The main goal of sheep breeding programs is to improve meat production and carcass traits (Kosgey et al. 2006). During the last forty years, the genetic improvement of livestock has been achieved by selection based on phenotypic information, but getting fast genetic improvement is difficult by this method. However marker-assisted selection (MAS) can increase the genetic improvement of polygenic traits which results from the presence of major genes and their effect on the performance of these traits. Therefore, this method has provided opportunities to increase response to selection, especially for traits with small heritability (Dekkers, 2004). Lori-Bakhtiari breed, with more than 1.7 million heads, is one of the most common indigenous sheep breeds in Iran. This breed adapted to the hilly and mountainous regions in the west of Iran and has been raised mostly in villages under semi-intensive systems. The Lori-Bakhtiari breed has high body weight and the largest fat tail compared to other Iranian Indigenous breeds (Kianzad et al. 2003; Vatankhah and Talebi, 2008). Zel breed, with about 1.5 million heads, is the only thin-tailed Iranian breed. Zelis raised in the north part of Iran located between the northern slopes of the Alburz mountain and the Caspian Sea (Kamalzadeh et al. 2008). Diacylglycerol acyltransferase1 (DGAT1) gene is involved in the metabolism of triglycerides and catalyzes the last step of the triacylglycerol synthesis (Hatzopoulos et al. 2011; Li et al. 2013). DGAT1 is extensively expressed in almost all tissues including the mammary glands, small intestine, adipose tissue, and skeletal muscle, with the highest expression level in adipose tissue and small intestine (Xu et al. 2008). The DGAT1 gene spans approximately 8.5 kbp and comprises 17 exons 16 introns, located on chromosome 14 of bovine (Grisart et al. 2002). The homologous sequence was found on chromosome 9 of sheep (Ovis aries). This gene contains about 1470 nucleotides that code a polypeptide with 489 amino acids (Xu et al. 2009). Specific polymorphisms in DGAT1 were detected and their association with some production traits were confirmed in farm animals, especially cattle. Then, DGAT1 was introduced as a candidate gene for milk production traits in dairy (Banos et al. 2008; Li et al. 2009; Signorelli et al. 2009; Cerit et al. 2014) and carcass traits (Anton et al. 2010; Souza et al. 2010; Pannier et al. 2010; Li et al. 2013; Tait et al. 2014; Ardicli et al. 2017) in cattle.Moreover, DGAT1 due to its key role in fat metabolism (Scata et al. 2009) is a candidate gene for milk fat content (Scata et al. 2009; Xu et al. 2009; Yang et al. 2011; An et al. 2013; Tabaran et al. 2014; Martin et al. 2017) and carcass traits (Xu et al. 2009; Mohammadi et al. 2013) in dairy sheep and goat. However, there are only a few studies on the association between DGAT1 polymorphisms and carcass traits in sheep (Xu et al. 2009; Mohammadi et al. 2013; Noshahr and Rafat, 2014; Nanekarani et al. 2016). Xu et al. (2009) sequenced the whole coding region of sheep DGAT1 and surveyed the associations between polymorphism of DGAT1 and 11 meat production traits. They reported thepositive effect of the T allele in exon-17 of DGAT1 on meat quality and quantity in three Chinese sheep breeds. Mohammadi et al. (2013) reported that there is a significant correlation betweenexon-17 of DGAT1 polymorphisms (T487C) and carcass weight and dressing percentage, and concluded that the CC genotypes had higher carcass weight and dressing percentage. Few studies have been carried out on the association of DGAT1 polymorphisms with carcass traits in sheep breeds. All of these studies suggested that further investigations are necessary to make a definitive statement about the association of this gene polymorphisms with carcass traits in sheep (Xu et al. 2009; Noshahr and Rafat, 2014; Mohammadi et al. 2013). The aim of this study is to investigate 5'UTR, exon-1 and exon-2 regions of DGAT1 polymorphisms and their association with carcass traits in Lori-Bakhtiari and Zel Iranian indigenous breeds.
MATERIALS AND METHODS
A total of 309 blood samples were collected from Lori-Bakhtiari (n=152) and Zel (n=157) sheep breeds. Lori-Bakhtiari and Zel samples were taken from two industrial slaughterhouses located in the Qom and Mazandaran provinces of Iran, respectively. Before slaughter, body weight (SBW) of lambs was measured. After slaughter, carcass weight (CW), fat-tail weight (FTW), carcass weight without fat-tail (CWFT), and back fat thickness (BFT) immediately were measured and fat-tail percentage (FTP), dressing percentage (DP) and dressing percentage without fat-tail (DPWFT) were calculated. Genomic DNA was extracted by the Salting-out procedure (Miller et al. 1998). The quality and concentration of extracted DNA were evaluated by visualizing on 0.8% agarose gel under UV light and spectrophotometer (Thermo, NanoDrop 1000).
DNA amplification with PCR
Two primer pairs DGAT1 (5'UTR and exon-1) F:5'-GGAACTACGCTTCCCAGGAC-3'; R:5'-ACGTCTCCGTCCTTGTCTGT-3'; and (exon-2) F:5'-GTCTTGCATCACCAGCTCCT-3'; R:5'-CAGGCATCTACTGGGATTCAG-3' were designed based on the published sheep sequences (GenBank accession no.EU178818) to amplify the determined fragment by Primer-BLAST (Ye et al. 2012). The length of the amplified fragments were 360 bp (5'UTR and partial exon-1) and 445 bp (exon-2) in both breeds. PCR was carried out on 25 µL volume of the following ingredients: 1 × PCR buffer (16 mM (NH4)2SO4, 67 mM Tris-HCl pH 8.8, 0.1% Tween-20), 2 mM MgCl2, 200 µM dNTP, 6 pmol of each primer, 1.5 unit Taq polymerase (GenetBio, Korea) and 50-100 ng of genomic DNA. PCR reactions were performed with a Genecycler (Bio-Rad, My Cycler, USA) with the amplification program consisted of an initial denaturation at 96 ˚C for 2 mins, then 32 cycles of 96 ˚C for 1 min, 57 ˚C for 45 seconds and 72 ˚C for 1 min, and a final extension of 72 ˚C was maintained for 10 mins for the exon-1 of DGAT1 gene. For exon-2 fragment, the thermal cycling conditions consisted of initial denaturation at 96 ˚C for 5 mins, followed by 30 cycles of denaturation at 96 ˚C for 1 min, annealing at 60 ˚C for 45 seconds, extension at 72 ˚C for 1 min, with a final extension at 72 ˚C for 10 mins. The PCR products were identified by 2% agarose gel electrophorese and visualized using UV light.
Single-strand conformation polymorphism
SSCP method was applied to detect SNPs in 5'UTR, partial exon-1 and exon-2 of the DGAT1 gene. For SSCP, 5µl PCR products were diluted in 5µl denaturing solution; the mixture was centrifuged and denatured at 98 ˚C for 10 mins, then rapidly chilled on ice for 5 mins and detected on 12% polyacrylamide gel. The electrophoresis was performed in a vertical unit (Bio-rad), in 1× TBE buffer at 4 ˚C. The gel running conditions for the 360 bp fragment of DGAT1 was 350 V for 18hours and 445 bp fragment of DGAT1 was 380 V for 21 hours, respectively. Then, DNA fragments were stained with 0.1% silver nitrate and visualized in 3% NaOH solution (Zhang et al. 2007). Two samples from each SSCP patterns were sequenced in both directions (Bioneer, Seoul, South Korea) and were determined by Bio Edit (Version 7.2.0) and Vector NT1 software. Different SSCP pattern sequences were aligned according to Genbank (EU178818.1) by BLAST algorithm to find homologous sequences in NCBI databases (http:// www.ncbi.nlm.hih.gov/).
Genotypes patterns or haplotypes frequencies were calculated using GENALEX6.4 software (Peakall and Smouse, 2006). Two fixed models were used to make an association between 360 bp fragment of DGAT1 variants and carcass traits using a generalized linear model (GLM) procedure of SAS software (SAS, 2007). Exon-2 of DGAT1 was eliminated from association analysis due to monomorphic state. Tukey–Kramer’s multiple range tests were used for least-squares means (LSM) comparison. The statistical model used for determining CW, DP, BFT and DPWFT traits in two breeds was as follows:
Another model used for FTW, FTP and DP traits in Lori-Bakhtiari sheep breed was as follows:
yijklm: phenotypic value of carcass traits.
µ: overall population mean for each trait.
Ai: ﬁxed effect of the ith age.
Sj: ﬁxed effect of the jth sex.
Bk: ﬁxed effect of the kth breed.
Gl: ﬁxed effect of the lth genotype.
covariate fixed effect of weight at slaughter.
Sj(Bk): effect of the jth sex in kth breed.
(AG(il: interaction between the ith age and the lth genotype. (SG(jl: interaction between the jth sex and the lth genotype. (BG)kl: interaction between the kth breed and the lth genotype.
eijklm: residual random effect.
RESULTS AND DISCUSSION
Single strand conﬁrmation polymorphism analysis
The results showed that both PCR products including 360 bp and 445 bp fragments, successfully amplified by the used primers. Five SSCP patterns were detected for exon-1 and its 5′UTR (Figure 1).
Figure 1 Five PCR-SSCP genotypes of exon-1 and its 5'UTR fragment (360 bp) of DGAT1 gene for Lori-Bakhtiari and Zel breeds
Furthermore, one SSCP pattern was observed for exon-2 and this segment was monomorphic in both breeds (Figure 2).
Figure 2 PCR-SSCP genotype of exon-2 fragment (445 bp) of DGAT1 gene for Lori-Bakhtiari and Zel breeds
The frequency of the observed genotypic patterns of 360 bp fragment for each breed is shown in Table 1. The G5 and G2 had higher frequency in Lori-Bakhtiari and Zel breeds, respectively. The G3 had the lowest frequency in both breeds. Also, the frequency of G2, G4, and G5 was different between Lori-Bakhtiari and Zelbreeds.
DNA sequence analysis
In this study, sequences of PCR amplicons from five different SSCP genotypic patterns of 360 bp fragment variants were analyzed and compared with the NCBI reference sequences (Table 2). For 360 bp fragment (including 5′UTR and exon-1), three novel single nucleotide polymorphisms (SNPs) were detected in the studied breeds. One of the discovered SNPs is located in the coding region (gA277G) and two of them located in the 5′UTR region (g.T101- and C129A) of DGAT1 (Table 2). Moreover, the protein sequences of exon-1 region were aligned to NCBI reference sequence (Figure 3). The only substitution in exon-1 of G3 pattern (A 277G) leads to the changes in amino acids (p.Arg54Gly) (Figure 3).
In the present study, exon-2 of DGAT1 was monomorphic and omitted from association studies. Association of the 360bp fragment of DGAT1 polymorphisms with carcass traits was analyzed (Table 3). Significant relationship (P<0.05) was found between FTW and BFT traits in the Lori-Bakhtiari breed, and animals with G5 had higher FTW and BFT compared to G1 pattern. Although no significant differences (P>0.05) were observed for DP and FTP among five identified genotypic patterns in Lori-Bakhtiari, animals with G3 pattern for DP and G5 pattern for FTP had the highest performance compared to other genotypes. No significant association (P<0.05) was found between observed genotypes of exon-1 of DGAT1 in Zel breed and the studied carcass traits. DGAT1 was selected as a candidate gene due to its effects on carcass traits that had been conﬁrmed in previous studies on cattle (Pannier et al. 2010; Souza et al. 2010; Li et al. 2013; Tait et al. 2014) and sheep (Xu et al. 2009; Mohammadi et al. 2013; Noshahr and Rafat, 2014). The detection of SNPs in 360 bp fragment was consistent with results of Scata et al. (2009) who amplified and sequenced whole of the DGAT1 gene using 18 primer pairs and reported novel SNPs at 5'UTR (g. C127A) in Sarda, Altamurana and Gentile di Puglia sheep breeds. But their result was not consistent with present study for exon-1. The detection of SNPs in 360 bp fragment was consistent with the results of Scata et al. (2009) who identified novel SNP in Sarda, Altamurana and Gentile di Puglia sheep breeds. Furthermore, results indicated that DGAT1 had significant genetic effects on fat content in Lori-Bakhtiari breed are consistent with the findings of Xu et al. (2009), Mohammadi et al. (2013) and Noshahr and Rafat (2014). Based on the sequence analysis, two differences, including one deletion and one substitution, were found in 5'UTR region between detected genotypes (Table 2). Regulation of DGAT1 expression in adipocytes mostly happens at the transcriptional and post-transcriptional steps (Yu et al. 2002), therefore mutations in 5'UTR region may influence DGAT1 expression and consequently fat content.
Table 1 Genotypic patterns frequencies of single strand conformation polymorphism (SSCP) variants of 5′ UTR and exon-1 of DGAT1 gene in Zel and Lori-Bakhtiari breeds
Table 2 Nucleotide substitutions and types of single-nucleotide polymorphisms (SNPs) for exon-1 of DGAT1 gene in Zel and Lori-Bakhtiari breeds
Table 3 Association of DGAT1 genotypes with carcass traits in Zel and Lori-Bakhtiari breeds
The means within the same row with at least one common letter, do not have significant difference (P>0.05).
SE: standard errors.
Figure 3 The amino acid sequence of exon-1 of DGAT1 gene for Lori-Bakhtiari and Zel breeds
Therefore, identiﬁed mutations in the 5'UTR region of DGAT1 could affect the function of DGAT1 enzyme in lipid metabolism. For instance, DGAT1 has considerable effect on fat deposition and fatty acid composition in sheep meat and milk (Scata et al. 2009). In the beginning cod on (ATG) has started from position 202, mutations have occurred before it was not able to change the protein sequence and only the mutation occurred in the exon-1 region of the DGAT1 gene which was able to change the amino acid sequence of translated protein derived from this gene. Therefore, mutation only occurred in position 277 of genotypic pattern G3 and led to the transformation of amino acid arginine to glycine (AGG to GGG). However, there was no significant difference (P>0.05) between this genotypic pattern and other genotypes. This result indicated that despite the change in the sequence of the enzyme, performance did not change significantly. Therefore, this SNP could not be introduced as a marker for the studied traits. By the way, future investigations can be useful to detect possible effects of this SNP on the production traits of sheep. Only CW, DP and BFT traits were analyzed in Zel breed because this breed does not have any fat-tail for measuring other traits. In Zel breed, no significant difference (P> 0.05) was found among the observed five genotypes in carcass traits, but G3 pattern showed higher DP and lower BFT compared to others. The lack of significant differences between most of the identified genotypic patterns may be due to the high standard errors found for some genotypic patterns, resulting from the low number of animals for these genotypes. Therefore, increasing the number of animals will probably change the results. The effects of different genotypes on common carcass traits in Zel and Lori-Bakhtiari breeds were similar, when analyzed separately or with each other. Possible contradictory results can be due to the influences of different genomic background and different genotypic frequencies.
In this study, five different PCR-SSCP genotypic patterns were detected for 360 bp fragment (including 5′UTR and exon-1) and three novel SNPs were detected for this region. There was a significant correlation (P<0.05) between FTW and BFT traits and observed genotypes in Lori-Bakhtiari breed, but there was no significant correlation (P<0.05) between detected genotypes of exon-1 of DGAT1 in Zel breed and carcass traits. These results indicated that DGAT1 is a potential candidate gene for carcass traits in MAS and breeding programs to improve FTW and BFT traits by selection in Lori-Bakhtiari breed. Although significant correlation (P<0.05) was found between carcass fat content and DGAT1 gene, but it can be due to the effect of other mutations in regions that were not investigated in present study (Xu et al. 2008) and also another QTLs close to the DGAT1 (Coppieters et al. 1998). Further investigation is necessary to make a conclusive decision about using the detected polymorphisms of this gene in breeding programs for carcass traits in sheep.
The authors thank all the teams who worked on the experiments and provided technical assistance in the laboratory during this study.