Mitochondrial diversity and phylogenetic structure of native Iranian goat population compared with 4 important livestock species

Introduction

The goat is the earliest ruminant to have been domesticated (Mason, 1984). The domestic goat Capra hircus is one of the most important livestock species in the world for providing good animal production even under harsh environmental conditions. Recently, molecular studies of goats based on mitochondrial DNA (mtDNA) sequences have been carried out to investigate the origin and phylogeny of goats (Luikart et al. 2001; Mannen et al. 2001; Mannen 2004; Naderi et al. 2007) [8,9,10] . Mitochondrial DNA is very useful for its multiple presences in cells. The most of animal mtDNA is coding 37 genes (Avise, 1994) [3] . One of them is gene for NADH dehydrogenase (ubiquinone) ND6 gene is a component of respiratory chain complex I (Howell, 1989, ESposti et al, 1993) [6,5] . Length of ND6 gene is 639 bp and has some stable sequences which were used for suggestion of universal primers and some variable sequences used for animal identification. The Mahabadi is the autochthonous goat from Iran and belongs to the same indigenous population that lives throughout west north Iran. It is a long haired and a small-sized goat and reared in extensive mixed farming systems, together with sheep and cows, or semi-intensive systems. The breed produces mainly meat, but it shows a high genetic potential for milk production. National projects for development of the small ruminant sector and biodiversity conservation strategies are currently developed in Iran for the native goat (FAO 2007). Goat milk can be used as food for people with cow milk and cheeses are appreciated by consumers (Boyazoglu et al. 2005) [4] . Furthermore, meat of suck ling kids is a delicacy and prices paid to farmers are constantly higher than that of lamb meat. Goat milk derived products are an important source of protein France and Greece, as these countries have started to exploit the value of their typical products. Indeed, under well-organized management, goat farming is a profitable way of marketing marginal natural resources without endangering the environment.

The study of autochthonous breeds can play an important role in the preservation of natural resources and the rural environment and landscape, in particular the protection of biodiversity. To extend the knowledge of goats reared in the Mediterranean area, we studied a particular gene of mitochondrial DNA (mtDNA), the ND6 gene To date, sequences from many species are known and the complete sequence of goat mitochondrial genome (Accession number: Gen Bank AF533441) was deposited in 2003 (Parma et al. 2003) [11] . Many studies used mtDNA as an important means of population studies. Luikart [2] . These studies confirmed a weak phylogeographic structure in goat species, when compared to cattle. This result has been explained by some authors (Luikart et al. 2001; Amills et al. 2009) [8,2] because goat, owing to its moderate size and ability to adapt to different environments, well-suited to the intercontinental transportation in ancient times. Based on previous literatures, in this study, molecular analysis of Mahabadi goat population based on ND6 gene of mitochondrial DNA were investigated to develop molecular markers for breed identification.

Materials and Methods

We collected blood samples of native goat from Mahabadi goat. Blood samples (5ml in EDTA Containing tubes) randomly collected from 20 animals and stored at -20 °C until used at biotechnology laboratory. Amplification and sequencing the complete ND6 gene was amplified by using forward primer ND6-F: 5'-CgATACATACACgCAAACggA-3' and reverse primer ND6-R: 5'AgAAggTTgTTTTCAATggTgC-3'. The forward and reverse primers were designed sequences of the mtDNA genome (Gen Bank accession no. V00654). Polymerase chain reaction (PCR) was carried out in a total volume of 25 ul, containing 10 ng of genomic DNA, 2.5 ul of 10ul buffer, 0.2 mM of dNTP, 10 pM of each primer and 1.5 units of Taq polymerase (TaKaRa, Japan). Therminal cycling was performed on a PTC-200 thermocycler (MJ Research Inc.) under the following conditions; 2 minute denaturation at94°C, followed by 35 cycles of 30 s at 94 °C, 30 s at 60 °C, 60 s at 72 °C, and a final 5 min at 72 °C before cooling to 4 °C for 10 min. The amplified products were separated by electrophoresis on 1% Agarose gels, and were visualized under UV illumination after staining with Ethidium Bromide. The PCR products were purified using a QIA quick PCR purification Kit (Qiagen, USA), and were directly sequenced on an ABI 3130xl Genetic Analyzer (PE Applied Biosystems, USA).C. Statistical and phylogenetic analyses. The sequences of the ND6 gene from different breeds were aligned in CLUSTAL W (Thompson et al. 1994). Numbers of nucleotide polymorphic sites(S) and haplotype (h), nucleotide diversity (Pi), haplotype diversity (Hd) and nucleotide divergence (Dxy) were performed in DNA sequence polymorphism Version 5.1 (Librado and Rozas, 2009) [7] . The Neighbor-joining (NJ) tree (Saitou and Nei, 1987) [12] among haplotypes based on the ND6 gene sequences was reconstructed in MEGA 5.05 package (Tamura et al. 2011), with the reliability of the tree topology assessed by 1,000 bootstrap replications (Felsenstein, 1985). The NJ tree among breeds was constructed in MEGA 5.05 package on the basis of divergence distances.

Results and Discussion

Sequence composition and variation of the ND6 gene the full length coding sequences of the ND6 genes in 30 individuals were determined. All sequences spanned 693 bp, started with an ATG translational start codon and ended with an AGA stop codon. Length variation was detected in these sequences (Fig. 2). According to the data in Fig. 3, to assume sequence index in Mahabadi native goat, we used consensus sequence using Bio Edit software in 639 pair bases. As presented in Fig. 3, the Composition procedure of Bio Edit software implied that 270 nucleotides was in group (A), 182 nucleotides in group (C), 50 nucleotides in group(G) and 137 nucleotides in group (T), respectively. Additionally, the G + C ratio was 36.31 and A+T was 63.69 percent. Furthermore, the molecular weight of this sequance was 197801 daltons and the Molecular weight of pairs was 386997daltons. These patterns were very similar to those of a previous report which analyzed goat breeds (Amer, 2014). Based on the alignment of the ND6 gene initial fragment, phylogenetic trees were constructed. Fig 5 . Demonstrates the diagram obtained by use of the method of minimal evolution. Clusterization of the samples in tree corresponded to their species affiliation. Currently, four tree branches can be distinguished. The ND6 gene sequences were not highly polymorphic. Our 30 sequences gave just 2 different haplotypes with 1 variable sites defined. The largest haplotype group consisted of 18 individuals, and other haplotypes included 11 individuals (table1). Mahabadi goat divided tow cluster within population (Fig 1).This result indicates that Mahabadi goat the between group distances were computed using the MEGA 5.0 software (Fig. 5). Iranian native Mahabadi goats are compered of 7 different livestock by drawing phylogenetic that is showed in bellow (Table 4). Distribution of the samples between the groups was made in accordance with the cluster obtained. Apparently, the longest distance separated the horse (Equus caballus) from the others and iranain native Mahabadi goat has shortest genetic distance with sheep (Ovis Aries) This is possible because of mtDNA of specific livestock, geographical distance and distributed.

Acknowledgement

AcknowledgementThe authors acknowledged the three reviewers for constructive comments on the manuscript. We gratefully acknowl-edge all farmers who took part in the present study, giving access to the animals. International Journal of Veterinary Sciences and Animal Husbandry

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