Art Genomic Evidence That Governmentally Produced Cannabis Sativa Poorly Represents Genetic Variation Available in State Markets

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Genomic evidence that governmentally produced Cannabis sativa

poorly represents genetic variation available in state markets
1 Daniela Vergara1*, Ezra L. Huscher1+, Kyle G. Keepers1+, Rahul Pisupati2, Anna L.

2 Schwabe3, Mitchell E. McGlaughlin3, and Nolan C. Kane1*
4 1Kane Laboratory, Department of Ecology and Evolutionary Biology, University of Colorado
5 Boulder, Boulder, Colorado, USA
6 2 Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Dr.
7 Bohr-Gasse 3, 1030 Vienna, Austria
8 3 University of Northern Colorado, School of Biological Sciences, Greeley, CO 80639, USA.
9 +Authors that contributed equally
10
11 * Correspondence:
12 [email protected] or [email protected]
13 Keywords: cannabinoids, copy number variation, genome diversity, hemp, repetitive genomic
14 content, marijuana, NIDA, THC
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bioRxiv preprint doi: https://doi.org/10.1101/2021.02.13.431041; this version posted February 14, 2021. The copyright holder for this preprint
available under aCC-BY Governmental Cannabis
4.0 International license . lacks genomic variation
26 Abstract
27 The National Institute on Drug Abuse (NIDA) is the sole producer of Cannabis for research
28 purposes in the United States, including medical investigation. Previous research established
29 that cannabinoid profiles in the NIDA varieties lacked diversity and potency relative to the
30 Cannabis produced commercially. Additionally, microsatellite marker analyses have established
31 that the NIDA varieties are genetically divergent form varieties produced in the private legal
32 market. Here, we analyzed the genome of multiple Cannabis varieties from diverse lineages
33 including two produced by NIDA, and we provide further support that NIDA’s varieties differ
34 from widely available medical, recreational, or industrial Cannabis. Furthermore, our results
35 suggest that NIDA’s varieties lack diversity in the single copy portion of the genome, the
36 maternally inherited genomes, the cannabinoid genes, and in the repetitive content of the
37 genome. Therefore, results based on NIDA’s varieties are not generalizable regarding the effects
38 of Cannabis after consumption. For medical research to be relevant, material that is more widely
39 used would have to be studied. Clearly, having research to date dominated by a single, non-
40 representative source of Cannabis has hindered scientific investigation.
41
42 Introduction
43 Public perception of recreational and medicinal Cannabis use has shifted, with Cannabis
44 derived products quickly becoming a multibillion-dollar legal industry. However, the National
45 Institute on Drug Abuse (NIDA), a United States (U.S.) governmental agency, continues to be the
46 sole producer of Cannabis for research. Additionally, high-THC producing Cannabis continues to
47 be classified as a Schedule I drug, along with heroin, LSD, and ecstasy, according to the DEA (DEA
48 2020). This schedule I classification restricts the acquisition of Cannabis from the private
49 markets, making NIDA the only federally legal source for research. In addition to this limitation,
50 research on Cannabis requires a multitude of permits and supervision (Nutt et al. 2013;
51 Hutchison et al. 2019). However, the medical and recreational Cannabis industry in North
52 America are predicted to grow to 7.7 and 14.9 billion dollars, respectively, by late 2021
53 (Hutchison et al. 2019).
54 Cannabis sativa L. (marijuana, hemp) is an angiosperm member of the family Cannabaceae
55 (Bell et al. 2010). It appears to be one of the oldest domesticated plants, utilized by numerous
56 ancient cultures, including Egyptians, Chinese, Greeks, and Romans (Li 1973, 1974; Russo 2007).
57 This versatile plant has many known uses, including fiber for paper, rope and clothing, oil for
58 cooking and consumption, and numerous medicinal applications. The plant produces secondary
59 metabolites known as cannabinoids that interact with the human body in physiological (Russo
60 2011; Swift et al. 2013; Volkow et al. 2014) and psychoactive (Russo and McPartland 2003;
61 ElSohly and Slade 2005) ways. Cannabinoids are terpenoid compounds (Zwenger and Basu
2
62 2008) that are concentrated in the trichomes of the female flowers (Sirikantaramas et al. 2005).
63 The remarkable properties of cannabinoids are partly responsible for driving the growth of the
64 thriving Cannabis industry. Two of the main cannabinoids-- Δ-9-tetrahydrocannabinolic acid
65 (THCA) and cannabidiolic acid (CBDA)--when heated are converted to the neutral forms Δ-9
66 tetrahydrocannabinol (THC) and cannabidiol (CBD), respectively (Russo 2011). The most well-
67 characterized enzymes responsible for the production of these cannabinoids in the plant are Δ-
68 9-tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS).
69 Despite the regulatory hurdles and the limited scope of contributions from the U.S.
70 government, Cannabis research is growing at a rapid pace (Vergara et al. 2016; Kovalchuk et al.
71 2020) and U.S. scientists have made significant advances in Cannabis research from multiple
72 disciplines. Researchers in the U.S. have produced one of the most complete publicly available
73 Cannabis genome assemblies to date, along with the locations of the cannabinoid family of genes
74 in the genome (Grassa et al. 2018). However, oversight is needed to assure the quality and
75 consistency of Cannabis testing across laboratories (Jikomes and Zoorob 2018). Regulation and
76 supervision will allow for a deeper understanding about all of the compounds produced by the
77 plant particularly minor cannabinoids which are not always measured (Vergara et al. 2020) and
78 that have multiple genes related to their production with complex interactions (Vergara et al.
79 2019). This is particularly important because medical Cannabis use has outpaced its research
80 (Hutchison et al. 2019). Collaborative research between American academic institutions and
81 private companies has shown that the cannabinoid content and genetic profile of Cannabis
82 provided by NIDA is not reflective of what consumers have access to from the private markets,
83 therefore research with these varieties is discordant (Vergara et al. 2017; Schwabe et al. 2019).
84 In 2017, we compared the cannabinoid chemotypes from the Cannabis produced in the
85 private market to the chemotypes from the governmentally produced Cannabis for NIDA by the
86 University of Mississippi (Vergara et al. 2017). We found that NIDA’s Cannabis lacked potency
87 and chemotypic variation and had an excess of cannabinol (CBN), which is a degradation product
88 of THC. The cannabinoid diversity from the governmentally produced Cannabis was a fraction of
89 that from the private markets. A study using microsatellite markers also showed that NIDA’s
90 Cannabis was genetically different from commercially available recreational and medical
91 varieties. This study concluded that results from research using flower material supplied by
92 NIDA may not be comparable to consumer experiences with Cannabis from legal private markets
93 (Schwabe et al. 2019).
3
94 Here, we present results of analysis to further examine the genetic diversity in

95 governmentally produced Cannabis. We acquired DNA from two NIDA-produced samples which
96 had been previously analyzed using ten variable microsatellite regions (Schwabe et al. 2019).
97 After sequencing, we compared their overall genomic diversity to that of previously sequenced
98 varieties including hemp and marijuana-types (Lynch et al. 2016; Vergara et al. 2019). We report
99 here the genomic characteristics of the two NIDA samples, including overall genetic variation, as
100 well as genetic variation within the cannabinoid family of genes, the maternally inherited
101 organellar genomes (mitochondrial and chloroplast), and the repetitive genomic content. We
102 compare this diversity to the publicly available genomes from other Cannabis lineages within the
103 species, to characterize the relationships with other well-studied lineages.
104
105 Materials and Methods

106 NIDA’s samples
107 Bulk Cannabis supplied for research purposes is referred to as “research grade marijuana”
108 by NIDA and is characterized by the level of THC and CBD (NIDA 2016). They offer 12 different
109 categories of Cannabis for research that vary in the levels of THC (low < 1%, medium 1-5 %, high
110 5-10 %, very high >10%) and CBD (low < 1%, medium 1-5%, high 5-10%, very high > 10%)”. The
111 high THC NIDA sample (Table S1) has an RTI log number 13494-22, reference number SAF
112 027355 and the high THC/CBD has an RTI log number 13784-1114-18-6, reference number SAF
113 027355. DNA from both samples was extracted by (Schwabe et al. 2019) and provided to the
114 University of Colorado Boulder. These two samples were sequenced using standard Illumina
115 multiplexed library preparation protocols as described in (Lynch et al. 2016) which yielded to
116 an approximate coverage of 17-20x (Table S1).
117
118 Genome assembly, whole genome libraries, and nuclear genome exploration
119 We aligned sequences from 73 different C. sativa plants to the previously developed
120 CBDRx assembly Cs10 (Grassa et al. 2018). These genomes were sequenced using the Illumina
121 platform by different groups (Table S1) and are, or will be, publicly available on GenBank. For
122 detailed information on sequencing and the library preparation of the 57 genomes sequenced by
123 our group at the University of Colorado Boulder please refer to Lynch et al., 2016. The remaining
4
124 16 genomes were sequenced and provided by different groups (Table S1), however most of these
125 genomes have been previously used in other studies (Lynch et al. 2016; Vergara et al. 2019).
126 We aligned the 73 libraries to the CBDRx assembly using Burrows-Wheeler alignment
127 (ver. 0.7.10-r789; Li and Durbin 2009), then calculated the depth of coverage using samtools (ver.
128 1.3.1-36-g613501f; Li et al. 2009) as described in Vergara et al. (2019). We used GATK (ver. 3.0)
129 to determine single nucleotide polymorphisms (SNPs). We filtered for SNPs lying in the single-
130 copy portion of the genome (Lynch et al. 2016) which resulted in 7,738,766 high-quality SNPs.
131 The single-copy portion of the genome does not include repetitive sequences such as
132 transposable elements or microsatellites. Subsequently, we were then able to estimate the
133 expected coverage at single-copy sites as in Vergara et al. (2019). We performed a STRUCTURE
134 analysis (ver. 2.3.4; Pritchard et al. 2000) with K=3 in accordance with previous research (Sawler
135 et al. 2015; Lynch et al. 2016). With these STRUCTURE results, we then classified the different
136 varieties into four different groupings: Broad-leaf marijuana type (BLMT), Narrow-leaf
137 marijuana-type (NLMT), Hemp, and Hybrid (Table S2). Hybrid individuals had less than 60%
138 population assignment probability to a particular population. We found 12 individuals in the
139 BLMT group, 16 in the Hemp group, 14 in the Hybrid group, and 31 in the NLMT group. We then
140 used SplitsTree (ver. SplitsTree4; Huson 1998) to visualize the relationships between the 73
141 individuals, VCFtools (ver. 4.0; Danecek et al. 2011) to calculate genome wide heterozygosity as
142 measures of overall variation, and PLINK (ver. 1.07; Purcell et al. 2007) for a principal
143 component analysis (PCA).
144 Cannabinoid gene exploration

145 Using BLAST, we found 12 hits for putative CBDA/THCA synthase genes in the CBDRx
146 assembly (Table S3) with more than 80% identity and an alignment length of greater than
147 1000bp. For this BLAST analysis, we used the CBCA synthase (Page and Stout 2017), the THCA
148 synthase with accession number KP970852.1, and the CBDA synthase with accession number
149 AB292682.1.
150 We estimated the gene copy-number (CN) for the cannabinoid genes (Vergara et al. 2019)
151 and calculated summary statistics of the CN for each of the 12 genes by variety (Table S1).
152 Differences in the estimated gene CN between the cultivars for each of the 12 cannabinoid
153 synthases gene family were determined using one-way ANOVAs on the CN of each gene as a
5
154 function of the lineages (BLMT, Hemp, Hybrid, NLMT), with a later post hoc analysis to establish
155 one-to-one group differences using the R statistical platform (Team 2013).
156 Maternally inherited genomes

157 We used the publicly available chloroplast (Vergara et al. 2015) and mitochondrial (White
158 et al. 2016) genome assemblies to construct haplotype networks using PopART (ver. 1.7; Leigh
159 and Bryant 2015) using only variants with a high quality score in the variant call file (VCF). The
160 chloroplast and mitochondrial haplotype networks comprised 508 and 1,929 SNPs, respectively.
161
162 Repetitive genomic content

163 We used Repeat Explorer (ver.2; Novak et al. 2010)to determine the repeat content in 71
164 of the 73 genomes (Pisupati et al. 2018). We decided to exclude ‘Jamaican Lion’ (NLMT) and
165 ‘Feral Nebraska’ (hemp) genomes due to low-quality reads that led to dubious results. We
166 estimated the repetitive content of the genome and annotating repeat families using custom
167 python scripts (https://github.com/rbpisupati/nf-repeatexplorer.git).
168
169 Results
170 Nuclear genome exploration
171 Our analysis of the nuclear genome used 7,738,766 high-quality SNPs from the inferred
172 single-copy portion of the genome. The STRUCTURE analysis (Figure 1, top panel) shows the
173 population assignment probabilities for all 73 different varieties including both of NIDA’s
174 varieties. This analysis established that NIDA’s samples cluster with both the hemp and NLMT
175 groupings, with less than 60% in either group and therefore we categorized them as ‘hybrid’
176 (Table S2). This classification led to 12, 16, 14, and 31 individuals from the BLMT, Hemp, Hybrid,
177 and NLMT groups, respectively. In other words, the 12 individuals that are part of the Hemp
178 group had a population assignment probability of more than 60% to this group, as well as those
179 assigned to the NLMT or BLMT groupings. However, those individuals with a probability of less
180 than 60% to a particular population were assigned to the ‘hybrid’ category, which includes both
181 of NIDAs samples. We color-code the hemp individuals in orange, the NLMT in blue, BLMT in
182 purple ,and the hybrid individuals in gray.
6
K=3
1.0
0.8
Population Assignment Probability
0.6
0.4
0.2
0.0
EuroOil_2
Mexican_E
Kandy_Kush
White_widdow
Super_Lemon_Haze
Auto_AK47
Harlequin
Dr._Grinspoon
MG_SVG_OG
Chocolope
Chocolope
US031
Durban_Poison
NIDA_THC2
Hindu_Kush
Afghan_Kush
Feral_Nebraska
Jamaica_Lion
Chemdawg
Girl_Scout_Cookies
Skunk_1
Somali_Taxi_Cab
Chinese_hemp
Carmagnola
Dagestani_hemp
Green_Crack
Recon
G13
Purple_Kush
Feral_Kansas
Feral_Nebraska
NIDA_THC_CBD2
Kunduz
Carmagnola
Hawaiian
Finola
Liberty_Haze
Afghan_Kush
Afghan_Kush
Durban_Poison
Rocky_Mountain_Bluberry
Grape_Ape
Colombia_Rio_Negro
bba_Kush
R4
Afghan_Kush
Alaskan_Thunderfuck
OG_Kush
Carmagnola
OG_Kush
Afghan_Kush
Kompolti
Lebanese
Tora_Bora
Original_Sour_Diesel
LA_confidential
Afghan_Kush
Cannatonic
Tunas_Banana
Blue_Dream
Carmagnola
Tangerine_Haze
Sievers_Infinity
Blueberry_DJ
Carmagnola
Carmagnola
Kompolti
Low_Ryder
Jack_Herer
Maui_Waui
Golden_Goat
0.2
BLMT
Hemp
0.1 Hybrid
PC2 (12.16% explained variation)
NIDA
NLMT
0.0
- 0.1
- 0.2
- 0.2 - 0.1 0.0 0.1
183
184 Figure 1. STRUCTURE and Principal Component Analyses. Top panel: Proportion of each color in the bar
185 indicates the probability of assignment to Hemp (orange), NLMT (blue), or BLMT (purple), groups. Both of NIDA’s
186 strains outlined with black margins are assigned to both NLMT and hemp groups with less than 60% probability,
187 and therefore we assigned them to the Hybrid group. Bottom panel: The two NIDA varieties in green cluster with
188 each other and away from other varieties.
189
7
190 In addition to clustering probability results (Figure 1 top panel) from STRUCTURE, we
191 colored the varieties in the PCA (Figure 1 bottom panel) and SplitsTree (Figure 2) according to
192 their population assignment probability. The first two principal components in the PCA explain
193 28.71% of the variation (Figure 1 bottom panel), and the two NIDA varieties cluster together,
194 also seen in the SplitsTree analysis (Figure 2). Both the PCA and SplitsTree indicate high genetic
195 similarity between the NIDA strains, and neither of them cluster with any other strains.
196
197 Figure 2. SplitsTree graph. Genetically similar individuals cluster together, such as the NIDA cluster, Afghan
198 Kush cluster, and Carmagnola cluster. NIDA’s varieties highlighted in green. Hemp, NLMT, and BLMT shown in
199 orange, blue, and purple respectively.
200 The hybrid group which contains NIDA’s varieties shows the widest range of
201 heterozygosity (μ= 0.131; s.d= 0.0545) in the single-copy portion of the genome. However, it is
202 not significantly different from any other group (Figure 3A). This wide range of heterozygosity
203 in the hybrid group is expected given that we are grouping individuals that do not belong one
204 particular genetic group but rather have some assignment probability to two or three genetic
205 groups. Therefore, varieties which are not related to each other, or that belong to more than one
8
206 group are found in the hybrid category. This is probably the reason why it is the highest of all
207 other groups (hemp: μ=0.0817; s.d=0.0352; BLMT μ=0.0959; s.d=0.0405; NLMT μ=0.112;
208 s.d=0.0411).
209
210 Figure 3. Genome wide heterozygosity. The hemp lineage differs significantly from the Hybrid grouping with a
211 P<0.03. NIDA’s two varieties are presented within the hybrid grouping by two green triangles
212
213 Cannabinoid gene exploration

214 Independent of which synthase we used for the BLAST analysis (either THCA, CBDA, or
215 CBCA), the BLAST results delivered the same hits on the CBDRx assembly with different percent
216 identities. Based on percent-identity scores, our BLAST results identified a hit in the CBDRx
217 assembly that appears to code for cannabichromenic acid synthase (CBCAS), and one that
9
218 possibly codes for CBDAS, but we did not find a hit for THCAS (Table S3). After calculating the
219 copy number variation, we found that most groups have one copy of the CBCAS gene (BLMT
220 μ=1.38; s.d=1.1; Hemp μ=1.88; s.d=2.15; Hybrid μ=1.56; s.d=1.33 and NLMT μ=1.44; s.d=2.57).
221 Despite the hemp group having the widest range, no group significantly differed from each other
222 (Figure 4A). For the CBCAS genes, the first NIDA sample has an estimated copy number of 0.37
223 and the second variety of 0.34. These values are on the lower side of the copy number
224 distribution. We include the copy number variation of an unknown cannabinoid, which was the
225 only other locus that had significant differences between groups (Figure 4B).
226 The copy number variation for the CBDAS gene was higher, ranging from 1-3 or more
227 copies (BLMT μ=3.24; s.d=1.23; Hemp μ=1.57; s.d=1.04; Hybrid μ=2.59; s.d=1.17 and NLMT
228 μ=2.97; s.d=3.15). The hemp group on average has a lower copy number of these genes, which is
229 significantly different from every other group (Figure 4C). For the CBDAS genes, the first NIDA
230 variety has an estimated copy number of 2.35 and the second one of 2.55. These copy number
231 estimates are close to the mean and median values.
10
232
233 Figure 4. Copy Number Variation in cannabinoid genes. The estimated copy number of the CBCAS-like genes
234 (A) is not different between groups despite the hemp lineage having the widest range. Another unknown
235 cannabinoid locus (B) shows significant differences between hemp and the other groups at the P< 0.001 level. The
236 hemp lineage also differs significantly with a P< 0.01 from all of the other lineages in the estimated copy number of
237 CBDAS-like genes (C). NIDA’s two samples are presented within the hybrid grouping by two green triangles.
11
238 Maternally inherited genomes

239 We analyzed both the chloroplast (Figure 5A) and mitochondrial (Figure 5B) haplotype
240 networks. The chloroplast haplotype network (Figure 5A) contains eight haplotypes, with a
241 common haplotype (I) that comprises 58 individuals (79%). Most of the individuals in the
242 haplotypes that diverge from the main haplotype (haplotypes II, V, VI) are hemp types. Both of
243 NIDA’s varieties are part of the main haplotype (I).
244 The mitochondrial haplotype network has a common haplotype with 60 individuals
245 (82%), and five additional haplotypes which are mostly comprised of hemp individuals (Figure
246 5B). As with the chloroplast, both of NIDA’s varieties are part of the common haplotype group.
247 The haplotype group for each individual for both the chloroplast and mitochondria is given in
248 columns 11 and 12 in table S1.
A. Chloroplast B. Mitochondria
NLMT
VIII Euro Oil BLMT
II N =8
Hemp Dagestani Hemp
Hybrid Lebanese
Feral Kansas
Sievers Infinity
Feral Nebraska
III N = 2 Feral Nebraska
Kompolti
Finola
USO31
III N = 2
Kompolti
II N = 8 VII Chinese Hemp

Dagestani Hemp
Lebanese
Feral Kansas
Sievers Infinity
Feral Nebraska
Feral Nebraska
Finola
USO31 VI
Euro Oil
I
N = 58 I
V Jamaican Lion N = 58
VI R4
IV Jamaican Lion
249 IV Cannatonic V Chinese Hemp

250 Figure 5. Chloroplast (A) and Mitochondrial (B) haplotype networks. Both haplotype networks are similar
251 with a common haplotype shared by most individuals (79% and 82% for the chloroplast and mitochondria
252 respectively) and smaller haplotypes that differ slightly, mostly comprised of hemp individuals.
253
254 Repetitive genomic content

255 We found that the 71 genomes analyzed had similar repetitive content in their genomes
256 (BLMT μ=62.9%; s.d=2%; Hemp μ=61.2%; s.d=2.6%; Hybrid μ=62.8%; s.d=2% and NLMT
12
257 μ=62.9%; s.d=3% ) with few outliers (Figure 6). The NLMT had the most variation in the repeat
258 fraction ranging from 58.6% to 70%. Both NIDA samples (showed as triangles in Figure 6) had
259 genome repeat fractions of 61.1%. As showed previously, the majority of repetitive content in
260 Cannabis is composed of Long Terminal Repeats (LTR) elements (Ty1 copia and Ty3 gypsy)
261 (Supplementary figure 1).
262
263 Figure 6. Repetitive Genomic content. The estimated repetitive genomic content by group which does not differ
264 significantly between groups. NIDA’s two varieties are presented within the hybrid grouping by two green
265 triangles.
266
267 Discussion
268 In this study, we analyzed the genomes of two Cannabis samples produced by the sole legal
269 provider of Cannabis for research in the U.S., the National Institute on Drug Abuse (NIDA). We
270 compared these two samples to the genomes of 71 commercially available varieties, many of
13
271 which are medicinally or recreationally consumed by the general public. A previous study has
272 shown that Cannabis provided by NIDA lacks diversity and cannabinoid potency compared to
273 commercially available Cannabis (Vergara et al. 2017), and microsatellite marker analysis also
274 shows that these differences extend to the genetic level (Schwabe et al. 2019). The results of this
275 study concur with previous studies that NIDA-produced Cannabis fundamentally differs from
276 Cannabis commonly consumed by the public.
277 Our whole-genome exploration suggests that the samples from NIDA are very similar to
278 each other, and not divergent to all other varieties in our analysis (Figures 1 and 2), including
279 the varieties commonly used for recreational and medical purposes (Figure 2). Therefore, the
280 samples from NIDA seem to be distantly related to those that are publicly available for
281 consumption.
282 Even though the two samples supplied by NIDA have high heterozygosity (Figure 3, Table
283 S1), they are comparable to other varieties from the hybrid group and from the NLMT group. The
284 high heterozygosity of both the samples from NIDA could be due to recent outcrossing, and
285 perhaps a recent hybrid origin. However, because we only sampled two individuals, this may not
286 represent the overall heterozygosity of all of the varieties produced for NIDA. Still, as previously
287 stated, research on the chemotypic variation of NIDA’s varieties show the limited cannabinoid
288 diversity (Vergara et al. 2017), supporting the possibility that these two samples are recent
289 hybrids and not bred for their chemotypic profiles including cannabinoids.
290 The copy number of the cannabinoid genes from the NIDA samples in some cases they fall
291 under the median (Figure 4A), above the median (Figure 4B) or near the median (Figure 4C).
292 However, there are some varieties that have up to 13 copies of some genes (Table S1), in
293 agreement with previous reports (Vergara et al. 2019). Additionally, in the maternally inherited
294 genomes, both NIDA samples have common haplotypes compared to other varieties in the
295 analysis, supporting recent research on the mitochondrial genome diversity in Cannabis (Attia et
296 al. 2020). Finally, the repetitive content in the samples from NIDA is comparable to that from
297 other varieties (Figure 6), which is mostly still unknown (Figure S1).
298 Although the NIDA material used for research does not represent the full range of genetic
299 variation, the results presented suggest that the cannabinoid synthase genes may be similar in
300 many respects to more widely used material. However, the lack of similarity at many other parts
301 of the genome, apparent in the genetic clustering illustrated in Figure 1, may help to explain why
14
302 the chemistry of NIDA material is so different (Vergara et al. 2017). Differences in cultivation,
303 storage, and processing may also play important roles.
304 One of the caveats of this investigation is that the Hybrid group is not a lineage of truly
305 related individuals, but a grouping of individuals whose population assignment probability is
306 less than 60% to any of the other groups hence is somewhat arbitrary. Had we chosen a higher
307 Hybrid assignment probability value, there would be fewer individuals in the NLMT, BLMT, or
308 Hemp groupings and more individuals in the Hybrid group. Had we chosen a lower value, there
309 would be fewer individuals in the Hybrid category and more individuals in the other groupings.
310 However, there are individuals with 100% assignment probability to one group, for example,
311 ‘Carmagnola’ has 100% genetic assignment to the Hemp group, ‘Afghan Kush’ has 100% genetic
312 assignment to the BLMT group, and ‘Super Lemon Haze’ has 100% genetic assignment to the
313 NLMT group. If we had chosen a value of 40% instead of 60%, both the NIDA varieties would
314 have grouped with the NLMT group (see Table S2 for the exact assignment probability).
315 In addition to limiting the research capacity on genetic and chemotypic variation by
316 restricting investigation to only Cannabis supplied by NIDA, medical research using this material
317 is also limited and inaccurate. Given that NIDA’s samples do not represent the genomic or
318 phenotypic variation found in Cannabis provided by the legal market, consumer experiences may
319 be different from that which is published in the scientific literature. Therefore, medical research
320 is hindered by using varieties that are not representative of what people are actually consuming,
321 making medical research less predictive. The use of NIDA’s Cannabis may be one of the reasons
322 why recent reviews have found therapeutic support for three medical conditions (Abrams 2018),
323 while efficacy as an appetite stimulant, as a relaxant, or to treat epilepsy were not supported
324 despite numerous patient reports.
325 Limiting Cannabis types available for study creates an obstacle for scientific discovery. It
326 has been proposed that Cannabis may be evolving dioecy from monoecious populations
327 (Divashuk et al. 2014; Razumova et al. 2016; Prentout et al. 2019) and cytonuclear interactions,
328 which could be involved in this transition to dioecy, may be also taking place. To understand
329 processes like these, scientists need access to a diverse and growing variety of Cannabis plants
330 which are not available through NIDA. Important discoveries in other plant groups such as
331 transposable elements (McClintock 1950) genes related to pathogen resistance (Leister et al.
332 1996), or genes related to yield (Sakamoto and Matsuoka 2008) would have not been possible
333 had there been similar restrictions on their research.
15
334 This limitation also affects the untapped possibilities of using Cannabis to treat a multitude
335 of illnesses, which is backed by a mass of anecdotes. These will continue to be anecdotes until
336 they are studied using rigorous scientific testing methods and scientists are able to provide
337 reliable answers to the community. Cannabis is the most widely consumed illicit substance in
338 both in the U.S. and worldwide (Gloss 2014), and therefore it is a matter of public health and
339 safety to provide honest and accurate information. This information is also crucial to policy
340 officials who rely on facts for laws and regulation. In conclusion, scientists must be allowed to
341 use all publicly available forms of Cannabis for research purposes in order to maximize scientific,
342 economic, and medicinal benefit to society.
343
344 Author Contributions

345 D.V. analyzed the single-copy portion of the genome, made figures, wrote the first draft of the
346 manuscript, conceived and lead the project; E.L.H. analyzed the single-copy portion of the
347 genome including STRUCTURE and splits tree graphs, wrote the bioinformatic pipelines; K.G.K.
348 wrote bioinformatic pipelines for the single-copy portion analysis and PCA; R.B.P. analyzed the
349 repetitive content of the genome; A.L.S, M.E.M. acquired DNA samples; and N.C.K. conceived and
350 directed the project. All authors contributed to manuscript preparation.
351
352 Funding
353 This research was supported by donations to the University of Colorado Foundation gift fund
354 13401977-Fin8 to NCK and to the Agricultural Genomics Foundation and is part of the joint
355 research agreement between the University of Colorado Boulder and Steep Hill Inc. which
356 made possible the sequencing of the two NIDA genomes.
357
358 Acknowledgments
359 We thank B. Holmes of Centennial Seeds; D. Liles, C. Casad, A. Ledden and J. Cole of The Farm;
360 MMJ America, Medicinal Genomics, A. Rheingold and M. Rheingold of Headquarters; D. Salama,
361 Nico Escondido, Sunrise Genetics, and B. Sievers for providing DNA samples or sequence
362 information.
363
364 Conflict of Interest

365 D.V. is the founder and president of the non-profit organization Agricultural Genomics
366 Foundation, and the sole owner of CGRI, LLC. N.C.K. is a board member of the non-profit
367 organization Agricultural Genomics Foundation.
16
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457
458
459 Supplementary Material
Genomic evidence that governmentally produced Cannabis sativa

poorly represents genetic variation available in state markets
460 Daniela Vergara1*, Ezra L. Huscher1+, Kyle G. Keepers1+, Rahul Pisupati2, Anna L.
461 Schwabe3, Mitchell E. McGlaughlin3, and Nolan C. Kane1*
462
463 1Kane Laboratory, Department of Ecology and Evolutionary Biology, University of Colorado
464 Boulder, Boulder, Colorado, USA
465 2 Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Dr.
466 Bohr-Gasse 3, 1030 Vienna, Austria
467 3 University of Northern Colorado, School of Biological Sciences, Greeley, CO 80639, USA.
468 +Authors that contributed equally
469
470 * Correspondence:
471 [email protected] or [email protected]
472 Keywords: cannabinoids, copy number variation, genome diversity, hemp, repetitive genomic
473 content, marijuana, NIDA, THC
474
475
19
0.80
0.70
0.60
Percent of the Genome
0.50
0.40
0.30
0.20
0.10
0.00
BLMT Hemp Hybrid NIDA NLMT
Repeat Family
18S rDNA LTR Contamination
25S rDNA TIR Plastid
5S rDNA Ty1 copia Simple
Repeat
Unknown Ty3 gypsy
476
477 Supporting Information Figure S1. Repetitive content characterization. The graph based
478 clustering algorithm Repeat Explorer characterized the percentage of the genome that belong
479 to the different repeat families. Exact numbers in table S4.
480
481 Supporting Information Table S1. Genetic and genomic information. Cultivar name
482 (column 1), Sample ID (column 2), classification based on Structure (Column 3), NCBI accession
483 number (column 4), provider (column 5), genome calculations (columns 6-10), haplotype
484 groups (columns 11-12), heterozygosity calculations (columns 13-20), PCA (columns 21-40),
485 cannabinoid loci statistics (columns 41-76).
20
486 Supporting Information Table S2. Population assignment probability. Sructure’s

487 population assignment probability. Individuals with an assignment probability of <60% to any
488 group were assigned to the ‘hybrid’ grouping.
489 Supporting Information Table S3. Cannabinoid BLAST results. Cannabinoid BLAST results
490 to the Cs10 assembly with more than 80% identity and an alignment length of greater than
491 1000bp.
492 Supporting Information Table S4. Repeat Families. Different families based on the
493 clustering algorithm used in Repeat Explorer.
494
495
21

Art Genomic Evidence That Governmentally Produced Cannabis Sativa Poorly Represents Genetic Variation Available in State Markets

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bioRxiv preprint doi: https://doi.org/10.1101/2021.02.13.431041; this version posted February 14, 2021.

The copyright holder for this preprint

Genomic evidence that governmentally produced Cannabis sativa

1 Daniela Vergara1*, Ezra L. Huscher1+, Kyle G. Keepers1+, Rahul Pisupati2, Anna L.

4 1Kane Laboratory, Department of Ecology and Evolutionary Biology, University of Colorado

5 Boulder, Boulder, Colorado, USA

7 Bohr-Gasse 3, 1030 Vienna, Austria

8 3 University of Northern Colorado, School of Biological Sciences, Greeley, CO 80639, USA.

9 +Authors that contributed equally

94 Here, we present results of analysis to further examine the genetic diversity in

105 Materials and Methods

144 Cannabinoid gene exploration

156 Maternally inherited genomes

162 Repetitive genomic content

- 0.2 - 0.1 0.0 0.1

213 Cannabinoid gene exploration

238 Maternally inherited genomes

II N = 8 VII Chinese Hemp

249 IV Cannatonic V Chinese Hemp

254 Repetitive genomic content

344 Author Contributions

364 Conflict of Interest

459 Supplementary Material

Genomic evidence that governmentally produced Cannabis sativa

464 Boulder, Boulder, Colorado, USA

466 Bohr-Gasse 3, 1030 Vienna, Austria

468 +Authors that contributed equally

486 Supporting Information Table S2. Population assignment probability. Sructure’s

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