Ancient DNA And The Origins Of Domestic Animals

The origin and evolution of domestic animals have become an important area of study for archaeologists and geneticists. As is known, the research on the origin of domestic animals will expand the knowledge for the origin and development of agriculture, the diffusion of animal epidemic and the genetic breeding of domestic animals. Previous studies were mainly based on two methods: 1) the analysis of the genetic structure of modern domestic animals; 2) the analysis of morphological variability of archaeological faunal remains. However, there are still some questions: 1) the studies were easily caught in the"time trap"by analyzing the genetic structure of modern domestic animals; 2) the poor quality and lower quantities of the remains belonging to the early period of domestication make it difficult to perform a morphological analysis. Ancient DNA technology provides an effective way to address above questions. As a carrier of genetic information, ancient DNA provides the most direct evidence to reveal the phylogenetic relationship between extinct and extant organisms through the reconstruction of past genetic structure and diversity of domestic animals, which can help to trace the evolution of domestic animals at a molecular level.Evidences from Near East archaeological sites suggested that sheep were probably first domesticated in the Fertile Crescent region of the Near East around 10,000 years ago. China has the glorious history of raising sheep. Some remains of sheep and earthen sheep were found in Neolithic sites dated to 7,000 years ago, such as Peiligang in Xinzheng, Henan Province; Cishan in Wuan, Hebei Province; Banpo in Xi'an, Shaanxi Province and Hemudu in Yuyao, Zhejiang Province. However, based on archaeological culture, Yuanjin considered that the earliest Chinese domestic sheep were found in Dahezhuang and Weijiazhuang sites in Yongjing, Gansu Provinc, dating to 4,000 years ago. Comparing to the domestication of sheep in the Near East, the short history of the domestication of sheep in China suggested Chinese domestic sheep probably were derived from Near East.A large number of remains of domestic horses and carriages suddenly appeared in the sites of the late Shang Dynasty (3,000 BP) in China, such as Yin Ruins in Anyang, Henan province; Laoniupo in Xi'an, Shaanxi province and Qianzhangda in Tengzhou, Shandong province. However, prior to the late Shang Dynasty, there were few records of the domestic horses. Excavations from thousands of Neolithic and early Bronze Age sites in China showed that only a few sporadic fragments of teeth and bones were discovered at limited sites, such as Banpo in Xi'an, Shaanxi province, Baiying in Tangyin, Henan province, Chengziya in Zhangqiu, Shandong province and Nansha village in Hua County, Shaanxi province. The lack of evidence at the early period of domestication of horses and the"sudden emergence"of domestic horses in the late Shang Dynasty makes the origin and history of Chinese domestic horses very confusing. There are mainly two hypothesis on the origin of Chinese domestic horses, One hypothesis thinks that the Chinese domestic horses were domesticated in China, and the Przewalski's horse has been proposed as ancestor. Another hypothesis suggests the Chinese domestic horses may be derived from other regions, not in China.In the present study, ancient DNA analysis was carried out on archaeological sheep and horse remains. Our results reveal the origin of Chinese domestic sheep and horses through the reconstruction of past genetic structure and diversity of Chinese sheep and horses.In summary:(1) In order to find an effective method for ancient DNA extraction, four methods based on silica particles (A: Modified Silica Particle Method; B: Modidied QIAquick Method) and silica-based spin columns (C: QIAamp Method; D: Modified QIAquick Method) were respectively used to extract mitochondrial DNA (mtDNA) from 5 ancient sheep dating to 4000 years ago. PCR amplification of mtDNA was performed, and the effects of 4 methods were determined by examining amplification success rates of DNA from 5 ancient sheep. Our results showed the amplification success rates of 4 methods were different from 20% to 100%, and the effects of extraction based on silica-based spin columns (60%, 100%)were distinctly better than those based on silica particles (20%, 40%). The effect of extraction based on modified silica particles was the lowest, and based on modified QIAquick was the highest among 4 methods. QIAquick Method is a highly effective method, with a 100% success rate, in which Centricon? ultrafiltration devices were used to concentrate teeth digest solution and purify DNA through silica-based spin columns. It can effectively remove the inhibitor as well as obtain preferable DNA template.(2) Recent research has thrown considerable light on the history of the domestic sheep, but not extended to ancient sheep specimens. In the present study, ancient DNA analysis was carried out on eight archaeological sheep remains recovered from Erlitou archaeological site in Henan Province dating to 4,000 years ago to explore the origin of Chinese domestic sheep. Ancient DNA sequences of 271 bp D-loop were successfully retrieved from 8 of 9 samples. The analysis of nucletotides variation position of 8 samples show that all Erlitou sheep belong to Lineage A. Previous studies indicated that the polymorphic HinfI recognition site to nucleotide positions 5562-5566 in the COI gene can be used to assay lineage A (–5562 HinfI) and B (+5562 HinfI ). Further identification of sheep lineage, HinfI-RFLP was performed. All samples showed the negative results (–5562 HinfI), all ancient sheep were identified as lineage A. In order to explore the phylogenetic relationship between ancient and modern sheep and wild sheep as well as to determine the phylogenetic position of ancient sheep, we reconstructed the phylogenetic tree and network based on 151 sequences representing a variety of domestic and wild breeds from all over the word. Phylogenetic tree and network profile showed that all the ancient sheep clustered into lineage A with small-tail Han, Hu, Mongolian and Tong sheep, and they were derived from a common maternal ancestor. The results also rejected the previous hypothesis that Tibet and Mongolia sheep group may be derived from argali and urial. In addition, our results suggest that ancient DNA analysis can serve as a powerful tool in tracing prehistoric population movement.(3) The origin and spread of domestic horses have become a core issue in evolutionary archaeology. In order to reveal the origin of Chinese domestic horses, ancient DNA analysis was performed on 46 horses excavated from 9 archaeological sites in Inner Mongolia, Henan Province, Ningxia and Shandong Province.In this study, most samples were recovered from Inner Mongolia. Therefore, it is very important to determine the condition of the burial environment of Inner Mongolia. The analysis of the highly conservative 16S rRNA gene sequences indicated that the dry and cold burial environment of Inner Mongolia is suitable for the preservation of ancient DNA.Ancient DNA sequences were successfully retrieved from 35 of 46 samples. Using the nomenclature from Jansen et al., and in comparison with the skeleton network described in McGahern et al., we determined the mtDNA lineage of 35 ancient horses. Lineage F represented a high frequency among Chinese ancient horses, suggesting lineage F was an important maternal lineage in China. Phylogenetic network showed that 35 horses were distributed into 7 horse mtDNA lineages (A-G). In addition, some samples also shared the founder haplotypes of cluster. All these suggested that the maternal genetic line of Chinese ancient horses was highly diversified and contributed to the gene pool of modern domestic horses. The network profile also showed that breeds from different geographical regions intermingled, and some haplotypes were shared by individuals from different geographical regions, illustrating that a significant gene flow could have occurred among the breeds in different geographical range, and reflecting the widespread culture exchange among ancient populations throughout the Euraisan range. The analyses of Fu's Fs test and mismatch distribution uniformly indicated the ancient horses had undergone population expansion events. According to above analysis, we think that the origin of Chinese domestic horses was more complex than previously thought. Two previous hypothesis on the origin of Chinese domestic horses were not reflected the maternal lineage diversity and population expansion events among Chinese ancient horses. Both the native origin and external mtDNA gene flow input have been probably involved in the domestication of Chinese domestic horses. In addition, the analysis of mtDNA lineage frequency, genetic distance and AMOVA consistently indicated Chinese ancient horses had closely affinity with horses from East Asia, which further supported the Chinese domestic horses had undergone the native domestication.Traditionally, the Przewalski's horses were considers as the ancestor of Chinese domestic horses. In this study, we hope to find the maternal genetic relationship between ancient horses and the Przewalski's horses. However, according to our results, none of 35 ancient horses and modern horses was observed in cluster A2 which belongs to Przewalski's horses. It says that there were not direct maternal genetic relationship between ancient horses and Przewalski's horses. In addition, the analysis of genetic distance and AMOVA consistently indicated that the Przewalski's horses are less related to Chinese ancient and modern horses. All these results suggest Przewalski's horse and domestic horse may be a sister taxa which have a common ancestor, Chinese domestic horses have other wild ancestors, other than the Przewalski's horse.