Introduction

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

Statistical Analysis

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).

Association Study

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 χ²_{cal (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

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

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.

Acknowledgement

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.

References

1. Falconer DS and Mackay TFC. 1996. Introduction to Quantitative Genetics. 4^th edn., pp. 56. Addison Wesley Longman Limited, English.
2. Kulig H, Kowalewska-Łuczak I, Żukowski K, Kunicka M, Kruszyński W. 2013. SLC27A1 SNPs in relation to breeding value of milk production traits in Polish Holstein-Friesian cows. Animal Science Papers and Reports. 31 (4): 273-279.
3. Lv Y, Wei C, Zhan L, Lu G, Liu K, Du L. 2011. Association between polymorphisms in the SLC27A1 gene and milk production traits in Chinese Holstein cattle. Animal Biotechnology. 22: 1-6.
4. Ordovás L, Roy R, Zaragoza P, Hayes H, Eggen A, Rodellar C. 2005. Assignment of the solute carrier family 27 member 1 (SLC27A1) gene to bovine chromosome 7. Animal Genetics. 36: 352-363.
5. Ordovás L, Zaragoza P, Altarriba J, Rodellar C. 2008. Identification of 14 new single nucleotide polymorphisms in the bovine SLC27A1 gene and evaluation of their association with milk fat content. Journal of Dairy Research. 75: 129-134.
6. Sambrook J and Russel DW. 2001. Molecular Cloning: A laboratory Manual. Cold Spring Harbour. Tab. Press, New York, pp. 6.4-6.11.
7. Snedecor GW and Cochran WG. 1989. Statistical Methods. 8th Ed. Ames: Iowa State Press.
8. Wiczer BM, Bernlohr DA. 2009. A novel role for fatty acid transport protein 1 in the regulation of tricarboxylic acid cycle and mitochondrial function in 3T3-L1 adipocytes. Journal of Lipid Research. 50: 2502-2513.