CannMed2016Molecular Phylotyping of Cannabis

Ryan Lynch PhD

Background

• Bacterial genomics since 2008

• Cannabis genomics since 2013, started in Nolan Kane’s Lab

• Medicinal Genomics scientist since late 2015

Cannabis diversity—phenotypic and genetic

What can we do with Cannabis DNA?

• Past, Present and Future

• Current efforts to unravel plant history– How many species?– ‘indica’ vs ‘sativa’? Hemp? ruderalis?

• Genetics of specific chemoprofiles of cannabinoids and terpenoids?

• Strain name issues– Is my OG Kush the real OG?

• Certifications set now

• Future prediction of traits from DNAseq– Custom strain development

DNA: genomes contain information about nearly all aspects of biology, even if we cannot yet interpret it.

≠Informationisnotknowledge:AlEinstein/FrankZappa

Step 1: Sample Collection

7

Restriction-siteassociatedDNAsequencing(RADseq)

http://www.floragenex.com/rad-seq/

Relatedness among strains--phylogenetics

Rootedv.Unrooted

The network of recent Cannabis breeding

https://www.icmag.com/ic/showthread.php?t=147494

MajorgroupsoftheCannabisFamilyTree(v3.1):

Lynchetal.GenomicandChemicalDiversityofCannabisNewPhytologist(2016)inrevieworpre-printisuponBioRxiv

Narrowleafletdrug typeI

Broadleafletdrug typeI

Narrowleafletdrug typeI

EuropeanHemp

=JackHererorJacksomething

11

Chemical Diversity of Genetic Groups

•Afterremovingtheindividualswithmissingvalues,wehadatotalof351individuals: 94BLDT,229NLDT,and28hemp.

Lynchetal.GenomicandChemicalDiversityofCannabisNewPhytologist (2016)inrevieworpre-printisuponBioRxiv

Scaling up with strainSEEKtm

• CO, CA, MA, ME, WA, Spain samples… so far• On average, our Rad seq protocol yields at least 10x

coverage of 625,000 sites of the current reference genome. These sites contain 25,000 to 30,000 informative single nucleotide polymorphisms (SNPs)

• This April will witness over 100,000 genetically certified METRC tracked products hit the CO market (~60 strains)

Kannapedia– TheDistributedConsensusonCannabisGenetics

• Optionalpublication toKannapedia• Allowspublishing of strainuser

comments/reviews• Allowspublicdistribution ofStrain

QRcode• Enablescommunityverificationof

strainqualities• Enablesratingsystemfordesired

qualities• Customerreviews• http://www.medicinalgenomics.co

m/kannapedia/

13Confidential

14

AK47

Recon

Felina322

USO312

Diana2

Carmagnola1

Monoica1Tygra1

Bialobreski1

Lovrin1

Ivory1

Ferimion1Santhica271

Fedora171Futura751

Dacia1

Tisza1

Mengm

a1Trainwreck

Otto

Ghost_Train_H

aze_X_Snow_Dog_TLE

Pineapple_tsu_EC

2nd_HarletTsu

Harlequin_Tsunam

i

CBD_Mango_H

aze

Critical_M

ass_EC

Cannatonic_E

C

Incredible_Power_E

C

ACDC2nd

Catatonic_ACDC_EC

Cannatonic

Bhutan_EC

CBDIACDC

Guerilla_Meds_EC

Moonshine_Haze124

Luvberry_EC

Pineapple_tsu2nd_EC

In_The_Pines

Landrace_Jamaican1_TLE

Space_Queen_TLE

Sierra_Gold

Arch_Angel_TLE

Green_Crack

Loven_Hope_EC

Blazin_GrapefruitGS

CXAK47Ha

rlequin_EC

Harlequin_TL

E

Green_

Crack_TLE

Critical_

Mass_T

LEDr_W

ho228

Purple_Ca

ndy_Cane

Cannatsu_TL

EDeath_S

tar_TLEKong_TLEBlue_Dream_TLE

Snoops_Dream

Blubonic_TLE

Cactus

Girl_Scout_Platinum

Dream_Beaver

CitriSoul_TLE

Banana_Kush_TLE

Charlie_Murphy_TLE

GrandDaddyPurple_ctrl2

Grand_Daddy_Purple_ctrl4

1988_Hash_Plant_TLE

Old_School_Blueberry_TLE

Unity_EC

Snowcap_TLE

Bubblegummer

Liberty_Haze_ctrl2

Liberty_Haze_ctrl

Lambs_Bread_TLEBreakthrough

Chernobyl

Harlet_Tsu

Northern_Lights

Cherry_Lim

eadeChocolope

Hash_P

lant_Puck_TLEAfghan_K

ush

Durban_TLE

Durban_P

oison_TLE

Super_Lemon_Haze_TLE

African_Haze_TLE

Lucky_Charms

NYC_Diesel

Girl_Scout_Cookies

Vitamin_CBD_EC

MangThai_Doo

Sugar_Cookies_TLE

White_Fire_TLE

Triangle_OG_TLE

Headband_TLE

Chemmy_Jones_TLE

Lemonhead_OG

El_Diablo_TLE

Face_Off_OG_TLE

Jesus_TLE

OG_Kush

Girl_Scout_Cookies_TLE

F-Cancer_EC

Trident_EC

Witches_W

eed_TL

E

Chedda

r_Head

Serious_H

appiness

GFU_x_Mr_

Nice_TLE

Sage-n-Sour

Corleone_Kush_T

LE

Bubba_Kush_TLE

Lemon_Skunk

0.01

strainSEEKTM Aprilupdate:sistersfromthesamemister?

=CBD:THCof2<31

Future Developments…

www.nationalgeographic.com

Summary

• DNA sequence analysis can tell you things about the plant (or plant microbial community) that are not otherwise apparent

• DNA data is an additional layer of information, not a substitute for other types of data like chemical analytics or patient responses.

• We have just scratched the surface of Cannabis potential as it produces at least 113 phytocannabinoids and at least 120 terpenoids, yet what is available on the market or for research today is quite limited!

MGC Acknowledgments

• KevinMcKernan• JeremyRualo• DouglasSmith• PeterDougherty• JessicaSpangler• YvonneHelbert• Wendall Orphe• LeiZhang• ChristinaSutera• AdrianDevitt-Lee• BenAmirault• JohnGeanacopoulos• Vasisht Tadigotla

17Confidential

DigiPath:CindyOrserGlenMarquez

TheListExchange:RobertoLopesino

OrrinDevinskyEricMarshRussel Saneto

UniversityofColorado:NolanKane

Kannapedia– TheDistributedConsensusonCannabisGenetics

19Confidential

MaleFemaleqPCR testing

• Worksona2nd Leaf• ProvidesXYanswer

inhours• $10/Test

20Confidential

Wholegenomeshotgunmethodfor43Cannabissamples

NolanKaneatworkonthefarm...