Single-nucleotide polymorphisms in FABP4 gene associated with growth traits in Egyptian sheep.

AIM: The present study was performed to assess the association of single-nucleotide polymorphisms (SNPs) in the fatty acid-binding protein 4 (FABP4) gene with birth weight (BW), final weight (FW), and average daily gain (ADG) in three Egyptian sheep breeds.
MATERIALS AND METHODS: Genomic DNA was extracted from the blood samples of 50 male and female individuals representing Ossimi, Rahmani, and Barki sheep breeds. A 407 bp nucleotide (nt) segment from the first intron of FABP4 was amplified by polymerase chain reaction, sequenced, and analyzed in the different samples.
RESULTS: Sequence analysis of the determined segment (407 bp) revealed four SNPs (all transition types) at nt position 372 (CP011894.1:g.57605471) A>G, nt position 211 (CP011894.1:g.57605632) A>G, nt position 143 (CP011894.1:g.57605700) T>C, and nt position 111 (CP011894.1:g.57605732) T>C. The allelic and genotypic frequencies for the identified SNPs in the sheep breeds were calculated. At nt positions 372 and 211, two alleles were identified (A and G). Only two genotypes were present at nt position 372 (AA and AG), while three genotypes were present at nt position 211 (AA, AG, and GG). Two alleles (T and C) and three identified genotypes (TT, TC, and CC) were detected at nt positions 143 and 111. Analysis of the results revealed that AA genotype at nt position 372 is associated with higher estimates for BW, FW, and ADG when compared to all the other genotypes. Very high correlation coefficients were found between the genotypes 143-TT and 111-TT and also between 143-TC and 111-TC. The genotypes 372-AG, 211-GG, 211-AA, 143-TT, 143-CC, 111-TT, 111-TC, and 111-CC were associated with negative effects on BW, FW, and ADG.
CONCLUSION: The detection of four SNPs in a partial sequence of the Egyptian ovine FABP4 gene intron 1 reflected that this gene harbors substantial diversity. In addition, a novel SNP at nt position 372 (CP011894.1:g.57605471) A>G was associated with higher estimates for BW, FW, and ADG. Copyright: © Shafey, et al.

Introduction

Since domestication in the 15th century, sheep is one of the most commonly recognized farm animal species worldwide. Sheep is an important agricultural animal and food source, especially in developing countries. Carcass composition is strongly related to sheep fat content. However, fat deposition in animals may be beneficial or undesirable depending on its quantity, location, and chemical composition. With the exception of intramuscular fat (IMF), excessive fat deposition has been recognized as detrimental to carcass quality and a causative agent of health risk to humans [1]. The IMF content, which is known as the total lipids extracted from muscle, has become one of the most important indicators of meat quality [2,3]. IMF is also associated with the expression of lipogenic generation [4]. Fatty acid-binding proteins (FABPs) are a family of homologous cytoplasmic low-molecular-mass proteins that are expressed in a highly tissue-specific manner and play an integral role in the balance between lipid and carbohydrate metabolism. Moreover, FABPs mediate fatty acid (FA) and/or hydrophobic ligand uptake, transport, and targeting within respective tissues. The mechanisms underlying these actions can give rise to both passive diffusional uptake and protein-mediated transmembrane transport of FAs. Nine types of FABPs have been identified in the mammalian FABP family [5,6]. Different types of FABPs are expressed in most mammalian tissues [7,8]. These hydrophobic ligand-binding proteins are encoded by a supergene family (FABP genes) [3,9].

FABP4 is a member of the FA-binding protein family. It is also known as adipocyte FA-binding protein (A-FABP) because it has been implicated in lipid metabolism in adipose tissue through regulating gene expression of enzymes involved in lipid ­metabolism and/or by transporting FAs [3,8]. This protein has a high affinity to FAs, participates in the transport of long-chain FAs, and is more highly expressed in mature adipocytes than other cells [10]. In bovine species, the FABP4 gene polymorphism has been associated with marbling, IMF, and meat quality [11,12]. However, FABP4 polymorphism Msp A1I previously identified in three Nellore lines of Zebu cattle was not significantly associated with evaluated growth and carcass traits [13]. The FABP4 protein content was also used as a marker of IMF content accretion in the longissimus thoracis muscle of sheep [14]. The nucleotide polymorphisms in FABP4 exons 1 and 2 and introns 1 and 2 affected growth and meat production traits in New Zealand Romney sheep [15]. Xu et al. [13] also reported a single-nucleotide polymorphism (SNP) in FABP4 intron 1 that affects meat tenderness and IMF content in Chinese local sheep breeds. However, information regarding genes affecting growth performance of Egyptian sheep are still scarce. The three major Egyptian sheep breeds are Barki, Ossimi, and Rahmani, representing about 65% of the total sheep population. They are well adapted to local environmental conditions, fat-tailed, small to medium in size, and have a twinning rate from 1.05 to 1.30. Barki sheep are reported to have the lowest birth and mature body weights, while the Rahmani breed has the highest body weights [16].

In the present study, we used the three main Egyptian sheep breeds (Ossimi, Rahmani, and Barki) to investigate the polymorphisms in intron 1 of the ovine FABP4 gene and to assess the potential association of SNPs detected in this gene with growth traits.

Materials and Methods

Ethical approval and informed consent

All necessary ethical and administrative approvals for performing this study have been obtained from the formal representatives of Faculty of Agriculture, Cairo University (CU). The collection of the blood samples was carried out by the veterinarian staff of the Agricultural Experiment Station of CU.

Study period

The samples were collected from September 2018 to November 2018. DNA isolation, PCR and Sequence analysis were carried out from December 2018 to August 2019.

Animals and samples collection

The current study was performed on 50 male and female individuals from Ossimi (14), Rahmani (18), and Barki (18) sheep breeds, where rams and ewes were assumed to be unrelated. The three breeds were reared as an experimental flock managed by the Agricultural Experiment Station, Faculty of Agriculture, Cairo University, Giza, Egypt. According to average daily gain (ADG), animals were classified into two distinct growth groups (low and high). Phenotypic records analyzed in the present study involved three traits: Birth weight (BW), FW at slaughter (FW), and ADG. Blood samples (5 ml) were taken from the jugular vein into vacuum tubes containing 0.25% ethylenediaminetetraacetic acid as an anticoagulant. Samples were shipped on dry ice for DNA extraction at the Laboratory of Cell Biology, National Research Centre, Giza, Egypt.

DNA extraction

Genomic DNA was extracted from whole blood as described by Miller et al. [17]. DNA concentration and purity were determined using a UV spectrophotometer, Thermo Scientific, at optical densities of 260 and 280 nm.

Polymerase chain reaction (PCR)

A DNA fragment covering 407 bp from the FABP4 first intron was selected for PCR amplification according to the previous study of Xu et al. [14]. A 20 ml PCR cocktail consisted of 20 pmol forward (5’-TGGTTATTCAAGAAGCAA-3’) and reverse (5’- ATCTAAGAAAGAGCAGGC-3’) primers (HVD Life Sciences), 0.2 mM dNTPs, and 1.25 U of Taq DNA polymerase (Fermentas). The cocktail was aliquoted into PCR tubes with 100 ng of ovine DNA. The PCR cycle conditions were initial denaturation for 5 min at 94°C, followed by 35 cycles of denaturation at 94°C, annealing at 60°C, and extension at 72°C, each step for 1 min and the final extension for 5 min at 72°C. The amplification was verified by electrophoresis on 2% agarose (GIBCO, BRL, England) gel (w/v) in 1× TBE buffer. The size of PCR product was measured using a 100 bp ladder (Gene Ruler, Fermentas). The gel was stained with ethidium bromide and visualized using a UV transilluminator.

Sequence analysis

The PCR products were purified and sequenced (Macrogen Incorporation, Seoul, South Korea). Sequence analysis and alignment to reveal nucleotide substitutions were carried out using nucleotide blast at CLUSTALW (http://www.genome.jp/tools/clustalw/) and NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) programs [18,19]. The SNP positions on sheep genomic DNA were identified based on the ovine chromosome 9 sequence in GenBank (accession number CP011894.1).

Statistical analysis

Population deviation from Hardy–Weinberg equilibrium was verified by a Chi-square test. Genotypic and allelic frequencies for each polymorphic locus were calculated using Microsoft Excel data analysis add-on software XLSTAT [20]. This software was also applied to determine the significance of the fixed effects (breed, sex, and growth class) on the studied traits (BW, FW, and ADG). A priori, genotypes were considered to be independent in all analyses performed. Association analysis of SNPs in the ovine FABP4 gene was performed separately for BW, FW, and ADG traits in the three sheep breeds studied. The model used to analyze the data was as follows:

Yijk = μ +Gi + Bj + Sk + ɛijkl

Where,

Yijk = the trait measured on each of the ijklth animal,

μ = the overall population mean,

Gi = the fixed effect associated with ith genotype,

Bj = the fixed effect of jth breed (j = 1, 2, 3), where 1 = Barki, 2 = Ossimi, and 3 = Rahmani,

Sk = the fixed effect of kth sex (k = 1, 2), where 1 = female and 2 = male, and

ɛijkl = random residual error.

Preliminary analysis of data showed that the interaction effects between two factors were non-significant. Therefore, interaction was excluded from the final statistical analysis. Effects associated with year of birth and season of birth were not included in the linear model, as the initial statistical analysis revealed that these effects did not significantly influence BW, FW, and ADG.

Taking into consideration the small sample size, a partial least squares regression procedure was used to create a set of h components with h