**2.2. Data collection**

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

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 polymerase (5 μL/U) (TaKaRa, China), and 16.8 μL ddH<sup>2</sup> O approximately. The PCR conditions 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 stored at 12°C until use.

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 sequencing ready reaction kit (Applied Biosystems, Darmstadt, Germany) in an automatic sequencer (ABI-PRISM 3730 genetic analyzer, Applied Biosystems, Foster City, California, United states of America). PCR for the sequencing was performed in an automatic sequencer with a total reaction volume of approximately 5 μL containing 3 μL genomic DNA (10 ng/μL), 1 μL (3 pM) of each sequencing primer, 0.5 μL BigDye, and 0.5 μL ddH2 O. The sequencing conditions were as follows: initial denaturation for 2 min at 95°C, 25 cycles of denaturation at 95°C for 10 s, and annealing at 51°C for 10 s. The final extension step was followed by a 190 s extension at 60°C. The PCR sequencing products were subsequently stored at 12°C until use.

and 43 haplotypes. The smallest haplogroup D consisted of 1 individual and 1 haplotype. The number of haplotypes, individuals, and frequency detected in each Tibetan sheep population of haplotype group varied from 1 to 49, from 0 to 62, and from 0 to 0.88, respectively. The haplotype diversity and nucleotide diversity were calculated separately for each Tibetan sheep population and were estimated to be 0.99 ± 0.01 and 0.02 ± 0.00, respectively. The values of haplotype diversity and nucleotide diversity ranged from 0.90 ± 0.16 to 1.00 ± 0.05 and from 0.01 ± 0.00 to 0.03 ± 0.00, respectively, thus demonstrating the high level of genetic diversity in the 15 Tibetan sheep populations. The nucleotide diversity value of the LZ and JZ populations was higher than that of the remaining 13 Tibetan sheep populations, indicating a relatively high level of diversity. Similarly, the haplotype diversity values were highest in LKZ and LZ

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

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

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The study presents the genetic distance and average number of nucleotide differences between and within the 15 Tibetan sheep populations. The genetic distance values ranged from 0.01 to 0.04 within the population diagonals, and the genetic distance values ranged from 0.01 to 0.04 among populations in Liu et al. [29]. Among the Tibetan sheep populations, the genetic distance within populations reached a maximum value in LZ and a minimum value in AW. Similarly, the genetic distance between the populations had a maximum value for LZ and JZ and a minimum value for AW and TJ. The average number of nucleotide differences values ranged from 10.00 to 29.81 within populations along the digital diagonal, and the average number of nucleotide difference values ranged from 10.73 to 30.99 between the populations below the diagonals. Among the Tibetan sheep populations, the average number of nucleotide differences within the populations reached its value maximum in LZ and its minimum value in AW. Similarly, the average number of nucleotide differences between populations reached a

value maximum in LZ and JZ and a minimum value in AW and TJ populations [29].

differences in altitudes (**Table 1**) has itself r = −0.65, one tailed P = 0.0044.

culated *FST* and *GST*. We also calculated *Nm*, *Dxy*, and *Da*

We test whether genetic distances between populations can be explained by absolute differences between altitudes for the 15 Tibetan sheep populations. Graphically, for the focal population of LZ, **Figure 1** plots the genetic distance between population LZ and each of the remaining populations as a function of the absolute difference in altitudes. Genetic distance tends to decrease with absolute difference in altitudes, as estimated by the Pearson correlation coefficient (r = −0.4136, two tailed P = 0.063, square root of 0.1711 indicated in **Figure 1**). This tendency is observed in 10 among the 15 sheep populations, but is never statistically significant at P < 0.05 (see **Table 1**). It is strongest (most negative) for high altitude populations and weakest (most positive) for populations living at low altitudes. This association between altitude and Pearson correlation coefficients obtained between genetic distances and absolute

To examine the genetic differentiation between the 15 Tibetan sheep populations, we cal-

among the 15 studied Tibetan sheep

populations and the lowest in the AW population.

**3.3. Genetic distances and altitude**

**3.4. Genetic differentiation**

**3.2. Genetic distance and average number of nucleotide differences**

#### **2.3. Data analysis**

The sequences were arranged for multiple comparisons using Clustal Omega [32] and were aligned using ClustalW and BLAST [33]. These results were compared with other sequences obtained from GenBank. The reference sequences for tree construction were taken from the maternal lineages of each tree: haplogroup A (AF039578), haplogroup B (AF039577, AY582801, and AY091487), haplogroup E (AY091490, AJ238300). The diversity parameters, including the haplotype diversity, nucleotide diversity and average number of nucleotide differences, were estimated using DnaSP (Sequence Polymorphism Software) 5.10.01 [34]. *GST*, *FST*, *Nm*, AMOVA test, and neutrality tests were estimated using Arlequin version 3.5.1.2 [35]. To identify differences between the geographic regions using the AMOVA program, four groups were established. The phylogenetic and molecular evolutionary relationships, *Dxy*, *Da* , ME phylogenetic haplotype and clustering tree, and genetic distance were assessed using MEGA version 6.0 [36]. We sketched the network and mismatched distribution graphs using the median-joining method implemented in the NETWORK version 4.6.1.2 software to assess the haplotype relationships [37].
