Investigation of Gene Polymorphisms in FGF2 and SΤAΤ5A Genes in the Greek Red Cow Breed ^†

Abstract

In the present study, we investigated the presence of single nucleotide polymorphisms at the Fibroblast Growth Factor 2 (FGF2) and Signal Transducer and Activator of Transcription 5A (STAT5A) genes loci in the Greek Red Cow breed, using RFLP-PCR analyzes. One hundred seventy-four Greek red cows were used for this study. PCR-RFLP analysis revealed that for FGF2, the frequencies of the genotypes were 0.25, 0.55 and 0.20 for the GG, GA and AA genotypes respectively and the alleles G and A sequences were 0.52 and 0.48, respectively. Genotyping of the GG, GC and CC genotypes of the STAT5A genes revealed that these frequencies were 0.22, 0.48 and CC in the investigated population, while the G and C allelic frequencies was 0.46 and 0.54, respectively. These genotyping analyses presented in this study revealed the presence of all three genotypes for the STAT5A and FGF2 genes loci, indicating that the investigated single nucleotide polymorphisms can be used in the future, in genetic selection improvements schemes, in order to improve the reproductive performance of the Greek Red Cow breed.

1. Introduction

There are many genes that can cause an impact in the cattle’s productivity. It is now well established that genetic technologies and genotyping of single nucleotide polymorphisms (SNPs) are very important tools regarding the improvement of productive and reproductive traits in the cattle industry [1,2]. Many studies have been focused on the detection of SNPs in traits of big economic importance in various bovine populations, such as milk and reproduction traits.

In this study we investigated for the first time the existence of single nucleotide polymorphisms in the Fibroblast Growth Factor 2 (FGF2) and Signal Transducer and Activator of Transcription 5A (STAT5A) genes loci in the Greek red cow breed. The Greek Red Cow breed is characterized by the adaptation and resilience in different environments, while these cows can utilize their feed from the less fertile soils. They are used more for their meat production than their milk production, while their milk quantity is not enough, but only for their calf’s breast feeding. It is very important to maintain the Greek red cow breed, because both for biodiversity and for tradition, as it is an integral part of the Greek livestock and cultural heritage.

The FGF2 gene is located in the bovine genome on chromosome 6 and has an important role in tissue development, homeostasis, in the regulation of the epithelial expansion in the mammary gland and plays and important role in the regulation of interferon-t (IFNT), a very important gene regarding cattle milk and reproductive traits [3]. The A/G substitution of the SNP 11646 in intron 1 at the FGF2 gene was investigated in this study.

STAT5A gene, located in the bovine genome on chromosome 19, and in dairy cattle participates in milk production [4]. It also affects the success of fertilization and fetal survival. In this study we also examined the presence of the SNP 12195 (C/G) of STAT5A gene in the investigated population.

The goal of this research was the determination and the establishment of the genes allelic, as well as the genotyping frequencies of the FGF2 and STAT5A gene loci, in the Greek Red cows. To our knowledge no similar studies regarding these genes have been reported in this breed, raised in Greece.

2. Materials and Methods

For the extraction of genomic DNA from blood from one hundred seventy-four Greek red cows was used the NucleoSpin Blood kit (Macherey–Nagel, Düren, Germany) according to manufacturer’s instructions. Prior the usage of the extracted DNA samples in the genotyping experiments, the quality and quantity of each sample was investigated using DNA electrophoresis on 1.5% agarose gels.

Genotyping analyses and specifically polymerase chain reactions and DNA digestions experiments were performed, using the conditions, enzymes and temperatures as previously detailed [5], and using the primer pairs as previously described [6,7]. Using these analyses the 207 base pair fragment, as well as the 820 base pair fragment of the FGF2 and STAT5A genes loci respectively were amplified.

In order to evaluate any potential genomic contamination of the extracted DNA, in every polymerase chain reaction that was performed, a negative control sample, with the completely absence of DNA samples was included in each reaction.

Following the successful determination of the genotypes in each DNA sample, for both the investigated single nucleotide polymorphisms, and using gene counting we also determined the allelic frequencies at each gene locus and we examined using chi-squared tests deviations from Hardy–Weinberg equilibrium.

3. Results and Discussion

Using the PCR analyzes described in the Section 2, amplification of the investigated 207 and 820 base pair fragments of the FGF2 and STAT5A genes loci respectively were successful, as shown in Figure 1 and Figure 2, which are representative PCR amplifications of 12 DNA Greek Red Cow breed DNA samples.

Following the successful PCR amplification of the FGF2 and STAT5A genes loci, PCR-restriction fragment length polymorphism analyses were performed in each amplified PCR DNA product, as described in materials and methods.

Using these PCR-RFLP analyses, the genotype of every investigated DNA sample, for both the two investigated gene loci were determined, as illustrated in Figure 3 and Figure 4, which are representatives of the different genotypes of both single nucleotide polymorphisms.

The frequency of genotypes, as well as the frequency of alleles of the single nucleotide polymorphisms, in the Fibroblast Growth Factor 2 (FGF2) and Signal Transducer and Activator of Transcription 5A (STAT5A) genes loci, which were detected in the present study are illustrated in the

Table 1. Frequency of genotypes and frequency of alleles for the FGF2 and STAT5A SNPs.

Gene Locus	Allele		Frequency of Genotypes (%)			Frequency of Alleles (%)
	0	+	00	0+	++	0	+
FGF2	G	A	0.25	0.55	0.20	0.52	0.48
STAT5A	G	C	0.22	0.48	0.30	0.46	0.54

. In the investigated population, consisting of one hundred seventy-four cows from the Greek red breed, both FGF2 gene locus and STAT5A gene loci were found to be in Hardy-Weinberg equilibrium

The molecular analyzes, which were performed for the first time to our knowledge in this study for the Greek Red Cow breed, lead in the determination of the SNP 11646 and the SNP 12195 genotypes for the FGF2 and STAT5A genes respectively.

Previous studies have reported that these gene mutations in these two genes can have important implications in the productive and reproductive performance, including traits such as conception, superovulatory responses, fertility and embryonic development in cattle breeds [8,9]. It was therefore of great importance the investigation and determination of these genotypes in the Greek Red Cow breed, in order to use this information in studies associated these single nucleotide polymorphisms and reproductive traits in this breed. As all three genotypes of these two genes were detected in the investigated population, similar to what has been previously published [8,9], further work is under way, in order to determine the association of these SNPs with various reproductive traits. These data will provide significant information for the use of the desired genotypes as functional genetic markers, for the genetic improvement of many reproductive parameters and in the selection of the most superior animals of this breed.