A Novel p.K116Q SNP in the OLR1 Gene and Its Relation to Fecundity in Awassi Ewes

Sheep's fecundity is determined by both twinning rate and litter size, both influenced by several genes, one of which is the OLR1 (oxidized low-density lipoprotein receptor) gene. This study aimed to determine the genetic variation of the OLR1 gene affecting the fecundity traits of Awassi ewes. The genomic DNA from 114 ewes with a single progeny and 86 ewes with twins was extracted. Polymerase chain reaction (PCR) was used to amplify three fragments (334 bp, 291 bp, and 274 bp) (exon 3, exon 4, and exon 6) of the OLR1 gene. Two genotypes of 334-bp amplicons - CC and CA - were detected. In a sequence reaction, the novel mutation p.K116Q was discovered in CA genotypes. There was a highly significant (P ≤ 0.01) association between the single nucleotide polymorphism (SNP) and reproductive traits, in that ewes with the p.K116Q SNP had lower litter size, twinning rate, fecundity, and lambing percentages than ewes with the CC genotype. These observations imply that the missense p.K116Q variant has an adverse effect on the traits under study and show that p.K116Q SNP has a negative influence on fecundity traits in Awassi sheep. Based on the findings of this study, it is clear that ewes with the p.K116Q SNP are associated with reduced litter size and reduced fecundity traits for this population.


Background
The fecundity in sheep ocks is an important determinant of pro tability [1].A sheep's fecundity is determined by twinning rate and litter size, which are affected by many different genes [2].One of these is the oxidized low-density lipoprotein receptor gene (OLR1) [3] that resides in chromosome 3 in sheep and contains 6 exons (Adopted from ncbi.nlm.nih.gov); in chromosome 5 in cattle with 5 exons [4].This gene encodes OLR1 protein and belongs to the C-type lectin family that functions to absorb and degrade low-density lipoproteins [5], which impacts energy balance, in turn in uencing reproductive characteristics [3].It is also possible that this encoded protein functions to regulate fecundity genes so that the OLR1 gene is involved in sheep fecundity [2].
Several studies have been reported the polymorphism of the OLR1 gene in livestock and its relationship to phenotypic traits.An SNP within the exon and intron regions of the OLR1 gene is associated with phenotypic traits in cattle, according to Komisarek and Dorynek [6] and Fonseca et al. [7].Furthermore, OLR1 is regarded as a potentially important gene in dairy cattle growth traits [8,9].The 3′ untranslated regions of OLR1 (NW_215807: g. 8238C>A) contain a polymorphism associated with the fat content of milk [10].Besides, the SNP in the bovine OLR1 gene (c.495 T> C) affects carcass traits, fat accumulation, and growth in Angus, Charolais, and hybrid cattle [11].The OLR gene is investigated in another study conducted on Dutch Holsteins/Friesians; the results indicated that OLR g.8232C>A signi cantly affected (P < 0.05) the fat content of milk [12].A genetic variant in buffalo OLR1 at position 8,232 in the 3′-UTR has been affected by fat content and yield in the Mehsana breed [13].Additionally, Javed et al. [14] provided evidence of the association between the polymorphisms of the OLR1 gene and growth traits, as well as dairy quality parameters in buffalo.The A/C SNP in the 3′-UTR fragment of the OLR1 gene has been linked to lactational traits in Iranian Holstein cattle [15].According to a recent study, the bovine OLR1 T10588C and T10647T mutations have signi cant associations with fat distribution, back fat thickness, and intramuscular fat accumulation [16].Additionally, there is a relationship between OLR1 gene genetic polymorphism and litter size in Xidu black pigs.The TT genotype exhibited signi cantly larger litter sizes in comparison to TC and CC genotypes (P< 0.01), indicating that the 7-bp mutation T/C at intron 4 had a signi cant effect on litter size traits [17].Considering the aforementioned studies, there has been little research conducted on the impact of genetic polymorphisms in the OLR1 gene on fecundity traits in livestock, and no research on their association with fecundity traits in Awassi sheep.

Animal
Two hundred mature ewes (114 with single progeny and 86 with twins) were used in this study, not pregnant or lactating, and aged between 2.5 and 5 years.Two herding stations (Babylon and Karbala, Iraq) were randomly selected to receive the animals.For the entire year, both grass and concentrated food (2.5 percent of their body weight every day, composed of barley, bran, salt (59%), (40%) and (1%) concentrates, respectively), as well as freshwater, were provided to the animals.Several fecundity traits were recorded at the stations, such as twinning rate, lambing percentage, and litter size.Litter size was determined by dividing the number of lambs born by the number of ewes lambing.

DNA and PCR
In the morning, before feeding the sheep, blood samples were drawn from the jugular vein.A vacutainer tube containing EDTA was used to collect blood for genetic analysis.To extract genomic DNA, a rapid salting-out technique was employed [18].The ampli cation of three different regions of the genetic code of OLR1 was achieved using NCBI Primer-BLAST [19], provided by Bioneer (South Korea).A PCR experiment was conducted using Bioneer's PCR premix (50 μM, 10mM, 30mM, 1.5mM for dNTPs, Tris-HCl, KCl, MgCl2, and 1 U Top DNA polymerase).Using the thermal gradient device (Eppendorf, Germany), the best PCR ampli cation conditions were determined (Table 1).The denaturation was carried out for 4 minutes at 94 °C, followed by 30 cycles of denaturation for 30 seconds, annealing for 45 seconds, and elongation for 30 seconds.Accordingly, the results were veri ed by electrophoresis of PCR products on agarose gels (1.5%) and determining the gel image with a Chemidoc Gel Imager (Bio-Rad, USA).

SSCP (single-strand conformation polymorphism)
All PCR products were genotyped according to the protocol of Imran et al. [20].The denaturing-loading buffer (95%, formamide, 0.05% xylene cyanol, and 20 mM EDTA, pH 8) was added to equal volumes for each PCR amplicon.Following 7 minutes of denaturation, the PCR amplicons were transferred onto wet ice and stored for 10 minutes.Using a 0.5 TBE buffer, samples denatured in neutral polyacrylamide gels were loaded on.Subsequently, the gels were electrophoresed for 4 hours at 200mA and 100V at room temperature.
To stain the gels, the rapid staining protocol developed by Byun et al. [21] was used.

Statistical analyses
Genotype and allele frequency were analyzed by PopGen32, version 1.31 [28].Using Hardy-Weinberg disequilibrium law, disequilibrium was calculated.According to Botstein et al. [29], polymorphism information content (PIC) was computed.Association analysis of the OLR1 genotypes was performed using IBM SPSS 23.0 (NY, USA), with a general linear model as follows: Where: Y ijkl = phenotype characteristics, μ = the mean of all traits, G i = xed effect of i th genotypes (i = CC, AC, AA) P j = xed effect of j th parity (j = 1, 2, 3, 4), A k = xed effect of k th age group (2.5,>2.5-3.5,>3.5-4.5,>4.5-5),and e ijkl = random residual error.Tukey-Krammer tests were performed to examine differences between means with a level of signi cance of (P ≤0.05).Reproductive traits of fecundity (the number of lambs weaned per ewe), lambing percentage (the number of lambs born per ewe lambed), and ewe birth type were analyzed using Chi-square test.Based on preliminary analysis, effects of interaction and lambing season were unaffected by the model ndings and were excluded.

Genotyping of OLR1 gene
The PCR ampli cation of three genetic fragments of 334 bp, 291 bp, and 274 bp was performed on the 3 OLR1 coding regions, along with their anking regions (Fig. 1, A).Although the 291 bp ampli cation of exon 4 by PCR was designed, PCR experiments turned out to be problematic since agarose gels showed no speci c bands.Following this, PCR-SSCP patterns were designed to cover exon 6 exhibited monomorphic electrophoretic migration and no heterogeneity was observed.Furthermore, two distinctive PCR-SSCP patterns were observed in the 334 bp amplicons designed for exon 3 (Fig. 1, B).The sequencing results showed that only one of the SSCP variants carried the C246A SNP, indicating exon 3 heterogeneity.Based on the C246A substitution, the detected SSCP variants were given genotypic values CC for homozygous C/C SSCP variants and CA for heterozygous C/A SSCP variants present at position 246 of PCR amplicons (Fig. 1, C).The Expasy software indicated that a missense (p.K116Q) occurred at the 116 th position of the mature OLR1, where lysine (Lys) was exchanged with glutamine (Glu) (Fig. 1, D). Regarding the genetic diversity of p.K116Q, the genotype CA was predominant with a total frequency of 0.75 (n = 149) and a lower prevalence for the genotype CC with a total frequency of 0.25 (n = 51) (Table 2).However, the Chi-square test showed that polymorphism in the OLR1 gene at the K116Q SNP locus deviated signi cantly from the HWE (P ≤ 0.05).
In silico prediction of p.K116Q All used in silico prediction tools were given neutral/non-deleterious signals for p.K116Q.The I-Mutant 2 tool further con rmed that the p.K112Q SNP does not function deleteriously, as it predicted increased stability of OLR1 upon modi cation of this SNP (Table 3).This non-deleterious effect on the structure, function, and stability of the analyzed protein may be attributed to the noncritical positions that it occupies on the OLR1 protein.

OLR1 gene analysis: statistical results
As such, based on the association analysis of p.K116Q SNP locus with fecundity traits, individuals with CA genotype had signi cantly (P ≤ 0.01) lower litter size, twinning rate, fecundity, and lambing percentage than individuals with CC genotype (Table 4).In comparison to that of CA genotype ewes, CC ewes had 1.98 lambs.Hence, the missense p.K116Q SNP had a negative impact on these traits.

Discussion of OLR1 gene and genetic diversity
The genetic diversity analysis of the OLR1 gene in Awassi ewes revealed that genotype CA was predominant with a total frequency of 0.75 (n = 149) and a lower prevalence for the genotype CC with a total frequency of 0.25 (n = 51).However, the Chi-square test showed that polymorphism in the OLR1 gene at the K116Q SNP locus deviated signi cantly from the HWE (P ≤ 0.05).Numerous studies have reported polymorphisms in the OLR1 gene in livestock in various regions.In direct genomic sequencing of OLR1, Khatib et al.
[8] identi ed 3 genotypes CC, AC, and AA in Holstein Dairy Cattle with 3′-UTR polymorphism.Using PstI/PCR-RFLP, Komisarek and Dorynek [6] identi ed 3 genotypes of OLR1 in Polish Holstein-Friesian bulls CC, CT, and TT.Further, Javed et al. [14] revealed the polymorphism in exonic regions of the OLR1 in Nili Ravi buffalo and determined 3 genotypes AA, AB and BB.Furthermore, a single nucleotide polymorphism (SNP) in the promoter region of the OLR1 gene (c.-495T>C) in hybrid, Angus, and Charolais beef cattle is identi ed and assigned CC, CT, and TT genotypes [11].Fonseca et al. [7] genotyped rs109019599 in OLR1 by PCR-RFLP assays and observed in the agarose gel AA (254 bp), CC (269 bp), and AC (269 and 254 bp).Moreover, the identi cation of OLR1 genotypes in the 3'UTR region of dairy cattle is performed using PCR-RFLP, i.e., AA, AC and CC genotypes [3].Recently, Anggraeni [4] identi ed the base mutations of the OLR1 gene at 3'UTR location by PCR-RFLP techniques in Holstein Friesian dairy cattle and found 3 genotypes CC, AC, and AA.The aforementioned studies indicated that no studies have been undertaken on OLR1 genotyping in Awassi sheep.Therefore, this study provides genotypic information and new associations that may prove more useful in selecting sheep and be recommended for measuring functional traits in future marker-assisted selection programs.

Sequencing and in silico analyses of OLR1 gene
By detecting the C246A SNP in only one of the SSCP mutations, a sequencing reaction con rmed the presence of heterogeneity in exon 3 (Fig. 1, C).A p.K116Q SNP was positioned in the 116 th lysine residue of the mature OLR1, where an amino acid substitution occurred from lysine (Lys) to glutamine (Glu) (Fig. 1, D).Some SNPs occur in non-coding regions, whereas others in coding regions [30].These non-synonymous SNPs modify amino acid sequences when present in a coding sequence (called mutant SNPs), altering protein activity and causing drastic phenotypic changes [31,32].The missense mutation can negatively impact function not only by changing the protein's stability but also by interfering with the structure and function of other biological molecules [33].Additionally, missense mutations can affect a molecule's exibility and alter its equilibrium or alter the dynamics of its conformation [34].All the in silico prediction tools used in the current study provided neutral or non-deleted signals for p.K116Q (Table 3).This non-deleterious effect in terms of the structural, functional, and structural stability of the analyzed protein occurs due to its non-critical positions where occupied in this protein.Studer et al. [35] note that mutations can change an organism's phenotype in any way, perhaps with a bene cial, detrimental or neutral effect on its tness.

Association analyses of OLR1 gene with fecundity traits
This study examined the association between p. K116Q SNP locus and fecundity traits and revealed that individuals with AC genotype had signi cantly (P≤0.01)lower litter size, twinning rate, fecundity, and lambing percentage compared to CC genotypes (Table 4).As a result, these traits were negatively affected by C246A SNP In this regard, only one article has addressed the potential association between polymorphisms in the porcine OLR1 gene and litter size.This study showed a T/C mutation at 7bp of intron4 with 3 genotypes TT, TC, and CC.The litter size of TT genotyped piglets is signi cantly higher than that of TC and CC genotyped piglets [36].
Importantly, the OLR1 degrades the oxidized forms of low-density lipoproteins (ox-LDL) and promotes cholesterol accumulation and adipocyte fatty acid uptake, thereby affecting liver fat metabolism [3,6].Although these roles are signi cant, changes in the lipid pro le or fat content could lead to clinical and physiological problems [37].Thus, by upregulating OLR1, adipocytes increase their total cholesterol content and capacity to absorb free fatty acids (FFA).If the adipocyte's lipid storage capacity is exceeded, it becomes unable to control the release of FFAs into the blood circulation, ultimately resulting in abnormal accumulation of lipid outside the adipocyte [38].Moreover, excess fatty acids in the ovary generate damage to the cumulus and granulosa cells responsible for normal steroidogenesis in the gonads [39].Furthermore, the accumulation of intracellular lipids causes high levels of free fatty acids to form, leading to oxidative damage and the production of highly active oxidative metabolites that cause irreversible cellular damage [40].It has been shown that these metabolic changes can damage oocytes directly or by affecting their follicular environment [41].Based on these data, OLR1 gene variation causes several damaging aspects on oocytes, which could adversely affect fecundity traits.Consequently, the Awassi breed is characterized by a high level of adaptability to harsh environments [42] with a lower reproductive capacity [43,44].
The low reproductive rate of this breed is a major concern for many breeders in the Middle East.Further, the low prevalence of the CC genotype with a total frequency of 0.25 (n = 51) in this study is likely to be one of several factors contributing to the low fertility of the Awassi breed.

Conclusion
A novel single nucleotide polymorphism (SNP), p. K116Q, was found in the OLR1 gene (exon 3), in the heterozygous CA genotype.The results of the association analysis between p. K116Q SNP locus and fecundity traits revealed that individuals with the CA genotype had signi cantly lower litter sizes, ewe birth types, fecundity, and lambing percentages than those with the CC genotype.Twin-producing ewes with CC genotypes showed more measured fecundity traits than single-producing ewes with CC genotypes.In light of these results, OLR1 is a promising candidate gene for sheep marker selection.

Declarations
Tables Table 1.The oligonucleotide primer sets designed for the amplification of the ovine OLR1 gene.The symbols "F" and "R" refer to forward and reverse primers, respectively.The design was based on the ovine NCBI Reference Sequence NC_040254.

Table 3 .
in silico prediction of p.K116Q on ovine OLR1 protein, in terms of structure and function.

Table 4 .
The association between OLR1 genetic polymorphism at locus p.116K>Q and fecundity traits in Awassi ewes.