Variation in the AA-NAT gene G203A is associated with Awassi and Hamdani sheep fertility.

Arylalkylamine-N-acetyl-transferase (AA-NAT) is one of several genes that influence sheep reproduction. Thus, the objective of this study was to investigate whether genetic variability within the AA-NAT gene influenced the reproductive performance of Awassi and Hamdani ewes. A total of 99 twin and 101 single-progeny ewes were analyzed for genomic DNA. Polymerase chain reaction (PCR) was used to produce amplicons of 300, 313, and 287 bp from exons 1, 2, and 3 of the AA-NAT gene. A 300-bp amplicon was genotyped, resulting in two genotypes: GG and GA. Through sequence analysis, a mutation 203 G > A was identified in the GA genotype. The statistical analysis revealed a strong correlation between the single nucleotide polymorphism (SNP) 203 G > A and reproductive performance. Ewes carrying this mutation showed significantly increased litter sizes, twinning rates, lambing rates, and fewer days to lambing compared to those carrying GG. These findings demonstrate that the presence of the 203 G > A SNP variant has a significant positive impact on litter sizes and enhances the fertility of Awassi and Hamdani sheep.

The Novel PTX3 Variant g.22645332G>T Is Strongly Related to Awassi and Hamdani Sheep Litter Size.

The detection of polymorphisms in genes that control livestock reproduction could be highly beneficial for identifying and enhancing economic traits. One of these genes is pentraxin 3 (PTX3), which affects the reproduction of sheep. Therefore, this study investigated whether the variability of the PTX3 gene was related to the litter size of Awassi and Hamdani ewes. A total of 200 ewes (130 Awassi and 70 Hamdani) were used for genomic DNA extraction. Polymerase chain reaction was used to amplify the sequence fragments of exons 1, 2, 3, and 4 from the PTX3 gene (Oar_v4.0; Chr 1, NC_056054.1), resulting in products of 254, 312, 302, and 253, respectively. Two genotypes, GG and GT, were identified for 302 bp amplicon. A novel mutation was discovered through sequence analysis in the GT genotype at position g.22645332G>T. The statistical analysis revealed a significant association between single nucleotide polymorphism (SNP g.22645332G>T; Oar_v4.0; Chr 1, NC_056054.1) and litter size. The presence of the SNP g.22645332G>T (Oar_v4.0; Chr 1, NC_056054.1) genotype in ewes resulted in a significant difference compared to ewes with GG genotypes. The discrepancy became apparent in several aspects, including litter sizes, twinning rates, lambing rates, litter weight at birth, and days to lambing. There were fewer lambs born to ewes with the GG genotype than to ewes with the GT genotype. The variant SNP g.22645332G>T (Oar_v4.0; Chr 1, NC_056054.1) has positive effects on the litter size of Awassi and Hamdani sheep. The SNP g.22645332G>T (Oar_v4.0; Chr 1, NC_056054.1 has been associated with an increase in litter size and higher prolificacy in ewes.

The novel C268A variant of BMP2 is linked to the reproductive performance of Awassi and Hamdani sheep.

BACKGROUND: Prolificacy-associated genetic markers can be utilized to enhance litter size in the sheep breeding industry. Sheep reproduction is influenced by a multitude of genes, including bone morphogenetic protein 2 (BMP2). This study aimed to explore the potential relationship between variability in the BMP2 gene and reproductive performance in Awassi and Hamdani ewes. METHODS AND RESULTS: The genomic DNA was extracted from 99 single-progeny ewes and 101 twin ewes. Polymerase chain reaction (PCR) was employed to produce an amplicon consisting of four sequence fragments: 277 bp, 251 bp, 331 bp, and 340 bp, from exons 1, 2, 3, and 4 of the BMP2 gene, respectively. Three genotypes were identified for amplicons in exon 4 with 340-bp lengths: CC, CA, and AA. Upon analyzing the sequence of the CA genotype 382 C > A, a novel mutation was discovered in this genotype. A robust association was identified between the single nucleotide polymorphisms (SNP) 382 C > A and reproductive performance through statistical analysis. An important distinction was discovered between ewes carrying SNP 382 C > A and those carrying CC in terms of litter sizes, twinning rates, lambing rates, and days to lambing. An analysis of logistic regression revealed a significant association between litter size and the 382 C > A SNP. There was a decrease in lamb production among ewes with the CC genotype compared to those with the CA and AA genotypes. CONCLUSIONS: These results indicate that the SNP variant 382 C > A has a positive influence on the reproductive performance of Awassi and Hamdani sheep. Sheep carrying the 382 C > A SNP exhibit increased litter size and overall productivity compared to those without the SNP.

The relationship between OXT gene polymorphisms and reproductive hormones in pregnant and lactating Awassi Ewes.

BACKGROUND: Numerous genetic loci interact intricately to control reproduction in mammals. The oxytocin gene (OXT) is a promising candidate for reproductive traits in mammals. Previously, sheep and goats have been studied for the presence of the OXT polymorphism. As of yet, no polymorphisms have been identified in the OXT gene of Awassi sheep. Thus, this study was conducted to determine the effects of OXT polymorphism and litter size on reproductive hormones in pregnant and lactating Awassi ewes. METHODS AND RESULTS: This study evaluated 232 ewes aged 3 and 4 years (123 single-progeny ewes and 109 twin-producing ewes). Serum was collected to measure reproductive hormones using ELISA kits manufactured by ELK Biotechnology. DNA was extracted from sheep blood for genotyping and sequencing to identify variations in OXT gene (exon 2, 266 bp). Genotyping analysis revealed three genotypes within 266 bp: CC, CA, and AA. Sequence analysis revealed a novel mutation in exon 2: 188 C > A. Statistical analysis showed significant associations between the 188 C > A SNP and phenotypic traits. Twin-pregnant ewes carrying CC genotypes had higher estrogen, progesterone, and follicle-stimulating hormone/luteinizing hormone levels (65.86 ± 3.87) (pg/mL), (6.51 ± 0.39) (ng/mL), and (20.22 ± 1.27) (ng/mL)/( 23.37 ± 2.14) (ng/mL) respectively, compared to CA and AA genotypes in the fourth month of twin-pregnant ewes compared to single-pregnant ewes. CONCLUSIONS: This study found that the 188 C > A SNP negatively affected reproductive hormone levels in Awassi sheep. These findings provide breeders with a new insight into the sheep OXT gene, useful for future breeding.

Association of Novel C319T Variant of PITX2 Gene 3'UTR Region With Reproductive Performance in Awassi Sheep.

Several genes influence sheep's reproductive performance, among them the paired-like homeodomain transcription factor 2 (PITX2) gene. Thus, this study aimed to examine whether the variability within the PITX2 gene is associated with the reproductive performance of Awassi ewes. A total of 123 single-progeny ewes and 109 twin ewes were used to extract genomic DNA. An amplicon of 4 sequence fragments from exons 2, 4, 5 (upstream portion), and 5 (downstream portion) of the PITX2 gene was generated by polymerase chain reaction (PCR), 228, 304, 381, and 382 bp, respectively. Three genotypes of 382 bp amplicons were identified: CC, CT, and TT. Sequence analysis revealed a novel mutation in the CT genotype 319C > T. Statistical analysis revealed that single-nucleotide polymorphism (SNP) 319C > T was associated with reproductive performance. Single-nucleotide polymorphism 319C > T-carrying ewes had significantly (P ⩽ .01) lower litter sizes, twinning rates, lambing rates, and more days to lambing than those carrying CT and CC genotypes. Based on a logistic regression analysis, it was confirmed that the 319C > T SNP decreased litter size. Ewes with TT genotype produced fewer lambs than ewes with CT and CC genotypes. According to these results, the variant 319C> T SNP negatively affects the reproductive performance of Awassi sheep. Ewes carrying the 319C > T SNP have a lower litter size and are less prolific than those without the SNP.

A Novel c.100C > G Mutation in the FST Gene and Its Relation With the Reproductive Traits of Awassi Ewes.

Reproductive traits are affected by many factors, including ovarian function, hormones, and genetics. Genetic polymorphisms of candidate genes are associated with reproductive traits. Several candidate genes are associated with economic traits, including the follistatin (FST) gene. Thus, this study aimed to evaluate whether the genetic variations in the FST gene are associated with the reproductive traits in Awassi ewes. The genomic DNA was extracted from 109 twin ewes and 123 single-progeny ewes. Therefore, 4 sequence fragments from the FST gene were amplified using polymerase chain reaction (PCR) (exon 2/240, exon 3/268, exon 4/254, and exon 5/266 bp, respectively). For a 254 bp amplicon, 3 genotypes were identified: CC, CG, and GG. Sequencing revealed a novel mutation in CG genotypes c.100C > G. The statistical analysis of c.100C > G showed an association with reproductive characteristics. Ewes carrying the c.100C > G had significantly (P ⩽ .01) lower litter sizes, twinning rates, lambing rates, and more days to lambing compared with CG and CC genotypes. Logistic regression analysis confirmed that the c.100C > G single-nucleotide polymorphism (SNP) is responsible for decreasing litter size. According to these results, the variant c.100C > G negatively affects the traits of interest and is associated with lower reproductive traits in Awassi sheep. As a result of this study, ewes carrying the c.100C > G SNP have lower litter size and are less prolific.

A Missense p.Q>R234 Mutation in the Osteopontin Gene Is Associated With the Prolificacy of Iraqi Awassi Ewes.

One of the most valuable traits in production and breeding is a sheep's prolificacy which is influenced by several genes, one of which is the osteopontin (OPN) gene. Thus, this study aimed to determine the effect of genetic variation within the OPN gene on Awassi ewe prolificacy. Genomic DNA was extracted from 123 single-progeny ewes and 109 twin ewes. Polymerase chain reaction (PCR) was used to amplify 4 sequence fragments (289, 275, 338, and 372 bp), representing exons 4, 5, 6, and 7 of the OPN gene. A 372 bp amplicon was identified with 3 different genotypes: TT, TC, and CC. Sequence analysis revealed a novel mutation in TC genotypes p.Q>R234. Statistical analysis revealed that the single nucleotide polymorphism (SNP) p.Q>R234 was associated with prolificacy. Ewes carrying the p.Q>R234 SNP had significantly (P ⩽ .01) lower litter sizes, twinning rates, and lambing rates, and more days to lambing than those with the TC and TT genotypes. The p.Q>R234 SNP was confirmed to be responsible for lower litter size through logistic regression analysis. From these results, we can conclude that the missense variant p.Q>R234 adversely affects the traits of interest and shows that the p.Q>R234 SNP negatively influences the prolificacy of Awassi sheep. Based on this study, it is evident that ewes in this population carrying the p.Q>R234 SNP have a lower litter size and are less prolific.

An efficient, fast and inexpensive method for genomic DNA extraction of fish tissue.

BACKGROUND: DNA extraction is an essential step for many genetic techniques like PCR and other molecular analyses. Based on the method of extraction and type of tissue used, the quality of extracted DNA for genetic studies varies. An appropriate extraction method is evaluated by the high concentration and purity of DNA. Thus, this study aimed to find a more efficient and effective method of DNA extraction from fish tissues and compare it to commercially available kits. METHODS AND RESULTS: A total of 200 fish tissue samples were extracted using each method and then validated with restriction enzymes and PCR amplification. The result revealed that the mean quantity of the isolated genomic DNA, when measured by Nanodrop for grass and common carp, was estimated at (624.41 ± 34.51) µg/ml and (651.27 ± 46.31) µg/ml, respectively, and the purity of this DNA was about (1.83 ± 0.04) and (1.88 ± 0.03) respectively, as compared to commercial extraction kits. Furthermore, gel electrophoresis was performed on the PCR-ready DNA, and the results were confirmed with restriction enzymes and PCR amplification. Based on results obtained from restriction enzymes and PCR analysis, it was determined that no significant inhibitors existed for the enzymes that were used in molecular biology reactions. CONCLUSION: As a result, this technique provides an efficient and versatile alternative to the traditional method for obtaining bulk amounts of highly qualified DNA from fish tissue and can be easily used for subsequent analyses such as PCR and several molecular experiments on other fish species.

Association of birth type and LHX4 gene polymorphism with reproductive hormones, growth hormone, and prolactin in Awassi ewes.

BACKGROUND: LIM homeobox transcription factor 4 (LHX4) is a promising candidate gene for mammalian reproductive traits. LHX4 polymorphism has previously been associated with phenotypic traits in goats and cattle. However, there have been no LHX4 gene polymorphisms identified in Awassi sheep. Therefore, this study investigated the effects of the LHX4 polymorphism on reproductive hormones, growth hormones, and prolactin in Awassi ewes. METHODS AND RESULTS: A total of 232 ewes between the ages of 3 and 4 years were selected for this study (123 single-progeny ewes and 109 twin-producing ewes). Serum was collected to measure reproductive hormones, growth hormone, and prolactin using ELISA kits made by ELK Biotechnology. Genomic DNA was extracted from sheep blood, genotyped, and sequenced to confirm variations in LHX4 (exon 1, 207 bp). Genotyping revealed three genotypes in 207 bp: AA, AG, and GG. Sequence analysis detected a novel mutation in exon 1: 160 A > G. Statistically, the 160 A > G SNP was significantly associated with the phenotypic traits. Ewes carrying AA genotypes had higher estrogen, progesterone, follicle-stimulating hormones/luteinizing hormones, and growth hormone, and lower prolactin levels (65.63 ± 3.84) (pg/mL), (6.67 ± 0.38) (ng/mL), (22.34 ± 1.27) (ng/mL)/(23.89 ± 2.13) (ng/mL), (1.30 ± 0.05) (ng/mL), and (13.16 ± 0.75) (pg/mL), respectively, compared to AG and GG genotypes in the fourth month of twin-pregnant ewes compared to single-pregnant ewes. CONCLUSION: This study suggests that the 160 A > G SNP negatively affects the Awassi sheep's hormone levels. It provides valuable insight into the sheep LHX4 gene, which could be an effective marker in marker-assisted selection.

A novel deleterious oxytocin variant is associated with the lower twinning ratio in Awassi ewes.

This study aimed to assess the possible association of oxytocin (OXT) gene with reproductive traits in two groups of Awassi ewes that differ in their reproductive potentials. Sheep were genotyped using PCR-single-stranded conformation polymorphism approach. Three genotypes were detected in exon 2, CC, CA, and AA, and a novel SNP was identified with a missense effect on oxytocin (c.188C > A → p.Arg55Leu). A significant (p < 0.01) association of p.Arg55Leu with the twinning rate was found as ewes with AA and CA genotypes exhibited, respectively a lower twinning ratio than those with the wild-type CC genotype. The deleterious impact of p.Arg55Leu was demonstrated by all in silico tools that were utilized to assess the effect of this variant on the structure, function, and stability of oxytocin. Molecular docking showed that p.Arg55Leu caused a dramatic alteration in the binding of oxytocin with its receptor and reduced the number of interacted amino acids between them. Our study suggests that ewes with AA and CA genotypes showed a lower reproductive performance due to the presence of p.Arg55Leu, which caused damaging impacts on oxytocin and is binding with the OXT receptor. The utilization of the p.Arg55Leu could be useful for improving Awassi reproductive potential.

Association of OLR1 gene polymorphism with live body weight and body morphometric traits in Awassi ewes: short communication.

BACKGROUND: Oxidized low-density lipoprotein receptor 1 (OLR1) is an endothelial receptor that binds and degrades oxidized low-density lipoproteins (Ox-LDL), thus having a physiological role in metabolism. Polymorphisms in the OLR1 gene are associated with animals with different production traits. Due to this, the study aimed to determine if OLR1 polymorphisms in Awassi ewes associate with live body weight and body measurement. METHODS AND RESULTS: In this study, 200 ewes between the ages of 2.5 and 5 years, not pregnant or lactating, were selected. Phenotypic measurements including live body weight and body measurements were collected. A sheep's blood was collected to extract genomic DNA, genotyped, and sequenced to confirm the presence of the variants that arose from the amplified fragments. One novel C246A single nucleotide polymorphism (SNP) was identified in the OLR1 gene (exon 3) that assigned two genotypes CC and CA. The study indicated significant differences (P ≤ 0.05) in live body weight and body measurements of the genotype CC compared with the genotype CA. The genotype CC correlated positively with live body weight, height at shoulder, height at hip, chest girth, and chest width (r = 0.67, P = 0.02), (r = 0.54, P = 0.03), (r = 0.61, P = 0.02), (r = 0.53, P = 0.01) and (r = 0.66, P = 0.04) respectively. CONCLUSIONS: Sheep with the CC genotype had a higher live body weight and larger body measurement, making them better for productivity. These genotypic data and associations can be used to better select sheep for future marker-assisted selection programs.

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.

Detection of a novel single nucleotide polymorphism in IGF2 gene with a negative impact on egg production and body weight in Japanese quail (Coturnix japonica).

BACKGROUND: Insulin-like growth factor 2 (IGF2) is one of three hormones that share high structural similarity to insulin. It is involved in several insulin-like growth-regulating and mitogenic activities. This study was conducted to genotype the coding regions of the IGF2 gene in Japanese quail (Coturnix japonica) using PCR-SSCP-sequencing, and to assess the possible association of the polymorphism of these regions with the main egg production traits. A total of 240 female birds with an equal number of three Japanese quail populations (Brown or BR, Black or BL, and White or WT) were included in this study. RESULTS: All the genotyped regions exerted no heterogeneity in their sequences with one exception detected in the exon 2. In this locus, a novel single nucleotide polymorphism (SNP) was detected in which "A" was substituted with "G" at 81 position with a silent effect (p.F79=SNP) on IGF2 protein. Association analyses indicated a significant (P < 0.05) relation of this SNP with egg number (EN) and bird weight (BW) in the analyzed populations, in which the birds with AG genotype had lower EN and BW than those with AA genotype. The p.F79=SNP was largely detected in the WT line than the other two lines. CONCLUSION: The detected p.F79=SNP has a highly negative effect on EN and BW in Japanese quail. Thus, the implementation of the variations of the IGF2 gene can be a useful marker in the marker-assisted selection of Japanese quail. This is the first report to describe IGF2 gene variations in Japanese quail, which strongly suggests raising the birds from the BR line with AA genotype when breeders desire to raise Japanese quail for large-scale egg production.

A Novel Missense Single Nucleotide Polymorphism in the GREM1 Gene is Highly Associated with Higher Reproductive Traits in Awassi Sheep.

GREM1 (gremlin1) is a known inhibitor for BMP15 (bone morphogenetic protein 15) family, but its genetic diversity in sheep is unknown. The present study was conducted to analyze the polymorphism of GREM1 gene using PCR- single-strand conformation polymorphism (SSCP) and DNA sequencing methods and to assess the possible association of GREM1 gene polymorphism with reproductive traits in Awassi ewes. A total of 224 ewes, 124 producing singles and 100 producing twins, were included in the study. Two SSCP patterns were detected in two amplified loci within the exon 2. Two exonic novel single nucleotide polymorphism (SNP)s were identified, c.74 T > G (the silent SNP p.Met123 =) and c.30 T > A with (the missense SNP p.Ile237Phe). Statistical analyses indicated a non-significant (P > 0.05) association of p.Met123 = with the analyzed reproductive traits of fecundity, prolificacy, litter size, and twinning rate. Meanwhile, p.Ile237Phe SNP exhibited a highly significant (P < 0.01) association with the measured reproductive traits, in which ewes with TA genotype (with p.Ile237Phe SNP) exhibited higher litter size, twinning ratio, fecundity, and prolificacy than those with TT genotype (without p.Ile237Phe SNP). The deleterious impact of p.Ile237Phe SNP was observed by the means of ten different state-of-the-art in silico tools that predicted a highly damaging effect of p.Ile237Phe SNP on the structure, function, and stability of gremlin1. In conclusion, the results of our study suggest that p.Ile237Phe SNP has a remarkable negative impact on the gremlin1 structure, function, and stability. Since gremlin1 is a known inhibitor of reproductive performance, a consequent higher reproductive performance was observed in ewes with damaged gremlin1 (with p.Ile237Phe SNP) than those with non-damaged gremlin1 (without p.Ile237Phe SNP). Therefore, it can be stated that the implementation of the novel p.Ile237Phe SNP in the GREM1 gene could be a useful marker in marker-assisted selection. This manuscript is the first one to describe GREM1 gene variations in sheep.

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.

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 r2 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.

Co-inherited novel SNPs of the LIPE gene associated with increased carcass dressing and decreased fat-tail weight in Awassi breed.

The lipase E hormone-sensitive (LIPE) enzyme is one of the lipolytic enzymes, and it plays a key role in the regulation of adipose tissue deposition. This study was conducted to investigate the possible association between the LIPE gene variations and the main body weight measurements in Awassi sheep. A total of 160 of sexually mature Awassi rams (Ovis aries) that aged between 2 and 3 years were included in the present study. Genomic DNA was extracted and two specific PCR amplicons were designed to amplify two coding regions within the LIPE gene. Genotyping experiments were performed using polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP). Two different SSCP banding patterns were identified, CC and CD in exon 2, and AA and AT in exon 9. Five novel single-nucleotide polymorphisms (SNPs) were detected by sequencing, namely g.151C > A and g.198C > T in exon 2, and g.213G > C, g.226G > T, and g.232A > C in exon 9. Haplotype block analysis showed strong linkage disequilibrium values between the two SNPs in exon 2 and the three SNPs in exon 9. Association analysis of haplotypes with carcass traits demonstrated a significantly higher dressing percentage (P < 0.05) and lower fat tail weight (FTW) in CACT and GCGTAC haplotypes made these haplotypes more favorable for human consumption. The current research is the first one to report a tight association between the LIPE genetic polymorphism and the dressing percentage and FTW traits, suggesting a pivotal role played by these co-inherited SNPs in the metabolism of carcass traits in sheep.

A novel T177P missense variant in the HSPA8 gene associated with the low tolerance of Awassi sheep to heat stress.

This study was conducted to identify the association of coding variations in the HSPA8 gene with heat stress in two different breeds of sheep. All the coding regions of the HSPA8 gene of Awassi and Arabi sheep were covered by amplifying nine exons. A single-strand conformation polymorphism (SSCP) was utilized to assess the genetic variations in both breeds. The possible association of the observed genotypes with rectal temperature (RT), respiratory rate (RR), and heat tolerance coefficient (HTC) was analyzed in different seasons. While all the coding regions of both sheep were monomorphous, a remarkable heterogeneity was observed in exon 4, of which two SSCP patterns, a normal TT and a mutant TG, were detected. The TG genotype was characterized by a missense variant of T177P with frequencies of 77% in Awassi and 54% in Arabi. Cumulative in silico tools indicated extremely deleterious consequences for T177P on protein structure, function, and stability. Results indicated that sheep with the TT genotype had significantly (P < 0.05) lower RT, RR, and HTC values than sheep with the TG genotype. Therefore, a significant association of T177P with a lower tolerance of Awassi to higher temperature conditions was revealed. In conclusion, the identified T177P may have damaging effects in the HSPA8, which affects the ability of Awassi sheep to cope up with elevated temperatures compared with Arabi sheep. This manuscript describes a novel description of a highly deleterious missense variant in the HSPA8 gene that may reduce the ability of sheep to withstand high-temperature conditions.

GHRL gene-based genotyping of ovine and caprine breeds reveals highly polymorphic intronic sequences in Awassi sheep with several RNA motifs.

BACKGROUND: The current study was conducted to identify the genetic polymorphism of ghrelin (GHRL) gene of sheep and goats, as well as to determine whether these polymorphisms were associated with the evolutionary genetic differences in the involved species. This study was performed on 233 sheep and 91 goats. Two genetic loci of 113 bp and 262 bp partially spanning over exon 2/intron 2 and intron 4/exon 5 of GHRL gene respectively were amplified and genotyped using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods. RESULTS: The SSCP banding pattern of 262-bp locus indicated the presence of four diplotypes (BC, BB, AC, and AB) in Awassi sheep, three diplotypes (BC, BB, and AB) in Karadi sheep, and only two diplotypes (BC and BB) in all goats' samples. The current study detected several novel SNPs in the ovine-caprine populations as well as two SNPs that are observed only in sheep, including intron4:119 C>A and intron4:123 T>G. The phylogenetic analysis revealed that the observed diplotypes resided within ovine sequences and were closely related to caprine counterparts. Computational analyses indicated the presence of various intronic RNA motifs. However, all these motifs were gathered in Awassi breed. CONCLUSION: It is stated that the intron 4 is highly diverse amongst goats and sheep as well as within sheep with a particular emphasis on Awassi. This genetic peculiarity may in turn suggest a high polymorphic pattern of this breed in comparison with other related counterparts.