SHORT COMMUNICATION

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 ÆXiao Li Æ Cheng-Sen Li Æ Subir Bera

Received: 28 September 2007 / Accepted: 21 April 2008 / Published online: 21 May 2008

� 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 intro-

duced into China from the European–Siberian center of

diversity.

Keywords Archaeological DNA �Hemp phylogeny � Humulus japonicus �Species identification � Yanghai Tombs

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

A. Mukherjee

Department of Botany, Dinabandhu Mahavidyalaya,

Bongaon, North 24 Parganas 743235, West Bengal, India

S. C. Roy (&) � S. De Bera � S. Bera

Department of Botany, University of Calcutta,

Kolkata 700019, India

e-mail: scroyind@yahoo.com

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

123

Genet Resour Crop Evol (2008) 55:481–485

DOI 10.1007/s10722-008-9343-9

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

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 archae-

ological 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 manufac-

turer’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

482 Genet Resour Crop Evol (2008) 55:481–485

123

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. Purifi-

cation 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 Maxi-

mum Parsimony of the software package MEGA 3.1

(Kumar et al. 2004) (obtained from http://www.

megasoftware.net). Bootstrap analysis has been car-

ried 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 respec-

tively. 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

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 pre-

served. 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 seq-

uence 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-Sibe-

rian 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 authen-

tication 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.

Genet Resour Crop Evol (2008) 55:481–485 483

123

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

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

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 indicavariety; AB035795:

Cannabis sativa strains

tochigishiro; AB033897:

Humulus japonicus and

AB036272: Humuluslupulus; 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

484 Genet Resour Crop Evol (2008) 55:481–485

123

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 species-

specific conserved DNA sequences in ancient DNA

research for better understanding of crop species of

multiple geographical origin.

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