**1. Introduction**

In China, Tibetan sheep (*Ovis aries*) play an important role in agriculture, economy, culture and religion in Qinghai-Tibetan Plateau areas, and provide meat, wool, and pelts for local populations [1]. Due to the rich Tibetan sheep genetic resources, there are approximately 17

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

indigenous sheep populations in the Qinghai-Tibetan Plateau area. All indigenous Tibetan sheep adapted to the local environment [2]. Moreover, they are considered as critical genetic resources, so they are one of the most important components in agro-animal husbandry societies. However, in reality, most indigenous Tibetan sheep suffer a serious situation because of the low numbers of each population, which decline steadily since 30 years [3]. Qinghai-Tibetan Plateau areas have special climate and landforms in geography, which provide a different livelihood for Tibetan nomads because of high altitude and cold mountains [4]. Besides, Tibetan sheep bind up with their herders in culture, religion, and social life. The fossil remains indicate that the domestic Tibetan sheep date their wild ancestors from the Pleistocene period [5]. Archeological evidences indicate that domestication of the yak is likely to have been performed approximately 5000 YBP (years before present) by the ancient Qiang people in Northern Tibet [6]. According to the temporal scale, indigenous animals are considered as an ideal model for cold and hypoxia environment adaption studies, on account of their adaptions for high-altitude hypoxia. In addition, Tibetan sheep are isolated from other local sheep in China, due to the unavailable traffic to other parts of China or external countries (Nepal, India, Pakistan, etc.). The severe changes of the ecological environment, value of Tibetan sheep in illegal commerce, and deficiency of animal conservation may result in extinction [7]. So far, the genetic diversity, phylogenetic relationship, and maternal origin of the Qinghai-Tibetan Plateau populations remain uncertain and controversial.

**2. Materials and methods**

We selected 15 Chinese local Tibetan sheep populations for investigation in this study. For analysis, 10 mL blood samples were collected from the jugular vein of each animal. From the 10 mL samples, 2 mL samples were quickly frozen in liquid nitrogen and stored at −80°C for genomic DNA extraction, as described previously [27]. The total DNA was extracted from the blood using the saturated salt method [28], quantified spectrophotometrically, and adjusted to 50 ng/ μL. Blood samples were collected from 636 sheep from 15 populations, living in the Qinghai-Tibetan Plateau areas in China. The genetic characteristics for these Tibetan sheep populations were analyzed in order to ascertain the phylogenetic evolution and phylogeography for the populations. The investigated populations included the following numbers and corresponding population types: 39 Guide Black Fur sheep (GD), 44 Qilian White Tibetan sheep (QL), 64 Tianjun White Tibetan sheep (TJ), 44 Qinghai Oula sheep (QH), 67 Minxian Black Fur sheep (MX), 58 Ganjia Tibetan sheep (GJ), 71 Qiaoke Tibetan sheep (QK), 52 Gannan Oula sheep (GN), 10 Langkazi Tibetan sheep (LKZ), 46 Jiangzi Tibetan sheep (JZ), 85 Gangba Tibetan sheep (GB), 34 Huoba Tibetan sheep (HB), 8 Duoma Tibetan sheep (DM), 5 Awang Tibetan sheep (AW), and 9 Linzhou Tibetan sheep (LZ) raised in China. The sampling information (population code, sample number, altitude, longitude and latitude, accession number, sampling location, and geographical

Phylogenetic Evolution and Phylogeography of Tibetan Sheep Based on mtDNA D-Loop Sequences

http://dx.doi.org/10.5772/intechopen.76583

137

location) for the 15 indigenous Tibetan sheep populations is shown by Liu et al. [29].

*ammon* mtDNA D-loops for the six individuals in GenBank [29, 30].

merase (5 μL/U) (TaKaRa, China), and 16.8 μL ddH<sup>2</sup>

To achieve good coverage of the tested populations, a dataset of six referenced breeds was completed using the six submitted sequences containing the *Ovis aries*, *Ovis vignei*, and *Ovis* 

Primers flanking sequences of the complete mtDNA D-loop was designed by an available genome sequence using the Primer Premier 5.0 software [31] and synthesized by BGI Shenzhen Technology Co., Ltd. (Shenzhen, China). The nucleotide sequence of reverse primer was 5'-GAACAACCAACCTCCCTAAG-3′, and the nucleotide sequence of forward primer was 5'-GGCTGGGACCAAACCTAT-3′. Polymerase chain reaction system (PCRs) took place in a 30 μL reaction system containing 2 μL genomic DNA (10 ng/μL) template, 2 μL dNTP (2.5 mM), 3 μL (3 pM) each primer, 3 μL 10× Ex Taq reaction buffer, 0.2 μL Taq DNA poly-

were as follows: initial denaturation for 5 min at 94°C, 36 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 1.5 min. The final extension step was followed by a 10 min extension at 72°C. The PCR amplification products were subsequently

The amplified D-loop fragment was purified using a PCR gel extraction kit from Sangon Biotech Co., Ltd. (Shenzhen, China) and sequenced directly using a BigDye Terminator v3.1 cycle

O approximately. The PCR conditions

**2.1. Sample collection**

**2.2. Data collection**

stored at 12°C until use.

The study of mitochondrial DNA (mtDNA) polymorphisms has been useful for describing the molecular phylogeny and diversity of this sheep [8–11], due to the extremely low rate of recombination of mtDNA, its maternal lineage heredity and its relatively fast substitution rate as compared to nuclear DNA [12]. In particular, the CR (the D-loop) is the main noncoding regulatory region for the transcription and replication of mtDNA [13]. Based on mtDNA sequence analysis, we investigate the history and phylogenic relationships of modern domestic Tibetan sheep populations [14].

It is possible to describe the genetic polymorphisms and maternal origin of Tibetan sheep, according to the variability and structure of the mtDNA control region because mtDNA merely has no recombination and follows simple maternal inheritance [14, 15] and evolves relatively rapidly [16]. The higher substitution rate of CR, compared with the heterogeneity rate in the other parts of mtDNA, can characterize intraspecific and interspecific genetic diversity optimally [17–21]. This high mutation rate is because the CR remains single stranded for long periods during mitochondrial replication and transcription [22–26].

Here, we investigate the mtDNA D-loop variability of Tibetan sheep indigenous to the Qinghai-Tibetan Plateau areas. We increase the number of Tibetan sheep samples by including six available reference genomes from GenBank for our population genetic and phylogenetic analysis of the 15 Tibetan sheep populations, based on completion of the mtDNA control region. Our results provide insight into the phylogenetic evolution and maternal origin of Tibetan sheep and improve the management of sheep genetic resources and conservation of their genetic diversity.
