Our study was approved by the Animal Care Committee of the Institute of Animal Science, Guangdong Academy of Agricultural Sciences (Guangzhou, People’s Republic of China) with approval number GAAS-IAS-2009-73. All birds were housed in individual battery cages with ad libitum access to food and water and humanely euthanized.
Two breeds of chicken were used, the Guangxi Huang (S4) chickens were bred by the Institute of Animal Science, Guangdong Academy of Agricultural Sciences (Guangzhou, China). The Qingjiao Ma (Q) chickens were bred by the Sichuan Agricultural University (Ya’an, China), and kindly providing the samples used in this study. All birds had free access to feed and water.
At the age of 91 days blood samples were collected in 1.5mL tubes with 1.5% EDTA and stored at -20℃. Birds were humanely euthanized and tissue samples including heart, liver, spleen, lungs, kidney, muscular stomach, glandular stomach, intestine, breast muscle, ovary, cerebrum, cerebellum, hypothalamus, hypophysis and back skin were collected. Tissue samples were collected for RNA isolation were put in 1.5mL tubes with RNA later (Sigma-Aldrich, MO, USA) and stored at -20℃. Skin tissue samples used for DNA isolation were stored at -80℃.
The DNA of reference breeds were collected at the Institute of Animal Science, Guangdong Academy of Agricultural Sciences. A total of 1054 DNA samples from seven different chicken breeds were genotyped and allele frequencies were determined: Huiyang Beard, Silkies, Mahuang, Youxi Ma, Qingjiao Ma, Fast-growing Lingnanhuang Line A and Guangxi Yellow Chicken. Samples for RNA isolation and DNA isolation were stored as described above until further use.
Measurement of Carotenoid Concentration in Chicken Skin
For the measurement of the carotenoid concentration in chicken skin, 0.5g back skin tissue was add to 5mL mixture reagent (CHCL3:CH3OH=2:1) in a 50mL tube and homogenized. Then 5mL mixture reagent was added to the homogenate followed by 2mL 0.9% (8.5g/L) NaCl after which the tube was vortexed for 2 minutes. Samples were centrifuge at 648g for 10 minutes at 4℃. After centrifugation, the CHCL3 layer was transferred to a new tube, while the water layer was transferred to another tube with 5mL hexane, vortexed for 2 minutes and centrifuged again at 648g for 10 minutes at 4℃. From this tube the hexane layer was removed and added to the tube with the CHCL3 phase [13, 14]. The content of the tube was put in a water bath at 50℃, and being dried by nitrogen using Rotary Evaporator (RE-3000A, Shanghai Yarong Biochemistry Instrument factory, Shanghai, China); the formed pellet was dissolved in 0.15~5mL ethanol after which 50ul of the ethanol samples was analyzed using High Performance Liquid Chromatography (LC-20AD, SHIMAZU Inc., Kyoto, Japan). Lutein and zeaxanthin were used as internal standard (Guangzhou Juyuan Biochemical Co., Guangzhou, China), and measured at a wavelength of 445nm and 451nm, respectively. Differential carotenoid concentrations in chicken skin, between the Guangxi Huang and Qingjiao Ma breeds, was determined using a t-test with SAS 8.0 software (SAS Institute, Cary, NC, USA).
Primer pairs were designed for 18 fragments (F1/R1-F18/R18) together responsible for amplification of the complete CDS of the BCO2 gene. These included primers for PCR-RFLP: YSD-F/R, primers for RT-PCR: BCO2-F/R and the housekeeping genes GAPDH-F/R and β-actin-F/R. TaqMan Real-time PCR: probes (BCO2-IN2, SOX5-IN2, shown in table 2) and primers (BCO2-C-F/R, SOX5-F/R). All primers were synthesis by Sangon Biotech (Shanghai) Co., Ltd. The primer sequences are shown in Table 1.
SNP Scanning and Genotyping
DNA was isolated from whole blood and tissue samples by SDS-proteinase K (ThermoScientific, Shanghai, China). The 18 fragments were amplified by PCR, using DNA as template. To check whether the correct products were amplified, the PCR products were sequenced (Sangon Biotech (Shanghai) Co., Shanghai, China). The sequences were aligned between the two breeds to identify the SNP site. Genotyping by PCR-RFLP and treatment with the restriction enzyme SduI (ThermoScientific, Shanghai, China) made it possible to recognize the SNP site.
Three female chickens from 91-days old of the Guangxi Huang and Qingjiao Ma breed were used for this analysis. Fifteen tissue heart, liver, spleen, lungs, kidney, gizzard (muscle part stomach), glandular stomach, intestine, breast muscle, ovary, cerebrum, cerebellum, hypothalamus, hypophysis and back skin were collected. Tissues were weighed (approximately 0.2 g if tissue was used), grounded into a powder in liquid nitrogen and homogenized in 2mL of TRIzol (Life Technologies, Rockville, MD USA) using a handheld electric homogenate instrument (Germany, Staufen, IKA). The homogenized samples were left for 5 min at room temperature and then centrifuged at 12,000 rpm (representative g value) at 4°C for 10 min. Total RNA was isolated using the RNAiso Plus kit (Takara Bio Inc., Dalian, China) according to the manufacturer’s instructions. The amount and quality of the samples was estimated using the NanoDrop ND-1000 spectrophotometer (ThermoScientific, MA, USA) and gel electrophoresis (Bio-Rad Laboratories, SYSTEM GelDoc XR+, California, USA ). First-strand cDNA synthesis and reverse transcriptase PCR were performed as described in the instructions for the PrimeScriptTM II reagent Kit with gDNA Eraser (TaKara, Dalian, China). Primer pairs BCO2-F/BCO2-R (Table 1) were used to determine the relative expression values of the BCO2 gene by qPCR. The PCR amplifications were performed in 20 μL reaction volumes comprising 0.5 μL of chicken cDNA, 0.5 μL of each primer and 10 μL of SYBR Green Real-time PCR Master Mix (TOYOBO, Tokyo, Japan) in a LightCycler 480 Real-Time PCR System (Roche Applied Science, Indianapolis, IN, USA). The PCR conditions were: 95°C for 1 min, followed by 40 cycles of 95°C for 15 s, 58°C for 15 s, and 72°C for 20 s. The level of fluorescence was used to calculate the threshold cycle (Ct) value for each sample. The relative gene expression levels were analyzed using the comparative Ct method, in which housekeeping genes β-actin and GAPDH (Table 1) were used as internal controls and the geometric averaging of those two internal control genes was calculated according a previously published method . The geometric mean of 2 reference genes according to the ΔCT , 2-ΔCT method were used to calculate the expression level . Expression abundances of BCO2 gene in 15 tissue samples, between the Guangxi Huang and Qingjiao Ma breeds, were determined and analyzed using a t-test with SAS 8.0 software (SAS Institute, Cary, NC, USA).
TaqMan Real-time PCR
Primer pairs BCO2-C-F/BCO2-C-R, SOX5-F/SOX5-R and probes BCO2-IN2, SOX5-IN2 (Table 1) were used to perform genomic copy number analysis by TaqMan Real-time PCR. Target probes were marked and labeled with 5’6-FAM/3’BHQ1, the reference probe located in an exon of SOX5 were labeled with 5’HEX/3’TAMRA (Sangon Biotech (Shanghai) Co., Shanghai, China). TaqMan RT-PCR system (TAKARA Inc.): Premix Ex Taq (Probe qPCR) (2x) 10 μL, PCR 2 Forward Primer 0.4 μL/each, 2 PCR Reverse Prime 0.4 μL/each, TaqMan® Probe1 0.6 μL, TaqMan® Probe2 0.6 μL, DNA 1 μL, ddH2O 6.2 μL, Total, 20 μL. Program: pre-degeneration, 95°C 30s 20°C/s, 1 Cycle; PCR, 95°C 5s 20°C/s, 60°C 20s 20°C/s, 40 Cycles; cooling, 50°C 30s (speed 2.2°C/s), 1 cycle. Data was analyzed using the ΔΔCt method by first normalizing the target Ct value to the reference Ct value within a sample . Error bars represent the minimum and maximum estimated copy number as calculated from technical replicates of each sample.