Two co‐inherited novel SNPs in the MC4R gene related to live body weight and hormonal assays in Awassi and Arabi sheep breeds of Iraq

Abstract Melanocortin‐4 receptor (MC4R) gene plays a key role in the regulation of body weight and energy homeostasis. This study aims to evaluate the association of single nucleotide polymorphisms (SNPs) of the MC4R gene with live body weight and hormonal assays in two breeds of sheep that differ in productive performance, Awassi and Arabi. All known coding sequences of the MC4R gene were covered in this study. DNA samples from 150 animals (Awassi and Arabi breed) were genotyped by PCR‐single‐strand conformation polymorphism (PCR‐SSCP) to assess their pattern of genetic variation. Concerning exon 1, clear heterogeneity was detected with three different SSCP‐banding patterns. The sequencing reactions confirmed these variations by detecting the presence of the two novel SNPs, 107G/C and 138A/C, and three genotypes, GC, AC and AA. The 107G/C SNP was detected in GC genotype, while the 138A/C was detected on both GC and AC genotypes. The other SSCP‐banding pattern (AA genotype) did not show any detectable unique variation. Both SNPs were closely and strongly linked in both breeds (D' and r 2 values were 1.00), which signifies that both loci were co‐inherited as one unit. Association analysis indicated that both breeds with GC/AC haplotype showed higher live body weight (37.250 ± 0.790) relative to the GG/AA (30.244 ± 0.968) and CC/CC (47.231 ± 1.230) haplotypes (p < .05). Concerning the genotyping of exon 2, only 362 bp showed heterogeneity with a missense mutation, with no significant association (p > .05) with the measured traits. In conclusion, the two novel SNPs (107G/C and 138 A/C) were highly associated with live body weight in both breeds. Haplotype analysis confirmed that these two novel SNPs were in strong linkage disequilibrium (LD) and could be used as genetic markers for sheep phenotypic trait improvement.


| INTRODUC TI ON
Body weight in sheep is a pivotal trait for economic breeding and is controlled by multiple genetic loci (Esmailizadeh, 2010). Among these loci, the melanocortin-4 receptor (MC4R) gene plays an important role in the regulation of body weight, energy balance and reproduction in humans and animals (Bakos et al., 2016;Siljee et al., 2013;Zeng et al., 2014). The ovine MC4R gene is mapped on chromosome 23 and consists of two exons (Shishay et al., 2019).
The MC4R gene is involved in the regulation of phenotype expressions of several economic traits in ruminant and non-ruminant animals. These traits are largely regulated by the binding of the MC4R gene-encoded receptors to four ligands of melanocyte-stimulating hormone (α-, β-and γ-MSH) and the adrenocorticotropic hormone (ACTH; Switonski et al., 2013).
The MCR family consists of five members (MC1R to MC5R); each of which has seven transmembrane domains belonging to the G protein-coupled receptor superfamily (Li & Li, 2006). Out of these five melanocortin receptors, MC4R has received more attention. It is expressed in the appetite-regulating areas of the brain that involve food intake, which can act as a critical mediator between appetite and reproduction in animals (Zandi et al., 2014). Thus, the MC4R gene can be a major mediator of leptin effects on food intake and body weight (El-Sabrout & Soliman, 2018;Hwa et al., 2001;Li & Li, 2006). The effects of the MC4R gene variation have been recently implicated in the rapid growth selection program of beef cattle (Prihandini & Maharani, 2019). On the other hand, it has been shown that some of the variations detected in the MC4R gene have been associated with economic traits in livestock (Shishay et al., 2019). It has been recently reported that the MC4R gene polymorphism has exhibited a noticeable association with weight, body length and chest circumference gains, in addition to the average daily gain in goats (Latifah et al., 2018). Likewise, several reports have also indicated a remarkable role of MC4R gene in live weight, backfat thickness, carcass traits and marbling score in several breeds of cattle (Lee et al., 2013;Liu et al., 2010;Seong et al., 2012). Additionally, genotyping studies of the MC4R gene have revealed a significant association of this gene with the milk yield and fat percentage in buffalos (Deng et al., 2016).
Moreover, the MC4R gene polymorphism has been associated with backfat thickness in sheep (Zuo et al., 2014). The combined effect of multiple mutation sites within MC4R gene has also been included as a valuable factor for assessing its effect on economic traits (El-Sabrout & Aggag, 2018). However, to accommodate such haplotyping studies, it is necessary to assess multiple genetic loci simultaneously (Lu et al., 2003).
Taking into consideration the above-stated data, the combining of the possible interaction between MC4R polymorphism in Awassi and Arabi breed with live body weight and hormonal assays may have some potential in the detection of the possible correlation between both patterns.
Awassi, followed by Arabi, is the most predominant one in the middle and southern portions of Iraq .
Though Awassi sheep have well-known living adaptation capacity, it has recently reported that Arabi sheep showed a higher genetic capability than Awassi sheep to cope up with harsh circumstances (Al-Thuwaini et al., 2020). Furthermore, it has recently been stated that genetic diversity is considerably correlated with the productive performance in both breeds (Aljubouri & Al-Shuhaib, 2020). Taking these genetic differences altogether, it is crucial to discriminate between both breeds to broaden our knowledge in critical production traits. Several biological functions have been associated with such adaptation conditions in sheep, such as live body weight and hormonal secretion (Marai et al., 2007). Thus, it is consequential to measure these values in both breeds taking advantage of the highly dynamic strategy sheep use to enhance their survival chances in harsh conditions (Niyas et al., 2015). Thus, it would be expected that Awassi and Arabi breeds could also exhibit different genetic variations in the MC4R gene in such a way it could affect functions associated with production traits. Therefore, the current study aims to describe the possible association between the MC4R gene polymorphism and the physiological differences between Awassi and Arabi breeds. Thus, single nucleotide polymorphisms (SNPs) of MC4R gene were identified, and association with live body weight was investigated to acquire possible molecular markers related to production traits for marker-assisted selection. According to our knowledge, no report has screened the association of the coding sequences of the MC4R gene with live body weight and hormonal assays in sheep.
Therefore, this research is the first one to describe such association in two breeds of sheep.

| Sheep population and ethical approval process
The study was conducted according to the international recommendations for the care and use of animals (Federation of Animal Science Societies, ), and the animal experimentations were approved by the Al-Qasim Green University (Approval No. 12.10.18). A total of 150 sheep (Ovis aries) were selected randomly from three sta- However, both breeds had slightly different head appearance and body characteristics (Alkass & Juma, 2005). Animals were fed ad libitum on seasonal grass during summer, while in winter, animals were kept indoors and fed with a concentrated mixture consisting of barley grain (59%), bran (40%) and salt (1%). The live body weight of the sheep was recorded in the morning before the animals were grazing using a suspended spring balance, while blood tests were assessed.
The descriptive statistics of estimated traits are shown in Table 1.

| Hormonal assay
Within 20 min of blood collection from the jugular vein, plasma was separated from peripheral blood by centrifugation at 3,500 g at 4°C for 15 min. Then, the plasma from each sample was collected and stored at -20°C. To assess the possible association between MC4R gene and sex hormones in both investigated breeds, testosterone and estradiol were measured using enzyme-linked immunosorbent assay (ELISA) kit based on the sandwich principle (cat. no. E0013Sh for testosterone and cat. no. E0047 Sh for estradiol, Bioassay Technology Laboratory Co.). The absorbance of hormones was measured at 450 nm using a microplate reader (ELx808 Ultraplate Reader, BioTek Instruments, Inc.). Concentrations were presented as pg/ml and ng/ ml for estradiol and testosterone, respectively. A standard curve was generated, and samples were interloped according to the manufacturer's instructions (Bioassay Technology Laboratory Co.).

| Genomic DNA extraction, primer design and PCR
Genomic DNA was isolated from the whole blood using a rapid and efficient salting-out method (Al-Shuhaib, 2017). Four pairs of specific polymerase chain reaction (PCR) oligonucleotides were designed to cover all the coding sequences of the ovine MC4R gene using NCBI Primer Blast online server (Ye et al., 2012). For exon 1, only one PCR primer pair was designed, while three pairs of primers were designed to adequately cover the coding sequences in the exon 2 ( Figure 1a). The lyophilized oligonucleotides were purchased from Bioneer Company (Bioneer, Korea). The PCR primers were designed based on GenBank accession no. NC_019480.2 and the details of the primer used in this study are shown in Table 2. PCR experiments were conducted using AccuPower ® PCR PreMix (Bioneer), and initiated by denaturation for 5 min, followed by 30 cycles (annealing at 57.8-59.1°C for 30 s each), and finalized with polymerase extension (72°C) for 5 min. The target amplified PCR products were confirmed by 1.5% agarose gel electrophoresis and then submitted to singlestrand conformation polymorphism (SSCP) protocols (Hashim & Al-Shuhaib, 2019).

| Single-strand conformation polymorphism (SSCP) and sequencing analysis
The initial denaturation of the PCR products, as well as SSCP pro-

| Genetic polymorphism and statistical analyses
The allele and genotype frequencies, observed heterozygosity (Ho), expected heterozygosity (He) and an effective number of alleles were analysed using PopGen32 software, ver. 1.31 (Yeh & Yang, 1999).
The polymorphism information content (PIC) was calculated by  utilizing the HET software ver. 1.8 (Ott, 2001). Pairwise linkage disequilibrium (LD) between SNPs was calculated by r 2 and D' values using SHEsis software (She & He, 2006).
The significant effects of breed, sex and SNP genotype on the various studied parameters were analysed by SPSS software ver.
23.0 (SPSS Inc), using the general linear model: where y ijkl is phenotypic traits, μ is the overall mean, B i is the fixed effect of ith breed (i = Awassi, Arabi), S j is the fixed effect of jth sex (j = ram, ewe), i k is the fixed effect of kth SNP genotype or combined genotype and e ijkl is the random error associated with y ijkl observation and assumed to be NID (0, σ2e). Means were compared using the Tukey-Kramer test with a significance level of p < .05. Preliminary statistical analysis indicated that the effects of age, parity, season, station and the interaction between these effects were not included in the final model because they did not have a significant effect on phenotypic traits.

| SNP genotype effects estimation
For the SNP that showed significant association with the phenotypic traits, differences between the means of each genotype and allelic frequencies were used to estimate additive effects (Hill & Mackay, 2004). The following formula was utilized to find additive genetic variance (Var A ) imputed to a SNP: where q and p were the allelic frequencies for the jth SNP predicted across the entire population, α i -SNP allele substitution effect obtained from a linear regression model in a statistical program, in which the genotypes recorded as a variate of 0, 1 and 2 copies of a particular allele.
The proportion of the phenotypic variance explained by SNP(s) was calculated by (%) varP = 100 × 2p i q i 2 i /V P , where p and q denote the SNP allele frequency; 2 i is the SNP allele substitution effect, 2p i q i 2 i is the additive genetic variance and V P is the phenotypic variance.  Table 3). All of these tools lead to neutral/ non-deleterious signals for both assessed R18G and H7Q. This

TA B L E 3
In silico prediction of H7Q and R18G on ovine MC4R protein, in terms of structure and function non-deleterious effect may be due to their non-critical positions in the generated 3D structure of MC4R protein (Figure 1d).

| Assessment of MC4R polymorphism and association analysis
Genotype, allele frequencies and genetic diversity parameters for exon 1 amplicon in the analysed populations of Awassi and Arabi sheep are presented in Table 4  and Arabi breeds, respectively. Furthermore, the genotype effect prediction confirmed that the GC/AC haplotype was associated with higher body weight (p < .05). Greater genetic variance percentage with the phenotypic traits (>1%) is detailed in Table 6A and B.
Meanwhile, the individuals with CC/CC haplotype had higher live body weight with a lower level of estradiol indicating that this haplotype should not be extensively selected in sheep breeding for litter size in Awassi breed. However, the association analysis in exon 2 showed no significant difference (p > .05) between 179G > A and 236G > A and phenotypic traits.

| D ISCUSS I ON
Association analyses for live body weight revealed that the Awassi breed showed higher live body weight in comparison to Arabi breed, indicating that Awassi breed had more body weight gain than Arabi breed. The breed of sheep was known to have an important impact on body weight (Aktaş et al., 2015). Awassi sheep were shown to respond favourably to body weight than other breeds (Galal et al., 2008). In regards to sex effect, this study found that ewes showed heavier weight compared to rams in both breeds.
These differences might be attributed to the physiological and sexual hormones of rams and ewes (Kratochvílová et al., 2002).
Indeed, oestrogen hormone is stimulating specific receptors in the pro-opiomelanocortin (POMC) neurons in the hypothalamus by the melanocortin system (Clegg et al., 2006). It has been reported that increased POMC levels result in more binding of α-MSH to MC4R, promoting increased food intake and reduced energy expenditure in ewes (El-Sabrout & Soliman, 2018;Hewagalamulage et al., 2015).
Furthermore, females are more severely affected than males in weight increase (Vaisse et al., 2000).
The MC4R gene/protein variation is a key component in the The g.998A/G SNP was significantly associated with weight gain, body length and chest circumference gain in Bligon goats (Latifah et al., 2018). Add to that, individuals with the AG and GG genotypes (−129A > G) in the MC4R gene had higher live body weight than individuals with AA genotype in cattle (Liu et al., 2010). In another study, individuals with heterozygous genotype at g.1104C > T of the MC4R gene showed better performance than individuals with homozygous genotype for milk yield in Chinese buffaloes (Deng et al., 2016).
Although the association study between single SNP with phenotypic traits is likely to be easier and more efficient in breeding programs, the haplotype analysis is valuable for assessing the effect of genes on phenotypic traits. In this study, statistical analysis showed that F I G U R E 2 Linkage disequilibrium plot of the observed four SNPs within the exon 1 and exon 2 amplicons of the ovine MC4R gene in Awassi, Arabi and two breeds. (a) D' value, (b) r 2 value. The red diamonds indicate strong LD between pairs of SNPs individuals with GC/AC haplotype were more associated with live body weight gain than those with GG/AA and CC/CC haplotypes, respectively. These results confirmed that the priority of genomic and inbreed selection should be held on heterozygous states compared to homozygous ones. The study of Cai et al., (2015) showed that individuals with CGACG and CTCCC haplotypes of the MC4R gene were associated with an increased body weight of animals aged 18 months in yaks. Based on previous information, Awassi sheep is a low prolific breed (Abdullah et al., 2002), and very low incidence of twinning (Al-Sa'aidi et al., 2018). The present study demonstrated that individuals with CC/CC haplotype had higher live body weight and lower levels of estradiol, which indicates that this haplotype has not been extensively selected in sheep breeding to enhance litter size and twinning ratio in Awassi breed. This fact was confirmed by Chen et al., (2017) who reported that the variation of MC4R gene implies a reduction of GnRH-LH secretion and dysfunction of the ovary in mice, resulting in a reduction in the number of developed corpora lutea and decreased litter sizes.

| CON CLUS ION
This study identified two co-inherited novel SNPs (107G/C and 138 A/C) of MC4R gene in two breeds of sheep. Both of the 107G/C and 138A/C SNPs were found to be highly associated with live body weight and hormonal (testosterone and estradiol) assays. We demonstrated that the GC/AC haplotype affects the sheep live body weight and hormonal assays and it is therefore highly recommended to be selected and fixed for sheep production.

ACK N OWLED G EM ENTS
This study was supported by the college of agriculture, Al-Qasim Green University. The authors are grateful to the staff of stations for raising sheep for their facilities that provided the sheep population.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

FU N D I N G I N FO R M ATI O N
The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

E TH I C A L A PPROVA L
The study was conducted according to international recommendations for the care and use of animals (Federation of Animal Science Societies, 2010) and the animal experimentations were approved by Al-Qasim Green University (Approval No. 12.10.18).

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/vms3.421.

TA B L E 5
Least square means ± SE for live body weight and sex hormone assay in association with the MC4R polymorphism in Awassi (A), Arabi (B) and both breeds (C) Note: varP proportion of phenotypic variance explained. The percentage of variance was calculated only for the SNPs that showed a significant association with the traits.