Morphological and molecular study of sex determination in Cannabis sativa L.

Document Type : Research Paper

Authors

1 MSc student, Department of Horticultural Science, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran.

2 Department of Horticultural Science, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iran.

Abstract

Cannabis sativa L. is an important plant with various uses in pharmaceutical and paper production industries. Due to the higher priority of female and male Cannabis plants for pharmaceutical uses and fibre industry, respectively, reduction of the quality of products after pollination, and also for detection of better genotypes before pollination for breeding purposes, early determination of sex in Cannabis is one of the major concerns of researchers and farmers. Seeds of 26 accessions from different regions of Iran along with one accession from Afghanistan were planted in the field based on randomized complete block design with 10 replications. Five female and five male plants were sampled from each accession for molecular and morphological analyses. Thirteen ISSR primers and two SCAR markers were used. Also, morphological differences, which possibly related to sex were measured in the Cannabis populations under study. The results showed significant differences among the accessions within female and male plants for plant height, number of days to flowering, days after full bloom and height of the first flowering node. Male plants were generally, but not always, taller than female plants and had a shorter life cycle. The highest expected heterozygosity was found in the ISSR3 primer and the lowest in the UBC825 primer. In total, 143 polymorphic bands and one polymorphic band were amplified using ISSR and SCAR primers, respectively. Out of 143 polymorphic ISSR bands, only 10 markers had significant relations with the gender of this plant. MADC6 SCAR marker was monomorph across the accessions. On the other hand, MADC5 was a polymorphic marker and showed a significant relation with gender. These markers, especially MADC5, have the potential to be used in sex determination of C. sativa.

Keywords


Article Title [فارسی]

مطالعه مورفولوژیکی و مولکولی تعیین جنسیت در شاهدانه

Abstract [فارسی]

شاهدانه (Cannabis sativa L.) یک گیاه مهم با کاربردهای مختلف در صنایع دارویی و تولید کاغذ است. با توجه به این که اغلب گیاهان ماده در صنایع دارویی و در گیاهان نر در تولید فیبر حایز اهمیت بیشتری هستند و گرده ­افشانی گیاه کیفیت فرآورده ­های تولیدی را کاهش می­ دهد، همیشه یکی از دغدغه­ های محققان و کشاورزان شناسایی زودهنگام جنسیت در شاهدانه بوده است. بذرها از 26 جمعیت از مناطق مختلف ایران به همراه یک جمعیت از افغانستان به صورت طرح بلوک های کامل تصادفی در 10 تکرار کشت شدند. پنج گیاه نر و پنج گیاه ماده از هر جمعیت برای تجزیه­ های مورفولوژیکی و مولکولی نمونه­ برداری شدند. از 13 آغازگر ISSR و دو SCAR استفاده شد. تفاوت مورفولوژیکی مرتبط با جنسیت در جمعیت­ های شاهدانه مورد مطالعه مشاهده شد. نتایج نشان داد که در برخی از صفات بین جمعیت­ ها در درون هر دو جنس تفاوت معنی­  داری وجود دارد. گیاهان نر عمدتا بلندتر و باریک­تر از گیاهان ماده بودند و چرخه زندگی کوتاه­تری نسبت به گیاهان ماده داشتند. بالاترین میانگین هتروزیگوسیتی در آغازگر ISSR3 و کم­ترین آن در آغازگرUBC825  مشاهده شد. به­ طور کلی 143 جایگاه پلی مورفیک و یک جایگاه پلی­مورفیک به ترتیب در آغازگرهای  ISSR و SCAR شناسایی شد. از بین 143 جایگاه پلی­ مورفیک در ISSR تنها 10 نشانگر رابطه معنی­ داری با جنسیت داشتند. آغازگر SCAR  MADC6مونومورف بود. از طرف دیگر آغازگر SCAR MADC5 پلی­ مورفیسم نشان داد و  رابطه معنی­ داری با نوع جنس داشت. بنابراین، به نظر می­ رسد که این نشانگرها، به ویژه  MADC5، دارای ارزش بالقوه برای تعیین جنسیت در C. sativa هستند.

Keywords [فارسی]

  • تعیین جنسیت
  • شاهدانه
  • ماده
  • نر
  • نشانگرهای مورفولیژیکی
  • نشانگرهای مولکولی
Andre CM, Hausman JF and Guerriero G, 2016. Cannabis sativa: the plant of the thousand and one molecules. Frontiers in Plant Science 7:19. doi:10.3389/fpls.2016.00019.
Dellaporta SL and Calderon-Urrea A, 1993. Sex determination in flowering plants. Plant Cell 5(10):1241-1251.
De Meijer EPM and Keizer LC, 1994. Variation of Cannabis for phonological development and stem elongation in relation to stem production. Field Crops Research 38(1): 37-46.
Doyle JJ, 1990. Isolation of plant DNA from fresh tissue. Focus 12: 13-15.
Divashuk MG, Alexandrov OS, Razumova OV, Kirov IV and Karlov GI, 2014. Molecular cytogenetic characterization of the dioecious Cannabis sativa with an XY chromosome sex determination system. PLoSOne 9(1): doi: 10.1371/journal.pone.0085118.
Flachowsky H, Schumann E, Weber WE and Peil A, 2008. Application of AFLP for the detection of sex-specific markers in hemp. Plant Breeding 12(4): 305-309.
Heikrujam M, Sharma K, Prasad M and Agrawal V, 2015. Review on different mechanisms of sex determination and sex-linked molecular markers in dioecious crops: a current update. Euphytica 201: 161-194.
Hill M, 1973. Diversity and evenness: a unifying notation and its consequences. Ecology, 54: 427-432.
Hoffmann W, 1947. Die Vererbung der Geschlechtsformen des hanfes (Cannabis sativa L.) I. Der Zuchter 17/18: 257-277.
Hormaza JI, Dollo L and Polito VS, 1994. Identification of a RAPD marker linked to sex determination in Pistacia vera using bulked segregant analysis. Theoretical and Applied Genetics 89: 9-13.
Mandolino G, Carboni A, Forapani S, Faeti V and Ranalli P, 1999. Identification of DNA markers linked to the male sex in dioecious hemp (Cannabis sativa L.). Theoretical and Applied Genetics 98: 86-92.
Mediavilla V, Jonquera M, Schmid-Slembrouck I and Soldati A, 1998. A decimal code for growth stages of hemp (Cannabis sativa L.). Journal of Industrial Hemp 5: 65-74.
Milewicz M and Sawicki J, 2013. Sex-linked markers in dioecious plants. Plant Omics Journal 6(2): 144-149.
Moliterni VMC, Cattivelli L, Ranalli P and Mandolino G, 2004. The sexual differentiation of Cannabis sativa L.: a morphological and molecular study. Euphytica 140: 95-106.
Nei M, 1973. Analysis of gene diversity in subdivided populations. PNAS USA 70(12): 3321-3323.
Nei M, 1978. Estimation of average heterozygosity and genetic distance from a small number of individuals.  Genetics 89(3): 583-590. 
Parker JS, 1990. Sex chromosomes and sexual differentiation in flowering plants. Chromosomes Today 10: 187-198.
Peil A, Flachowsky H, Schumann E and Weber WE, 2003. Sex-linked AFLP markers indicate a pseudoautosomal region in hemp (Cannabis sativa L.). Theoretical and Applied Genetics107(1): 102-109.
Pérez-Llorca M and Sánchez Vilas J, 2019. Sexual dimorphism in response to herbivory and competition in the dioecious herb Spinacia oleracea. Plant Ecology 220: 57-68.
Rode J, In-Chol K, Saal B, Flachowsky H, Kriese U and Weber WE, 2005. Sex-linked SSR markers in hemp. Plant Breeding124: 167-170.
Sakamoto K, Akiyama Y, Fukui K, Kamada H and Satoh S, 1998. Characterization; genome sizes and morphology of sex chromosomes in hemp (Cannabis sativa L.). Cytologia 63: 459-464.
Sakamoto K, Shimomura K, Komeda Y, Kamada H and Satoh S, 1995. A male- associated DNA sequence in dioecious plant, Cannabis sativa L. Plant and Cell Physiology 36(8): 1549-1554.
Samantaray S, Phurailatpam A, Bishoyi AK, Geetha KA andMaiti S, 2012. Identification of sex-specific DNA markers in betel vine(Piper betleL.). Genetic Resources and Crop Evolution 59: 645-653.
Shannon CA, 1948. Mathematical theory of communication. Bell System Technical Journal 27: 379-423.
Thomas B and Vince-Prue D, 1997. Photoperiodism in Plants. Second edition. Academic Press, USA.
Torjek O, Bucherna N, Kiss E, Homoki H,  Finta-Korpelová Z,  Bócsa I,  Nagy I and Heszky LE, 2002. Novel male-specific molecular markers (MADC5, MADC6) in hemp. Euphytica 127: 209-218.