Associations of IGF2 and DRD2 polymorphisms with laying traits in Muscovy duck

Ethics statement

Ethical approval for all animal experiments was granted by the Animal Care Committee of South China Agricultural University (Guangzhou, People’s Republic of China) with approval number 20131019002.

Sample collection

Eight hundred white Muscovy ducks were offered by Wens Nanfang Poultry Breeding company (Yunfu, Guangdong, China) which were in the same run. All Muscovy ducks were reared under identical conditions of management and feeding. Ducks were maintained outside on the ground from four to 12 weeks of age, after which they were transferred to individual cages in a semiconfined house. Feed was provided by Wens (Xinxing, Guangdong, China). The first egg age (FEA), egg numbers at 300 days (E300D), and egg numbers at 59 weeks (E59W) were recorded for each female duck. Genomic DNA from each individual at 59 weeks was isolated from 0.5 ml blood stored with EDTA as an anticoagulant, using E.Z.N.A NRBC Blood DNA Kit (Omega, Norcross, GA, USA) according to the manufacturer’s instructions.

RNA isolation and cDNA synthesis

Muscovy duck tissues including pituitary, brain, lung, abdominal fat, liver, ovary, subcutaneous fat, spleen, kidney, leg muscle, hypothalamus, cerebellum, heart and breast muscle used for expression pattern analysis of the IGF2 and DRD2 genes, were sampled at first egg age. These ducks were raised under the same conditions, but in different batches from the eight hundred Muscovy ducks mentioned above. Total RNA was isolated from tissues using a TRIZOL Reagent kit (TaKaRa, Dalian, China) according to the manufacturer’s protocol. RNA quality was evaluated by 2% agarose gel electrophoresis and then was reverse transcribed using Takara reverse transcription Kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions. The cDNA was used as template to amplify the coding region of the IGF2 and DRD2 genes from Muscovy duck.

Cloning of Muscovy duck IGF2 and DRD2 genes

The Muscovy duck IGF2 and DRD2 genes were identified using Mallard duck gene sequences as a reference (Gene Bank accession No. XM_005019778 and XM_013109685). Primers were designed to amplify the coding regions of Muscovy duck IGF2 and DRD2 using Primer 5.0 (Primer IGF2-CDS and Primer DRD2-CDS; Table S1). PCR amplifications were conducted in a final volume of 50 µl with 2 µl cDNA, 25 µl 2 × Easy Taq SuperMix (TransGen, Beijing, China), and 0.5 µl each pair of primers, and 22 µl double distilled H₂O. Optimum PCR amplification conditions were programmed as pre-denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s, and a final extension at 72 °C for 10 min. The PCR products were evaluated by electrophoresis using a 2% agarose gel and then gel purified using a HiPure Gel Pure DNA kit (TransGen, Beijing, China). The amplified fragments were cloned into pMD-18T vector (TaKaRa, Dalian, China), and sequenced by Majorbio, Shanghai, China. Sequence alignment and phylogenetic trees are constructed using MEGA5.

Expression pattern analysis of IGF2 and DRD2 mRNA

Total mRNA from 14 different tissues was extracted to investigate the mRNA expression profiles of Muscovy duck IGF2 and DRD2 genes using real-time qPCR. Muscovy duck β-actin gene was used as the internal reference gene. Primers for the IGF2, DRD2 and β-actin genes were designed using Primer 5.0 (Primer β-actin-duck, IGF2-Q and DRD2-Q; Table S1). The qPCR was performed using a standard SYBR Premix Ex Taq II (TaKaRa, Dalian, China) on a BioRad CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol. The thermal cycling was 95 °C for 2 min, followed by 39 cycles of 95 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s, and final cycle of 72 °C for 7 min. Relative expression of IGF2 and DRD2 genes was calculated relative to the expression of β-actin. Real-time PCR data were analyzed using the 2^−ΔΔCt method.

SNPs detection by sequencing

We designed 7 primers to identify potential SNPs of IGF2 and DRD2 (Primer IGF2-P1, IGF2-P2, DRD2-P1, DRD2-P2, DRD2-P3, DRD2-P4 and DRD2-P5; Table S1). Twenty white Muscovy ducks were sampled and five individuals were selected as a mixed pool. PCR reactions were performed in a 50 µl final volume, containing 2 µl DNA, 25 µl 2 × Easy Taq SuperMix (TransGen Biotech, Beijing, China), 0.5 µl each pair of primers, and 22 µl double distilled H₂O. PCR parameters were 3 min at 94 °C followed by 37 cycles of 94 °C for 30 s, annealing temperature for 60 s, 72 °C for 30 s and a final extension at 72 °C for 10 min. PCR products were evaluated by electrophoresis using 2% agarose gel and sequenced as described above. SNPs were identified by the Seqman program of DNASTAR 7.1.0 software (DNASTAR, Inc., Madison, WI, USA).

Genotyping and association analysis

The SNPs were genotyped in 800 female ducks with egg production records via sequencing. We designed 3 primers to genotyping SNPs of IGF2 and DRD2 (Primer IGF2-SNP, DRD2-SNP1 and DRD2-SNP2; Table S1). PCR reactions were identical to those used in SNP detection as described above. Genotypes were tested for Hardy-Weinberg equilibrium with the chi-square test. Linkage analysis was performed using Haploview software (Barrett et al., 2005). The associations between SNPs and egg production traits were calculated using the general linear model procedure of SAS v. 9.2 with the following model: ${\displaystyle Y_{ij}=\mu +G_i+e_{ij}}$ where Y_ij is the observed value of different egg production traits, μ is the overall population mean, G_i is the effect of each genotype, and e_ij is the random error. For each egg production trait, the least-squares mean was estimated and differences between the genotypes were analyzed using a Bonferroni adjustment for multiple comparisons. Difference with P value ≤0.05 was considered to be significant in analyses.

Characterization of Muscovy duck IGF2 and DRD2 coding region

We obtained a 311-bp partial cDNA of the IGF2 gene that was 98% and 95% identical to Anas platyrhynchos (XM_013191560.1) and Anser cygnoides domesticus (XM_005019778.2), respectively. We obtained the full-length cDNA of DRD2 including a 52-bp 5′-untranslated region (UTR), an 1,104-bp open reading frame (ORF) containing 368 codons and a 294-bp 3′-UTR. The Muscovy duck DRD2 cDNA sequence was 98% and 96% to Anas platyrhynchos (XM_013109686.1) and Anser cygnoides domesticus (XM_013187289.1), respectively. A phylogenetic tree constructed based on the DRD2 gene also revealed that the Muscovy duck was closely related with both animals above (Fig. 1).

Tissue expression of IGF2 and DRD2 genes

We examined tissue-specific expression of IGF2 and found that it was expressed in most tissues. The highest expression levels were found in the kidney and ovaries (Fig. 2). DRD2 expression was the highest in ovary, but it was also expressed in the cerebrum, cerebellum, hypothalamus and pituitary at lower levels. However, other tissues also had expression levels near detection limits including abdominal fat, sebum and breast and leg muscle. Expression in the spleen was negligible (Fig. 3).

Polymorphisms of IGF2 and DRD2 Genes

We identified 5 SNPs in the 5′  flanking region of IGF2, a level of one SNP per 449 bp on average. These SNPs were A-1864G, C-1704G, A-584G, A-227G and A-183G (Table 1). We found 28 SNPs in DRD2 giving rise to one SNP per 317 bp on average. Among them, the SNP C+7T in exon 1 was a missense mutation resulting in a P to S amino acid change (Table 1). We selected 2 SNPs of IGF2 and 11 SNPs of DRD2, based on mixed pool sequencing results which indicated that these were more likely to be associated with egg laying traits, for further association analysis.

Association of IGF2 and DRD2 with egg production traits

Association analysis indicated that the A-1864G and C-1704G SNPs of IGF2 gene were both significantly associated with E59W (P < 0.05) (Table 2), and linkage disequilibrium analysis indicated a high linkage block between A-1864G and C-1704G for IGF2 (Fig. 4). Multiple comparisons of different genotypes showed that the AG genotype individuals of A-1864G had 6–7 eggs more than GG genotype individuals for E59W (P < 0.01). The GG genotype individuals of C-1704G had 7–8 eggs more than individuals with the CC genotype for E59W (P < 0.05).

Association analysis for DRD2 gene further showed that C+7T and C+364G had highly significant associations with FEA and E300D (P < 0.01) and were significantly associated with E59W (P < 0.05) (Table 3). A+3489G, A+3484T and T+3428C were significantly associated with FEA and E300D (P < 0.05), and highly associated with E59W (P < 0.01). T+3423C and A+3262G were significantly associated with FEA (P < 0.05) and highly significantly associated with E59W (P < 0.01). A+3183C was significantly associated with E59W (P < 0.05), and T+3024C has no significant association with any of the three egg production traits. Moreover, it was notable that C+3301G and C+3545G were highly significantly associated with FEA, E59W and E300D (P < 0.01). Multiple comparisons among different genotypes showed that the GG genotypes of C+3301G and C+3545G were advantageous for earlier egg laying and egg production. There were two high linkage blocks (C+7T and C+364G, A+3183C and A+3262G) for DRD2 (Fig. 5).