Introduction

Over the course of the past decades, scientists have made great progress in the field of animal genetics and breeding by screening candidate genes as markers of animal productivity (Meuwissen 2007). In China, the growth rates of most local chicken breeds are low, and their production performance is poor, which restricts the development of the poultry industry to a certain extent. To fully exploit the excellent traits of the local chicken populations and improve their production performance, it is necessary to search for major effect genes that control growth and carcass traits to support marker-assisted breeding.

The prolactin receptor (PRLR), a type I cytokine receptor characterized by its ability to activate associated signaling proteins such as JAK2 and STATs, is the specific receptor for prolactin (PRL) (Fleenor et al. 2006; Morammazi et al. 2016). PRL, also known as luteotropic hormone or luteotropin, is best known as a single-chain peptide hormone that enables mammals, usually females, to produce milk (Evans et al. 1989; Mihailov et al. 2014). PRL plays a vital role in metabolism, regulation of the immune system and pancreatic development and performs multiple functions in the central nervous system from physiology to pathology (An et al. 2015; Bole-Feysot et al. 1998; Goffin et al. 2002). To mediate the various physiological functions mentioned above, PRLR must bind PRL as a transmembrane receptor to act on target cells (Bole-Feysot et al. 1998).

In view of the important physiological functions of PRLR in animal reproduction and the differences in its genetic effects in numerous studies (Drogemuller et al. 2001; Linville et al. 2001; Rens et al. 2003; Vincent et al. 1998), scholars worldwide have conducted a great deal of research on polymorphisms in PRLR. Considering the vital role of PRLR in animal mammary gland development and milk synthesis, researchers have adopted the PRLR gene as an important candidate gene for dairy cow milking traits. In terms of growth and development, a study has shown that the S18N variation site significantly affected body height, body length, chest circumference, hip width, body weight and average daily gain in Nanyang cattle (Lü et al. 2011). In the study of sheep breeds, the PRLR gene has primarily been used as a candidate gene for reproductive traits, milk production and cashmere quality. In Small-Tail Han sheep, 2 polymorphic sites in PRLR intron 1 and 1 polymorphic site in exon 10 were identified. Further analysis showed that these different genotypes of the polymorphic loci significantly affected lambing number, and the dominant genotypes were BB and AB (Chu et al. 2011). In addition, the PRLR gene in domestic pigs has been studied extensively as a candidate gene for litter size. The PCR-SSCP method was used by Mihailov et al. (2014) to detect polymorphisms of the PRLR gene in three groups of sows: Large White, Danish Landrace (LD), and Landrace × Yorkshire × Duroc hybrids (L × Y × D). It was found that the BB genotype was associated with the largest number of viable offspring. In LD sows, the AB genotype was associated with the shortest number of days to reach 100 kg; in L × Y × D hybrids, the AA genotype was associated with better meat traits than BB genotype. At present, studies of PRLR gene polymorphisms in poultry are mainly focused on egg-laying performance and broodiness. In some poultry breeds, a single nucleotide polymorphism (SNP) in the exon of the PRLR gene has been shown to result in significant differences in egg production among different genotypes (Rashidi et al. 2012; Chen et al. 2012). However, there are few studies on the effects of PRLR gene variants in the growth and development of poultry.

In this study, the full-length coding sequence (CDS) region of the PRLR gene was analyzed using bioinformatic methods. This study aimed to simultaneously identify indel polymorphisms in the PRLR gene in different poultry breeds and to analyze its genetic effect on growth and carcass traits. These results will illuminate the biological roles of the PRLR gene in the growth and development of poultry and serve as a theoretical reference for molecular breeding of poultry.

Materials and methods

Ethics approval

All animal experiments and animal care methods were approved by the Institutional Animal Care and Use Committee (IACUC) of Henan Agricultural University, Zhengzhou, P.R. China (Permit Number: 11–0085) and were performed in accordance with the protocols outlined in the “Guide for Care and Use of Laboratory Animals” (Henan Agricultural University). Chickens were allowed access to feed and water ad libitum under normal conditions. All animal experiments and methods were carried out in accordance with the approved guidelines, and all efforts were made to minimize suffering.

Animal sampling and trait measurement

In total, 3736 chickens were used as study subjects: 797 in the F2 resource population; 1854 from dual-purpose Chinese native chicken breeds [Gushi blue-shell chicken (GS, n = 272, 16 weeks), Xichuan black-bone chicken (XC, n = 393, 6 weeks), Changshun blue-shell chicken (CS, n = 142, 16 weeks), Dongxiang blue-shell chicken (DX, n = 172, 16 weeks), Lushi blue-shell chicken (LS, n = 132, 6 weeks and 14 weeks) and Wuhei chicken (WH, n = 479, 28 weeks)]; two cultivated lines [Xiaohuayu chicken (H-lines, n = 170, 72 weeks), Hengbanluhua chicken (B-lines, n = 94, 72 weeks)]; 704 commercial broilers [recessive white chicken (RW, n = 96, 6 weeks), Arbor Acres broiler (AA, n = 169, 1 day), Ross308 (n = 172, 1 day), Hubbard broiler (HBD, n = 267, 21 day)]; and 381 commercial hens [Lohmann brown laying hen (LB, n = 63, 1 day) and Hyline brown hen (HB, n = 318, 1 day)]. Among these populations, the breeding methods of the two cultivated lines (H-lines and B-lines) have been described previously (Wang et al. 2019a, b). The F2 resource population was produced after two hatchings from an F1 generation constructed via reciprocal crossing between slow-growing Chinese native GS chickens (2 roosters and 24 hens) and fast-growing Anka broilers (4 roosters and 12 hens). In total, 836 chickens from the F2 resource population were slaughtered at 84 days of age. The growth and carcass traits of the F2 individuals were measured for further analysis. The measuring methods and additional details were performed as described by Han et al. (2011).

DNA samples, polymerase chain reaction (PCR), and genotyping

Blood samples were collected from the jugular vein, and genomic DNA was isolated using the phenol–chloroform extraction method. The blood samples of 836 F2 individuals were collected during slaughtering and stored at − 80 °C until further analysis. All genomic DNA samples were quantified and subsequently diluted to 50 ng/μL for use as working solutions.

According to the published Gallus PRLR gene sequence (NC_006127.5) in GenBank, a pair of primers for PRLR was designed using Oligo 7 software and synthesized by Sangon Biotech Co. Ltd. (Shanghai, China). The primer sequence used to amplify the specific DNA fragment is shown in Table 1. The 10 µL volume PCRs contained 50 ng of template DNA, 3 µL of ultrapure water, 5 µL of 2 × Taq Master Mix (Kangwei, Beijing, China), and 0.5 μM of each primer. An initial denaturation at 95 °C for 5 min; 35 cycles of denaturation at 95 °C for 30 s, followed by annealing at 63 °C for 30 s and extension at 72 °C for 30 s; and a final extension at 72 °C for 10 min constituted the PCR amplification procedure. Subsequently, the PCR products were electrophoresed on 2.0% agarose gels stained with DNAgreen in 1 × TBE buffer. Different genotypes of the indel were confirmed by sequencing.

Table 1 Details of primer pairs
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RNA isolation, cDNA preparation, and real-time quantitative PCR

Total RNA was extracted from different tissues (including the heart, liver, spleen, lung, kidney, duodenum, skin, muscular stomach, jejunum, ileum, cecum, breast muscle, and leg muscle) of 4 LS chickens aged 20 weeks and 30 weeks and treated as previously described (Li et al. 2019). A pair of specific Q-PRLR primers was designed to detect different gene expression levels in different tissues and stages, and its sequence is shown in Table 1. The GAPDH gene was selected as an internal control, and two pairs of primers for GAPDH were synthesized by Sangon Biotech Co. Ltd. (Shanghai, China). The composition of the real-time quantitative PCR (qPCR), the reaction conditions, and the method of calculating the relative expression have been described in a previous study (Han et al. 2019). All data are presented as the mean ± standard deviation (mean ± SD).

Bioinformatics analysis

As shown in Table S1, the mRNA and amino acid sequences of the PRLR gene from 16 species were searched and downloaded from the National Center for Biotechnology Information (NCBI). Using MEGA 7.0 (Molecular Evolutionary Genetics Analysis) software, the amino acid sequences from 16 species were analyzed, and a phylogenetic tree was constructed using the neighbor-joining method. A bootstrap test with 1000 replicates was used to estimate the reliability of the constructed phylogenetic tree, and Poisson correction was used in the construction of the model. DNAMAN (version 6) was used to analyze the chicken PRLR gene DNA, mRNA, and amino acid sequences. Moreover, the amino acid sequences of the chicken PRLR genes were analyzed using a series of bioinformatics websites, as shown in Table S2.

Statistical analysis

Hardy–Weinberg equilibrium (HWE) and the allelic frequencies and genotypes of these breeds were calculated using the SHEsis program (http://analysis.bio-x.cn), and χ2 tests performed with SPSS software (Version 24.0; NY, USA) were used to analyze the differences in frequencies among individuals. In addition, Nei’s methods were used in Popgene software (Version 1.3.1) to compute the colony genetic indices, including heterozygosity (He), effective allele numbers (Ne), and polymorphism information content (PIC) (Nei and Roychoudhury 1974; Yeh 1999).

SPSS 24.0 was used to perform all the statistical analyses according to the following two linear mixed models. For growth traits, model I (Yijklm= µ + Gi + Sj + Hk + fl+ eijklm) was used. In the case of the effects of body weight on carcass traits, model II (Yijklm= μ + Gi + Sj + Hk + fl+ b (Wijklm − \(\overline{W}\)) + eijklm) was applied to the association analysis of carcass traits, in which carcass weight was used as a concomitant variable. In these two models, Yijklm represents the observed value; µ is the overall population mean; Gi is the fixed effect of the genotype; Sj is the fixed effect of sex; Hk is the fixed effect of hatching; fl is the fixed effect of family; b represents the regression coefficient for carcass weight; \(\overline{W}\) represents average slaughter weight; Wijklm represents individual slaughter weight; and eijklm is the random error (Ren et al. 2019). All data for each trait obtained by statistical analysis are presented as the mean ± standard error (mean ± SE). P < 0.05 was considered to be statistically significant, and Bonferroni’s test was performed to control for multiple comparisons (Li et al. 2019).

Results

Bioinformatic analysis of the chicken PRLR gene

The PRLR gene of chickens has 25 exons and is located on chromosome Z. Multiple transcript variants of the PRLR gene exist. The mRNA sequence (Accession: NM_204854) was chosen for bioinformatic analysis in this study. The full-length CDS region of the PRLR gene (Accession: MG517522.1) was 2496 bp, encoding 831 amino acids (Accession: NP_990185).

The results of physical and chemical analyses showed that the molecular weight of the PRLR protein (molecular formula: C4166H6472N1106O1298S40) was 94102.25 kD, and the isoelectric point was 5.41, indicating that the protein was acidic. There are 831 amino acids in the protein, of which 84 are positively charged (Arg, Lys) and 110 are negatively charged (Asp, Glu). The total average hydrophilic index presented in Fig S1 was − 0.590, suggesting that the protein was hydrophilic. The signal peptide prediction result yielded C, Y, and S values of 0.297, 0.478, and 0.910, respectively. As shown in Fig S2, this amino acid sequence has a potential signal peptide splice site located at amino acid 24, and the signal peptide length is 1–23. However, two transmembrane domains were predicted (Fig S3), the first of which (5–27) might be the signal peptide sequence. Protein phosphorylation sites were predicted, and those with scores above 0.7 are shown in Fig S4. The results of the secondary structure prediction (Fig S5) are as follows: random coil, 53.67%, 446 amino acids; extended strand, 23.83%, 198 amino acids; α helix, 17.21%, 143 amino acids; and β turn, 5.29%, 44 amino acids.

Molecular evolution and genetic conservation analysis

The genetic conserved synteny analysis shown in Fig. 1 revealed that the PRLR gene was highly conserved in diverse species, with a consistent position on the chromosome and transcriptional orientation, which suggested that PRLR has essential functions in organism. As shown in Fig. 2, the phylogenetic tree was mainly divided into two categories. Humans, rats, mice, pigs, cows, sheep, platypus, etc., can be grouped together, and chickens and ducks can be grouped together. In particular, Xenopus laevis was grouped separately, and these species above constitute the first category. Moreover, Zebrafish and Salmo salar were distantly related to the first category and were classified as the second category. Interestingly, as shown in Fig. 2 and Table S1, the mRNA sequence lengths of the PRLR genes of some closely related species are fairly similar; otherwise, the mRNA sequence lengths vary.

Fig. 1
figure 1

Conserved genetic synteny for the genomic region of the PRLR gene in different species. Species and their corresponding chromosomes are listed on the left and right sides of the figure, respectively, and the gene symbols are listed at the top. Boxes of the same color represent the same gene. The transcriptional direction of each gene is shown by the direction of the gene boxes. The figure is drawn and edited by Microsoft PowerPoint 2016

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Fig. 2
figure 2

Phylogenetic relationships among 16 species

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Relative expression in different tissues and ages

To further identify the physiological function of PRLR, the gene expression in different tissues of 20-week-old and 30-week-old LS chickens was measured by qPCR. The results in Fig. 3 show that PRLR gene expression in spleen and duodenum tissues was significantly higher at 30 weeks than at 20 weeks. In contrast, expression increased significantly at 20 weeks compared to that at 30 weeks in the heart and cecum tissues. Additionally, the alteration of the PRLR gene relative expression values between 20- and 30 weeks in the spleen and heart tissues was very significant (P < 0.01). In general, PRLR gene expression was highest in the lung, duodenum and ileum, and the tissues with the lowest expression were the breast and leg tissues.

Fig. 3
figure 3

Gene expression levels in different tissues of 20 weeks and 30 weeks LS chickens. * Represents a significant difference (P < 0.05); ** represents a very significant difference (P < 0.01)

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Genotyping and sequencing confirmation

A novel 80-bp indel mutation in the 3′-UTR of the PRLR gene was detected by whole-genome resequencing and was described as NC_006127.5 g. 10604211ins cacaaatgcc actcttcatc tggaatggca cagccatcag ttttgtaaat gcattatatt tctcagagat10604290. This indel polymorphism was genotyped in the 3736 studied individuals by agarose gel electrophoresis, and three genotypes were identified, as shown in Fig. 4. The sizes of the DNA fragments of these three genotypes were as follows: the II genotype was 219 bp, the ID genotype was 219 bp and 139 bp, and the DD genotype was 139 bp. Furthermore, the different variants of the PRLR gene were sequenced to confirm the presence of the 80-bp indel. As shown in Fig. 5, the sequence resulting from the PCR product also confirmed that an 80-bp insertion was located in the downstream region of the PRLR gene.

Fig. 4
figure 4

Electrophoresis pattern of the 80-bp indel within the chicken PRLR gene

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Fig. 5
figure 5

Sequencing comparison of the 80-bp indel polymorphism of the chicken PRLR gene. The results above were obtained by reverse sequencing. a is the partial sequence of the I allele; b is the partial sequence of the D allele

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Genotypic distribution and allelic frequencies

The frequencies of the three genotypes and two alleles, as well as polymorphic indicators associated with the 80-bp indel of the PRLR gene in 15 breeds, were calculated and analyzed by HWE tests to identify the distribution of genotypes among different breeds. The results shown in Table 2 indicate that in Chinese native chicken breeds, the frequency of genotype II was extremely higher than that of genotype DD, except in the DX breed. Similarly, the I allele frequency was much higher than the D allele frequency. Notably, only the II genotype of the PRLR gene was found in commercial laying hens and broilers, which suggested that the 80-bp indel of the PRLR gene was artificially selected during the breeding process of these commercial chickens.

Table 2 Genotypic and allelic frequencies and related genetic parameters for the chicken PRLR gene in 15 breeds
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Association between the PRLR gene 80-bp indel and growth and carcass traits

To detect the functional effects of the 80-bp indel polymorphism of the PRLR gene, the association between this indel and the phenotypic traits of the F2 resource population was analyzed. As shown in Table 3, the indel was significantly associated with carcass weight (CW), semi-evisceration weight (SEW), evisceration weight (EW), head weight (HW), claw weight (cW), wing weight (WW) and leg weight (LW) (P < 0.05), and ID genotype individuals had the highest values for these carcass traits. Furthermore, among these carcass traits, HW, cW, WW and LW were strongly correlated with the 80-bp indel (P < 0.01).

Table 3 Effect of the 80-bp indel of the PRLR gene on carcass traits in chickens
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An analysis of the effect of this indel on chicken growth traits was performed (Table 4), and interestingly, body weight (BW), shank length (ShL), shank girth (SG), sternal length (StL), and body slanting length (BSL) were significantly different (P < 0.05) at the ages of 8 weeks and 12 weeks. In addition, the effects of the indel genotypes on phenotypic traits were observed, and the results show that the values of these traits were highest in ID individuals. The difference between the ID and DD genotypes was significant (P < 0.05), while the difference between the ID and II genotypes was not significant (P > 0.05).

Table 4 Effect of the 80-bp indel of the PRLR gene on growth traits in chickens
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Discussion

Recently, genetic variation has been observed both in nucleotide sequences and in the length of coding sequence (Zhou et al. 2015). However, compared to SNPs, indel variants have higher efficiency and wider application because of their advantages of convenient detection and strong effects (Li et al. 2017). Genome size can also be affected by the relative incidence of indels, which have been recognized as a key parameter governing genome size evolution. In addition, due to their usage in phylogenetic reconstruction and their essential role in genomic evolution, indels are attracting increasing interest (Paśko et al. 2011). The fact that indel variation plays crucial roles in many aspects of animal growth and carcass traits has been revealed by related studies (Wang et al. 2019a, b). In this study, an 80-bp indel polymorphism in the 3′-UTR of the chicken PRLR gene was revealed for the first time.

In the artificial breeding process of commercial laying hens, particular attention is paid to reproductive traits, while the selection of commercial broilers focuses on meat quality traits. Therefore, along with the genetic changes caused by population differentiation that occurred during the domestication process, various beneficial alleles have become fixed in these specialized commercial breeds. We can conclude that during the poultry domestication process and in the recent development of breeds and commercial production populations, artificial selection has shaped the amount and distribution of genetic variation (Jia et al. 2016). Remarkably, the results of this study indicated that only II genotypes were identified in almost all commercial laying hens and broiler chickens, suggesting that during the breeding process of these commercial chickens, the 80-bp indel of the PRLR gene was artificially selected, leading to the appearance of obvious genetic differentiation.

As mentioned above, due to their effects on economic value (Gaya et al. 2006), the growth and carcass traits of chickens are the main reference indicators for molecular breeding. An 80-bp indel genetic variation in the chicken PRLR gene and its association with various growth and carcass traits in an F2 generation resource population are described in this study. Our results indicate that the 80-bp indel polymorphism is significantly or very significantly associated with multiple growth and carcass traits, such as BW, SL, and LW. Interestingly, this study revealed that the associations between the indel and BW, ShL, SG, StL, and BSL were significant (P < 0.05) at the ages of 8 weeks and 12 weeks but not at the age of 4 weeks or younger. At different ages, the hormone expression levels in chickens change to adapt to biological behavior that occurs during specific periods. For example, LS chickens enter the laying period at 20 weeks, and that period peaks at 30 weeks. Based on these results, we can speculate that the effect of the 80-bp indel polymorphism of the PRLR gene on growth and carcass traits at different ages is influenced by changes in PRL expression levels during specific growth stages. The association analysis showed that for most of the phenotypic traits, the values of the ID genotype individuals were highest, and the differences between the ID and DD genotypes were significant (P < 0.05), which illustrated that the I allele was the dominant allele. This study demonstrates that this indel polymorphism has great potential as a molecular marker to eliminate or choose preferred individuals in the breeding of local chickens. In terms of the relative expression of the PRLR gene, gene expression in different tissues and stages was analyzed, and the results suggested that chicken PRLR gene expression was highest in the lung, duodenum and ileum, whereas the tissues with the lowest expression were the breast muscle and leg muscle. In addition, the results of genetic conservation analysis revealed that the PRLR gene was conserved in diverse species but may have functional differences between mammals and nonmammals. Previous research has shown that in accordance with the various actions of PRL in vertebrates, PRLR is widely expressed in diverse tissues, including the brain, ovary, placenta, and uterus (Binart et al. 2010; Bole-Feysot et al. 1998). However, unlike PRLR gene expression in mammals, which is controlled by multiple promoters (Hu et al. 1998; Zhang et al. 2002), PRLR is also widely expressed in various tissues of avian species, such as chickens (Ohkubo et al. 1998) and pigeons (Chen and Horseman 1994). Moreover, we found that PRLR gene expression in the lung, duodenum and jejunum tissues was much higher at 30 weeks than at 20 weeks. Conversely, PRLR gene expression was greater at 20 weeks than at 30 weeks in the ileum and cecum. This result may indicate that the expression of PRLR changes as the expression of PRL changes in different tissues before and after the laying period, thereby better exerting its physiological functions. However, it is necessary to perform a further functional analysis of PRLR at the molecular level to gain a better understanding of the biological function and regulatory mechanisms of PRLR in chickens.

In summary, the chicken PRLR mRNA was characterized at the molecular level using bioinformatics methods. We found that during the breeding process of commercial laying hens and broiler chickens, the 80-bp indel had been artificially selected, leading to obvious genetic differentiation. Importantly, this study revealed for the first time that the 80-bp indel polymorphism of the PRLR gene is significantly associated with growth and carcass traits in chickens. Taken together, these results indicate that the PRLR gene can be used as a candidate gene for not only reproductive traits but also growth and carcass traits in chickens.