【作者】 韩璐；

【导师】 周慧；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 家养动物作为全球生物多样性的重要组成部分,在人类文明的起源与传播中起着重要的作用,是人类生活方式由散居、渔猎转变成群居、农业的必然产物。羊是人类最早驯化的家养动物之一,也是最重要的家畜,为人类提供了肉、奶、皮、毛等资源,在中国的农业、经济、文化甚至宗教中起到了非常重要的作用。研究古代家养绵羊和山羊的起源与进化,不仅有助于了解农业和畜牧业的起源与发展,还对了解古代人类的生活方式和迁移历史有着重要的参考价值。因此,家养绵羊和山羊的起源与进化研究一直以来都是生物学家和考古学家共同关注的热点问题。家养绵羊和山羊的起源与进化研究主要有动物考古学和分子生物学两种方法。动物考古学的方法主要是根据形态特征对古代动物遗骸进行观察和测量,系统分类鉴定动物的属种,计算种类数量、最小个体数,从而反应出古代人群的生活方式和经济类型。然而,很多遗址中出土的古代动物遗骸由于保存不完整,很难通过形态特征准确地鉴定出动物的属种,尤其是绵羊和山羊这两个近缘物种。分子生物学方法将为鉴定古代遗骸提供更为真实有效的手段。本文以内蒙古中南部凉城县忻州窑子和小双古城两个墓地出土的35个家养羊的古代遗骸为例,结合古动物形态学分析,通过分子生物学方法,提供区分鉴定古代遗骸山羊和绵羊的分子遗传证据。结果表明:线粒体DNA控制区序列和细胞色素b基因片段可以为鉴定绵羊和山羊古代遗骸提供更为详尽的分子遗传信息。其中,细胞色素b基因更适合作为DNA基因条码来区分鉴定古代动物遗骸是绵羊还是山羊。我们从35个古代样本中成功获得了33个真实可靠的古DNA序列,并对这33个样本进行分子鉴定。绝大多数古代样本的分子鉴定结果和形态学鉴定结果一致,只有3个样本的形态学鉴定和分子鉴定结果不一致,这3个样本最初均经形态学鉴定为山羊,最终分子鉴定结果为绵羊。分子鉴定结果纠正了形态学鉴定结果的误差。另外2个古代样本由于没有获得古DNA序列,以形态学鉴定种属结果为准。形态学和分子生物学鉴定35个古代动物遗骸的最终结果为:24个古代样本为绵羊,11个古代样本为山羊。对古代遗骸进行种属的区分鉴定,形态学方法和分子生物学方法各有自己独特的优点,相互补充对同一个样本进行两种方法的区分鉴定是最为理想的选择,不仅降低了古代样本保存不好给种属鉴定带来的难度,而且增加了种属鉴定结果的真实性、准确性和可靠性。分子生物学方法研究绵羊和山羊的起源与进化,大多集中在对现代家养羊遗传多样性的研究,目前,对古代绵羊和山羊遗传结构的研究还很少。而保存在古代动物遗骸中的古DNA能够提供古代家养动物的遗传信息,摆脱研究现代家养动物受到的“时间陷阱”的束缚。因此,重建古代绵羊和山羊的遗传结构,将有助于了解古代绵羊和山羊的谱系发育关系,追踪家养动物分子进化的轨迹。我们对内蒙古凉城县忻州窑子墓地和小双古城墓地出土的距今约2,500年的24个古代绵羊样本进行了线粒体DNA分析。成功获得了23个古代绵羊的线粒体DNA控制区271bp序列和细胞色素b基因300bp序列。线粒体DNA控制区和细胞色素b的系统发育树结果相一致,均清晰地显示出中国内蒙古东周时期的古代绵羊存在3个分支明显的单倍型类群,分别为单倍型类群A、B和C。这三个单倍型类群在古代绵羊中含有不同的分布频率:单倍型类群A占主要的优势,分布频率最显著,占样本总数的73.9%;单倍型类群B的分布次之,占样本总数的17.4%;单倍型类群C的分布频率最低,占样本总数的8.7%。细胞色素b的分析结果显示这3个母系世系的分化时间远远早于动物的驯化时间,揭示了中国绵羊有3个不同的母系起源。单倍型类群A和单倍型类群C的起源分析暗示了包括中国在内的亚洲地区很可能是绵羊的驯化中心之一。我们的研究还发现,中国古代绵羊群体与中国现代绵羊群体的遗传距离最近,并且二者之间的遗传结构极为相似,这表明2,500年前中国古代绵羊的遗传结构就已经趋于稳定,并对现代绵羊起着非常重要的基因贡献作用。我们对内蒙古凉城县忻州窑子墓地和小双古城墓地出土的距今约2,500年的11个古代山羊样本进行了线粒体DNA分析。成功获得了10个长度为289bp的山羊线粒体DNA控制区序列。系统发育树结果显示这10个不同单倍型的古代山羊序列分别属于已定义的单倍型类群A、单倍型类群B和单倍型类群D。其中7个样本属于个体数目最大也是分布最广泛的单倍型类群A,比例占总样本数的70%,2个样本属于单倍型类群B,比例占总样本数的20%,1个样本属于比较稀少的单倍型类群D,比例占总样本数的10%,这表明中国古代山羊有多个母系来源。单倍型类群B的中介网络图分析结果显示单倍型类群B被进一步分为两个亚群B1和B2,中国古代山羊的1个样本与亚群B1的中心建立者类型分享同一个单倍型,中介网络图分析结果暗示了山羊单倍型类群B,包括两个亚群B1和B2可能都起源于中国。这进一步支持了中国可能是山羊驯化中心之一的假说。我们的研究为解决古代遗骸是山羊还是绵羊的难题提供了分子鉴定的遗传依据。内蒙古东周时期古代绵羊和山羊的分子遗传学研究,为探索绵羊和山羊的起源与进化提供了分子遗传学信息。我们的研究同时也反映了古DNA技术在研究动物驯化、农业和畜牧业的起源与发展及古代人类活动的历史等领域的应用前景。更多还原

【Abstract】 Domestic animal as an important component of global biodiversities played an important role in the origin and spread of human civilization, which resulted lifestyle of human changed from scattering and hunting to gathering and farming. Sheep and goats as important livestock were the earliest animals to be domesticated, providing people with many resources, such as meat, milk, skin and fiber, which have played very important roles on agricultural, economic, cultural, and even religious in China. Study on the origin and evolution of ancient domestic sheep and goats would not only help to better understand the origin and development of agriculture and animal husbandry, but also help to understand the lifestyle and migratory history of ancient people. Consequently, the origin and evolution of domestic sheep and goat have long been an area of interest in genetics and archaeology.Animal archaeology and molecular biology are two main methods for studying the origin and evolution of domestic sheep and goats. Animal archaeology is based on morphological characters of animal remains to identify and classify species, the number of species and the minimum number of individuals would reflect the lifestyle and economic type of the ancient population in the archaeological site. However, it is difficult to identify the animal species accurately by morphological characters when the animal remains were damaged in many archaeological sites, especially, two closely related species, such as sheep and goats.Molecular biology would provide an accurate and effective method for identifying the ancient remains. In this study, 35 animal remains from Xinzhouyaozi cemetery and the cemetery of Xiaoshuang city-site of south central Inner Mongolia were exampled by molecular identification of sheep and goat remains. The result showed that both mitochondrial DNA (mtDNA) control region sequences and cytochrome b gene fragments were good DNA markers to identify the animal remains of sheep and goats, cytochrome b gene is more suitable as DNA barcoding to distinguish the animal remains whether sheep or goats. 33 authentic ancient DNA sequences were successfully obtained from 35 samples. Among of the 33 samples, molecular identification of the major ancient samples were consistent with that of morphological identification, except 3 samples identified as goats by morphological method, but identified as sheep by molecular identification. Combined with the morphological analysis, the molecular identification of sheep and goat remains would provide more detailed genetic information, which is a powerful tool to identify the animal remains whether sheep or goats. The other 2 samples were not obtained ancient DNA sequences, so the identification species depended on morphological characters. Finally, among of the 35 samples, 24 samples were identified as sheep, and 11 samples were identified as goats. Both morphological and molecular biological methods have their own unique advantages, so it is the best choose to identify the animal remains by two methods, which would not only reduce difficult of species identification, but also increase accurate and reliable of the species identification result.To explore the origin and evolution of domestic sheep and goats, many studies have focused on genetic diversity of modern samples, but so far, a few studies have focused on genetic structure of ancient samples. While the ancient DNA of animal remains can cast off“time trap”, and provide the genetic information of ancient domestic animals. The genetic structure of ancient sheep and goats would reveal the phylogenetic relationships of sheep and goats, which would help us to trace the origin and evolution of sheep and goats on molecular level.We analyzed mtDNA D-loop control region, cytochrome b (Cyt b) gene, and CO I gene of 24 ancient sheep, which were excavated from Inner Mongolia of northern China and dated about 2,500 years before present. Three maternal lineages with different frequencies (74% for lineage A, 17% for lineage B, and 9% for lineage C) were present in the ancient sheep population. Our results showed a close genetic relationship between ancient and modern sheep, which suggested that the genetic structure of Chinese sheep has been stabilized since 2,500 years before present. The divergence time of lineage A, B and C indicated that at least three independent sheep domestication events have occurred. Significantly, lineage A was predominant in the ancient sheep, which may contribute to further understanding about the origin and development of Chinese domestic sheep. We analyzed the 289bp fragments of mtDNA control region of 11 ancient goats, which were excavated from two archaeological sites in Inner Mongolia of northern China and dated about 2,500 years before present. 10 haplotypes were successfully obtained from 11 ancient goats. Phylogenetic analysis revealed the multiple maternal origins of Chinese domestic goats, three mtDNA lineages A, B and D were identified in the Chinese ancient individuals, in which lineage A was predominant (70%), lineages B was moderate (20%), and lineage D was present at low frequency (10%).The network analysis showed that lineage B was subdivided into two subgroups B1 and B2. One of the Chinese ancient goats shared the founder haplotype in the center of subgroup B1, and the shared sequences of the founder haplotypes of subgroups B1 and B2 distributed mainly in China. These results implied that lineage B including subgroups B1 and B2 probably originated from China, and further supported the hypothesis that China may be one of the goat domestication centers.In this study, molecular technology was used to analyze the ancient sheep and goat remains from Inner Mongolia during Eastern Zhou period. We developed a molecular identification of sheep and goat remains by mtDNA analysis, the molecular analysis of Chinese ancient sheep and goats would contribute to the phylogenetics of domestic sheep and goats in the global and shed light on tracing the evolution of Chinese ancient domestic animals. This study also reflects the prospect of ancient DNA technology in the area of origin and development of domestic animals, agriculture, and history of human activities.更多还原