Animal samples and trait measurement
DNA samples of 766 chickens from eight populations, Lushi chickens (LS, n = 39, 6 weeks), Ningdu chickens (ND, n = 95, 12 weeks), Wenchang chickens (WC, n = 65, 7 weeks), Qingyuan Partridge chickens (QY, n = 70, 7 weeks), Recessive White Rock chickens (RW, n = 55, 7 weeks), ISA Brown laying hen (ISA, n = 54, 20 weeks), Guangxi chickens (GX, n = 71, 12 weeks) and F2 population (F2, n = 360, 13 weeks) were used. These DNA samples are all from the chicken breed resource library kept in our laboratory. In eight different breeds, LS, ND, WC, QY and GX are domestic chicken breeds in China, RW and ISA are commercial broilers and layer hens, respectively. And the F2 resource population is a hybrid strain of RW and Xinghua (XH) chickens, XH chickens represent a slow-growing Chinese domestic chicken. In our laboratory, 2 mL of 5% Pentobarbital was injected intraperitoneally into the chicken (No. 57–33-0 of Chinese Academy of Sciences, Beijing Siyuan Technology Co., Ltd.). After 2-3 minutes, the chicken was sacrificed by bleeding through the carotid artery. All F2 population had data records about economic traits, and detailed information on measuring methods is as previously described [30].
12 different tissues were obtained from four QY chickens. Moreover, breast muscle of six embryonic periods (E10-15) and leg muscle of four embryonic periods (E12-15) was used to detect the relative GNB1L expression.
cDNA synthesis and qRT-PCR
RNA extraction using the TRIzol (Takara, Dalian, China) method, reverse transcription by the cDNA reverse transcription kit (Takara, Dalian, China) followed by PCR. Relative gene expression was calculated by the 2–ΔΔCt method, and significance using ANOVA followed by Duncan's test. All reactions using three biological and technical repetitions. The relative expression of GNB1L in different tissues and embryo ages were analyzed by qRT-PCR. Primers of GNB1L qRT-PCR and internal control β-actin are listed in Table S3.
Indel detection and diversity analysis of different breeds
A 31-bp Indel in the GNB1L gene from whole-genome re-sequencing data of ten XH and ten RW chickens (unpublished data). Genotyping of GNB1L 31-bp Indel by PCR amplification and gel electrophoresis in eight diverse populations. Blood samples were used for the extraction of DNA, and the final concentration of DNA used for amplification was diluted to 50 ng/µL. GNB1L PCR primers based on the genome is listed in Table S3. Each 15-µL PCR amplification volume contained 1µL DNA, 1.5 µL primer, 7.5 µL 2×Taq Master mix (TSINGKE, Beijing, China), and 5µL double-distilled water. PCR procedure included at 95°C for 3 min, 35 cycles at 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 10 min. The PCR products after amplification were separated by 3.0% gel electrophoresis.
The genotypes and allele frequencies of the mutation were calculated directly in different breeds. Hardy-Weinberg equilibrium (HWE) was analyzed using the SHEsis online website (http://analysis. biox.cn). Moreover, allele numbers (Ne), genetic indices of heterozygosity (He), effective polymorphism information content (PIC) and population differentiation were analyzed by PopGene software (Version 1.3.1) [31, 32].
Transcription Factor Prediction
The transcription factors (TFs) in the 31-bp Indel mutation of 5′ UTR regions of the GNB1L gene were predicted by online AliBaba software (Version 2.1) [24].
Statistics
Association analysis of F2 population by SPSS 22.0 software, and was used two different models in the analysis. All growth traits use Model Ⅰ (Yijkl=µ+ Gi + Sj + Hk + fl + eijkl), and all carcass traits use Model II (Yijkl=μ + Gi + Sj + Hk + fl + b (Wijkl - W(—)) + eijkl), and carcass weight serve as a concomitant variable of Model II. Yijkl represents the observed value, µ is the overall population mean, fl is the fixed effect of family, Gi is the fixed effect of genotype, Hk is the fixed effect of hatch, Sj is the fixed effect of sex, b is the regression coefficient for carcass weight, W(—) is average slaughter weight, Wijkl represents the individual slaughter weight, and eijkl represents the random error in two Models. Significance was set at P-value < 0.05, and the Bonferroni’s test serve as multiple comparisons [18].