by using SPSS17.0, then adopted one-way ANOVE analyses to compare the difference among the different tissues, periods and breeds, respectively. Comparisons of genotypes between the different breeds were evaluated by Chi-square (x
2) tests. P < 0.01 was considered statistically very significant in all.