Cannabis is an annual plant genus belonging to the Cannabaceae family. Cannabis, whose fibers in its stems are used in yarn, weaving, and fabric production, while the pulp part is used in making paper, is known as one of the oldest vegetable raw materials in human history. Cannabis, originating from Central Asia, is hard, bushy, hollow stem, palm-leafed, dioecious, and one-year-old. In addition, its fibers are durable and quite long. So would you like to take a closer look at the genetics of cannabis?
Cannabis plants are usually dioecious, and plants bearing male or female flowers form separate individuals. Males are heterogametic (XY), females are homogametic (XX). The cannabis genome has a karyotype consisting of 9 autosomes and a pair of sex chromosomes (female “2n = 18 + XX”; male “2n = 18 + XY”). Although the size difference between the X and Y chromosome is small, cannabis is one of the few dioecious species with heteromorphic sex chromosomes. It is claimed that changing sex chromosomes during the developmental stages in many dioecious plants is a vital strategy.
Genetic Structure of Cannabis
Although the cannabis plant is widely cultivated in various parts of the world for its medicinal use, food raw material, different industrial uses, and pleasurable properties, we do not have sufficient knowledge of its genetics, including the molecular determinants of cannabinoid content. Hemp is a diploid species (2n = 20). Estimated haploid genome sizes are 818 Mb for female plants and 843 Mb for males.
While the sex of a cannabis plant is determined by the X and Y chromosomes, there are rarely monodic (mo-no + Oikos = i.e. flowers bearing male and female organs on different branches of the same plant) plants that produce flowers of both sexes in nature. The sex-determining system is of Drosophila type and the ratio of X to autosomes is determinant. Unlike other dioecious plants with heteromorphic sex chromosomes, the Y chromosome of cannabis probably does not contain genes necessary for male fertility.
As stated, XX chromosomes are found in female plants and XY in male plants; The Y chromosome is slightly larger than the X chromosome and autosomes. Genome sizes of diploid female and male plants in fiber hemp were calculated using flow cytometry, and these ratios for females and males were determined as 1636 and 1683 Mbp, respectively. The size difference between both genomes (47 Mbp, ie 2.8% of the genome size of male plants) was attributed to the long arm of the Y chromosome.
It is thought that the X chromosome and the autosome balance play a role in determining sex in dioic cannabis, not a Y-active mechanism. On the other hand, the factors in the sex determination of monoic forms of fiber hemp are unknown. Although researchers assume the existence of XX, XY, and YY forms, recent studies have observed the XX chromosome in monofilament cannabis plants, and recent studies have not revealed the presence of DNA markers associated with male sex (MADC, male individual-related DNA in cannabis). ). However, so far very few monoic plants have been screened with MADC markers, but karyotype analysis has been limited to the “Kentucky” variety.
Fiber Genetics of Cannabis
The researchers noted that there was no specific report about the chromosome set of monoic fiber hemp. All these studies, including monoic fiber cannabis varieties, accepted monoicity as a qualitative feature. In this context, the evaluation of the genotypic variability of the sexual phenotype expressed as a quantitative variable and the determination of the composition of the sex chromosomes of a large number of monoic fiber cannabis plants will enable the determination of preliminary information about the sex determination of monoic fiber cannabis that may be of interest for further research.
Given the size of the Y chromosome of fiber hemp, the presence of a Y chromosome in the monoic fiber hemp similar to that found in male plants can only be understood by comparing the genome sizes of female and male monoic plants. In addition, several DNA markers closely related to the male phenotype have been developed in fiber hemp. Among these markers, MADC2 has been proven to be fully associated with the male phenotype, so it is assumed that this marker should be in a region on the Y chromosome that is out of recombination during meiosis.
Propagation of said reagent by monoic plants may indicate the presence of a male-related fragment on the sex chromosomes of monoic fiber hemp. In the study of this researcher on the sex determination of monoic fiber hemp, it was determined that gender expression in monoic fiber hemp varied significantly quantitatively between cultivars. The genome size of plants is not significantly different from that of female plants but is lower than that of the male plant genome.
The MADC2 marker is not found in monoic fiber hemp. This supports the fact that monoic fiber hemp has the XX structure in the sex chromosomes and has a genetic basis for gender expression. When the genetic basis of the cannabinoid variation in cannabis was examined, it was shown that the amount of THC versus CBD was controlled by a locus with two codominant alleles, B (d) and B (t).
According to the first possible explanation of this situation; these two alleles code for THCA or CBDA synthase so that homozygous plants contain either tetrahydrocannabinolic acid (THCA) or cannabidiolic acid (CBDA) as the major cannabinoid. On the other hand, heterozygotes will have an almost equal mixture of these two substances. The second explanation is that THCA and CBDA synthases are produced by closely linked genes, perhaps as a result of gene duplication.
In a study, THCA synthase sequence analyzes obtained from the drug (high THC) and fiber (low THC) varieties of cannabis reported that the amino acid sequence of THCA synthase from high THC varieties differed by 37 major changes compared to low THC. The draft genome and transcriptome of Purple Kush, the drug type of cannabis announced in 2011, and the re-sequence analysis of the fiber hemp variety “Finola” were compared, but due to the high fragmentation, no distinction could be made between these two models.
However, it has been reported that about 70% of the cannabis draft genome consists of repetitive sequences. In a later study, analysis of 4 different types of single nucleotide variants (SNV) showed a heterozygote rate ranging from 0.18-0.26%. Moreover, it has been found that drug-type and fiber-type cannabis strains can differentiate well from each other by SNVs, and this ratio can reach up to 0.64% between the two cannabis types.
The cytogenetic analysis also showed a high degree of intra- and inter-cultivar karyotype polymorphism, which could further complicate the genome, at least among the fiber hemp varieties. The physical and genetic mapping by researchers shows that cannabinoid biosynthesis genes are generally not linked, but the total cannabinoid content of aromatic prenyltransferase (AP), which produces the substrate for THCA and CBDA synthases (THCAS and CBDAS) reveals that it is tightly bound to a known token.
These two enzymes (THCAS and CBDAS), which determine the chemotype of drug and fiber cannabis, are found in highly inhomogeneous, large (> 250 kb) retrotransposon-rich regions between drug and hemp-type alleles. Breeding trials of determining the cannabinoid synthase mechanism have shown that crossing between a cannabinoid-free plant and a plant with a high cannabinoid content results in an F1 with a low cannabinoid content. Subsequently, these F1s produce F2s in a monogenic opening ratio of 1: 2: 1, divided into separate chemotypes as “cannabinoid free”, “low containing” and “high containing”.
This triple dissociation can also be shown in binary form in a 1: 3 aspect ratio as “non-cannabinoid-containing” and “cannabinoid-containing” chemotypes. Plants that grow from cannabinoid-free plants are not always cannabinoid-free. These results can be explained by proposing a single allele locus with a common functional allele that allows cannabinoid synthesis and a rare null or nonfunctional ale that inhibits it.
The fact that 25% of F2 plants do not contain cannabinoids means that this empty allele is recessive and functionally dominant. However, the fact that 2/3 of the cannabinoid-containing plants have a lower content than the cannabinoid content in the parent plant indicates that the empty allele strongly suppresses the expression of the functional allele in heterozygotes.
Cannabis Genetics: Cannabis Species and Miracle Properties of Cannabinoids
Sativa is the most common cannabis strain used worldwide today. It is longer and narrower compared to Indica. Its leaves are relatively thin and long. Sativa originates in Ecuador – Colombia, Mexico, Thailand, and Southeast Asia. Indica is the second most common cannabis strain in the world. It grows at lower altitudes and is wider than Sativa. It has relatively short and broad leaves. Its origin is the Middle East and the region of Afghanistan and Pakistan.
Ruderalis is a relatively rare species originating in Russia. It grows relatively steadily and is less affected by the light-dark ratio. It blooms only from the tip and has low levels of psychoactive substance THC. Industrial hemp seed oil (Cannabis Sativa Seed Oil) does not contain THC and is non-psychoactive. It is extremely beneficial for children’s brain development and offers solutions to many health problems.
CBD is the most abundant cannabinoid found in cannabis, and unlike THC, it is legal and non-psychoactive. The strain of Cannabis indica is known as cannabis. Its active ingredient is THC, namely Tetrahydrocannabinol. Both hemp and cannabis contain cannabidiol (CBD), a non-psychoactive substance. However, THC is the substance that provides a “high” or psychoactive effect to users.
CBD has many similarities to THC when it comes to potential health benefits, but the main difference is that it is a non-psychoactive substance so it does not give inherent value to users. Also, CBD does not cause anxiety, paranoia, or dry mouth and eyes associated with THC, even when consumed in high concentrations.
Evidence from the tombs of India, Egypt, and ancient Assyrian kings showed that cannabis was used for medical purposes even 5000 years ago. Ceremonies that took place in India 2700 years ago probably point to the use of a combination of wine and hemp for surgical anesthesia purposes. In China, there is evidence that cannabis was used as a pain reliever 2000 years ago. The cannabis plant probably came to Europe in the 13th century and was first used only in the 17th century for agriculture, clothing, sail making, oil drilling, and food products.
After the worldwide spread of the cannabis plant in the 18th century, doctors in the USA began to recommend the use of cannabis for medical purposes against urinary incontinence, venereal diseases, and skin infections. In England, while cannabis is used as an arthritis treatment and a nausea drug, it has also been seen to be used in the treatment of tetanus, cholera, and rabies.
Chemogenetics of Cannabis: Chemical Compounds and Cannabinoids
There are more than 461 different chemical components in the cannabis plant, most of which are in three plant metabolite families: terpenoids, flavonoids, and cannabinoids as the most well-known chemical group. These plant metabolite groups have a unique set of functions related to plant growth, protection from insects, openness to pollen sources, and a broad spectrum of therapeutic effects on human health.
Terpenoids and flavonoids are components in many plants that make a great contribution to the functioning and development of the plant, and they also contribute to many properties such as smell and taste. In addition, these ingredients are also associated with many therapeutic effects. However, this time a specific effect for any of the components has not been revealed and its action mechanisms cannot be described.
Phyto-cannabinoids are recognized as pharmacologically active ingredients. It is synthesized in microscopic granular secretions that accumulate at the extreme point of resinous growths called “trichomes”. Trichomes are found in every plant and express themselves in high density in unfertilized female flowers. Cannabinoids primarily accumulate in the pollination area; however, they can be found in lower density in other plant parts.
These plant cannabinoid groups have more than 100 components unique to the cannabis plant, and as botanical and medical research on the plant and its components is carried out, new components are also emerging. “Medical cannabis products” approved for medical use are defined and classified according to the ratio and amount of THC, CBD, and CBN cannabinoids. However, it should be remembered that besides THC, CBD, and CBN, the cannabis plant contains dozens of different ingredients and there is less empirical information on the individual and individual pharmacological effects of these ingredients.
The genetic, morphological, and chemical difference between the original species of Sativa and Indica is also defined by distinctive physiological effects that are not only caused by the concentrations of THC and CBD. It is common practice today to attribute the terms “Sativa character source” and “Indica character source” to the decomposition of the “cannabis product” according to a broader genetic source and the effect on the patient.
Cannabis Seed Genetics
When purebred cannabis breeds are not renewed with certified seeds every 5-10 years, it means that the seeds used will both expand and disappear due to the heterozygous structure due to the loss of their characteristics. Seeds should be kept in a gene bank and propagated periodically under ideal conditions. In the last 50 years, the genetic diversity of the Cannabis genome has been decreasing. Despite the danger of generations being lost, care must be taken to preserve and reproduce what remains.
As the researchers point out, many local pure breeds developed for local use as a result of hundreds of years of selection have disappeared due to the neglect of the cannabis farming industry, the repression of cannabis cultivation by law, the anti-hemp propaganda, and the decline in fiber hemp breeding and research. As is known, genetic materials are a living heritage and must be protected by humanity. Therefore, the remaining cannabis genetic resources need to be collected, preserved, characterized, and used, and evaluated if necessary, before it is too late.
As the decline in cannabis diversity continues worldwide, the importance of genetic conservation becomes more evident. Unfortunately, comprehensive collections of cannabis genetic resources are not available today. The majority of the small number of seed varieties are stored in national gene banks, which may or may not share these valuable stocks with producers in other countries. The largest collection of hemp fiber genetic resources, Russia’s St. Provided by the Vavilov Plant Research Institute (VIR) in St. Petersburg.
Currently, there are 563 cannabis seed varieties in total, while all remaining fiber hemp and wild varieties are Armenia, Bulgaria, Chile, China, Czechoslovakia, Estonia, France, Germany, Hungary, Italy, Latvia, Moldova, Poland, Portugal, Romania, Russia, Spain, It is in Sweden, Ukraine, the United States, and the former Yugoslavia.
Since the late 1980s, political, technical, and financial difficulties in Russia have resulted in a low population ratio and incomplete isolation. As a result, there was a significant loss of genetic diversity and purity in the VIR collection. As many varieties are now very similar, this could lessen their importance in future breeding programs. In 1992, cannabis genetic resources at Wageningen University in the Netherlands have more than 156 varieties from 22 countries, largely obtained from other collections and research institutes.
Almost half of these varieties come from the former USSR and Hungary. The gene bank collections of the Institute of Natural Fibers and Medicinal Plants in Poland contain 139 varieties from France, Hungary, and Ukraine, which contribute 54.7% of the collections. The collection in China’s Yunnan province has about 350 varieties, most of East Asian origin, while the Ecofibre Global Gene Resources Collection in Australia includes additional Eurasian varieties. However, extensive variety data are missing in many of these collections; this limits the value of the respective varieties to producers.
In addition, there are a total of 43 cannabis varieties in the Svalbard Global Seed Vault and three other seed banks on the Norwegian island of Spitsbergen. North Korea, the Netherlands, Spain, 5 of them from Syria and Turkey is probably varieties of Cannabis marijuana; 21 of them from Argentina, Austria, China, Croatia, France, Georgia, Germany, Italy, Poland, Romania, Slovakia, Spain, and Sweden are fiber hemp varieties, and the origin of 16 varieties is unknown.
Cannabis Seed Gene Banks
There is a single Slovak cannabis variety at the Wellcome Trust Millennium Building in West Sussex, near London, the world’s largest seed warehouse specializing in wild plants. Considering the importance of cannabis as a traditional and current crop plant, and especially in the light of recent research on medical hemp, it is seen that the biodiversity of this genus (especially among drug varieties) is fairly underrepresented in seed banks.
Given this imperfect diversity in seed banks and factors such as genetic instability and low seed counts, it will appear that there is no reliable source of cannabis seed protection. The primary goal of genetic resource conservation is to protect the entire genome of each population. It is especially important for open-pollinated and hybridized plants that each gene pool has a population size large enough to allow as many alleles to be propagated in the seed.
A minimum of 1000 plants for monoic varieties and 2000 plants for dioecious varieties make it possible to reproduce 99% of the cannabis alleles. Unfortunately, seed reserves of many cannabis seed bank varieties consist of less than 1000 viable seeds; therefore, genetic diversity is limited by the number of seeds that can be preserved. The secondary purpose of genetic conservation is to ensure that a sufficient quantity of varieties is a reserve for future propagation and breeders.
The common goal of cannabis growers should be to create a more comprehensive center collection of cannabis varieties that have been comprehensively characterized, agronomically in field conditions, at the molecular level in the laboratory, genetically, and chemically. Only then will researchers be able to work with a rich gene pool in the future. This central collection should be maintained with appropriate propagation and storage methods and made accessible to growers so that each variety can be valued.
The situation has only stagnated in the last 20 years. The researchers examined collections of ex-situ cannabis genetic resources and highlighted the importance of “comprehensive conservation and characterization of cannabis by coordinating ex-situ sources, preservation of gene pool diversity and cultivation”. However, for more than 50 years, legal restrictions imposed by international narcotics conventions have had an impact on the erosion of public ex-situ cannabis genetic resources. Restrictions on the legal exchange of bona fide research materials continue to limit the establishment of physical and centralized ex-situ gene bank collections.
Genomic Approaches in Cannabis Cultivation
The result of the first genome sequence of cannabis was published in 2011 (Van Bakel et al., 2011). The haploid cannabis genome is from 818 Mbp (female) to 843 Mbp (male). Usable results were obtained in a blueprint genome of cannabis, the drug type Purple Kush (clonally propagated), and DNA sequences of the industrial types Finola and USO-31. It has been reported that the genomic sequences of these three varieties were compared and many or many SNPs supporting the separation of drug-type hemp from fiber types were detected.
Whole-genome sequencing and genome comparisons provide information about the magnitude of genetic variation found in cannabis genetic resources. The physical map of the cannabis genome with DNA polymorphisms provides new targets for the development of molecular markers in the breeding period. Cannabis grown in three different locations were phenotyped, and with the genome-wide association map, important alleles, quantitative trait loci, and associated DNA polymorphisms in the main genes were determined.
By using next-generation sequencing technologies in certain target genes known to have important features; By screening large numbers of plants for rare, induced, or natural genetic diversity, mutagenic tendencies were investigated. Targeted mutations can remove or alter the functionality of genes. It was also stated that these selected mutant genotypes are suitable for direct use as breeding material.
Genetic Cannabinoid Profile of Cannabis
In fiber hemp breeding, the main goal was to develop varieties with low THC content, and levels below THC (<0.2%) were reached for some cultivars. In 2001, only plants with a THC content of 0.2% were allowed to be grown in the European Union. Since then, a further steady reduction of THC has become important as a breeding goal. While maintaining efficiency and some other positive characters; Planning breeding studies with lower THC content emerges as a great necessity.
In the former Soviet Union (USSR), studies on the reduction of cannabinoids started in the 1970s and as a result, THC-free varieties were obtained. While several new monoic varieties were developed with the joint studies of scientists from France and Ukraine; It was determined that these varieties have very low THC levels (THC <0.07%) and do not contain a typical flavor (USO-45 variety). The distinction between fiber and drug-type cannabis varieties can only be made on the basis of the cannabinoid profile (chemotype). The three main “chemotypes” hemp, THC and CBD, between the two main cannabinoids are considered fiber hemp in terms of flower status and dry matter content;
- It is a type of drug with the presence of THC and is grown for marijuana. In this type of flower state, it is a form with a THC amount above 0.30% of dry weight and a CBD content of less than 0.50% (low CBD / THC ratio).
- It is an intermediate type that contains both THC and CBD in the same amounts; It has a complementary ratio (0.5-2.0%) of substances.
- It is a fiber type with CBD content; it is grown economically for its fiber and seeds. It has a high rate of CBD and an undetectable THC (<0.30%) level. Two allele genes (BT and BD) at the B locus control this feature. The fourth chemotype is the main cannabinoid, a precursor to cannabigerol (CBG), THC, and CBD. CBG widely (> 0.30%); CBD is less common (<0.50%). This chemotype is likely to be controlled by the Bo allele, a mutant form of the BD locus.
There is an undetectable amount of cannabinoids (zero cannabinoids). All cannabinoids that cannot be detected practically by standard gas chromatography analyzes are considered under this group. This feature is controlled by the single locus (O) running upstream of the B locus. It is reported that this zero cannabinoid chemotype is likely caused by inhibition in the metabolic pathway leading to the production of cannabinoids and preventing a change in glandular trichomes.
A particular morphological phenotype that produces CBC is found in plants with an “extended vegetative period chemotype”. The genetic factors (locus C or Bc) that control this trait are independent of the B loci encoding THC and CBD synthase.
Cannabinoid-free cannabis chemotypes or chemotypes with CBG or CBC, simply because of their pharmaceutical value, are gaining attention. The CBC has a young cannabis plant cannabinoid fraction, which decreases with maturation towards harvest. Chemotype-related molecular markers can be used for elimination to aid in the selection of THC-producing plants. To identify such markers, a cross is made between natural lines with opposite chemotypes (THC: CBD).
While F1 was almost completely hybrid (CBD / THC; chemotype II), F2 chemotypes showed a 1: 2: 1 Mendelian distribution. THC [BT, BT]: CBD / THC [BD, BT]: CBD [BD, BD) while these two codominant alleles (BT and BD) are in agreement with one gene; The F2 population is useful for chemotype identification in a particular population. Genes for THC and CBD syntheses have been isolated and specific primers have been developed that allow identification of the allele composition at locus B.
In the cannabis plant, substances such as cannabinoid, cannabidiolic acid (CBDA), and 9-tetrahydrocannabinolic acid (THCA) are synthesized and accumulated as acids. When the herbal product is dried, stored, or heated, the acids are partially or completely decarboxylated (subjected to heating) to give their neutral form (for example, from CBDA to CBD; from THCA to THC). To date, 66 different cannabinoids have been reported in cannabis; Mostly CBD, 9-THC or THC, CBG, and CBC were detected.
Based on a genetic model with two codominant alleles CT and BD at the same location, drug-type cannabis is expected to be homozygous for CT (BT / CT) and harbor only THC synthase. The publication of the draft genome of the “Purple Kush” variety with high THC content in 2011 facilitated the analysis of genes effective in cannabinoid biosynthesis. Although only a single THC synthase gene (THCAS) with a full open reading frame has been identified in this cannabis genome, as the first step for the biosynthesis of 9-THC, CBD, and CBC cannabinoids, the result of the condensation of geranylgeraniol diphosphate (GPP) and OA olive, CBG is produced.
The next step is through the action of THC- and CBD-synthases that convert CBG to THC (BT locus), CBD (BD locus), and in some cases CBC-synthase, which catalyzes the conversion of CBG to cannabinoid cannabichromene (CBC). . Markers such as BC, BT, BD, and Bo are alleles of the B locus that explain the different chemotypes. However, the genomic organization of the locus is more complex, as pseudogenes with premature stop codons and frameshift mutations have been noted to be part of a small gene family of cannabinoid synthase genes for both genes.
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