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Aug 1, 2013 - Abstract Accessions of wild and domesticated hemp. (Cannabis sativa L.) originating from Colombia,. Mexico, California, Bolivia, Thailand, ...

Genet Resour Crop Evol (2013) 60:2331–2342 DOI 10.1007/s10722-013-0001-5

RESEARCH ARTICLE

Differentiation between fiber and drug types of hemp (Cannabis sativa L.) from a collection of wild and domesticated accessions G. Piluzza • G. Delogu • A. Cabras S. Marceddu • S. Bullitta



Received: 21 December 2012 / Accepted: 15 April 2013 / Published online: 1 August 2013 Ó Springer Science+Business Media Dordrecht 2013

Abstract Accessions of wild and domesticated hemp (Cannabis sativa L.) originating from Colombia, Mexico, California, Bolivia, Thailand, Afghanistan, Serbia, Hungary, south Africa and different regions of China, were studied by means of DNA polymorphisms in order to discriminate between drug and fiber types. Analysis of molecular variance (AMOVA) was used to partition the total genetic variance within and among populations. The significance of the variance components was tested by calculating their probabilities based on 999 random permutations. AMOVA revealed 74 % variation among accessions and 26 % within accessions, all AMOVA variation was highly significant (P \ 0.001). The cluster analysis of molecular data, grouped accessions into eight clusters and gave a matrix correlation value of r = 0.943, indicating a very

good fit between the similarity values implied by the phenogram and those of the original similarity matrix. In this study, DNA polymorphisms could discriminate the fiber and drug types, and accessions were grouped in accordance to their classification and uses. In addition, seed size variation and micromorphological characters of seeds were studied by means of a scanning electron microscope (SEM). Seeds varied significantly in size, and were bigger in the fiber types. SEM analysis exhibited variation of micromorphological characters of seeds that could be important for discriminating the fiber or drug types.

G. Piluzza  S. Bullitta (&) CNR-ISPAAM u.o.s. Sassari, Traversa La Crucca 3, localita` Baldinca, 07100 Li Punti-Sassari, Italy e-mail: [email protected]

Introduction

G. Delogu CNR-ICB u.o.s. Sassari, Traversa La Crucca 3, localita` Baldinca, 07100 Li Punti-Sassari, Italy A. Cabras SBA, University of Sassari, Via Enrico de Nicola 1, 07100 Sassari, Italy S. Marceddu CNR-ISPA u.o.s. Sassari, Traversa La Crucca 3, localita` Baldinca, 07100 Li Punti-Sassari, Italy

Keywords Cannabis sativa  DNA polymorphisms  Drug type  Fiber type  Seeds  SEM

Cannabis sativa L. (Cannabaceae), is considered one of the oldest cultivated plant species, its use is documented by several archeological evidences since the Neolithic period (Rivoira 1981; Li 1974). Hemp is economically important for its multiple uses. It has been utilized as a source of fiber, fuel and nutrition for thousands of years. Cloth, rope, paper and canvas are produced from its fibers, oil is produced from seeds to be utilized in the cosmetic industry and also for production of colors and varnishes. In the past, hemp purified oil mixed with colza oil, was also used for

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lighting purposes. The importance of the crop reached a maximum level in the XIX century, while after that period, a decline started for the crop, either for the introduction of alternative fibers such as cotton and synthetic fibers, and for the diffusion of steam boats and the consequent reduction in the request for ropes and sails (Rivoira 1981). Nowadays new perspectives for hemp as a crop that can be grown for food and nonfood purposes are arising. According to Hansen (2009), as a result of its numerous nutritional benefits, many new food products containing hemp seed and its oil are finding their way into the marketplace, including pasta, tortilla chips, salad dressings, snack products and frozen desserts. Non-dairy hemp ‘‘milk’’ beverages, which provide significant amounts of omega 3 essential fatty acids (EFAs) and protein, are also available. Hemp oil is also used in nutraceuticals to supplement diets poor in EFAs and in health care products. Particularly, hemp oil is considered an ideal topical ingredient in lotions, lip balms, conditioners, shampoos, soaps and shaving products. Hansen (2009) also indicates potential health benefits of hemp seed oil for diabetes, cancer, lupus, asthma, rheumatoid arthritis, depression and hypertension. The review of Caffarel et al. (2012) summarizes the current knowledge on the antitumor potential of cannabinoids in breast cancer. As a fiber crop, hemp is gaining new perspectives for production of biomaterials for construction; technical nonwovens for applications in automotive composites, insulation products and geotextiles; fabrics for landscaping, tree planting, mulching, erosion control and horticulture; hemp logs suitable for burning on wood and coal burning stoves (www.hemcore.co.uk). Other recent uses of hemp fibers are the making of new nanostructural polymers (Pommet et al. 2008) and bioremediation through biosorption of heavy metal ions in aqueous solutions (Pejic et al. 2009). More traditional products made from hemp fiber are cigarette paper, bank notes, technical filters and hygiene products, art papers and tea bags. The taxonomic treatment of Cannabis is problematic (Hillig 2005). The name C. sativa or ‘‘cultivated cannabis’’ was probably first employed by Fuchs in his herbal of 1,542 accompanied by a illustration of European hemp (Fuchs 1542), thus pre-dating the monotypic assignation of Linneaus in his Species Plantarum of 1,753 (Russo 2007). Lamarck (1785) determined that Cannabis strains from India are

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distinct from the common hemp of Europe, and named the new species Cannabis indica Lam. Distinguishing characteristics include more branching, a thinner cortex, narrower leaflets and the general ability of C. indica to induce a state of inebriation. Other species of Cannabis have been proposed (reviewed in Schultes et al. 1974; Small and Cronquist 1976), including Cannabis chinensis Delile, and Cannabis ruderalis Janisch. Vavilov (1926) considered C. ruderalis to be synonymous with his own concept of C. sativa L. var. spontanea Vav. He later recognized wild Cannabis populations in Afghanistan to be distinct from C. sativa var. spontanea, and named the new taxon C. indica Lam. var. kafiristanica Vav. (Vavilov and Bukinich 1929). Small and Cronquist (1976) proposed a monotypic treatment of Cannabis, which is a modification of the concepts of Lamarck and Vavilov. They reduced C. indica in rank to C. sativa L. subsp. indica (Lam.) and differentiaded it from C. sativa L. subsp. sativa primarily on the basis of ‘‘intoxicant ability’’ and purpose of cultivation. Small and Cronquist bifurcated both subspecies into ‘‘wild’’ (sensu latu) and domesticated varieties on the basis of achene size and other achene characteristics. This concept was challenged by other botanists who used morphological traits to delimit three species: C. indica, C. sativa and C. ruderalis (Anderson 1974, 1980; Emboden 1974; Schultes et al. 1974). Schultes et al. (1974) and Anderson (1974) circumscribed C. indica to include relatively short, densely branched, wide-leafleted strains from Afghanistan. In spite of the numerous new applications nowadays possible for hemp oil and fibers, a drawback for the diffusion of hemp cultivation is still represented by the presence of hemp psychoactive components. Hemp plants are characterized by the presence of terpenophenolic substances known as cannabinoids, which accumulate mainly in the glandular trichomes of the plant (Mechoulam 1970), the most abundant are cannabidiol (CBD) and D-9-tetrahydrocannabinol (THC). Hemp varieties used for drug consumption, characterized by a high content of D-9-tetrahydrocannabinol (THC), are often not distinguishable morphologically from fiber varieties with a lower content of THC. C. sativa subspecies are divided into several chemical phenotypes. The relative proportions of THC, CBN (cannabinol) and CBD have been used by various authors for distinguishing three predominant chemotypes: I (drug type, the predominant

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cannabinoid is THC) II (intermediate type, the predominant cannabinoids are THC and CBD) and III (fibre type, the predominant cannabinoid is CBD) (Broseus et al. 2010). The D-1-tetrahydrocannabinolic acid synthase is considered to be a key enzyme controlling the psychoactivity (i.e. level of THC) of Cannabis because this enzyme mediates the biosynthetic step of THC (Shoyama et al. 2012). The Italian law (D.P.R. 309/90) does not allow the cultivation of drug type cannabis, while the fiber type plants are legally cultivated in some regions of the Mediterranean area. During the past there was some uncertainty in the Italian law concerning the content of psychoactive substances in cannabis plants, which led to a number of interventions of the legal authorities against the cultivators of the fiber type cannabis, therefore it is of relevant importance to determine which growing conditions produce the lowest levels of psychoactive constituents. Since 2001 the European Union (EC 2860/2000) allows cultivation of fiber-type hemp varieties with THC content \0.2 %. If THC concentration is higher than 0.2 % of the dried material, the Cannabis product is considered a narcotic substance, whereas cannabis with \0.2 % THC is considered agricultural cannabis, growing and possession of which is legal. The drug type plant usually contains up to 5 % of THC, though higher percentages (up to 10 %) have been reported (Bruci et al. 2012). Cannabis sativa is grown widely throughout the world, in temperate and tropical countries and according to the World Drug Report 2005 of the United Nations Office on Drugs and Crime (UNODC), cannabis cultivation is widespread in Africa, the Americas, Asia and Europe. Identifying a total of 86 countries where the cannabis plant is grown, the World Drug Report 2005 states that world cannabis production in 2004 was 47,000 tons, compared with 687 tons of cocaine and 565 tons of heroin. A total of 7,206 tons of cannabis products were seized in 2003, which is 15 times the total of cocaine seized and about 65 times the total of heroin seized (Stambouli et al. 2005). According to Datwyler and Weiblen (2006), drug enforcement efforts could benefit from DNA fingerprinting technology capable of separating marijuana from hemp, pinpointing geographic sources, and establishing conspiracy in illicit distribution networks. Drug type hemps are typically characterized by elevated levels of psychoactive cannabinoid compounds

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leading to C. sativa being the most used illicit drug world-wide (Howard et al. 2008). Marijuana trafficking is a thriving, multibillion dollar industry (Datwyler and Weiblen 2006) and the ability to identify and/or link organized crime syndicates by determining the likely origin of seized drugs and to distinguish between legalized fiber crops and drug crops are highly sought by the international forensic community (Howard et al. 2008). To pinpoint the hemp geographic origin through molecular markers and to identify psychoactive and non-psychoactive types can be useful for forensic purposes in order to ascertain the origin of seized samples but can also help to identify hemp cultivars and help the diffusion of the crop in those countries where the controversial borders between licit and illicit crops have determined a decline in hemp cultivation. We utilized DNA polymorphisms in order to discriminate between drug type and fiber type accessions from a collection of hemp of different origin. In order to confirm the molecular data, THC content and seed-size variations were considered. We also examined micro-morphological characters of seeds by means of a scanning electron microscope (SEM) in order to obtain a differentiation between fiber and drug types of hemp.

Materials and methods Plant materials Import of hemp seeds was regularly authorized by the competent Authorities. Table 1 shows the hemp accessions and related uses. CMTI and CMTI2 are sister lines from ‘‘Haze’’ that is a multhybrid fairly true-breeding cultivar in which landraces are combined from Colombia, Mexico, Thailand and southern India. The ancestral landraces are all C. sativa types used for marihuana production. TAI and TAI2 are the full-sib F1 progeny from a female and a male plant both selected from one (TAI) and two different (TAI2) Thai landraces, the ancestral landraces are C. sativa types for marihuana production. AMC is the full-sib F1 progeny from a female and a male plant selected from ‘‘Skunk’’. Skunk is a true-breeding indica 9 sativa hybrid cultivar based on landraces from Afghanistan (indica, hashish type), Mexico and Colombia (sativa, marihuana type). AMC2 and AMC3 are half-sib F1

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progenies from Skunk. CAL is the full-sib F1 progeny from female and a male plant selected from ‘‘Early California’’. Early California is a cross-progeny from various indica and sativa drug strains, inbred and selected for earliness. AFG is a half-sib progeny from selected parents from an Afghani landrace, a pure C. indica type traditionally used for hashisch production. SER is a Serbian fibre cultivar selected from ‘‘Fleischmann hemp’’. BOL is a drug type landrace from Bolivia unselected. The accessions UNG-UNG2 correspond to an Hungarian fibre cultivar selected from ‘‘Fleischmann’’ hemp, UNG2 is the F1 seed harvested from the original cultivar which was grown in China. CYU and CHO are fibre landraces respectively from Yunnan, China and Hohhot, Inner Mongolia, China. Other four Cannabis sp. accessions had no recorded use: SA was from South Africa while the location of reproduction was Holland; the geographical origin of CIM, CYUH, CIM2 are respectively China-Inner Mongolia, China-Yunnan and China-Shandong. Hemp seeds were kindly provided by dr. E.P.M. De Meijer from Hortapharm BV., Amsterdam, except for the accessions SA, CIM, CYUH, CIM2 (Table 1) that

were provided by dr R. Clarke from International Hemp Association (IHA) Amsterdam. Scarified seeds were sown in Petri dishes, seedlings were transplanted in pots and transferred in a climatic chamber at 20 °C under a light/dark cycle of 15 and 9 h respectively. DNA extraction from leaves and analysis Leaf samples from individual plants were collected after 26 days of growth and were shock-frozen in liquid nitrogen and stored at -80 °C until DNA isolation that was performed according to Piluzza et al. (2005). Two DNA bulks were prepared from each accession. Each bulk was obtained by mixing equal amounts of DNA from ten single plants. DNA polymorphisms were detected by using the Random Amplified Polymorphic DNA (RAPDs) method (Williams et al. 1990). According to Dulson et al. (1998), by means of RAPD it is almost always possible to determine the overall profile of a bulked sample from several individual plants, and hence the identification of variety-specific DNA bands. The 10-mers primers

Table 1 List of hemp accessions and related uses Acronymsa

Species

Accessions

CMTI

Cannabis sativa

Haze 45.2

Marijuana

CMTI2

Cannabis sativa

Haze H3

Marijuana

TAI TAI2

Cannabis sativa Cannabis sativa

91.171.18 9 93.171.2 92.172.9 9 93.171.2

Marijuana Marijuana Marijuana/hashish

Uses

AMC

Cannabis indica 9 sativa

White 9 91.1.1

AMC2

Cannabis indica 9 sativa

Red 9 Skunk pollen mix

Marijuana/hashish

AMC3

Cannabis indica 9 sativa

Blue 9 Skunk pollen mix

Marijuana/hashish

CAL

Cannabis indica 9 sativa

92.201.1 9 201.1001

Drug

AFG

Cannabis indica

92.26.11 9 A1 mix

Hashish

BOL

Cannabis sp.

Bolivian

Drug

SER

Cannabis sativa

cv. Novosadska (IHA 22)

Fiber

UNG

Cannabis sativa

cv. Kompolti (IHA 63)

Fiber

UNG2

Cannabis sativa

cv. Kompolti (IHA 6)

Fiber

CYU

Cannabis sp.

Yunnan 2 (IHA 2)

Fiber

CHO

Cannabis sp.

Hohhot (IHA 13)

Fiber

SA

Cannabis sp.

Kaas #4

Unknown

CIM

Cannabis sp.

Hohot #1

Unknown

CYUH

Cannabis sp.

#4

Unknown

CIM2

Cannabis sp.

#97

Unknown

a

Acronyms related to geographic origin of accessions (see ‘‘Materials and methods’’)

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utilised for DNA amplification were from Operon Technologies, Alameda, California; a previous screening allowed to select six primers suitable to show DNA polymorphism. The sequence of each primer is shown on Table 2, amplification and electrophoresis conditions were as described in Piluzza et al. (2005). Each DNA fragment generated was treated as a separate character and scored as a discrete variable, using 1 to indicate presence and 0 for absence. Accordingly, a rectangular binary data matrix was obtained and statistical analysis was performed using the NTSYS-pc (Rohlf 1992) statistical package. A pairwise similarity matrix was generated using Jaccard’s coefficient (Dunn and Everitt 1982) by means of SIMQUAL procedure of NTSYS-pc. The Jaccard’s coefficient (Sneath and Sokal 1973) was used as the association coefficient. This coefficient is the ratio of the number of positive matches (a) to the total number of characters (n) minus the number of negative matches (d) between any two entries: J ¼ a=ðn  dÞ Then, cluster analysis was performed (by means of SAHN procedure of NTSYS-pc) via unweighted pairgroup method using arithmetic average (UPGMA) to develop a dendrogram. The COPH and MXCOMP options were used to test for the existence of cluster using the computer program NTSYS. The analysis of molecular variance (AMOVA, Excoffier et al. 1992) was performed using GENALEX 6 (Peakall and Smouse 2006) to partition the total molecular variance between and within populations. Significance level was detected via permutation test (n = 1,000). Extracts preparation and chemical analysis Samples were extracted for the analysis of the cannabinoid components according to Brennelsen

(1984). A 200 mg of dried (40 °C, 24 h) pulverized sample was extracted with 1 ml methanol-chloroform (9:1 v/v) by sonication (BRANSON 3200) for 15 min and then in a centrifuge 2,000g for 5 min. The surnatant was utilized for chromatographic analysis. Analyses with HPLC were performed on a VARIAN 3090 HPLC with UV detector at 254 nm, using a RP WATERS SPHERISORB (4.6 mm 9 250 mm, 5 l S5 ODS2) column, in isocratic environment with a solution 8.64 g/l H3PO4 (30 %) and acetonitrile (70 %) at 254 nm. A cluster analysis for grouping the different accessions according to their THC content obtained from HPLC analysis, was performed by means of the NTSYS-pc (Rohlf 1992) statistical package. The SIMINT option was used to generate a similarity matrix based on DIST coefficient (Sokal 1961), the SAHN option was utilized to cluster the different accessions according to the UPGMA method. The COPH and MXCOMP options were used to test for the existence of clusters. Seed morphology For the SEM study, seeds of each accession were mounted directly on aluminium stubs and sputter coated with gold. For evaluation of uniformity, seed samples were placed on the stubs with their dorsal, ventral and lateral side upwards so that characteristic features of all different sides could be scanned and photographed using a DSM 962 Zeiss SEM. To achieve better resolution the accelerating voltage was 20 kV. The length (mm) and width (mm) of seeds was also measured on twenty seeds per accession. Seed of each accessions were weighted to complete the seed characterization with the thousand-seed weight.

Table 2 Selected primers and their sequences, total number of bands, number of polymorphic bands, range of DNA fragments produced Primers

Sequence

OPA-20

50 -GTTGCGATCC-30

6

5

2,027–400

OPB-08

50 -GTCCACACGG-30

12

12

2,040–300

OPG-17

50 -ACGACCGACA-30

15

14

2,030–564

OPG-19

50 -GTCAGGGCAA-30

10

8

2,030–500

OPH-05

5 -AGTCGTCCCC-3

0

12

11

1,800–400

OPH-08

50 -GAAACACCCC-30

12

11

2,060–700

0

Total bands

Polymorphic bands

Amplified DNA fragments range (bp)

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Results and discussion Six decamer primers were selected for the production of polymorphic bands able to discriminate among the accessions. The total number of bands produced by each primer, the number of polymorphic bands and the range of the amplified DNA fragments are shown in Table 2. The six random primers revealed the presence of polymorphisms in the amplified DNA fragments in the range from 2,060 to 300 bp. A maximum of 15 bands, 14 of which polymorphic were produced by the primer OPG-17. Analysis of molecular variance (AMOVA) was used to partition the total genetic variance within and among accessions. The

Table 3 Summary of AMOVA results Source

Df

SS

MS

Est. Var.

Among populations

18

335.789

18.655

7.920

74 \0.001

Within populations

19

53.500

2.816

2.816

26 \0.001

Total

37

389.289

10.735

%

P

100

SS sum of squares, MS mean squares, Est. Var. estimated variability; %: proportion of genetic variability; P significance level Fig. 1 UPGMA clustering using Jaccard coefficient of association on DNA amplification patterns of the hemp accessions

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significance of the variance components was tested by calculating their probabilities based on 999 random permutations. AMOVA (Table 3) revealed 74 % variation among accessions and 26 % within accessions, all AMOVA variation was highly significant (P \ 0.001). The cluster analysis performed by using Jaccard coefficient of association across the selected primers, is represented in the dendrogram shown on Fig. 1, where, according to the adopted software package, ‘‘1’’ corresponds to the maximum genetic similarity. The cophenetic correlation (that is the correlation between the cophenetic value matrix and the matrix upon which the clustering was based) was used as a measure of the goodness of fit for the cluster analysis. The matrix correlation value of r = 0.943 was found, it indicates a very good fit between the similarity values implied by the phenogram and those of the original similarity matrix. As no standard objective procedure exists for selecting the number of clusters, the distance among clusters at successive step was used as a guide. Following the advise by Afifi and Clark (1984) grouping of genotypes was stopped when the differences in distance between steps made a sudden jump. Eight groups were found cutting the dendrogram at the value 0.50. A first cluster includes (CMTI) and (CMTI2) sister lines from Haze, a multihybrid fairly true breeding cultivar in which

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landraces are combined from Colombia, Mexico, Thailand and Southern India, the ancestral landraces are all C. sativa types used for marijuana production. A second cluster includes (TAI) and (TAI2) the full-sib F1 progenies from plants selected from Thai landraces (C. sativa type for marijuana production). A third cluster includes (AMC) the full sib F1 progeny from two plants selected from ‘‘Skunk’’, a true-breeding indica 9 sativa hybrid cultivar based on landraces from Afghanistan (indica, hashish type), Mexico and Colombia (C. sativa, marijuana type) and (AMC2) and (AMC3) the F1 half sib progenies from ‘‘Skunk’’, it also includes (CAL) the full sib F1 progeny from plants selected from ‘‘Early Californian’’ a cross-progeny from various indica and sativa drug strains. A fourth cluster includes one of the two bulked samples from accession (AMC), it differed from the other bulk of ten plants that was included in another cluster. A fifth cluster includes (AFG) the half sib progeny from selected parents from an Afghani landrace, pure indica type traditionally used for hashish production. All the fiber types under study were included in the sixth cluster, a Serbian fiber cultivar (SER) selected from ‘‘Fleischmann hemp’’, an Hungarian fiber cultivar (UNG) selected from ‘‘Fleishmann hemp’’ and (UNG2) the F1 seed of the same cultivar grown in China (CYU) a Chinese fiber landrace from Yunann region and (CHO) a Chinese fiber landrace from Inner Mongolia region. In the same cluster it is also included (BOL) a Cannabis sp. accession recorded as a landrace from Bolivia for drug use. A seventh cluster includes Cannabis sp. accessions from South Africa (SA), from China (Shandong Province) (CIM2) and Yunnan province (CYUH), all accessions with unknown use. The eighth cluster includes accessions from China (Inner Mongolia) (CIM) and the other bulk sample of accession (CIM2) from Shandong Province, all with unknown use. The six selected decamer primers were useful to discriminate among the accessions. The cluster analysis of molecular data according to the UPGMA method, discriminate the fiber and drug types among the different accessions and grouped the accessions in accordance to the classification, origin and use. RAPD markers allow random sampling of markers over whole genomic DNA and do not require any previous information on the genome of the organism under investigation respect to other molecular markers as AFLP, STR. According to

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Mandolino and Ranalli (2002) the use of RAPD markers coupled with the use of specific population analysis softwares can describe the materials examined with good accuracy and agreement with the known characteristics of the varieties. Gillan et al. (1995) reported the differentiation of C. sativa samples with the use of RAPDs when HPLC analysis was inefficient. Faeti et al. (1996) assessed genetic diversity of C. sativa cultivars/accessions (from 5 European countries, and one accession from Korea) by using RAPD markers and reported high levels of polymorphism. In a study of genetic structure and degree of variability of six C. sativa varieties via RAPD markers, it was reported that 5 varieties were properly identified with the scored loci (Forapani et al. 2001). Jagadish et al. (1996) with RAPD analysis distinguishes between C. sativa samples from distinct Australian sources and they concluded that RAPD markers are useful for forensic studies of plant materials. Pinarkara et al. (2009) reported a study of RAPD analysis of psychoactive type Cannabis samples from 29 different locations of Turkey. Such authors studied Australian accessions (Jagadish et al. 1996), Italian and French cultivars (Forapani et al. 2001), accessions from European countries and one accession from Korea (Faeti et al. 1996). In this paper, we studied hemp accessions originating from Colombia, Mexico, California, Bolivia, Serbia, Hungary, Thailand, Afghanistan, south Africa and different regions of China: either fiber or drug types. According to De Backer et al. (2009), Cannabis can be considered as the most controversial plant in our society: next to the important medical use, Cannabis is also the most frequently consumed drug of abuse in Europe. It has been estimated that about four million European adults (&1 % of all 15-to 64-year-olds) are using Cannabis each day or almost daily; and that around 23 million Europeans (&7 % of all 15-to 64-year-old) have consumed Cannabis at least one time during the past years. The whole HPLC profiles of all cannabinoids were obtained within 55 min of elution. Taking into account HPLC data, two retention time (RT) intervals might be identified in order to classify fiber hemps (RT 7–14 min) and drug hemps (RT 38–48 min), respectively. The chromatograms obtained from fiber types of hemp (UNG, UNG2, CYU, CHO, SER) are characterized by peaks in the first 15 min of elution, an example of such pattern is shown on Fig. 2, while in such time interval, almost no peaks were detected for

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Fig. 2 Chromatogram obtained from a fiber type of hemp

Fig. 3 Chromatogram obtained from a drug type of hemp

drug types (CMTI, CMTI2, TAI, TAI2, AMC, AMC2, AMC3, AFG, BOL). After the first 38 min of elution, peaks appear for the drug types, an example on Fig. 3. The accessions (SA, CIM, CYUH,) provided by the IHA without indication about uses, were characterized

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by the presence of peaks with retention times (RTs) typical of drug types and the lack of dominant peaks in the RTs typical of fiber types. Lehmann and Brenneisen (1995) obtained whole HPLC profiles of all cannabinoids within 55 min of elution. They found the

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dominant key cannabinoids THC and THCA-A in the drug type at the RT between 40 and 46 min; while they found the major cannabinoids CBD and CBDA in the fiber type at the RT between 30 and 32 min. The clustering of the different hemp accessions obtained utilizing the THC content is shown on Fig. 4. Cutting the dendrogram at the value 10, four clusters are evident. The first cluster includes four Cannabis

Fig. 4 UPGMA clustering of hemp accessions based on THC data processed by DISTcoefficient of association

sativa drug types (CMTI, CMTI2, TAI and TAI2), three Cannabis sp. accessions (SA, CYUH, CIM) that were provided without record about uses and the Cannabis sp. accession (BOL) that is an unselected drug type landrace from Bolivia. The second cluster includes three Cannabis indica 9 sativa (AMC, AMC2, AMC3) and the C. indica accession AFG, all for drug uses. A third cluster groups two C. sativa fiber type accessions (SER and UNG) a Cannabis sp. accession without record about uses (CIM2) and a C. indica 9 sativa accession CAL for drug use. A fourth cluster includes two Chinese Cannabis sp. fibre landraces (CYU and CHO) and the accession UNG2 (F1 seed harvested from the original Hungarian fiber cultivar grown in China). The cophenetic correlation (that is the correlation between the cophenetic value matrix and the matrix upon which the clustering was based) was used as a measure of the goodness of fit for the cluster analysis. The matrix correlation value of r = 0.84 was found, it indicates a good fit between the similarity values implied by the phenogram and those of the original similarity matrix. Seeds varied significantly in size (Table 4). The seed length of the accessions ranged from 3.44

Table 4 Seed dimensions and weight of the hemp accessions Accessions acronyms

Uses

Length (mm)

Width (mm)

Thousand seed weight (g)

CMTI

Marijuana

3.66

2.78

12.28

CMTI2

Marijuana

3.44

2.77

11.26

TAI TAI2

Marijuana Marijuana

4.28 4.17

3.15 3.09

14.45 13.87

AMC

Marijuana/hashish

3.81

2.78

10.67

AMC2

Marijuana/hashish

4.05

3.13

14.12

AMC3

Marijuana/hashish

4.03

3.06

13.02

CAL

Drug

4.29

3.29

14.17

AFG

Hashish

3.93

3.29

BOL

Drug

4.59

3.66

16.6

SER

Fiber

4.76

3.73

21.33

UNG

Fiber

4.93

3.85

20.64

UNG2

Fiber

4.91

3.85

18.97

CYU

Fiber

4.69

3.61

21.13

CHO

Fiber

5.68

4.14

26.89

SA

Unknown

4.88

3.85

16.15

CIM

Unknown

5.50

4.0

26.22

CYUH

Unknown

5.12

3.74

18.40

CIM2 LSD

Unknown

4.92 0.39

3.67 0.30

16.5 1.12

9.72

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Genet Resour Crop Evol (2013) 60:2331–2342 b Fig. 5 Scanning electron microscopy of seeds of Cannabis

indica drug (A, B, C, D); Cannabis indica 9 sativa drug (E, F, G, H); Cannabis sativa fiber type (I, L, M, N); Cannabis sativa drug type (O, P, Q, R); Cannabis sativa fiber type (S, T, U, V). Micrographs of seeds OA, E, J, O, S; micrographs of seed surface OB, F, L, P, T (91,000) and OC, G, M, Q, U (9120); micrographs of hilum D, H, N, R

(CMTI2, drug type) to 5.68 mm (CHO, fiber type), width from 2.77 (CMTI2) to 4.14 mm (CHO). The thousand seed weight ranged from 9.72 (AFG, drug type) to 26.89 g (CHO, fiber type). Seeds of the fiber types were bigger. The study of seed surface by means of SEM, revealed micro morphological characters, which exhibited interesting variation that could be important for the identification (Fig. 5). Seed surface pattern could be categorized into smooth (Fig. 5B, C), more or less papillate (Fig. 5F, L, P, T). The hilum is located in an apical position and is almost circular or oval. Seed morphology has provided useful characteristics for the analysis of taxonomic relationships in a wide variety of plant families (Buss et al. 2001; Zhang et al. 2005; Gontcharova et al. 2009). The importance of ultrastructural pattern analysis of the seed surface observed under the SEM has been well recognized as a reliable approach for assessing phenetic relationship and identification of species or taxa (Koul et al. 2000; Yoshizaki 2003; Javadi and Yamaguchi 2004; Gandhi et al. 2011; De Queiroz et al. 2013). To our knowledge, there is no literature available about hemp seed micromorphology detected by means of SEM.

Conclusion DNA polymorphisms can be useful for the obtainment of information at an early stage of growth about the hemp type, fiber or drug. The DNA polymorphisms generated by the selected primers appear to be quite effective in resolving Cannabis type determinations. The clustering of hemp accessions by means of DNA polymorphism data, showed four groups of drug type hemp and one group of fiber type hemp, only the unselected drug type landrace (BOL) appears to be misplaced within the fiber types. The Cannabis sp. that did not have a recorded use, are separated from all the others and grouped in two clusters. The clustering of accessions according to THC content gave four groups, two included fiber types of hemp and two

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included drug types of hemp, except for the CAL accession that although recorded as drug type was included among the fiber types. Three of the four accessions with no recorded use (SA, CYUH, CIM2) grouped together with the drug types, while a fourth accession (CIM2) grouped with the fiber types. The grouping of the accessions with no recorded use was not in agreement with the grouping based on molecular data. Micromorphological features of seed surface and seed sizes are useful in differentiating drug and fiber types and could be helpful to discriminate in doubtful cases. Acknowledgments Thanks are due to dr. E.P.M. De Meijer from Hortapharm B.V., Amsterdam, and Dr. R. Clarke from International Hemp Association (IHA) Amsterdam for kindly providing the hemp accessions.

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