ГЕНЕТИКА, 2012, том 48, № 10, с. 1185-1189
ГЕНЕТИКА ЖИВОТНЫХ =
УДК 575.17
HAPLOTYPE VARIATION OF ESTROGEN RECEPTOR-a (ER-a) GENE EXON 4 IN TURKISH SHEEP BREEDS
© 2012 O. Ozmen1, I. Seker2, B. C. Kul3, O. Ertugrul3
1 Firat University, Faculty of Veterinary, Medicine Department of Genetic, Elazig, Turkey e-mail: oozmen@firat.edu.tr; ozgeozmen@gmail.com 2Firat University, Faculty of Veterinary, Medicine Department of Animal Breeding, Elazig, Turkey 3 Ankara University, Faculty of Veterinary, Medicine Department of Genetic, Ankara, Turkey
Received January 30, 2012
Estrogen receptor a (ERa) gene has previously been found to related with sexual development and reproduction. In this study, on the basis of the sequences of human, cattle and caprine estrogen receptor a (ERa) genes, available in the GenBank database, sets of PCR primers were designed and used to amplify the ovine ERa gene exon 4 region. We identified six single nucleotide polymorphisms (SNP) in the ERa exon 4. Some variations determined for exon 4 g.43A > G, p.T43A; g.49C > T, p.L49F; g.178A > T, p.T178S led to changes in the amino acids, but no amino acid changes were determined in g.18G > C, g.27C > T, g.96G > A. These fragments were deposited in the GenBank database under accession number: JF262030—JF262035. It was noted in particular that White Karaman and Awassi breeds were similar to each other, whereas the Chios breed had a different variation.
Estrogens play a crucial role in sexual development and reproduction in various tissues. In mammals, estrogens are responsible for development of female secondary sex characteristics and function as reproductive hormones in both males and females. However, these are not the only roles of estrogens [1] but it is most important role is the development and functioning of the female reproductive system. Due to numerous functions that estrogens play in the animal organisms, estrogen receptors and their genes are considered candidates for the markers of production and functional traits in farm animals [2].
The effects of estrogens are mediated by spesific receptor subtypes, which is ERa and ERp, known as ESR1 and ESR2 respectively [3]. In the ovine genome, ERa and ERp are encoded by a separate genes, localized on chromosome 8 and 7, respectively. It has been suggested that ERa and ERp may play different roles in estrogen-responsive tissues. For example, the mRNA for ERa is highly expressed in the rat brain areas responsible for reproduction, whereas ERa mRNA is present mainly in regions of non-reproductive function [4, 5]. Of the estrogen receptor (ER) genes, expression levels of ERa are much higher than those of ERp in the uterus. In addition, ERa knock-out mice demostrate complete uterine dysfunction and resistance to estrogen while ERp knock-out mice do not [6, 7]. Interestingly, only ERa is regulated by estradiol in the utrerus [8]. That is the reason our studies focus on ER-a.
A few candidate genes for litter size have been already identified according to their roles in the physiology of reproduction and their position within chromo-
somal regions containing quantitative trait loci (QTL) for reproductive traits. To use these markers in MAS, it is necessary to verify whether these markers are associated with the traits in the specific population under selection. However, as a preliminary step, it is important to evaluate whether the markers are polymorphic in the investigated populations [9, 10]. The Chios sheep breed has a high milk yield and an outstanding prolificacy. The average litter size is 2.3. The Awassi is principally a milk breed, but meat production from this breed is also important and the twinning rate is 10—20%. The White Karaman is a breed indigenous to Turkey with a twinning rate of 20—30% [11]. It is well known that Chios sheep are highly prolific in comparison with many other breeds. The purpose of this study is to reveal the genetic polymorphism of ERa gene in high prolificacy Chios and low prolificacy White Karaman and Awassi sheep breeds.
MATERIALS AND METHODS
Jugular blood samples (2 ml per ewe) were collected from 48 Chios, 41 White Karaman, 48 Awassi sheeps using EDTA as an anticoagulant. Those ewes were chosen at random. Genomic DNA was extracted from the whole blood using the Phenol-chloroform method and then it was dissolved in 10 mM Tris-HCl (pH 8.0) buffer and kept at -20°C.
PCR amplification and sequence analysis. For this study, according to the human (AF123496), cattle (AY538775) and caprine (GQ358923) sequence homology of exon 4 of ERa gene, following PCR primers were designed: F: 5'-GAGGGAGAATGTTGAAGC-3'
Table 1. Polymorphic sites (excluding the ambigous ones) and amino acid changes at the ERa gene exon 4 for the Turkish sheep breeds
Haplotypes (GeneBank no.) Haplotype frequency Position
WK (n = 41) AW (n = 48) CH (n = 48) 18 27 43 49 96 178
Amino T L T
acid 4 4 1
change 3 9 7
A F 8
S
H1 (JF262030) 41 29 9 G C A C G A
H2 (JF262031) 0 16 5 A
H3 (JF262032) 0 1 0 T A
H4 (JF262033) 0 2 4 T
H5 (JF262034) 0 0 1 T
H6 (JF262035) 0 0 29 C G T
Notes: Nucleotides are numbered from 1 to 258 (WK, AW, CH represent White Karaman, Awassi and Chios respectively).
and R: 5'-GCCCAGTTGATCATGTGTA- 3'. For primer design, Primer3 software [26] was used. PCR reaction was carried out in 50 ^l of total volume, containing 10x PCR Buffer (50 mM/l KCl, 10 mM/l Tris-HCl (pH 8.0), 0.1% Triton X-100), 1.5 mM MgCl2, 0.2 mM of each dNTP, 10 pM/l of each primer, 50 ng ovine genomic DNA and 1U Taq DNA polymerase (Eppendorf AG, Hamburg, Germany). PCR conditions were as follows: denaturation at 94°C for 5 min, followed by 34 cycles of denaturation at 94°C for 1 min, annealing at 62°C for 1 min, extention at 72°C for 1 min and final extention at 72°C for 10 min, on Mastercycler® (EppendorfAG, Hamburg, Germany). The PCR products were separated by electrophoresis on 2% agarose gels (Promega, Madison, WI, USA) in paralel with a 100 bp DNA marker. Then PCR products were purified with the PCR purification kit and sequenced in an ABI 310 Prism Sequencer.
Statistical analysis. Sequences were analysed using the BIOEDIT software (http://www.mbio.ncsu.edu./ BioEdit/bioedit.html) for sequence alignment. NETWORK 4.5.1.6. (http://www.fluxus-engineering.com) was used to build the network of exon 4 haplotype groups using the median joining algorithm. MEGA version 4.1 [12] was used for the phylogenetic sequence analysis of haplotypes by the Neighbour-Joining method based on Kimura-2P model and the reliability of the inferred tree was assessed by bootstrap (1000 replicates) (data not shown).
The DnaSP software 5 [13] was used to calculate haplotype diversity (Hd) and nucleotide diversity (n); Watterson's theta estimator for the studied species separately using a haplotype sequence was obtained. Pi (n) is based on the average number of nucleotide differences between the sequence, and theta is based
on the total number of segregating sites in the sequence [14].
To estimate the effect of selection, we calculated Tajima's D [15], Fu and Li's D* and F* test [16] and Fu's Fs test [17] for each group separately. Tajima's D test compares the difference between the number of segregating sites and average number of pairwise [15]. Under neutrality Tajima's D value is assumed to be zero; under positive selection there is an excess of rare polymorphisms and Tajima's D value is negative. Negative D values can also be due to population expansion. If there is balancing selection, intermediate frequency genetic variants are kept and Tajima's D value is positive [14]. The statistical analysis package DnaSP 5 [13] was used for the neutrality tests.
The impact of amino acid variants on protein structure via analysis of multiple sequence alignments was done with Sorting Intolerant From Tolerant (SIFT), which uses sequence homology to predict whether an amino acid substitution will affect protein function and hence, potentially alter the phenotype. It gives a normalized probability score value that the amino acid change is tolerated. If the score value is less than 0.05, the amino acid change is predicted to be deleterious. The median conservation value for the diversity of the sequence in the alignment is measured as well, and the default value is 3.0. Higher conservation values can lead to higher false positive error [18].
RESULTS
In this study, the sequence analysis of the exon 4 of the ERa gene revealed interesting variations in the studied populations and subpopulations. For exon 4, six different haplotypes were obtained (Table 1). The most common haplotype was OVIS_ER6 for the
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Chios breed and OVIS_ER1 for White Karaman and Awassi. OVIS_ER2-6 haplotypes were not detected in the White Karaman breeds.
Six variations were determined in exon 4, of which three were non-synonymous mutations: g.43A > G, p.T43A; g.49C > T, p.L49F; g.178A > T, p.T178S; while three variations (g.18G > C, g.27C > T, g.96G > > A) were determined as synonymous mutations (Table 1). The ERa haplotype sequences from these sheep breeds have been deposited in the GeneBank database (http://www.ncbi.nlm.nih.gov) under the accession numbers JF262030-JF262035.
Based on the observed mismatch distributions and the constructed radiation tree (data not shown), two main groups (group A and B) were determined (Table 2). Neutrality tests were applied separately to these haplotype groups.
Neutrality tests at ovine ERa gene, Tajima's D value, Fu and Li's D* and F* values are shown in Table 2. Tajima's D value, Fu and Li's D* and F* values for exon 4 both group are negative. Only the Fu and Li's F* and D* values for group B deviate statistically significantly from zero. Haplotype diversity (Hd), nucleotide diversity (n) and Watterson's theta estimator were calculated separately for the studied species using the haplotype seque
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