Genetic Diversity and Phylogenetic Analysis of the mtDNA D-loop Region in Tibetan Sheep

Seventeen haplotypes were detected from the complete mitochondrial DNA control region sequences analyzed from eighty individuals of two Tibetan domestic sheep breeds. The nucleotide composition of all the sequences was 33.0% A, 29.7%T, 22.9%C and 14.4%G; G+C was 37.3%. The length of the sequences ranged from 1,107 bp to 1,259 bp. The difference between them was primarily due to 3-5 copy numbers of a 75 bp tandem repeat sequence. The NJ phylogenetic tree (the number of replications of bootstrap test is 1,000) presented three major domestic sheep lineages, which suggested that modern Tibetan sheep breeds are derived from three maternal sources. (


INTRODUCTION
Mitochondrial DNA is the genetic material that exists outside the nucleus of eukaryotic cells.It has a doublelinked annular structure which is covalent and close.Including coding and non-coding regions, its length ranges from 15 kb to 20 kb in different species.The non-coding region is the control region.The rate of mtDNA evolution is about 5 to 10 times faster than nuclear DNA, and its genes do not usually recombine (Upholt et al., 1977).Animal mtDNA is deemed to strictly follow maternal inheritance and is highly variable within a species, so mtDNA is an important material for phylogenetic inference and for analyzing genetic diversity (Wolf et al., 1999).
The climate and landform of Tibet are different from other areas of China.Traffic from other parts of China is blocked, so the region's livestock are seldom influenced by external breeds.In this study we investigated two sheep breeds indigenous to Tibet which have not been studied or crossbred yet.By using the entire sequence information of the mtDNA D-loop, we analyzed the genetic diversity of two breeds and inferred their phylogenetic status.Therefore, the results are useful for the conservation and utilization of Chinese sheep genetic resources.

Sample collection and DNA extraction
Ear samples from eighty individuals (40 from each breed) of two Tibetan sheep breeds were collected from local Tibetan sheep production centers according to the simple random sampling method.The breeds were chosen from the Linzhou and Gangba areas of Tibet.DNA was extracted from these specimens using phenol/chloroform as described by Sambrock et al. (2001).

Amplification, purification, cloning and DNA sequencing
The sequences of the primers for the mtDNA control region between tRNA Phe and tRNA Pro are forward: 5'CTCACCATCAACCCCCAAAGC3'; reverse: 5' TCATCTAGGCATTTTCAGTG 3' (Hiendleder et al., 2002).The PCR amplification reaction system consisted of genomic DNA 50 ng, dNTPs 200 µM, primers 10 pmol, MgCl 2 250 µM, Taq DNA polymerase 1 U.The reaction profiles included an initial denaturation at 95°C for 5 min, followed by 30 cycles, each consisting of 30 sec denaturation at 94°C, 45 sec primer annealing at 55°C, 60 sec extension at 72°C, and then a final 8 min extension at 72°C.The PCR products were electrophoresed through 2.0% (wt/vol) agarose gel which was stained with ethidium bromide solution.
The amplified products were purified with a DNA purification kit according to the manufacturer's instructions (TW-Biotech.CO., LTD.).The purified fragments were cloned into pGM-T easy vector and subsequently transformed into E. coli Top10.After 16 to 20 h, the single clone was cultured, and the recombinant plasmid was obtained.The recombinant DNA was extracted and then sequenced using an ABI model 3,730 automated sequencer.The unique polymorphisms in the results were confirmed by resequencing of independent clones.

Data analysis
All the sequences were multiple aligned using the BioEdit 7.0 software.Identical sequences were considered as the same haplotype.The polymorphism of the haplotypes was analyzed with the DnaSP 4.1 software.The neighborjoining (NJ) phylogenetic tree was constructed using the MEGA 2.0 program (Kumar et al., 2001) with a Kimura 2parameter model and the bootstrap value was 1,000.At the same time, we selected the complete sequences of B. taurus and B.indicus from GenBank as controls.

Sequencing analysis of the complete control region
With the sequence of AF010406 (GenBank accession number) as a criterion, all the sequences were aligned and seventeen haplotypes were obtained from the sequenced individuals (GenBank accession numbers: DQ903190-DQ903200 and DQ903202-DQ903207).The haplotype diversity was 1.0.The average number of nucleotide differences was 33.81.Except for minor deletions and insertions, 114 variable sites were obtained in the sequences, including 109 transitions and 5 transversions.The nucleotide composition of all the haplotypes was 33.0%A, 29.7%T, 22.9%C and 14.4%G.The nucleotide diversity was 3.06%.The mean pair-wise genetic distance of all the haplotypes calculated using the Kimura 2-parameter model was 0.034.

Haplotype analysis in Chinese Tibetan sheep
The length of the sequences varied considerably between 1,107 bp and 1,259 bp, but most of the sequences were 1,181 bp.The length variations were caused by different copy numbers of a 75 bp tandem repeat except for minor insertions and deletions.The copy numbers of the tandem repeat were present randomly in the haplotypes, so they could not be seen as being characteristic of the haplotypes.The variations in the three haplotypes mainly occurred in the region of high variability (from 640 bp to 1,140 bp), and haplotype C had greater variation than haplotypes A and B. Fu's test showed that the populations had not been expanded historically (0.10>p>0.05).

Phylogenetic analysis of Tibetan sheep
The neighbor-joining, UPGMA and minimum evolution (ME) dendrograms were generated based on the sequences obtained in this study together with the sequences from GenBank using the MEGA 2.0 program (Bootstrap value is 1,000).The results were identical.We only presented the NJ phylogenetic tree (Figure 1).Three distinct lineages were observed.B.taurus and B.indicus were first gathered as an outgroup.O. musimon and European sheep were in the same lineage showing that the relationship between them was close.In addition, the number of individuals in lineage A was more than in lineages B and C. Lineage C was only present in Tibetan sheep breeds, but not in European sheep and O. musimon.

DISCUSSION
Mitochondrial DNA has been very widely used by geneticists to analyze the phylogenetic relationships at inter-or intra-species level in cattle, swine, goats and water buffalo.It has also been used to investigate the genetic variation of species (Watanbe et al., 1989;Tanaka et al., 1996;Giuffra et al., 2000;Sultana et al., 2003;Sasazaki et al., 2006).The composition of the nucleotides in this study was similar to that of Hiendleder's (1998a).The shortest The use of the mtDNA PCR-RFLP and sequence analysis methods has led to the prevailing opinion that domestic sheep breeds are derived from two maternal resources (Wood and Phua, 1996;Hiendleder et al., 1991Hiendleder et al., , 1998bHiendleder et al., , 1999Hiendleder et al., , 2002)).The limit of their study is that the genetic material was collected only from European, African, New Zealand and central Asian, but not Chinese sheep breeds.China has numerous domestic sheep breeds, but until now they have not been studied comprehensively for genetic diversity or phylogenetic analysis at the molecular level.Li (1993) pointed out that Mongolian and Tibetan sheep were the ancestors of Chinese sheep, and that they were distinct in appearance.Linzhou and Gangba sheep are the oldest breeds in the remote areas of Tibet.They are barely influenced by external breeds, so they could represent the genetic background of Tibetan sheep.They have not been studied since the breeds came into being except at the morphological level.In this study, we are the first to investigate their genetic diversity at the molecular level.The results are useful for the further utilization of Tibetan sheep resources.Three lineages could be identified among the haplotypes in this study, which indicated that Tibetan sheep breeds were derived from three maternal sources.This finding is not consistent with previous research.Our study only involved a molecular investigation.Additional archaeological and geographical evidences are required to reach a definite conclusion.The results should be useful to help determine the origins of modern domestic sheep breeds.

Figure 1 .
Figure 1.The NJ phylogenetic tree of all the mtDNA haplotypes of Tibetan sheep and a goat as an outgroup.LIN stands for Linzhou sheep, and GB for Gangba sheep.The numbers following LIN or GB are the individual number of the two Tibetan breeds.Others numbers are the accession numbers from GenBank for European sheep and cattle.A, B and C stand for lineages A, B and C respectively.D stands for an outgroup.