The SREBP1 84 bp-indel polymorphism has been already discovered and genotyped by a conventional PCR amplification and gel electrophoresis [15]. In the present study, the HRM technique was applied, which is a fast, simple and highly reliable method developed from previously established genotyping technologies [37]. An optimized protocol with a new screening method to genotype the insertion/deletion of the polymorphism of interest (84 bp-indel) by real-time PCR-HRM analysis has been successfully performed (Figure 1).
Transcription regulations of SREBP1 and SREBP2 are preferentially specified for FA and cholesterol metabolism, respectively [38], while gene duplication occurred when independent regulation of FA and cholesterol was required [9]. However, little is known about the subcutaneous fat tissue in bovine and other mammals. The maximum-likelihood mRNA tree of SREBP1 showed a phylogenetic organization (Figure 2) similar to previous studies performed with mitochondrial DNA of ruminants and mammals [39]. Alignments of bovine SREBP1 full-length gene, mRNA, intron 5, and the 84 bp-indel sequences might give an insight into the significance of the indel in other species. The SREBP1 full-length gene alignment showed that similarity among mammals in intron 5 was higher than in other introns (Figure 2b). In ruminant mammals, the homology of bovine SREBP1 mRNA coding sequence was lower (85–99 %, Figure 2a) in comparison with the homology of intron 5 (96–99 %, Figure 2b). This result was unexpected since mRNA is normally translated to protein, which is better conserved compared to non-coding introns. Homology between Bos taurus and Bos indicus was the highest (99 %) in all alignments (Figure 2a and 2b). S allele (deletion) seems to be absent in Bos indicus sequences of GenBank which is in agreement with LL genotype observed in Asian zebu breeds [18]. On the other hand, the 84 bp-indel region showed the highest homology among ruminants (≥ 99 %, Figure 2b), compared to mRNA or intron 5 regions. In non-ruminant mammals, homology was also higher in the 84 bp-indel region (56–76 %) compared with intron 5 (45–71 %). And our results also showed higher homology in the 84 bp-indel region compared to full intron 5 (Figure 2b), especially in ruminants, suggesting that this could be a well conserved region throughout evolution with an undetermined functions in SREBP1 or that even could act regulating transcription levels of downstream genes. Similarly, intron 16 of SREBP1 has been described to be highly conserved among mammals [40], which encodes a non-coding RNA (miR–33) in cattle [41]. Accordingly, alignment analyses concluded that 84 bp-indel region seems to be highly conserved in mammals. However, the absence of S allele in mammals other than Bos taurus suggests that the 84 bp-indel polymorphism could have appeared after Bos indicus and Bos taurus species differentiation, reported to occur 1.7–2 million years ago [39].
In terms of bovine commercial types studied, Pirenaica is the most important beef cattle breed in northern Spain, highly appreciated for its value as a genetic resource as well as for its production traits. But French Salers cattle and Holstein-Friesian cull cows are also an integral part of the regional beef supply. This study characterized the 84 bp-indel polymorphism of the SREBP1 gene and showed, for the first time, the presence of S allele in Salers and Pirenaica cattle breeds (Table 1). In previous studies with bigger sample size, only SL and LL genotypes were reported in Simmental and crossbred beef breeds [23, 25]. In Salers bulls and Pirenaica heifers, the observed frequency of SS and LL genotypes was higher than the expected which could indicate an excess of homozygote individuals, although the Hardy-Weinberg equilibrium did not show significant differences for any of the commercial types studied (Table 1). The frequency of S allele in Salers bulls was 0.385, higher than Pirenaica heifers (0.214) and bulls (0.135). In contrast, all Holstein-Friesian individuals showed LL genotype as already reported [18, 20, 21] which could have been the consequence of a high selection pressure towards milk production. The scientific literature shows that beef breeds seem to have higher S allele frequency compared to dairy breeds. Interestingly, S allele frequency of Salers was similar to Limousin (0.335; [20]) breed, which might be an effect of the phylogenetically and geographically close relationship between the two breeds [42]. In general, these results indicate higher S allele frequencies in European beef cattle breeds, although the highest frequency values were reported in Japanese Black (0.450; [18]).
This polymorphism has been associated to FA composition. First, Hoashi et al. [15] indicated that S allele of the 84 bp-indel contributed to a higher (1.3 %) intramuscular MUFA content in Japanese Black. In terms of individual MUFAs, SL genotype was associated with significantly higher contents of 9c-14:1 compared to LL genotype in subcutaneous fat of Simmental bulls [23], while in intramuscular fat, LL showed higher 9c-16:1 and 9c-17:1 contents than SL genotype of Simmental bulls [27]. In the present study, only Pirenaica bulls showed slightly higher backfat MUFA content in SS/SL compared to LL genotype (p > 0.05). Bhuiyan et al. [43] reported higher stearic acid (18:0) content in muscle fat of Korean Hanwoo bulls with LL genotype compared to LS and SS genotype. However, in the present study, a higher but not significant 18:0 content was observed in SS/SL compared to LL genotype in all commercial types (Table 2) which was in agreement with the results obtained by Xu et al. [27] in muscle fat of Simmental bulls. In terms of PUFA content, the higher content in 18:3n–3 and total n–3 in SS/SL than LL genotype of Pirenaica bulls may suggest an effect of this polymorphism on PUFA content in this commercial type (p ≤ 0.05; Table 2), although SS was the highest compared to SL and LL genotypes (p > 0.05; data not shown). Only Bhuiyan et al. [43] reported higher muscle 18:2n–6 and total PUFA contents in SS compared to LL genotype. The high correlations between SREBP1 gene expression and UFA contents in Salers bulls (Figure 3), together with their highest S allele frequency compared to other European cattle breeds (0.385; Table 1), could indicate a greater influence of S allele in SREBP1 and/or FA content. When all individuals with SS genotype were considered together, positive correlations were observed between gene expression and PUFAs, while negative correlations were observed with the rest of the FAs. Some of these associations could be also explained by the linkage between this DNA marker and other causative polymorphisms [25].
In other studies, associations of rs419112290, located in exon 14 of SREBP1, with fat content and trans-MUFA of milk in Holstein-Friesian have been found [16], but also with 14:0, 16:0, 9c-18:1, 18:2n–6, 18:3n–3, SFA and MUFA of muscle fat in Asian cattle [26]. Holstein-Friesian cows, with only LL genotype, different correlation patterns in SFAs compared to the other commercial types were observed. The different correlations in Holstein-Friesian cows could be related to a less homogeneous diet and older age of these animals, but also their selection towards milk production compared to Pirenaica and Salers which are specifically raised for meat production.
In previous publications, SCDs have been associated with FA composition of adipose tissues [16, 17, 23, 44, 45, 46]. The fact that the 84 bp-indel of SREBP1 is associated with FA content would be possible because the downstream genes, such as SCDs, could be regulated through the transcriptional regulation. SREBP1 can directly activate the expression of over 30 genes [38]. As expected, several patterns observed between SREBP1 and FAs were also noted in correlations with SCDs (Figure 4). For example, positive correlations observed between SREBP1 and PUFAs in SS genotype of Salers (p > 0.05; Figure 3), became significant when SCD1 and PUFA correlations were calculated for same genotype (p < 0.05; Figure 4). Thus, this might suggest that the regulation through SREBP1 to SCD1 can affect PUFA content in SS genotype of Salers bulls. However, this statement should be carefully considered, since Salers bulls with SS genotype only permits correlation analysis without covariate (SREBP1 gene expression; see statistical analysis section). Interestingly, negative correlations observed between SREBP1 and SFA in LL genotype of Salers (p > 0.05; Figure 3), also became significant for SCD1 and SFA correlations (p < 0.05; Figure 4), indicating a stronger effect of SCD1 than SREBP1 on SFA in LL genotype of Salers. These results are not unexpected since SCD1 desaturates SFA to MUFA and PUFA, and the relationship between SREBP1 and SCD1 in Salers was previously demonstrated [17]. These results indicate that in Salers the effect of SREBP1, and particularly SCD1, on SFA and PUFA seems to be different depending of the 84 bp-indel genotype. However, this assertion could not be corroborated through differences in SFA and PUFA contents reported (Table 2).
On the other hand, the 18:0, 18:3n–3 and total n–3 content differences observed between SS/SL and LL genotypes of Pirenaica bulls (Table 2), should be further investigated with a larger sample size of Pirenaica bulls that could increase the S allele frequency to sufficiently evaluate its effect. Nevertheless, significant correlations between SCD1 and SFAs (including 16:0) were observed in SL genotype of Pirenaica bulls, but also 9c-16:1 in Pirenaica breed. In our previous study [17], 9c-16:1 desaturation index was significantly correlated with SCD1 in Pirenaica bulls while these results suggested that relationship between SCD1 and 16:0/9c-16:1 FA contents might be different depending on the indel genotype. Differences between Pirenaica bulls and heifers might occur as a result of a stronger lipogenic gene regulation in a sex dependent manner. In Pirenaica bulls, SS/SL had significantly higher 18:0 content than LL genotype (Table 2) and this higher 18:0 content might indirectly increase the expression of SCD1 giving rise to a positive correlation between SCD1 and 9c-18:1 observed in Pirenaica breed and all individuals (Figure 4). In Pirenaica heifers and all individuals group, SS and SS/SL genotypes showed a positive correlation between SCD5 and 18:0, whereas it was negative between SCD5 and 9c-18:1. Therefore the relationship between SCD5 and 18:0/9c–18:1 content could also be quite dependent on the indel genotype. Overall, the content of several SFAs and corresponding UFAs (i.e., 16:0/9c-16:1 and 18:0/9c-18:1) are related as a result of the unsaturation by SCD1 or SCD5. Moreover, opposite correlations between these lipogenic genes with 16:0 and 9c-16:1, but also 18:0 and 9c-18:1, seems to be dependent on the indel genotype, especially in Pirenaica breed. Indeed, in Pirenaica, it might be more evident the transcriptional feedback regulation of SREBP-SCAP-INSIG complex, where SREBP1 promotes the desaturation of SFA into UFAs by SCDs. Whereas, UFAs stabilize INSIG1 protein which acts as an inhibitor of SREBP1 activity by retaining the inactive SREBP/SCAP complex in the ER membrane [6]. Consequently, expression of SREBP1 produces a negative feedback regulation, influencing its own activity and SCD desaturation. In a previous study, we reported a novel genetic compensation mechanism between SCD1 and SCD5 that showed that one SCD isoform could well be compensated by upregulation the other isoform [17]. This opposite pattern has been consistently detected even when individuals were separated by the 84 bp-indel. Thus, this mechanism might strongly regulate expression of both SCDs, although the pathway by which it occurs remains unclear. Several transcription factors apart from SREBP1 such as LXR, PPARα, C/EBPα, NF–1, NF-Y, and Sp1, have also been revealed to bind to the SCD1 promoter region [47]. It has been proposed that SCD1 is mainly regulated by a wide variety of hormones and nutrients, while SCD5 is not affected by external inputs like food sources [48]. Discrepancies of SCD5 regulation among species and tissues showed a SREBP1 binding site only in human SCD5 but not in other mammals [49], while other studies support a direct regulation of SCD5 by SREBP1 in skeletal muscle cells [50] or even directly binding to the promoter of SCD5 in cattle [51]. Further studies are necessary to establish the mechanism by which both SCD isoforms are compensated and clarify their regulation by SREBP1 in beef (muscle and fat), leading to meat production with favorable characteristics.