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Multiplex-Polymerase Chain Reaction을 이용한 대마의 조기 성판별 Sequence Characterized Amplified Region 마커 개발

Development of Sequence Characterized Amplified Region Marker for Early Sex Determination of Cannabis sativa L. using Multiplex-Polymerase Chain Reaction

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Background: Cannabis are typically dioecious, with female and male reproductive parts located on separate plants. Female cannabis plants are preferred for the production of cannabinoids, ter- penes, and other valuable compounds synthesized within the female floral tissue. Female and male plants look identical during seedling and vegetative phases, and only after transition to flowering phase can male, female, and monoecious plants be distinguished. Early identification of the sex of cannabis plant can conserve time and money by avoiding the undesired sex of the plant. The objec- tive of the present study was to provide a sex determination method for cannabis plants using the sequence characterized amplified region (SCAR) marker, and set multiplex-polymerase chain reac- tion (PCR) conditions for more accurate early sex determination of cannabis to overcome the dis- advantages of SCAR markers. Methods and Results: Cannabis seeds were collected and cultivated for several purposes. Leaf samples were harvested and stored during vegetative growth phase. In silico mapping of three male-specific sequences (MADC3, MADC4, and MADC6) revealed through random amplified polymorphic DNA, showed that MADC3 has a very similar sequence with the female cannabis genome. Primers were produced based on the sequences (SCAR1, SCAR2, and SCAR3, respec- tively) and subjected to gradient PCR. Only SCAR3 produced a male-specific band with an anneal- ing temperature of over 61.4 ℃. PCR was performed on cannabis plants produced for several purposes by mixing SCAR3 and internal standard 3 (IS3). Result showed that all cannabis plants produced an IS3 band of 197 bp, but only male cannabis plants produced a SCAR3 band of 118 bp. Conclusions: Multiplex-PCR primer produced a male-specific band of 119 bp in cannabis bred for several purposes, and the presence of the 197 bp cannabis common band confirmed that PCR pro- ceeded normally for all samples. The multiplex-PCR primer is greatly beneficial because the IS3 band is a specific sequence found only in cannabis, making it possible to differentiate cannabis from other plants in addition to determining the sex from with a single PCR run.

Abstract

Background: Cannabis are typically dioecious, with female and male reproductive parts located on separate plants. Female cannabis plants are preferred for the production of cannabinoids, ter- penes, and other valuable compounds synthesized within the female floral tissue. Female and male plants look identical during seedling and vegetative phases, and only after transition to flowering phase can male, female, and monoecious plants be distinguished. Early identification of the sex of cannabis plant can conserve time and money by avoiding the undesired sex of the plant. The objec- tive of the present study was to provide a sex determination method for cannabis plants using the sequence characterized amplified region (SCAR) marker, and set multiplex-polymerase chain reac- tion (PCR) conditions for more accurate early sex determination of cannabis to overcome the dis- advantages of SCAR markers. Methods and Results: Cannabis seeds were collected and cultivated for several purposes. Leaf samples were harvested and stored during vegetative growth phase. In silico mapping of three male-specific sequences (MADC3, MADC4, and MADC6) revealed through random amplified polymorphic DNA, showed that MADC3 has a very similar sequence with the female cannabis genome. Primers were produced based on the sequences (SCAR1, SCAR2, and SCAR3, respec- tively) and subjected to gradient PCR. Only SCAR3 produced a male-specific band with an anneal- ing temperature of over 61.4 ℃. PCR was performed on cannabis plants produced for several purposes by mixing SCAR3 and internal standard 3 (IS3). Result showed that all cannabis plants produced an IS3 band of 197 bp, but only male cannabis plants produced a SCAR3 band of 118 bp. Conclusions: Multiplex-PCR primer produced a male-specific band of 119 bp in cannabis bred for several purposes, and the presence of the 197 bp cannabis common band confirmed that PCR pro- ceeded normally for all samples. The multiplex-PCR primer is greatly beneficial because the IS3 band is a specific sequence found only in cannabis, making it possible to differentiate cannabis from other plants in addition to determining the sex from with a single PCR run.

참고문헌 (55)

  1. Alstrom-Rapaport C, "Identification of a RAPD marker linked to sex determination in the basket willow(Salix viminalis L. )", Journal of Heredity, 89, pp.44~49 (1998)
  2. Aryal R, "Sex determination in flowering plants : Papaya as a model system", Plant Science, 217, pp.56~62 (2014)
  3. Atalay S, "Antioxidative and anti-inflammatory properties of cannabidiol", Antioxidants, 9, pp.21~ (2019)
  4. Bai Q, "The sex expression and sex determining mechanism in Pistacia species", Breeding Science, 69, pp.205~214 (2019)
  5. Boggs DL, "Clinical and preclinical evidence for functional interactions of cannabidiol and Δ9-tetrahydrocannabinol", Neuropsychopharmacology, 43, pp.142~154 (2018)
  6. Chesney E, "Cannabidiol(CBD) as a novel treatment in the early phases of psychosis", Psychopharmacology, pp.1179~1190 (2022)
  7. Consroe P, "Cannabidiol--antiepileptic drug comparisons and interactions in experimentally induced seizures in rats", Journal of Pharmacology and Experimental Therapeutics, 201, pp.26~32 (1977)
  8. Crippa JA, "Translational investigation of the therapeutic potential of cannabidiol (CBD): Toward a new age", Frontiers in Immunology, 9, pp.2009~ (2018)
  9. de Backer B, "Evolution of the content of THC and other major cannabinoids in drug-type cannabis cuttings and seedlings during growth of plants", Journal of Forensic Sciences, 57, pp.918~922 (2012)
  10. Doh EJ, "DNA markers to discriminate Cannabis sativa L. 'Cheungsam' with low tetrahydrocannabinol(THC)content from other South Korea cultivars based on the nucleotide sequences of tetrahydrocannabinolic acid synthase and putative 3-Ketoacyl-CoA synthase genes", Evidence-Based Complementary and Alternative Medicine, 2019, pp.8121796~ (2019)
  11. D'Souza DC, "Delta-9-tetrahydrocannabinol effects in schizophrenia : Implications for cognition, psychosis, and addiction", Biological Psychiatry, 57, pp.594~608 (2005)
  12. ElSohly MA, "Phytocannabinoids", pp.1~36 (2017), Springer
  13. Flajšman M, "Production of Feminized Seeds of High CBD Cannabis sativa L. by Manipulation of Sex Expression and Its Application to Breeding", Frontiers in Plant Science, 12, pp.718092~ (2021)
  14. Freeman D, "How cannabis causes paranoia : Using the intravenous administration of Δ9-tetrahydrocannabinol(THC)to identify key cognitive mechanisms leading to paranoia", Schizophrenia Bulletin, 41, pp.391~399 (2015)
  15. Go YY, "Cannabidiol enhances cytotoxicity of anti-cancer drugs in human head and neck squamous cell carcinoma", Scientific Reports, 10, pp.20622~ (2020)
  16. Guimarães FS, "Antianxiety effect of cannabidiol in the elevated plus-maze", Psychopharmacology, 100, pp.558~559 (1990)
  17. Gupta SK, "Development and validation of SCAR markers co-segregating with an Agropyron elongatum derived leaf rust resistance gene Lr24 in wheat", Euphytica, 150, pp.233~240 (2006)
  18. Heikrujam M, "Review on different mechanisms of sex determination and sex-linked molecular markers in dioecious crops : A current update", Euphytica, 201, pp.161~194 (2015)
  19. Hindley G, "Psychiatric symptoms caused by cannabis constituents : A systematic review and meta-analysis", The Lancet Psychiatry, 7, pp.344~353 (2020)
  20. 허만규, "식물에서 분자 마커의 동향", 생명과학회지, 25(7), pp.839~848 (2015), 한국생명과학회
  21. 조익현, "Applications of molecular markers in the discrimination of Panax species and Korean ginseng cultivars (Panax ginseng)", Journal of Ginseng Research, 41(4), pp.444~449 (2017), 고려인삼학회[DOI]
  22. Khampila J, "Identification of RAPD and SCAR markers linked to northern leaf blight resistance in waxy corn(Zea mays var. ceratina)", Euphytica, 164, pp.615~625 (2008)
  23. 김문교, "국내 대추 품종의 구분을 위한 InDel 마커의 개발", 한국약용작물학회지, 29(4), pp.282~292 (2021), 한국약용작물학회[DOI]
  24. Kim WJ, "Rapid identification of Paeoniae radix and moutan radicis cortex using a SCAR marker-based conventional PCR assay", Plants, 11, pp.2870~ (2022)
  25. Koltai H, "Anti-cancer activity of Cannabis sativa phytocannabinoids: Molecular mechanisms and potential in the fight against ovarian cancer and stem cells", Cancers, 14, pp.4299~ (2022)
  26. Lacombe JP, "Discrimination of sexes as a function of early vegetative processes in dioecious hemp(Cannabis sativa L. )", Physiologie Vegetale, 18, pp.419~430 (1980)
  27. 박희운, "SCAR 마커를 이용한 황기와 몽고황기 판별", 한국약용작물학회지, 15(1), pp.51~55 (2007), 한국약용작물학회
  28. Linacre A, "Detection and identification of cannabis by DNA", Forensic Science International, 91, pp.71~76 (1998)
  29. Lubell JD, "Foliar sprays of silver thiosulfate produce male flowers on female hemp plants", HortTechnology, 28, pp.743~747 (2018)
  30. Maliepaard C, "Aligning male and female linkage maps of apple(Malus pumila Mill.) using multi-allelic markers", Theoretical and Applied Genetics, 97, pp.60~73 (1998)
  31. Mansouri H, "Ethephon application stimulats cannabinoids and plastidic terpenoids production in Cannabis sativa at flowering stage", Industrial Crops and Products, 46, pp.269~273 (2013)
  32. Mohan Ram HY, "Induction of fertile male flowers in genetically female Cannabis sativa plants by silver nitrate and silver thiosulphate anionic complex", Theoretical and Applied Genetics, 62, pp.369~375 (1982)
  33. Mohan Ram HY, "Modification of growth and sex expression in Cannabis sativa by aminoethoxyvinylglycine and ethephon", Zeitschrift Für Pflanzenphysiologie, 105, pp.165~172 (1982)
  34. Moliterni VMC, "The sexual differentiation of Cannabis sativa L. : A morphological and molecular study", Euphytica, 140, pp.95~106 (2004)
  35. Naqvi NI, "Development of a sequence characterized amplified region(SCAR)based indirect selection method for a dominant blast-resistance gene in rice", Genome, 39, pp.26~30 (1996)
  36. Ohmori T, "Molecular characterization of RAPD and SCAR markers linked to the Tm-1 locus in tomato", Theoretical and Applied Genetics, 92, pp.151~156 (1996)
  37. Paran I, "Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce", Theoretical and Applied Genetics, 85, pp.985~993 (1993)
  38. 박춘근, "청도라지와 백도라지의 구분을 위한 SCAR 마커 개발", 한국약용작물학회지, 15(1), pp.1~5 (2007), 한국약용작물학회
  39. Rodriguez-Morrison V, "Cannabis yield, potency, and leaf photosynthesis respond differently to increasing light levels in an indoor environment", Frontiers in Plant Science, 12, pp.646020~ (2021)
  40. Rodziewicz P, "Cannabinoid synthases and osmoprotective metabolites accumulate in the exudates of Cannabis sativa L. glandular trichomes", Plant Science, 284, pp.108~116 (2019)
  41. Russo EB, "Phytochemical and genetic analyses of ancient cannabis from Central Asia", Journal of Experimental Botany, 59, pp.4171~4182 (2008)
  42. Ryu BR, "Conversion characteristics of some major cannabinoids from hemp(Cannabis sativa L.) raw materials by new rapid simultaneous analysis method", Molecules, 26, pp.4113~ (2021)
  43. 류재혁, "엽록체 DNA (trnL-trnF, rps16-trnK) 염기서열에 의한 국내 민들레속 유전자원의 유전적 변이와 유연관계 분석", 한국자원식물학회지, 30(5), pp.522~534 (2017), 한국자원식물학회[DOI]
  44. Sakamoto K, "RAPD markers encoding retrotransposable elements are linked to the male sex in Cannabis sativa L", Genome, 48, pp.931~936 (2005)
  45. Salentijn EMJ, "New developments in fiber hemp(Cannabis sativa L. )breeding", Industrial Crops and Products, 68, pp.32~41 (2015)
  46. Schaffner JH, "Influence of environment on sexual expression in hemp", Botanical Gazette, 71, pp.197~219 (1921)
  47. Schultes RE, "Cannabis and Culture", pp.21~38 (1975), De Gruyter Academic Publishing
  48. Small E, "Cannabinoid phenotypes in Cannabis sativa", Nature, 245, pp.147~148 (1973)
  49. Tahir MN, "The biosynthesis of the cannabinoids", Journal of Cannabis Research, 3, pp.7~ (2021)
  50. Tajik T, "Extracellular vesicles of cannabis with high CBD content induce anticancer signaling in human hepatocellular carcinoma", Biomedicine and Pharmacotherapy, 152, pp.113209~ (2022)
  51. Törjék O, "Novel male-specific molecular markers(MADC5, MADC6)in hemp", Euphytica, 127, pp.209~218 (2002)
  52. Toth JA, "Development and validation of genetic markers for sex and cannabinoid chemotype in Cannabis sativa L", GCB-Bioenergy, 12, pp.213~222 (2020)
  53. Urits I, "Use of cannabidiol(CBD)for the treatment of chronic pain", Best Practice and Research Clinical Anaesthesiology, 34, pp.463~477 (2020)
  54. Xu J, "Screening genetically modified organisms using multiplex-PCR coupled with oligonucleotide microarray", Biosensors and Bioelectronics, 22, pp.71~77 (2006)
  55. Zhang J, "Genetic diversity and population structure of cannabis based on the genome-wide development of simple sequence repeat markers", Frontiers in Genetics, 11, pp.958~ (2020)

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