Gene 537 (2014) 214–219
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When and how did Bos indicus introgress into Mongolian cattle? Xiangpeng Yue a, Ran Li a, Li Liu b, Yunsheng Zhang a, Jieping Huang a, Zhenhua Chang c, Ruihua Dang a, Xianyong Lan a, Hong Chen a, Chuzhao Lei a,⁎ a b c
College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China Department of East Asian Languages and Cultures, Stanford University, CA 94305, USA Weinan Vocational and Technical College, Weinan, Shaanxi, 714000, China
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Article history: Accepted 31 December 2013 Available online 11 January 2014 Keywords: Mongolian cattle Bos taurus Bos indicus mtDNA Y chromosome Archeology
a b s t r a c t The Mongolian cattle are one of the most widespread breeds with strictly Bos taurus morphological features in northern China. In our current study, we presented a diversity of mitochondrial DNA (mtDNA) D-loop region and Y chromosome SNP markers in 25 male and 8 female samples of Mongolian cattle from the Xinjiang Uygur autonomous region in Western China, and detected 21 B. taurus and four Bos indicus (zebu) mtDNA haplotypes. Among four B. indicus mtDNA haplotypes, two haplotypes belonged to I1 haplogroup and the remaining two haplotypes belonged to I2 haplogroup. In contrast, all 25 male Mongolian cattle samples revealed B. taurus Y chromosome haplotype and no B. indicus haplotypes were found. Historical and archeological records indicate that B. taurus was introduced to Xinjiang during the second millennium BC and B. indicus appeared in this region by the second century AD. The two types of cattle coexisted for many centuries in Xinjiang, as depicted in clay and wooden figurines unearthed in the Astana cemetery in Turfan (3rd–8th century AD). Multiple lines of evidence suggest that the earliest B. indicus introgression in the Mongolian cattle may have occurred during the 2nd–7th centuries AD through the Silk Road around the Xinjiang region. This conclusion differs from the previous hypothesis that zebu introgression to Mongolian cattle happened during the Mongol Empire era in the 13th century. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cattle is one of the most economically important livestock in China and other parts of the world, and can be classified into two subspecies, Bos taurus (BTA) and Bos indicus (BIN, or zebu). The Fertile Crescent is considered as the primary center of taurine cattle domestication, whereas evidence for independent domestication events in other locales is currently debated (Edwards et al., 2011). mtDNA sequences revealed that indicine cattle originated from a different wild aurochs population, Bos primigenius namadicus, in the Indus Valley approximately 8000 years before present (Chen et al., 2009; Troy et al., 2001). It has turned out that there are five haplogroups (P, E, Q, R, and T) in taurine cattle and two (I1 and I2) in indicine cattle (Achilli et al., 2009). In addition, there are three paternal haplogroups (Y1, Y2 and Y3) detected in cattle based on Y chromosome single nucleotide polymorphism (Y-SNP) markers. Of these, Y1 and Y2 belong to BTA, and Y3 belongs to BIN (Götherström et al., 2005). As for 28 cattle breeds and many local populations in China, they can be divided into three groups based on their geographic distribution, morphological characteristics and sex chromosome polymorphisms: the northern group in North China, the central group in the middle
Abbreviations: mtDNA, mitochondrial DNA; BTA, Bos taurus; BIN, Bos indicus. ⁎ Corresponding author. Tel.:+86 29 87092004; fax: +86 29 87092164. E-mail address: [email protected] (C. Lei). 0378-1119/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.12.066
and lower areas of the Yellow River and the southern group in South China (Chen et al., 1993; Qiu et al., 1998). The studies of sex chromosome and mtDNA polymorphisms revealed a declining south-to-north gradient of female zebu introgression and a geographical hybrid zone in central China of BTA and BIN (Cai et al., 2007; Chen et al., 1993; Lei et al., 2006). The Mongolian cattle breed, with strictly BTA morphological features, is one of the most commonly distributed breeds among many Chinese indigenous cattle breeds. It is herded mainly in the Inner Mongolian region, and is also widely distributed in the northeast, north and northwest of China (Qiu et al., 1998). It is suggested that the Mongolian cattle originated from BTA based on the Y chromosome karyotype (Chen et al., 1993). However, previous studies detected BIN lineage in Mongolian cattle (Lei et al., 2006; Mannen et al., 2004). Therefore, Mannen et al. (2004) agreed with the hypothesis that the import of zebu and other cattle from Southeast Asia, Southwest Asia and Northern India during the Mongol Empire era in the 13th century with subsequent crossing with native taurine cattle (Rupen, 1979), which could explain the BIN introgression in the Mongolian cattle. Meanwhile, although Cai et al. (2007) did not find any zebu mtDNA introgression in Mongolian cattle, they found lower percentages of zebu mtDNA introgression in Kazakh and Zaosheng cattle, which also belonged to North cattle group. In contrast, other studies speculated that the introgression of zebu cattle in China may originate from North Africa and the hybridization between African zebu and indigenous taurine cattle through the Silk Road of ancient China (Chen and Cao, 2001; Chen et al., 1990).
X. Yue et al. / Gene 537 (2014) 214–219
In order to clarify the above debates regarding the BIN introgression in the Mongolian cattle, we analyzed the mtDNA D-loop and Y-SNP polymorphisms of 33 Mongolian cattle samples from Bayanbulak steppe in Xinjiang, China. Furthermore, we present the DNA results incorporating relevant historical and archeological information about the first appearances of cattle in Xinjiang, with an attempt to determine when and how the earliest BIN may have introgressed into the Mongolian cattle. Particular attention is given to the context of the ancient southern Silk Road, which connected northwestern China with India.
2. Materials and methods 2.1. Samples collection, DNA extraction and GenBank sequence mining Blood samples of 33 Mongolian cattle (25 males, 8 females) were collected from Bayanbulak steppe in Hejing County, Xinjiang Uygur autonomous region, China (Fig. 1). The sample collection was permitted by the owner of cattle, and the owners were interviewed in detail to ensure unrelatedness among the sampled individuals. The present study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Northwest A&F University. Genomic DNA was extracted using standard phenol–chloroform protocol. Fifteen mtDNA D-loop sequences, of which 6 sequences have been assigned to BIN I1 haplogroup (GenBank accession nos: AB268563, EF417976, EF417979–EF417980, DQ166127–DQ166128) (Chen et al., 2009; Jia et al., 2010), 8 sequences to I2 haplogroup (GenBank accession nos: AB268560–AB268562, AB268568–AB268570, AB268574–AB268575) (Chen et al., 2009), and one reference mitochondrial DNA genome sequence (GenBank accession no: NC_006853) of B. taurus, were collected to use as phylogenetic analysis. A complete mtDNA sequence (GenBank accession no: EU780708) of buffalo was used as an outgroup in the phylogenetic tree construction.
2.2. PCR amplification and sequencing The PCR protocol was as follows: each 25 μl reaction contained 20 ng of genomic DNA, 20 pmol of each primer, 0.2 mM of dNTPs, 1× PCR buffer (including 2.5 mM Mg2+), and 1.0 U Taq DNA polymerase (Tiangen Biotech, Beijing, China). Thermocycling consisted of 4 min of denaturation at 95 °C, 35 cycles of 30 s at 94 °C, 60 s at 54 °C (condition for Y-markers see Table S1) and 90 s at 72 °C, and a final extension for 10 min at 72 °C. PCR products were purified with Watson PCR Purification Kit (Watson Biotechnologies, Shanghai) and sequenced using an ABI model 3730 automated sequencer (Applied Biosystems).
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2.3. Analysis of bovine mtDNA sequences and bovine Y-SNP diversity The complete mtDNA D-loop was amplified using a pair of primers: 5-CTGCAGTCTCACCATCAACC-3 and 5-GATTATAGAACAGGCTCCTC-3 (Loftus et al., 1994). mtDNA sequences were edited using DNASTAR 5.0 (DNASTAR, Madison, WI) and aligned using ClustalX (Thompson et al., 1997). All positions containing gaps were eliminated from the analysis. The polymorphisms in the analyzed segments were exported using MEGA 5.0 (Tamura et al., 2011). A Maximum likelihood (ML) tree, using Hasegawa–Kishino–Yano model with an additional parameter of 1000 bootstrapping replicates, Gamma distribution (+ G) was constructed in MEGA 5.0 (Tamura et al., 2011). The Bayesian phylogenetic tree was also constructed using TOPALi 2.5 (Milne et al., 2004). The haplotype diversity and nucleotide diversity for Mongolian cattle breed were estimated using Arlequin 2.0 package (Schneider et al., 2000). The pairwise mismatch distribution between mtDNA sequences was generated using DnaSP 5.0 program. The divergence time and most recent common ancestor were calculated using MEGA 5.0. The mutation rate was assumed to be 32%/million years (Troy et al., 2001). Four Y-SNP markers (DDX3Y-7, ZFY-9, ZFY-10 and UTY-19) were selected (Ginja et al., 2009; Götherström et al., 2005) to distinguish BTA and BIN Y chromosome haplotypes in 25 male Mongolian cattle samples. Y chromosome regions, primer sequences, fragment sizes, and annealing temperatures of the above four markers are shown in Table S1 (Ginja et al., 2009; Götherström et al., 2005). The PCR products of the 25 male samples were sequenced in both directions and the sequences were analyzed with SEQMAN TM II v6.1 (DNASTAR Inc.). Y chromosome haplogroup assignment was performed following Ginja et al. (2009). 3. Results 3.1. Variation of mtDNA D-loop sequences in Mongolian cattle Within the 33 complete mtDNA D-loop sequences analyzed in this study, 25 haplotypes (Fig. S1) were defined by the polymorphisms at 79 sites: 57 transitions, 20 transversions, and 2 coexistence sites of transition and transversion. Mongolian cattle showed high haplotype diversity of 0.978 and nucleotide diversity of 0.017. The haplotypes H1, H4, H6, H13 and H19 represented 2, 3, 2, 4 and 2 individuals, respectively, whereas the remaining 20 haplotypes occurred only once, respectively (Fig. S1). 3.2. Phylogenetic tree of mtDNA in Mongolian cattle The ML (Fig. 2) and Bayesian (Fig. 3) trees were constructed based on 25 mtDNA haplotypes in Mongolian cattle and combined with 15 published BIN (14) and BTA (1) mtDNA sequences as controls, and buffalo mtDNA sequence as an outgroup. Both of the ML and Bayesian trees
Fig. 1. The location of samples and Astana cemetery and the map of partial Silk Road route. (A) The map of Xinjiang Uygur autonomous region, it lists the sample location, and the location of Ancient Astana Cemetery. (B) The map of the partial Silk Road route.
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Fig. 2. The maximum likelihood tree of 25 mtDNA haplotypes in this study and 15 published mtDNA sequences. It obviously revealed that 25 haplotypes can be divided into Bos taurus branch and Bos indicus branch (I1 and I2).
illustrated that the 25 haplotypes examined fell into two distinct mtDNA haplogroups: taurine and zebu, of which 21 haplotypes could be assigned to BTA and four haplotypes (H22, H23, H24 and H25) to BIN (Fig. 2). Within four BIN haplotypes, H22 and H23 fell into I1
haplogroup, while H24 and H25 into I2 haplogroup (Figs. 2 and 3). We found this to be the first time that the I2 haplogroup was detected in Mongolian cattle when we compared our results with previous studies (Table 1).
Fig. 3. The Bayesian tree of 25 mtDNA haplotypes in this study and 15 published mtDNA sequences. The Bayesian tree showed the similar pattern as Maximum likelihood tree.
X. Yue et al. / Gene 537 (2014) 214–219 Table 1 The genetic diversity analyses for Mongolian cattle in recent studies. Animal number
mtDNA B. taurus
B. indicus I1
B. indicus I2
B. taurus
Y-chromosome B. indicus
33 44 51 10 7
29 35 46 9 7
2 9 5 1 0
2 0 0 0 0
25 35 – – –
0 0 – – –
References
This study Mannen et al. (2004) Jia et al. (2010) Lei et al. (2006) Cai et al. (2007)
3.3. Population expansion and haplotype divergence The sign of a population expansion was demonstrated by the bellshaped curve of the mismatch pairwise distributions (Fig. 4), indicating that the studied Mongolian cattle has undergone the population expansion. Meanwhile, there were a negative Fu's Fs value (− 0.396) and Tajima's D value (− 1.044) for the 33 Mongolian cattle investigated, which can be interpreted as a signal of purifying selection or alternately as demographic expansion. Based on the mutation rate of 0.32 per million years (MYA) in mtDNA, the estimated divergence time of B. taurus and B. indicus was 9379 years ago. In the B. indicus branch, two separate lineages I1 and I2 have been identified with an estimated divergence time of 3610 years. 3.4. The Y chromosome haplotypes in Mongolian cattle Taurine Y2 haplogroup was observed in all 25 male Mongolian cattle samples and no indicine Y chromosome haplotype was detected in this study based on four Y chromosome SNP markers (Ginja et al., 2009; Götherström et al., 2005). This result is consistent with previous studies that no indicine Y chromosome haplotype was detected (Table 1). 3.5. Historical and archeological information Perhaps the initial cultural encounter between China and India can be traced to the second century BC when Zhang Qian (167?–114 BC) was send by the Western Han dynasty (202 BC–AD 23) to Central Asia for military exigencies (according to the textural record). Upon his return, Zhang Qian reported to the Han court about the existence of a trade route linking southwestern China to India. Zhang Qian's travel in those regions established the official trade routes between China and the West with an extension to India, known as the Silk Road. By the end of the first century AD, the Chinese already gathered detailed information about the southern Hidukush region in north India, where the kingdom of Jibin (Kapisa–Gandhara) was of great importance to the Han court (Sen, 2003). The chapter “the Western Regions” in Hanshu
Fig. 4. Pairwise mismatch distribution test of 33 Mongolian cattle. The test demonstrated the population expansion in studying cattle.
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(Book of the Han Dynasty, compiled by Ban Gu, 32–92 AD), provided an account of flora and fauna from Jibin. Among the many animals mentioned, there was zebu, referred to as fengniu (humped ox). In the Eastern Han period, recorded in chapter “Emperor Shun” of Hou Hanshu (Book of the Later Han Dynasty, composed by Fan Ye, 398–446 AD), a king of the Shule kingdom (Kashgar in western Xinjiang) sent zebu with a lion to the Han court as tribute in 133 AD. Kashgar was one of the major trade cities connecting Xinjiang with India along the southern Silk Road (Liu, 1988). These accounts suggest that zebu, originating in India, were introduced to Xinjiang through the Silk Road by the second century AD. The earliest archeological remains of cattle in China have been discovered at the Bronze Age sites of Gumugou (ca. 1800 BC) and Xiaohe (ca. 1650–1450 BC) in Lop Nur, eastern Xinjiang. Complete cattle skulls from Xiaohe, apparently belonging to B. taurus, and artifacts made of horn from Gumugou, were buried with the deceased in tombs (Wang, 2001; Yidilisi et al., 2007). The archeological evidence for the first appearance of zebu in Xinjiang, however, is much later. At the Astana cemetery in Turfan, eastern Xinjiang (Fig. 3), archeologists have discovered several clay and wooden ox figurines. One of them is a clay humped ox, apparently depicting a zebu. A wooden ox figurine, on the other hand, shows morphological features of taurine cattle (Yao, 2009). These two artifacts were on display in the Turfan City Museum in 2008 (Mu, 1983) (Fig. 5). The Astana cemetery was associated with Gaochang, a strategically important city along the Silk Road (Ding, 2001). Some of the residents in the city were traders who transported goods along the Silk Road (de la Vaissiere, 2005). This cemetery dates to an era between the Western Jin period (AD 265–316) and the middle of the Tang Dynasty (AD 618–907). While the wooden taurine figurine belongs to the Western Jin period, the clay zebu, as well as two other clay taurines, dates to the Xizhou period (AD 640–791) (dates based on personal communication with Xingcan Chen and Ling Chen, 2012). In summary, the archeological and historical information suggests that taurine cattle was first brought to Xinjiang by people in the early second millennium BC, the Bronze Age, as evidenced at the Gumugou and Xiaohe sites. By the second century AD, indicine cattle were introduced into the Xinjiang region, as mentioned in Hou Hanshu. Taurine and indicine seem to have coexisted for many centuries in Xinjiang before their images appeared together in the Astana cemetery in the seventh century AD. 4. Discussion This study advanced knowledge on the phylogenetics of Mongolian cattle in mtDNA and Y chromosome markers and revealed that the Mongolian cattle have two maternal origins (BTA and BIN) (Figs. 2 and 3) and one paternal origin (BTA). The absence of zebu Y chromosome haplotype contrasts with the presence of indicine mtDNA haplotypes in Mongolian cattle suggested that the earlier Mongolian cattle in Xinjiang region were mainly the pure BTA origin and the introgression of BIN was a secondary phenomenon. This complexion has been observed elsewhere in cattle, particularly within Africa (MacHugh et al., 1997). It can be explained that the local people preferred bulls of BTA morphology in the breeding program and the introduced zebu were mainly female, which can profoundly affect mtDNA distribution while having no influence on Y chromosomes in cattle. As for maternal origin, in previous studies on Mongolian cattle, only indicine I1 haplogroup was reported detected (Jia et al., 2010; Lei et al., 2006; Mannen et al., 2004). However, in this study, the I2 haplogroup was first detected in Mongolian cattle, which is a rare and more ancient origin than I1 haplogroup (Chen et al., 2009; Jia et al., 2010). Interestingly, the distribution of I2 haplogroup in Chinese local cattle breeds was not random. I2 haplogroup was only detected in five cattle populations (Liping, Sinan, Yunnan, Apeijiaza, Xigaze) located in Yunnan–Guizhou Plateau and Tibet region, which are close to India and Nepal (Jia et al.,
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Fig. 5. Cattle wooden carving (left) and clay figurine (right) from Astana cemetery in Turfan City, Xinjiang region, China. The date for these two figurines is provided by archeologists working at site, they are validated from Xizhou period (640–791 AD). The archeological code for wooden carving figurine (Bos taurus) is 72TAM233:2 and clay figurine (Bos indicus) is 72TAM187:133.
2010; Lei et al., 2006). It is believed that zebu cattle were domesticated in the Indus Valley approximately 8000 years before present (Chen et al., 2009; Troy et al., 2001). However, zebu cattle are thought to have been introduced to South China from domestication centers not earlier than 2000 BC, and perhaps, closer to 1500–1000 BC (Higham, 1996). The dramatic predominance of the I1 haplogroup and low frequency of I2 haplogroup in Chinese cattle seem to suggest a later incorporation of the I2 haplogroup into the domestic pool. Culturally established cattle breeds in China by this time would have precluded the simple spread of I2 haplogroup, whose scattered presence in the region, is more readily explained as resulting from later diffusion by trade (Chen et al., 2009). The discoveries of taurine remains at the Bronze Age Gumugou and Xiaohe sites and the clay figurine of indicine cattle in the Astana cemetery, together with ancient textual record, suggest that there was a long history of coexistence of two cattle species in Xinjiang beginning no later than the second century AD. It is very possible, therefore, that the BIN introgression into Mongolian cattle occurred during the period between the second and the seventh centuries AD. This conclusion differs from the hypothesis of Mannen et al. (2004) that the Mongolian conquest in the 13th century AD catalyzed the introgression of cattle with a more occidental origin into the Mongolian population. How did zebu cattle introgress into Mongolian cattle in a broader region of north China? Archeological evidence indicates that domesticated BTA cattle were introduced to north China from the West about 5400–4700 years ago (Rowan et al., 2009). It is currently unclear when BIN was first introduced to south China from India due the lack of systematic faunal studies in the region. Nevertheless, zebu images were commonly depicted on bronze artifacts from the Dian kingdom in Yunnan, indicating that this animal was brought to south China no later than the third century BC (Liu and Chen, 2012). Accordingly, we suggested two hypothetical migratory routes for zebu introgression in Mongolian cattle, described as follows: Sex chromosome and mtDNA polymorphisms revealed that the geographical distributions of the BTA and BIN were predominant in north China and south China respectively, central China was the hybrid zone of BTA and BIN (Cai et al., 2007; Chen et al., 1993; Jia et al., 2010). Hypothesis one is that the zebu cattle were first introduced to south China from the domestication center in the Indus Valley and then gradually expanded and dispersed northward, finally introgressing into Mongolian cattle. However, we cannot determine when zebu introgression in Mongolian cattle occurred through this route. There are some problems with this hypothesis. First of all, the northern cooler climate does not offer a selective advantage for the heat-tolerant zebu cattle and the Qinling Mountains constituted the main natural barriers to their northward expansion. On the other hand, cattle was mainly used as a tool to cultivate crops in southern China, thus they have slower pace of expansion than cattle in the steppe region. Hypothesis two proposes that the introgression occurred along the Silk Road in northwestern China, as discussed early in this paper. This
trade route system started from ancient Chang'an City (now Xi'an, Shaanxi province) in the east and reached west to the Mediterranean. To the south, it extended to India (Fig. 1). Based on mtDNA data, the ethnic populations in the Silk Road region exhibit genetically intermediate values between those of Europe and East and Southeast Asia, suggesting extensive gene admixture. This pattern of genetic admixture is in general agreement with the results of previous osteological studies (Yao et al., 2000). Similarly, the domestic animals, such as the Mongolian cattle presented in the current research, also show characteristics of genetic admixture. Ancient people in the Xinjiang region were agropastoralists, and herding domesticated animals, such as cattle and sheep/goats, was an important part of their subsistence economy (Gong et al., 2011; Wang, 2001). The advent of the Silk Road certainly availed them to introduce cattle breeds from other countries, especially from India or Centre Asia countries. The coexistence of two types of cattle prior to the Tang dynasty constituted a necessary condition for interbreeding. Thus the Indian zebu is likely to have introgressed into Mongolian cattle during the early period of the Silk Road. This hypothesis is consistent with the fact that in China, the I2 haplogroup was only detected in cattle breeds from the Yunnan–Guizhou Plateau and Tibet, near India and Nepal. Therefore, the alternative and more plausible explanation is that the earliest zebu introgression in Mongolian cattle occurred in Xinjiang between the second and the seventh centuries AD, after zebu was brought to China through the southern Silk Road, and the hybrid cattle subsequently spread over other parts of north China. 5. Conclusion This study analyzed the diversity of mtDNA and Y chromosome haplotypes in Mongolian cattle. The results revealed that Mongolian cattle have two maternal origins (BTA and BIN) and one paternal origin (BTA). BIN I2 haplogroup first detected in Mongolian cattle indicated that female zebu cattle were introgressed into Mongolian cattle in ancient times. However, one BTA paternal origin detected in Mongolian cattle indicated that the Indian zebu introgression was a secondary phenomenon, with the earlier Mongolian cattle in the region being pure BTA. Based on historical texts and archeological evidence, BTA and BIN coexisted in Xinjiang by the second century AD, providing a necessary condition for interbreeding. We conclude that the BIN origin in Mongolian cattle may have derived from Indian zebu in the Xinjiang region around 2nd–7th centuries AD, as a part of the Silk Road history which has not been told until now. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2013.12.066. Conflict of Interest statement The authors declare that they have no competing interests.
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Acknowledgments We are grateful to Xincan Chen and Ling Chen for supplying the archeological date of wooden carving and clay cattle in Turfan Museum. We are thankful to Albert Dien and Thomas Bartlett for their constructive suggestions regarding the historical record of zebu in Xinjiang. This work was supported by the National Natural Science Foundation of China (Grant Nos. 31272399, 31072001), the Agricultural Science & Technology Project of Shaanxi Province (No. 2012K02-02) and the Program of National Beef Cattle Industrial Technology System (CARS-38). References Achilli, A., et al., 2009. The multifaceted origin of taurine cattle reflected by the mitochondrial genome. PLoS ONE 4, e5753. Cai, X., Chen, H., Lei, C.Z., Wang, S., Xue, K., Zhang, B., 2007. mtDNA diversity and genetic lineages of eighteen cattle breeds from Bos taurus and Bos indicus in China. Genetica 131, 175–183. Chen, Y.C., Cao, H.H., 2001. Diversity of Chinese yellow cattle breeds and their conservation. Sheng Wu Duo Yang Xing 9 (3), 275–283 (in Chinese). Chen, Y.C., Wang, Y.Y., Cao, H.H., Zhang, Y., 1990. Characteristics of Chinese Yellow Cattle Ecospecies and Their Course of Utilization. Agricultural Publishing House, Beijing. Chen, H., Qiu, H., Zhan, T.S., Jia, J.X., 1993. Studies on sex chromosome polymorphism of four local cattle breeds in China. Yi Chuan 15 (4), 14–17 (in Chinese). Chen, S., et al., 2009. Zebu cattle are an exclusive legacy of the South Asia Neolithic. Mol. Biol. Evol. 27 (1), 1–6. de la Vaissiere, Etienne, 2005. Sogdian Traders: A History. Brill, Leiden. Ding, X.L., 2001. Gaochang Gucheng (Ancient City of Gaochang). Xinjiang Meishu Sheying Press, Wulumuqi (in Chinese). Edwards, C.J., et al., 2011. Dual origins of dairy cattle farming-evidence from a comprehensive survey of European Y-chromosomal variation. PLoS ONE 6 (1), e15922. Ginja, C., Telo da Gama, L., Penedo, M., 2009. Y chromosome haplogroup analysis in Portuguese cattle breeds using SNPs and STRs. J. Hered. 100, 148–157. Gong, Y.W., et al., 2011. Investigation of ancient noodles, cakes, and millet at the Subeixi Site, Xinjiang, China. J. Archaeol. Sci. 38, 470–479. Götherström, A., et al., 2005. Cattle domestication in the Near East was followed by hybridization with aurochs bulls in Europe. Proc. Biol. Sci. 2 (272), 2345–2350. Higham, C., 1996. The Bronze Age of Southeast Asia. Cambridge University Press, Cambridge. Jia, S., et al., 2010. A new insight into cattle's maternal origin in six Asian countries. J Genet Genomics 37, 173–180.
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Gene journal homepage: www.elsevier.com/locate/gene
When and how did Bos indicus introgress into Mongolian cattle? Xiangpeng Yue a, Ran Li a, Li Liu b, Yunsheng Zhang a, Jieping Huang a, Zhenhua Chang c, Ruihua Dang a, Xianyong Lan a, Hong Chen a, Chuzhao Lei a,⁎ a b c
College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China Department of East Asian Languages and Cultures, Stanford University, CA 94305, USA Weinan Vocational and Technical College, Weinan, Shaanxi, 714000, China
a r t i c l e
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Article history: Accepted 31 December 2013 Available online 11 January 2014 Keywords: Mongolian cattle Bos taurus Bos indicus mtDNA Y chromosome Archeology
a b s t r a c t The Mongolian cattle are one of the most widespread breeds with strictly Bos taurus morphological features in northern China. In our current study, we presented a diversity of mitochondrial DNA (mtDNA) D-loop region and Y chromosome SNP markers in 25 male and 8 female samples of Mongolian cattle from the Xinjiang Uygur autonomous region in Western China, and detected 21 B. taurus and four Bos indicus (zebu) mtDNA haplotypes. Among four B. indicus mtDNA haplotypes, two haplotypes belonged to I1 haplogroup and the remaining two haplotypes belonged to I2 haplogroup. In contrast, all 25 male Mongolian cattle samples revealed B. taurus Y chromosome haplotype and no B. indicus haplotypes were found. Historical and archeological records indicate that B. taurus was introduced to Xinjiang during the second millennium BC and B. indicus appeared in this region by the second century AD. The two types of cattle coexisted for many centuries in Xinjiang, as depicted in clay and wooden figurines unearthed in the Astana cemetery in Turfan (3rd–8th century AD). Multiple lines of evidence suggest that the earliest B. indicus introgression in the Mongolian cattle may have occurred during the 2nd–7th centuries AD through the Silk Road around the Xinjiang region. This conclusion differs from the previous hypothesis that zebu introgression to Mongolian cattle happened during the Mongol Empire era in the 13th century. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cattle is one of the most economically important livestock in China and other parts of the world, and can be classified into two subspecies, Bos taurus (BTA) and Bos indicus (BIN, or zebu). The Fertile Crescent is considered as the primary center of taurine cattle domestication, whereas evidence for independent domestication events in other locales is currently debated (Edwards et al., 2011). mtDNA sequences revealed that indicine cattle originated from a different wild aurochs population, Bos primigenius namadicus, in the Indus Valley approximately 8000 years before present (Chen et al., 2009; Troy et al., 2001). It has turned out that there are five haplogroups (P, E, Q, R, and T) in taurine cattle and two (I1 and I2) in indicine cattle (Achilli et al., 2009). In addition, there are three paternal haplogroups (Y1, Y2 and Y3) detected in cattle based on Y chromosome single nucleotide polymorphism (Y-SNP) markers. Of these, Y1 and Y2 belong to BTA, and Y3 belongs to BIN (Götherström et al., 2005). As for 28 cattle breeds and many local populations in China, they can be divided into three groups based on their geographic distribution, morphological characteristics and sex chromosome polymorphisms: the northern group in North China, the central group in the middle
Abbreviations: mtDNA, mitochondrial DNA; BTA, Bos taurus; BIN, Bos indicus. ⁎ Corresponding author. Tel.:+86 29 87092004; fax: +86 29 87092164. E-mail address: [email protected] (C. Lei). 0378-1119/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.12.066
and lower areas of the Yellow River and the southern group in South China (Chen et al., 1993; Qiu et al., 1998). The studies of sex chromosome and mtDNA polymorphisms revealed a declining south-to-north gradient of female zebu introgression and a geographical hybrid zone in central China of BTA and BIN (Cai et al., 2007; Chen et al., 1993; Lei et al., 2006). The Mongolian cattle breed, with strictly BTA morphological features, is one of the most commonly distributed breeds among many Chinese indigenous cattle breeds. It is herded mainly in the Inner Mongolian region, and is also widely distributed in the northeast, north and northwest of China (Qiu et al., 1998). It is suggested that the Mongolian cattle originated from BTA based on the Y chromosome karyotype (Chen et al., 1993). However, previous studies detected BIN lineage in Mongolian cattle (Lei et al., 2006; Mannen et al., 2004). Therefore, Mannen et al. (2004) agreed with the hypothesis that the import of zebu and other cattle from Southeast Asia, Southwest Asia and Northern India during the Mongol Empire era in the 13th century with subsequent crossing with native taurine cattle (Rupen, 1979), which could explain the BIN introgression in the Mongolian cattle. Meanwhile, although Cai et al. (2007) did not find any zebu mtDNA introgression in Mongolian cattle, they found lower percentages of zebu mtDNA introgression in Kazakh and Zaosheng cattle, which also belonged to North cattle group. In contrast, other studies speculated that the introgression of zebu cattle in China may originate from North Africa and the hybridization between African zebu and indigenous taurine cattle through the Silk Road of ancient China (Chen and Cao, 2001; Chen et al., 1990).
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In order to clarify the above debates regarding the BIN introgression in the Mongolian cattle, we analyzed the mtDNA D-loop and Y-SNP polymorphisms of 33 Mongolian cattle samples from Bayanbulak steppe in Xinjiang, China. Furthermore, we present the DNA results incorporating relevant historical and archeological information about the first appearances of cattle in Xinjiang, with an attempt to determine when and how the earliest BIN may have introgressed into the Mongolian cattle. Particular attention is given to the context of the ancient southern Silk Road, which connected northwestern China with India.
2. Materials and methods 2.1. Samples collection, DNA extraction and GenBank sequence mining Blood samples of 33 Mongolian cattle (25 males, 8 females) were collected from Bayanbulak steppe in Hejing County, Xinjiang Uygur autonomous region, China (Fig. 1). The sample collection was permitted by the owner of cattle, and the owners were interviewed in detail to ensure unrelatedness among the sampled individuals. The present study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Northwest A&F University. Genomic DNA was extracted using standard phenol–chloroform protocol. Fifteen mtDNA D-loop sequences, of which 6 sequences have been assigned to BIN I1 haplogroup (GenBank accession nos: AB268563, EF417976, EF417979–EF417980, DQ166127–DQ166128) (Chen et al., 2009; Jia et al., 2010), 8 sequences to I2 haplogroup (GenBank accession nos: AB268560–AB268562, AB268568–AB268570, AB268574–AB268575) (Chen et al., 2009), and one reference mitochondrial DNA genome sequence (GenBank accession no: NC_006853) of B. taurus, were collected to use as phylogenetic analysis. A complete mtDNA sequence (GenBank accession no: EU780708) of buffalo was used as an outgroup in the phylogenetic tree construction.
2.2. PCR amplification and sequencing The PCR protocol was as follows: each 25 μl reaction contained 20 ng of genomic DNA, 20 pmol of each primer, 0.2 mM of dNTPs, 1× PCR buffer (including 2.5 mM Mg2+), and 1.0 U Taq DNA polymerase (Tiangen Biotech, Beijing, China). Thermocycling consisted of 4 min of denaturation at 95 °C, 35 cycles of 30 s at 94 °C, 60 s at 54 °C (condition for Y-markers see Table S1) and 90 s at 72 °C, and a final extension for 10 min at 72 °C. PCR products were purified with Watson PCR Purification Kit (Watson Biotechnologies, Shanghai) and sequenced using an ABI model 3730 automated sequencer (Applied Biosystems).
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2.3. Analysis of bovine mtDNA sequences and bovine Y-SNP diversity The complete mtDNA D-loop was amplified using a pair of primers: 5-CTGCAGTCTCACCATCAACC-3 and 5-GATTATAGAACAGGCTCCTC-3 (Loftus et al., 1994). mtDNA sequences were edited using DNASTAR 5.0 (DNASTAR, Madison, WI) and aligned using ClustalX (Thompson et al., 1997). All positions containing gaps were eliminated from the analysis. The polymorphisms in the analyzed segments were exported using MEGA 5.0 (Tamura et al., 2011). A Maximum likelihood (ML) tree, using Hasegawa–Kishino–Yano model with an additional parameter of 1000 bootstrapping replicates, Gamma distribution (+ G) was constructed in MEGA 5.0 (Tamura et al., 2011). The Bayesian phylogenetic tree was also constructed using TOPALi 2.5 (Milne et al., 2004). The haplotype diversity and nucleotide diversity for Mongolian cattle breed were estimated using Arlequin 2.0 package (Schneider et al., 2000). The pairwise mismatch distribution between mtDNA sequences was generated using DnaSP 5.0 program. The divergence time and most recent common ancestor were calculated using MEGA 5.0. The mutation rate was assumed to be 32%/million years (Troy et al., 2001). Four Y-SNP markers (DDX3Y-7, ZFY-9, ZFY-10 and UTY-19) were selected (Ginja et al., 2009; Götherström et al., 2005) to distinguish BTA and BIN Y chromosome haplotypes in 25 male Mongolian cattle samples. Y chromosome regions, primer sequences, fragment sizes, and annealing temperatures of the above four markers are shown in Table S1 (Ginja et al., 2009; Götherström et al., 2005). The PCR products of the 25 male samples were sequenced in both directions and the sequences were analyzed with SEQMAN TM II v6.1 (DNASTAR Inc.). Y chromosome haplogroup assignment was performed following Ginja et al. (2009). 3. Results 3.1. Variation of mtDNA D-loop sequences in Mongolian cattle Within the 33 complete mtDNA D-loop sequences analyzed in this study, 25 haplotypes (Fig. S1) were defined by the polymorphisms at 79 sites: 57 transitions, 20 transversions, and 2 coexistence sites of transition and transversion. Mongolian cattle showed high haplotype diversity of 0.978 and nucleotide diversity of 0.017. The haplotypes H1, H4, H6, H13 and H19 represented 2, 3, 2, 4 and 2 individuals, respectively, whereas the remaining 20 haplotypes occurred only once, respectively (Fig. S1). 3.2. Phylogenetic tree of mtDNA in Mongolian cattle The ML (Fig. 2) and Bayesian (Fig. 3) trees were constructed based on 25 mtDNA haplotypes in Mongolian cattle and combined with 15 published BIN (14) and BTA (1) mtDNA sequences as controls, and buffalo mtDNA sequence as an outgroup. Both of the ML and Bayesian trees
Fig. 1. The location of samples and Astana cemetery and the map of partial Silk Road route. (A) The map of Xinjiang Uygur autonomous region, it lists the sample location, and the location of Ancient Astana Cemetery. (B) The map of the partial Silk Road route.
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Fig. 2. The maximum likelihood tree of 25 mtDNA haplotypes in this study and 15 published mtDNA sequences. It obviously revealed that 25 haplotypes can be divided into Bos taurus branch and Bos indicus branch (I1 and I2).
illustrated that the 25 haplotypes examined fell into two distinct mtDNA haplogroups: taurine and zebu, of which 21 haplotypes could be assigned to BTA and four haplotypes (H22, H23, H24 and H25) to BIN (Fig. 2). Within four BIN haplotypes, H22 and H23 fell into I1
haplogroup, while H24 and H25 into I2 haplogroup (Figs. 2 and 3). We found this to be the first time that the I2 haplogroup was detected in Mongolian cattle when we compared our results with previous studies (Table 1).
Fig. 3. The Bayesian tree of 25 mtDNA haplotypes in this study and 15 published mtDNA sequences. The Bayesian tree showed the similar pattern as Maximum likelihood tree.
X. Yue et al. / Gene 537 (2014) 214–219 Table 1 The genetic diversity analyses for Mongolian cattle in recent studies. Animal number
mtDNA B. taurus
B. indicus I1
B. indicus I2
B. taurus
Y-chromosome B. indicus
33 44 51 10 7
29 35 46 9 7
2 9 5 1 0
2 0 0 0 0
25 35 – – –
0 0 – – –
References
This study Mannen et al. (2004) Jia et al. (2010) Lei et al. (2006) Cai et al. (2007)
3.3. Population expansion and haplotype divergence The sign of a population expansion was demonstrated by the bellshaped curve of the mismatch pairwise distributions (Fig. 4), indicating that the studied Mongolian cattle has undergone the population expansion. Meanwhile, there were a negative Fu's Fs value (− 0.396) and Tajima's D value (− 1.044) for the 33 Mongolian cattle investigated, which can be interpreted as a signal of purifying selection or alternately as demographic expansion. Based on the mutation rate of 0.32 per million years (MYA) in mtDNA, the estimated divergence time of B. taurus and B. indicus was 9379 years ago. In the B. indicus branch, two separate lineages I1 and I2 have been identified with an estimated divergence time of 3610 years. 3.4. The Y chromosome haplotypes in Mongolian cattle Taurine Y2 haplogroup was observed in all 25 male Mongolian cattle samples and no indicine Y chromosome haplotype was detected in this study based on four Y chromosome SNP markers (Ginja et al., 2009; Götherström et al., 2005). This result is consistent with previous studies that no indicine Y chromosome haplotype was detected (Table 1). 3.5. Historical and archeological information Perhaps the initial cultural encounter between China and India can be traced to the second century BC when Zhang Qian (167?–114 BC) was send by the Western Han dynasty (202 BC–AD 23) to Central Asia for military exigencies (according to the textural record). Upon his return, Zhang Qian reported to the Han court about the existence of a trade route linking southwestern China to India. Zhang Qian's travel in those regions established the official trade routes between China and the West with an extension to India, known as the Silk Road. By the end of the first century AD, the Chinese already gathered detailed information about the southern Hidukush region in north India, where the kingdom of Jibin (Kapisa–Gandhara) was of great importance to the Han court (Sen, 2003). The chapter “the Western Regions” in Hanshu
Fig. 4. Pairwise mismatch distribution test of 33 Mongolian cattle. The test demonstrated the population expansion in studying cattle.
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(Book of the Han Dynasty, compiled by Ban Gu, 32–92 AD), provided an account of flora and fauna from Jibin. Among the many animals mentioned, there was zebu, referred to as fengniu (humped ox). In the Eastern Han period, recorded in chapter “Emperor Shun” of Hou Hanshu (Book of the Later Han Dynasty, composed by Fan Ye, 398–446 AD), a king of the Shule kingdom (Kashgar in western Xinjiang) sent zebu with a lion to the Han court as tribute in 133 AD. Kashgar was one of the major trade cities connecting Xinjiang with India along the southern Silk Road (Liu, 1988). These accounts suggest that zebu, originating in India, were introduced to Xinjiang through the Silk Road by the second century AD. The earliest archeological remains of cattle in China have been discovered at the Bronze Age sites of Gumugou (ca. 1800 BC) and Xiaohe (ca. 1650–1450 BC) in Lop Nur, eastern Xinjiang. Complete cattle skulls from Xiaohe, apparently belonging to B. taurus, and artifacts made of horn from Gumugou, were buried with the deceased in tombs (Wang, 2001; Yidilisi et al., 2007). The archeological evidence for the first appearance of zebu in Xinjiang, however, is much later. At the Astana cemetery in Turfan, eastern Xinjiang (Fig. 3), archeologists have discovered several clay and wooden ox figurines. One of them is a clay humped ox, apparently depicting a zebu. A wooden ox figurine, on the other hand, shows morphological features of taurine cattle (Yao, 2009). These two artifacts were on display in the Turfan City Museum in 2008 (Mu, 1983) (Fig. 5). The Astana cemetery was associated with Gaochang, a strategically important city along the Silk Road (Ding, 2001). Some of the residents in the city were traders who transported goods along the Silk Road (de la Vaissiere, 2005). This cemetery dates to an era between the Western Jin period (AD 265–316) and the middle of the Tang Dynasty (AD 618–907). While the wooden taurine figurine belongs to the Western Jin period, the clay zebu, as well as two other clay taurines, dates to the Xizhou period (AD 640–791) (dates based on personal communication with Xingcan Chen and Ling Chen, 2012). In summary, the archeological and historical information suggests that taurine cattle was first brought to Xinjiang by people in the early second millennium BC, the Bronze Age, as evidenced at the Gumugou and Xiaohe sites. By the second century AD, indicine cattle were introduced into the Xinjiang region, as mentioned in Hou Hanshu. Taurine and indicine seem to have coexisted for many centuries in Xinjiang before their images appeared together in the Astana cemetery in the seventh century AD. 4. Discussion This study advanced knowledge on the phylogenetics of Mongolian cattle in mtDNA and Y chromosome markers and revealed that the Mongolian cattle have two maternal origins (BTA and BIN) (Figs. 2 and 3) and one paternal origin (BTA). The absence of zebu Y chromosome haplotype contrasts with the presence of indicine mtDNA haplotypes in Mongolian cattle suggested that the earlier Mongolian cattle in Xinjiang region were mainly the pure BTA origin and the introgression of BIN was a secondary phenomenon. This complexion has been observed elsewhere in cattle, particularly within Africa (MacHugh et al., 1997). It can be explained that the local people preferred bulls of BTA morphology in the breeding program and the introduced zebu were mainly female, which can profoundly affect mtDNA distribution while having no influence on Y chromosomes in cattle. As for maternal origin, in previous studies on Mongolian cattle, only indicine I1 haplogroup was reported detected (Jia et al., 2010; Lei et al., 2006; Mannen et al., 2004). However, in this study, the I2 haplogroup was first detected in Mongolian cattle, which is a rare and more ancient origin than I1 haplogroup (Chen et al., 2009; Jia et al., 2010). Interestingly, the distribution of I2 haplogroup in Chinese local cattle breeds was not random. I2 haplogroup was only detected in five cattle populations (Liping, Sinan, Yunnan, Apeijiaza, Xigaze) located in Yunnan–Guizhou Plateau and Tibet region, which are close to India and Nepal (Jia et al.,
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Fig. 5. Cattle wooden carving (left) and clay figurine (right) from Astana cemetery in Turfan City, Xinjiang region, China. The date for these two figurines is provided by archeologists working at site, they are validated from Xizhou period (640–791 AD). The archeological code for wooden carving figurine (Bos taurus) is 72TAM233:2 and clay figurine (Bos indicus) is 72TAM187:133.
2010; Lei et al., 2006). It is believed that zebu cattle were domesticated in the Indus Valley approximately 8000 years before present (Chen et al., 2009; Troy et al., 2001). However, zebu cattle are thought to have been introduced to South China from domestication centers not earlier than 2000 BC, and perhaps, closer to 1500–1000 BC (Higham, 1996). The dramatic predominance of the I1 haplogroup and low frequency of I2 haplogroup in Chinese cattle seem to suggest a later incorporation of the I2 haplogroup into the domestic pool. Culturally established cattle breeds in China by this time would have precluded the simple spread of I2 haplogroup, whose scattered presence in the region, is more readily explained as resulting from later diffusion by trade (Chen et al., 2009). The discoveries of taurine remains at the Bronze Age Gumugou and Xiaohe sites and the clay figurine of indicine cattle in the Astana cemetery, together with ancient textual record, suggest that there was a long history of coexistence of two cattle species in Xinjiang beginning no later than the second century AD. It is very possible, therefore, that the BIN introgression into Mongolian cattle occurred during the period between the second and the seventh centuries AD. This conclusion differs from the hypothesis of Mannen et al. (2004) that the Mongolian conquest in the 13th century AD catalyzed the introgression of cattle with a more occidental origin into the Mongolian population. How did zebu cattle introgress into Mongolian cattle in a broader region of north China? Archeological evidence indicates that domesticated BTA cattle were introduced to north China from the West about 5400–4700 years ago (Rowan et al., 2009). It is currently unclear when BIN was first introduced to south China from India due the lack of systematic faunal studies in the region. Nevertheless, zebu images were commonly depicted on bronze artifacts from the Dian kingdom in Yunnan, indicating that this animal was brought to south China no later than the third century BC (Liu and Chen, 2012). Accordingly, we suggested two hypothetical migratory routes for zebu introgression in Mongolian cattle, described as follows: Sex chromosome and mtDNA polymorphisms revealed that the geographical distributions of the BTA and BIN were predominant in north China and south China respectively, central China was the hybrid zone of BTA and BIN (Cai et al., 2007; Chen et al., 1993; Jia et al., 2010). Hypothesis one is that the zebu cattle were first introduced to south China from the domestication center in the Indus Valley and then gradually expanded and dispersed northward, finally introgressing into Mongolian cattle. However, we cannot determine when zebu introgression in Mongolian cattle occurred through this route. There are some problems with this hypothesis. First of all, the northern cooler climate does not offer a selective advantage for the heat-tolerant zebu cattle and the Qinling Mountains constituted the main natural barriers to their northward expansion. On the other hand, cattle was mainly used as a tool to cultivate crops in southern China, thus they have slower pace of expansion than cattle in the steppe region. Hypothesis two proposes that the introgression occurred along the Silk Road in northwestern China, as discussed early in this paper. This
trade route system started from ancient Chang'an City (now Xi'an, Shaanxi province) in the east and reached west to the Mediterranean. To the south, it extended to India (Fig. 1). Based on mtDNA data, the ethnic populations in the Silk Road region exhibit genetically intermediate values between those of Europe and East and Southeast Asia, suggesting extensive gene admixture. This pattern of genetic admixture is in general agreement with the results of previous osteological studies (Yao et al., 2000). Similarly, the domestic animals, such as the Mongolian cattle presented in the current research, also show characteristics of genetic admixture. Ancient people in the Xinjiang region were agropastoralists, and herding domesticated animals, such as cattle and sheep/goats, was an important part of their subsistence economy (Gong et al., 2011; Wang, 2001). The advent of the Silk Road certainly availed them to introduce cattle breeds from other countries, especially from India or Centre Asia countries. The coexistence of two types of cattle prior to the Tang dynasty constituted a necessary condition for interbreeding. Thus the Indian zebu is likely to have introgressed into Mongolian cattle during the early period of the Silk Road. This hypothesis is consistent with the fact that in China, the I2 haplogroup was only detected in cattle breeds from the Yunnan–Guizhou Plateau and Tibet, near India and Nepal. Therefore, the alternative and more plausible explanation is that the earliest zebu introgression in Mongolian cattle occurred in Xinjiang between the second and the seventh centuries AD, after zebu was brought to China through the southern Silk Road, and the hybrid cattle subsequently spread over other parts of north China. 5. Conclusion This study analyzed the diversity of mtDNA and Y chromosome haplotypes in Mongolian cattle. The results revealed that Mongolian cattle have two maternal origins (BTA and BIN) and one paternal origin (BTA). BIN I2 haplogroup first detected in Mongolian cattle indicated that female zebu cattle were introgressed into Mongolian cattle in ancient times. However, one BTA paternal origin detected in Mongolian cattle indicated that the Indian zebu introgression was a secondary phenomenon, with the earlier Mongolian cattle in the region being pure BTA. Based on historical texts and archeological evidence, BTA and BIN coexisted in Xinjiang by the second century AD, providing a necessary condition for interbreeding. We conclude that the BIN origin in Mongolian cattle may have derived from Indian zebu in the Xinjiang region around 2nd–7th centuries AD, as a part of the Silk Road history which has not been told until now. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2013.12.066. Conflict of Interest statement The authors declare that they have no competing interests.
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Acknowledgments We are grateful to Xincan Chen and Ling Chen for supplying the archeological date of wooden carving and clay cattle in Turfan Museum. We are thankful to Albert Dien and Thomas Bartlett for their constructive suggestions regarding the historical record of zebu in Xinjiang. This work was supported by the National Natural Science Foundation of China (Grant Nos. 31272399, 31072001), the Agricultural Science & Technology Project of Shaanxi Province (No. 2012K02-02) and the Program of National Beef Cattle Industrial Technology System (CARS-38). References Achilli, A., et al., 2009. The multifaceted origin of taurine cattle reflected by the mitochondrial genome. PLoS ONE 4, e5753. Cai, X., Chen, H., Lei, C.Z., Wang, S., Xue, K., Zhang, B., 2007. mtDNA diversity and genetic lineages of eighteen cattle breeds from Bos taurus and Bos indicus in China. Genetica 131, 175–183. Chen, Y.C., Cao, H.H., 2001. Diversity of Chinese yellow cattle breeds and their conservation. Sheng Wu Duo Yang Xing 9 (3), 275–283 (in Chinese). Chen, Y.C., Wang, Y.Y., Cao, H.H., Zhang, Y., 1990. Characteristics of Chinese Yellow Cattle Ecospecies and Their Course of Utilization. Agricultural Publishing House, Beijing. Chen, H., Qiu, H., Zhan, T.S., Jia, J.X., 1993. Studies on sex chromosome polymorphism of four local cattle breeds in China. Yi Chuan 15 (4), 14–17 (in Chinese). Chen, S., et al., 2009. Zebu cattle are an exclusive legacy of the South Asia Neolithic. Mol. Biol. Evol. 27 (1), 1–6. de la Vaissiere, Etienne, 2005. Sogdian Traders: A History. Brill, Leiden. Ding, X.L., 2001. Gaochang Gucheng (Ancient City of Gaochang). Xinjiang Meishu Sheying Press, Wulumuqi (in Chinese). Edwards, C.J., et al., 2011. Dual origins of dairy cattle farming-evidence from a comprehensive survey of European Y-chromosomal variation. PLoS ONE 6 (1), e15922. Ginja, C., Telo da Gama, L., Penedo, M., 2009. Y chromosome haplogroup analysis in Portuguese cattle breeds using SNPs and STRs. J. Hered. 100, 148–157. Gong, Y.W., et al., 2011. Investigation of ancient noodles, cakes, and millet at the Subeixi Site, Xinjiang, China. J. Archaeol. Sci. 38, 470–479. Götherström, A., et al., 2005. Cattle domestication in the Near East was followed by hybridization with aurochs bulls in Europe. Proc. Biol. Sci. 2 (272), 2345–2350. Higham, C., 1996. The Bronze Age of Southeast Asia. Cambridge University Press, Cambridge. Jia, S., et al., 2010. A new insight into cattle's maternal origin in six Asian countries. J Genet Genomics 37, 173–180.
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