(Cannabis sativa L.) DNA sample from North West China - Springer Link

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May 21, 2008 - species with a long history of cultivation and used as a medicinal, a fibre and ... Beijing Museum of Natural History, Beijing 100050,. China. 123.
Genet Resour Crop Evol (2008) 55:481–485 DOI 10.1007/s10722-008-9343-9

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Results of molecular analysis of an archaeological hemp (Cannabis sativa L.) DNA sample from North West China Ashutosh Mukherjee Æ Satyesh Chandra Roy Æ S. De Bera Æ Hong-En Jiang Æ Xiao Li Æ Cheng-Sen Li Æ Subir Bera

Received: 28 September 2007 / Accepted: 21 April 2008 / Published online: 21 May 2008 Ó Springer Science+Business Media B.V. 2008

Abstract Hemp (Cannabis sativa L.) cultivation and utilization is an ancient practice to human civilization. There are some controversies on the origin and subsequent spread of this species. Ancient plant DNA has proven to be a powerful tool to solve phylogenetic problems. In this study, ancient DNA was extracted from an archaeological specimen of Cannabis sativa associated with archaeological human remains from China. Ribosomal and Cannabis specific chloroplast DNA regions were PCR amplified. Sequencing of a

species-specific region and subsequent comparison with published sequences were performed. Successful amplification, sequencing and sequence comparison with published data suggested the presence of hemp specific DNA in the archeological specimen. The role of Humulus japonicus Sieb. et Zucc. in the evolution of Cannabis is also indicated. The identification of ancient DNA of 2500 years old C. sativa sample showed that C. sativa races might have been introduced into China from the European–Siberian center of diversity.

A. Mukherjee Department of Botany, Dinabandhu Mahavidyalaya, Bongaon, North 24 Parganas 743235, West Bengal, India

Keywords Archaeological DNA  Hemp phylogeny  Humulus japonicus  Species identification  Yanghai Tombs

S. C. Roy (&)  S. De Bera  S. Bera Department of Botany, University of Calcutta, Kolkata 700019, India e-mail: [email protected] H.-E. Jiang  C.-S. Li Laboratory of Systematic and Evolutionary Botany, Chinese Academy of Sciences, Beijing 100093, China H.-E. Jiang Graduate School, Chinese Academy of Sciences, Beijing 100039, China X. Li Bureau of Cultural Relics of Turpan Prefecture, 838000 Xinjiang, Turpan, China C.-S. Li Beijing Museum of Natural History, Beijing 100050, China

Introduction Cannabis sativa L. (hemp) is one of the few plant species with a long history of cultivation and used as a medicinal, a fibre and seed oil plant. It has probably been used for at least 10,000 years (Schultes et al. 1974). The use of Cannabis as medicine is found in India in the medical work Susruta, compiled around 1000 BC (Bouquet 1950; Schultes 1970). The ancient Chinese also knew its medicinal value, as it was referred to in the herbal Pen ts-ao Ching (Li 1974, 1978; Touw 1981). The taxonomic treatment of the species is problematic. Linnaeus was of the opinion

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that it is a single species, whereas Lamarck (1785) determined that the Indian strains of the species are different from the hemp of Europe and gave a new specific name to the Indian Cannabis as C. indica. Small and Cronquist (1976) treated Cannabis as a single species and divided it into subspecies Cannabis sativa L. subsp. indica (Lam.) Small and Cronquist and C. sativa L. subsp. sativa. Recently, Hillig (2005), on the basis of allozyme data, showed that Cannabis has derived from two major gene pools and on the basis of this data, he recognized C. sativa and C. indica as separate species. The centre of origin of Cannabis is believed to be in Central Asia, from where it subsequently spread to Mediterranean countries as well as to Eastern and Central European countries (Faeti et al. 1996). The genus may have two centers of diversity, Hindustani and European–Siberian (Zeven and Zhukovsky 1975). It is difficult to mention the exact place of origin of the plant due to its long history of cultivation. The molecular analysis and the study of sequence homology from ancient samples have considerable value in phylogenetic studies (Kim et al. 2004). The materials from which aDNA analyses have been done include pollen grain (Suyama et al. 1996; Parducci et al 2005), charred wheat (Blatter et al. 2002), ancient wood (Liepelt et al. 2006) and compressed leaf fossil (Kim et al. 2004). Recently, Jiang et al. (2006) discovered ancient 2500 years old hemp remains from Yanghai Tombs, Turpan, Xinjiang, China which provides the evidence for the ancient hemp utilization in Chinese history. In the present investigation, ancient DNA was extracted and analyzed for the first time from this plant material collected from Yanghai Tombs, China. The objective of the present study was to investigate the status of the aDNA in this C. sativa sample by comparing with the DNA from the extant one and also to throw some light on the evolution of the species with the help of aDNA data.

Materials and methods At the Yanghai Tombs, Xinxiang, China, a mummy of a Caucasoid man, about 40 years of age, was found along with plant remains of ancient Cannabis sativa, which are believed to be grave gifts (Jiang et al. 2006). The seeds, leaves and shoots of Cannabis were placed

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in a wooden bowl and a leather basket near the head of the man. Light as well as electron microscopic studies revealed excellent preservation of the materials (Jiang et al. 2006). Leaves, fruits and shoots of the archaeological Cannabis sativa materials were collected from this material. Leaves of one extant material of Cannabis sativa growing as weed were also collected from the adjoining field of the tomb. DNA was extracted from both these materials. Extractions of DNA from ancient materials were done by DNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s protocol. We also performed a CTAB based extraction as used for the modern DNA. The isolated DNA of this second extraction was brown in colour due to the presence of some contaminants. Then QIAquick spin column (Qiagen) was used to purify the DNA. PCR amplification of regions from both nuclear and chloroplast DNA was performed to investigate the status of these genomes in the ancient materials. To verify the species authenticity of the DNA of ancient materials, non-coding spacer region of the chloroplast DNA was selected. Specific PCR was done for the Inter Transcribed spacer (ITS) region of the ribosomal DNA with the primers ITS4 (50 -TCCTCCGCTTATTGA TATGC-30 ) and ITS5 (50 -GGAAGTAAAAGTCGT AACAAGG-30 ) as developed by White et al. (1990) to amplify the nuclear rDNA ITS region. PCR reactions were done in 25 ll of PCR reaction mix containing 4 ll of extracted DNA, 0.2 lM of each primer, 100 lM of dNTPs, 10 mM Tris (pH 8.3), 3.0 mM MgCl2 and 1U Taq polymerase. During amplification of DNA, the initial denaturation was done at 94°C for 5 min followed by 35 cycles each at 94°C for 1 min, 55°C for 1 min, 72°C for 2 min, and final extension for 5 min at 72°C. Amplified products were separated on 1.4% agarose gel. In the gel, a faint band appeared approximately of 700 bp in case of the archaeological sample. The first PCR product was then used as template for a second round of amplification which resulted in a prominent band. Additionally, we used a set of Cannabis sativa specific primers designed by Linacre et al. (1998) to amplify a portion of chloroplast intergenic spacer between trnL and trnF region. The amplification condition was as follows: initial denaturation at 94°C for 5 min and 35 cycles each at 94°C for 1 min, 57°C for 1 min, 72°C for 2 min, followed by 5 min final extension at 72°C. PCR reaction volume and quantity of contents were the same as in case of ITS region

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amplification. Amplified products were separated on 1.4% agarose gel. In gel, a faint band appeared approximately of 200 bp in case of the archaeological sample. The first PCR product was then used as template for a second round of amplification. Purification of PCR products was done by PCR purification kit from QIAGEN. Sequencing was done using the PCR primers in ABI 3100 automatic sequencer (Applied Biosystems). Sequences were submitted to GenBank (Accession No. EF547125 for aDNA sequence and EF552430 for DNA sequence of extant material). Searches for similar published sequences were done thereafter using BLAST (Altschul et al. 1990) from the website http://www.ncbi.nlm.nih.gov/ blast/Blast.cgi. Four such published sequences were obtained from the BLAST analysis including a sequence of Cannabis sativa subsp. indica (Lam.) E. Small et Cronquist (AB035797), one Cannabis sativa strain tochigishiro (AB035795) and sequences from two Humulus species (AB033897: Humulus japonicus Sieb. et Zucc. and AB036272: Humulus lupulus L.), which belong to the family Cannabaceae. Sequence alignment of these four sequences and the two sequences of the present study was done using algorithms of CLUSTALW (Thompson et al. 1994) from the website http://www.ebi.ac.uk. Phylogenetic analysis was conducted with the algorithm of Maximum Parsimony of the software package MEGA 3.1 (Kumar et al. 2004) (obtained from http://www. megasoftware.net). Bootstrap analysis has been carried out with 1000 replicates.

Results and discussion Both the nuclear ribosomal and chloroplast DNA region were successfully amplified. The ribosomal primers amplified ca. 700 bp products in both the ancient and present day samples. The Cannabis specific regions also amplified ca. 200 bp products. After sequencing, we obtained 185 bp and 186 bp DNA in ancient and present day specimens respectively. DNA analysis from ancient plant samples is largely dependent on the condition of the botanical remains. Naturally, well-preserved plant remains should contain better quality DNA. Preservation of organelle DNA and genomic DNA also varies with the preservation condition. Successful amplification of mitochondrial and chloroplast DNA was reported from

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compressed leaf fossil (Kim et al. 2004), whereas preservation of nuclear DNA was observed in charred wheat (Banerjee and Brown 2002). The successful amplification of ribosomal and chloroplast aDNA region in the present investigation showed that the nuclear as well as organellar DNA were well preserved. Additionally, sequence comparison of the Cannabis specific trnL-trnF region from the ancient specimen with the present day specimen along with other published sequences showed high level of sequence similarity (Fig. 1). These published sequences include a sequence of Cannabis sativa subsp. indica (Lam.) E. Small et Cronquist (AB035797), one Cannabis sativa strain tochigishiro (AB035795) and sequences from two Humulus species (AB033897: Humulus japonicus Sieb. et Zucc. and AB036272: Humulus lupulus L.), which belongs to the family Cannabaceae. The high level of sequence similarity also indicates the authenticity of the aDNA. The phylogenetic tree shows that the ancient material and the present day material from China are in the same clade. Cannabis sativa subsp. indica and C. sativa strain tochigishiro forms different clades (Fig. 2) indicating some differences from the Chinese material. The work of Jiang et al. (2006) suggested that the deceased man was a shaman and was aware of the intoxicant and/or the medicinal value of Cannabis. This is also evident from the smooth inner surface of the wooden bowl which is due to its prolonged use as a pestle. The shaman with knowledge of herbal medicine also played the role of physician in ancient times (Li 1974). These evidences show that these ancient materials were used as psychoactive drugs in ritual purposes. Two centers of diversity namely Hindustani and European-Siberian was proposed for this species (Zeven and Zhukovsky 1975). Hillig (2005) showed that through human vectored dispersal, C. indica, originating from Afghanistan, dispersed to places like China, Japan, Africa and other South East Asian countries and C. sativa, originating from Central Asia, dispersed into Europe. However, the present study of authentication of aDNA from 2500 years old samples of Yanghai tomb indicated the migration of C. sativa might take place through the European-Siberian center of diversity. Considering the geographical position of Turpan, which is very close to Central Asia, there is a probability that these plant materials may have originated from C. sativa subsp. sativa.

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Fig. 1 Sequence alignment of Cannabis sativa specific region from modern, archaeological DNA and four published sequences (EF552430: modern DNA sequence of our study; EF547125: archaeological DNA sequence; AB035797: Cannabis sativa indica variety; AB035795: Cannabis sativa strains tochigishiro; AB033897: Humulus japonicus and AB036272: Humulus lupulus; members of the family Cannabaceae)

Fig. 2 Phylogenetic tree obtained from the sequences of Cannabis and Humulus spp. using Maximum Parsimony with 1000 bootstrap replications. Branch lengths are shown at the nodes

Additionally, the molecular data of the present investigation shows the unique genetic nature of these plants which is maintained till now. Humulus japonicus Sieb. et Zucc., a member of the family Cannabaceae, forms a cluster with Chinese materials. Previously, it was also noted that the 26s rDNA region of H. japonicus is almost identical to C. sativa (Pillay and Kenny 2006). Additionally, cross grafting of H. japonicus and H. lupulus with

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C. sativa was successful (Crombie and Crombie 1975) indicating the very close relationship between these two genera. Considering these factors, it can be said that there may be a possibility of gene transfer between C. sativa and H. japonicus in ancient times though Humulus lupulus L. forms a different line. Comparative DNA analysis of ancient and present materials showed homology of ancient DNA with present modern taxa as well as related materials

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which may help in the study of phylogenetic relationships among taxa. Phylogenetic analysis through dendrogram showed that the origin of C. sativa of North West China is different from Cannabis sativa subsp. indica (Lam.) E. Small et Cronquist as shown by forming separate clades which corroborates the two centers of origin of the genus. The present study shows the importance of speciesspecific conserved DNA sequences in ancient DNA research for better understanding of crop species of multiple geographical origin.

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