This study investigated the status of solute carrier 27A1 (SLC27A1) polymorphism in Indian cattle breeds (n = 102) using PCR-RFLP assay and its association with milk production and reproduction traits. Two region of SLC27A1 gene consisting of exon 3 and exon 4 revealed 261 and 160 bp products, respectively. The digestion of 261 bp product using SacII restriction enzyme (RE) resulted in monomorphic pattern; only GG genotype. The BsaHI/PCR-RFLP assay revealed three types of genotypes, namely, TT (160 bp), CC (106 and 54 bp) and heterozygous CT (160, 106 and 54 bp) genotypes with frequencies 18.62, 59.80 and 21.56 %, respectively. The allelic frequency of C and T alleles were 0.71 and 0.29, respectively. Association study revealed that lactation period (LP), milk yield in 300 days (MY300), calving interval (CI), peak yield (PY) and days to reach peak yield (DRPY) had non-significant difference among all the three genotypes in first and second lactations while, significant difference was (P=0.028) observed for total milk yield (TMY), where TMY of CC genotype was significantly higher than CT and TT genotypes in only first lactation. Results indicated that C allele for SLC27A1 gene can be used as candidate gene for marker assisted selection in Sahiwal and Hariana cattle.
Solute carrier family 27 member 1 (SLC27A1) is a member of the fatty acid transport protein (FATP) family. It is the transmembrane protein that facilitates long chain fatty acid (LCFA) transport across the cytoplasmic membrane. It also plays a role as the regulator of Krebs’ cycle activity and therefore assists in mitochondrial function. SLC27A1 is expressed in various tissues, predominantly in skeletal, heart muscle and adipose tissue, which are characterized by rapid fatty acids metabolism (Wiczer and Bernlohr, 2009). SLC27A1 encoding gene was mapped to bovine chromosome 7, where QTLs for milk production traits have been identified (Ordovás et al.,2005). Various SNPs were identified within the bovine SLC27A1 gene, significantly associated with milk production and composition traits in exotic cattle (Ordovás et al., 2008, Lv et al., 2011 and Kulig et al., 2013). There are no reports on SLC27A1 polymorphisms and their association with milk production traits in Indian breed of cattle. Therefore, the aim of this study was to establish possible associations between the SLC27A1 genotypes and milk production traits in Sahiwal and Hariana cows.
Materials and Methods
Animal Source, DNA Extraction and SacII, BsaHI /PCR-RFLP
A total of 102 adult females including Sahiwal (n = 52) and Hariana (n = 50) breed of cattle, maintained at ILFC, DUVASU, Mathura were utilized in the present investigation. Approximately 3-5 ml venous blood was collected from jugular vein into EDTA vacutainer tubes. Genomic DNA was extracted using the standard protocol of Sambrook and Russel (2001). Two SNPs containing regions of SLC27A1 gene fragments have been amplified from isolated DNA as per Kulig et al.(2013). Restriction digestion of PCR product was done at 370C for 14-16 h in a total volume of 15 µl containing 5.0 µl of PCR product, 1.5 µl of 10X RE buffer and 10 units of 1.0 µl RE (Table 1).
Table 1: SNPs, primers, annealing temperature and restriction enzyme details of amplified regions of SLC27A1 gene
The data was generated by estimating the frequency of different SLC27A1 genotypes. The allelic and genotypic frequencies were estimated by standard procedure (Falconer and Mackay, 1996) using following formulae-
Gene frequency = (2D + H)/2N
where, D is number of homozygote of particular gene; H is number of heterozygote having that gene and N is total no. of individuals.
Total no. of individual of particular genotype
Genotypic frequency = ———————————————————
Total no. of individuals of all genotype
The chi square (χ2) test (P ≤ 0.05) was also performed to test whether the distribution of the genotype frequencies was in the Hardy-Weinberg equilibrium (Snedecor and Cochran, 1989).
The association study of different SLC27A1 genotypes with the following milk production and reproduction traits- Lactation Period (LP = date of drying –date of calving), Total Milk Yield (TMY = calculated by totalling of daily milk records of individual cow after completion of their lactation), Milk Yield at 300 days (MY300 = calculated by totalling of daily milk records of individual cows up to 300 days of lactation), Calving Interval (CI = difference between two successive calving), Peak Yield (PY) and Days to reach Peak Yield (DRPY) was performed. Statistical analysis of milk production traits in relation to different SLC27A1 genotypes was carried out using the General Linear Model (GLM) using SPSS software (ver. 16). The following linear model was applied-
Yij = μ + Gi + eij
Where, Yij– observed trait value in animal; μ – mean trait value; Gi – effect of genotype; eij– random error. Significant differences among least square means of different genotypes were calculated using Duncan’s multiple-range test, and P values of 0.05 were considered statistically significant.
Results and Discussion
Two amplified fragments of the SLC27A1 gene revealed 261 and 160 bp products (Fig. 1 and 2). The SacII/PCR-RFLP assay revealed only one type of banding pattern (uncut); which was of 261 bp (GG genotype) (Fig. 1). This revealed that the cattle used in the present study were monomorphic in nature for C>G SNP. The BsaHI /PCR-RFLP assay revealed three types of banding pattern (genotypes); one of them was of 160 bp (TT genotype); second of 106 and 54 bp (CC genotype) and heterozygous pattern had 160, 106 and 54 bp bands (CT genotype) (Fig. 2). It indicated that the cattle population used in the present study were polymorphic in nature with two types of alleles C and T. The genotypic and allelic frequency of BsaHI PCR-RFLP assay (C>TSNP) are presented in Table 2.
The monomorphic pattern of C>G SNP in the present study on Hariana and Sahiwal cattle was not in agreement with the findings of Kulig et al. (2013) in polish Holstein Frisian cattle as they found polymorphism for this SNP with genotypes GG, GC and CC having genotypic frequencies as 63%, 35% and 2%, respectively and allelic frequency of G and C alleles as 0.8 and 0.2, respectively.
Fig. 1: SLC27A1-I/SacII PCR-RFLP assay pattern in 2.0% agarose gel; Lane 1: Undigested PCR product, M: Marker (50 bp ladder), 2, 3 & 4: RE digested uncut PCR products
Fig. 2: SLC27A1-II/BsaHI PCR-RFLP assay showing genotype pattern in 2.0% agarose gel; Lane 1: CC genotype (106 & 54 bp), 2: CT genotype (160, 106 & 54 bp), 3: TT genotype (160 bp), M: Marker (50 bp ladder) and 4: Undigested PCR product
The BsaHI/ CC genotypic frequency observed in present study (59.80%) was not in agreement with the previous reports of Lv et al. (2011) and Kulig et al. (2013) in Chinese (15.76%) and Polish Holstein Frisian (8.0%) cattle, respectively. The CC genotypic frequency observed in present study was higher than the other two genotypes. In the present investigation, the genotypic frequency of CT and TT genotype was 21.56 and 18.62%. In contrast, Lv et al. (2011) and Kulig et al. (2013) observed higher value for CT (45.76 and 56.0%) and TT (38.48 and 36.0%), respectively. Moreover, the observed allelic frequency of C allele (0.71) in present study was higher than the frequency observed by Lv et al. (2011); 0.38 and Kulig et al. (2013); 0.36 and that of T allele (0.29) was lower than previous reports 0.61; (Lv et al., 2011) and 0.64; Kullig et al., 2013). Chi square test revealed that χ2cal (23.99) > χ2 tab (5.99) at 5% level of significance for degree of freedom 1 indicating that screened cattle population was not in Hardy-Weinberg equilibrium (Table 2).
Table 2: Genotypic and allelic frequencies of C>T SNP/BsaHI genotypes in Indian Sahiwal and Hariana cattle breeds
|Breed||Genotypic Frequency (%)||Allelic Frequency|
|Number||Observed||61||22||19||χ2 cal = 23.99
χ2 tab = 5.991 (P<0.05)
χ2 cal > χ2 tab
Where; N= Sample size, n= Number of animals in particular genotype
The overall means with standard errors (Mean ± S.E) for milk production traits related to different BsaHI genotypes in first and second lactations are presented in Table 3.
Table 3: Association studies of SLC27A1-II/BsaHI genotypes with milk production and reproduction traits
|Lactation||Genotype||n||LP (days)||TMY (liters)||MY300 (liters)||CI (days)||PY (liters)||DRPY (Days)|
Different letters in superscript of a given row indicates significant (P<0.05) difference between genotypes, n — number of individuals in particular genotype, N — total number of individual in particular lactation, values present in Mean ± SEM (Standard error of mean)
Association study showed that LP, MY300, CI, PY and DRPY had non-significant difference among all the three genotypes in both the lactations. However, significant difference was (P=0.028) was observed for TMY where TMY of CC genotype (2037.3±141.6 liters) was significantly higher than CT and TT genotypes (1470.9±172.8 and 1482.2±28.5 liters) in only first lactation. There was no significant difference (P=0.43) was observed for the TMY among all the genotypes in second lactations. This result indicated that allele C of C>T SNP was associated with positive effect on milk yield. Similarly, Lv et al. (2011) also found significant associations between C>T SNP and EBV for milk yield in Chinese Holstein cattle. They observed CC animals having significantly (P ≤ 0.05) higher value of milk compared with the CT and TT individuals while, Kulig et al. (2013) could not observe any association of C>T SNP with milk yield in Polish Holstein Frisian cattle.
This is our first report of SLC27A1 polymorphisms and their association with milk production traits in Indian cattle. In the present study, we reported that the screened cattle were found monomorphic for C>G SNP in SLC27A1 exon 3 region. Furthermore, we also identified the C>TSNP polymorphisms in SLC27A1 exon 4 region with two types of alleles C and T in screened cattle. Association study revealed that the CC genotype performed better in total milk yield trait as compared to CT and TT genotypes. So, Sahiwal and Hariana cattle with predominantly C allele for SLC27A1gene can be used as candidate gene for marker assisted selection in these breeds.
This work was carried out under the project funded by University, DUVASU, Mathura. The authors are thankful to Vice Chancellor, DUVASU, Mathura, (U.P.) for providing necessary facilities during entire research work at this esteemed university. The assistance of Instructional Livestock Farm Complex (ILFC), DUVASU, Mathura in providing blood samples of Sahiwal and Hariana cows are duly acknowledged.