Polymorphisms within CEBPA, PRKAG3 and SREBF1 Genes Associated with Fat Deposition in Fat-tail Altay Sheep

All procedures involving animals were approved by the animal care and use committee at the respective institutions where the experiment was conducted. All procedures involving animals were approved and authorized by the Chinese Ministry of Agriculture.

For SNP analysis study, 200 ewes of two different breeds reared in China were selected. The animals were distributed as follows: 100 Altay sheep in Fuyun Breeding Farm (Fuyun County, Xinjiang Uygur Autonomous Region, P.R. China), 100 White Suffolk in Beijing Aoxin Stud Farm Co. Ltd. (Beijing, P.R. China). All the sheep were in a good state of health and nutrition. Ear tissue taken from each Altay sheep was immersed in 70% ethanol under 4°C and stored at -20°C pending for DNA isolation. Venous jugular blood of White Suffolk was collected using acid citrate dextrose as an anticoagulant. Genomic DNA was extracted from ear tissue or whole blood by the phenol-chloroform method, and then dissolved in TE buffer (10 mmol/l Tris-HCl (pH 8.0), 1 mmol/l EDTA (pH 8.0)) and kept at -20℃.

As shown in Supplementary Table S1, primers of CEBPA, PRKAG3 and SREBF1 genes were designed according to the mRNA sequences of sheep derived from GenBank database. Polymerase chain reactions and were carried out as previously described (Liu et al., 2015).

The PCR products were separated by electrophoresis on 2% agarose gels (Promega, Madison, WI, USA) in parallel with DNA markerⅠ (Tiangen, Beijing, P.R. China). Gels were visualized using a 1.5% agarose gel that contained ethidium bromide, photographed, and analyzed using an AlphaImagerTM 2200 and 1220 Documentation and Analysis Systems (Alpha Innotech Corporation, San Leandro, CA, USA).

For SNP analysis 10 individuals for each sheep breed were selected randomly. Genomic DNA from Altay sheep and White Suffolk sheep was used as template to amplify with primers as shown above and sequences were aligned to search for the base pair variations. PCR products were separated on 2% agarose gels and recovered using Geneclean Ⅱ kit (Promega, Madison, WI, USA). Each DNA fragment was sequenced in both directions using an automatic ABI 3730 sequencer (Perkin Elmer Applied Biosystems, Foster City, CA, USA) by SinoGenoMax Co. Ltd. (Beijing, China).

Sequence analysis, and amino acid determination were performed with the program DNAMAN version 9.0 and DNAstar lasergene version 7.1.

For genotyping analysis three SNPs were selected for genotyping by using 200 samples from both Altay and White Suffolk sheep. Genotyping was performed using primer extension chemistry and mass spectrometric analysis (iPlex assay, Sequenom, San Diego, CA) on the Sequenom MassArray according to the manufacturer’s instructions (http://www.sequenom.com). Only those samples with a > 95% success rate and only those SNPs with a genotype success rate of > 95% were included in the analysis.

Allele and genotype frequencies were estimated by direct counting. Statistical analyses were performed by use of the SAS (Ver 8.1) (SAS Institute Inc., Cary, NC, USA). Differences between two groups of samples were accessed by t-tests assuming unequal variances. P values less than 0.05 were considered to be significant. Chi-square test was applied to analyze the statistical significance of loci genotype distributions of two sheep breeds.

Zero SNP was identified for CEBPA. Two SNPs were identified and genotyped for PRKAG3, and one SNP was identified and genotyped for SREBF1. SNPs were selected for genotyping by using 200 samples from Altay and White Suffolk sheep on the Sequenom MassARRAY plateform (Gabriel et al., 2009). As shown in Supplementary Figure S1, for PRKAG3 gene, a c.1744C>T SNP and a c.1840C>T SNP (GenBank accession no. NM_001122692; Both are synonymous) have been genotyped. For SREBF1 gene, an unknown synonymous A>G SNP was also detected (c.2878A>G, GenBank accession no. XM_004013336).

The allele and genotype frequencies of PRKAG3 and SREBF1 genes in Altay and White Suffolk sheep were calculated respectively as shown in Table I after genotype detection. As shown in Table II, allele C is dominant allele at the c.1840C>T of PRKAG3 gene in both two breeds, while in the c.2878A>G locus of SREBF1 gene, allele G is dominant allele. It was also shown that the genotype distributions in above two loci were significantly different between fat tail and thin tail breeds by chi-square test (P<0.05).

Altay sheep chosen for this study has a large rump composed entirely of white adipose tissue which is known for their ability to cope with harsh environmental conditions such as drought and famine in northern part of Xinjiang Uygur Autonomous Region. Due to improved forage availability and healthy issue, fat tail trait is commercially undesirable now. For this trait breeders are interested in looking for useful molecular markers to serve sheep breeding program via marker- assisted selection, so searching gene variants affecting the phenotypic expression of fat-tailed trait in sheep are becoming a hot topic in molecular genetics.

Up to now, there is little published information related to tail fatness especially for Chinese local breeds.

Note: The numbers in the brackets are the genotype individuals. * P<0.05; ** P<0.01 (χ20.05,5.99; χ20.01,9.21)

Table II. Test of difference of loci genotype distributions of PRKAG3 and SREBF1 in Altay and Suffolk sheep breeds. GenBank accession numbers for these SNPs can be found in Supplementary Table S1.

Note: * P < 0.05, ** P < 0.01, *** P <0.001

Several candidate genes have been studied to associate with fat deposition and lipid metabolism in domestic animals. PPARG and its target genes is one factor leading to greater intramuscular fat deposition in cattle (Moisa et al., 2014). FABP3 gene plays in fat deposition and the regulation of fatty acid metabolism in the Lanzhou fat-tailed sheep (Bai et al., 2013). FABP4 gene mRNA and protein have no significant differences between control and continuous starvation groups which means that FABP4 may not be the key gene in fat depositon in Altay sheep (Ruixia et al., 2015). The mRNA abundance of G-protein coupled receptor 41 (GPR41), Adiponectin receptors 1 and 2 (ADIPOR1/2) and LEPTIN are divergent in different fat depots from sheep (Lemor et al., 2010). There were novel associations of DGAT1 gene in which the C allele had a positive effect on fat-tail weight and backfat thickness in fat-tailed sheep (Mohammadi et al., 2013). CAST gene being a potential candidate gene for growth and meat quality traits has been detected for novel SNPs and breed-specific haplotypes, and CAST-10 and CAST-8 might be breed-specific haplotypes that distinguish between fat-tailed and thin-tailed sheep breeds (Aali et al., 2014).

It has been reported that CEBPA highly expressed in fat-rumped sheep while lower expressed in thin-tailed sheep breeds which had significant correlations with fat deposition in tail tissues of sheep (Wei et al., 2014). Polymorphisms of sheep CEBPA, PRKAG3 and SREBF1 genes and their association with fat deposition between fat-tailed (rumped) and thin-tailed breeds were firstly investigated in the current study. New polymorphic sites of PRKAG3 gene (c.1744C>T SNP and c.1840C>T SNP) and SREBF1 gene (c.2878A>G SNP) were detected in our study. Genotype distributions were significantly different between fat tail and thin tail breeds. It may indicate that those two loci may be associated with fat deposition in fat-tail breed.

This work was supported by Joint Funds of the National Natural Science Foundation of China (Grant No. U1130302), by the Earmarked Fund for China Agriculture Research System (Grant No. CARS-38), by Special Fund for Basic Scientific Research of Institute of Animal Science, Chinese Academy of Agricultural Sciences (Grant No. 2013ywf-zd-1) and by Agricultural Science and Technology Innovation Program of China (Grant No. ASTIP-IAS13) to Mingxing Chu.

There is supplementary material associated with this article. Access the material online at: https://dx.doi.org/10.17582/journal.pjz/20190121080154

The authors have declared no conflict of interest.