An Introduction to Cannabis Genetics, Part III

By Dr. CJ Schwartz

Polyploidy in Cannabis

Polyploidy is defined as containing more than two homologous sets of chromosomes. Most species are diploid (all animals) and chromosomal duplications are usually lethal, even partial duplications have devastating effects (Down’s syndrome). Plants are unique as in being able to somewhat “tolerate” chromosomal duplications. We often observe hybrid vigor in the F1, while the progeny of the F1 (F2) will produce mostly sickly or dead plants, as the chromosomes are unable to cleanly segregate.

Chromosomal duplications, either one chromosome or the whole genome, happen frequently in nature, and actually serves as a mechanism for evolution. However the vast majority (>99.99%) results in lethality.

Thus there is polyploidy in Cannabis, and a few examples are supported by scientific evidence. The initial hybrid may show superior phenotypes and can be propagated through cloning, but there may be little potential for successful breeding with these plants.

Epigenetics and Phenotypic Consistency in Clones

One mechanism of turning off genes is by the DNA becoming physically inaccessible due to a structure resembling a ball. In addition, making molecules similar to DNA (RNA) that prevents expression of a gene can turn off certain genes. Both mechanisms are generally termed epigenetics.

These mice are all genetically identical yet they manifest different phenotypes for fur color. — These mice are genetically identical, yet their coat color phenotype is variable. Something above or beyond (epi) the gene (genetic) is controlling the phenotype.

Epigenetic regulation is often dependent on concentrations of certain proteins. Through the repeated process of cloning, it is possible that some of these proteins may be diluted, due to so many total cell divisions and epigenetic control of gene expression can be attenuated and results in phenotypic variability.

Sexual reproduction, and possibly tissue culture propagation, may re-establish complete epigenetic gene regulation, however the science is lacking. Epigenetic gene regulation is one of the hottest scientific topics and is being heavily investigated in many species including humans.

Hermaphrodites and Sex Determination

Cannabis is an extremely interesting genus (species?) for researching sex determination. Plants are usually either monoecious (both male and female organs on a single plant), or dioecious, separate sexes. Sex determination has evolved many times in many species. Comparing the mechanisms of sex determination in different organisms provides valuable opportunities to contrast and compare, thereby developing techniques to control sex determinations.

The sex organs on a Cannabis plant identified.

Cannabis is considered a male if it contains a Y-chromosome. Females have two X chromosomes. Even though female Cannabis plants do not have the “male” chromosome, they are capable of producing viable pollen (hermaphrodite) that is the source of feminized seeds. Therefore, the genes required to make pollen are NOT on the Y-chromosome, but are located throughout the remainder of the Cannabis genome. However, DNA based tests are available to identify Male Associated Sequence (MAS) that can be used as a test for the Y-chromosome in seedlings/plants.

Natural hermaphrodites may have resulted from Polyploidization (XXXY), or spontaneous hermaphrodites could be a result of epigenetic effects, which may be sensitive to the environment and specific chemical treatments.

Feminized seeds will still have genes segregating, thus they are not genetically identical. This shouldn’t lead to a necessary decrease in health, but could. A clone does not have this problem.

The other issue is that “inbreeding depression” is a common biological phenomenon, where if you are too inbred, it is bad…like humans. Feminized seeds are truly inbred. Each generation will decrease Heterozygosity, but some seeds (lines) may be unhealthy and thus are not ideal plants for a grower.

GMO– The Future of Cannabis?

Is there GMO (genetically modified organism) Cannabis? Probably, but it is likely in a lab somewhere…deep underground! Companies will make GMO Cannabis. One huge advantage to doing so is that you create patentable material…it is unique and it has been created.

The definition of a GMO is…well, undefined. New techniques exist whereby a single nucleotide can be changed out of 820 million and no “foreign” DNA remains in the plant. If this nucleotide change already exists in the Cannabis gene pool, it could happen naturally and may not be considered a GMO. This debate will continue for years or decades.

Proponents of GMO plants cite the substantial increase in productivity and yield, which is supported by science. What remains to be determined, and is being studied, are the long-term effects on the environment, ecosystem and individual species, in both plants and animals. Science-based opponent arguments follow the logic that each species has evolved within itself a homeostasis and messing with its genes can cause drastic changes in how this GMO acts in the environment/ecosystem (Frankenstein effect). Similarly, introducing an altered organism into a balanced ecosystem can lead to drastic changes in the dynamics of the species occupying those ecological niches. As in most things in life, it is not black and white; what is required is a solid understanding of the risks of each GMO, and for science to prove or disprove the benefits and risks of GMO crops.

May 13, 2016

An Introduction to Cannabis Genetics, Part II

By Dr. CJ Schwartz

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Plants and animals have roughly 25,000 to 30,000 genes. The genes provide the information needed to make a protein, and proteins are the building blocks for all biological organisms. An ideal analogy is a blueprint (DNA) for an alternator (the protein) in a car (the plant). Proteins are the ‘parts’ for living things. Some proteins will work better than others, leading to visible differences that we call phenotypes.

Many traits, and the genes controlling them, are of interest to the cannabis industry. For hemp seed oil, quality, quantity and content can be manipulated through breeding natural genetic variants. Hemp fibers are already some of the best in nature, due to their length and strength. Finding the genes and proteins responsible for elongating the fibers can allow for the breeding of hemp for even longer fibers. In cannabis, the two most popular genes are THCA and CBDA synthases. There are currently over 100 sequences of the THCAS/CBDAS genes, and many natural DNA variations are known. We can make a family tree using just the THCAS, gene data and identify ‘branches’ that result in high, low or intermediate THCA levels. Generally most of the DNA changes have little to no effect on the gene, but some of the changes can have profound effects.

In fact, CBDAS and THCAS are related, in other words, they have a common ancestor. At some point the gene went through changes that resulted in the protein producing CDBA, or THCA or both. This is further supported by the fact that certain CBDAS can produce some THCA, and vice-versa. Studies into the THCAS and CBDAS family are ongoing and extensive, with terpene synthase genes following close behind.

Identifying gene (genetic) variants and characterizing their biological function allows us to combine certain genes in specific combinations to maximize yield, but determining which genes are important (gene discovery) is the first step to utilizing marker-assisted breeding.

Gene Discovery & Manipulation

The term genetics is often misused in the cannabis industry. Genetics is actually “the study of heredity and the variation of inherited characteristics.” When people say they have good genetics, what they really mean is that they have good strains, presumably with good gene variants. When people begin to cross or stabilize strains, they are performing genetic manipulation.

A geneticist will observe or measure two strains of interest, for example a plant branching and myrcene production. The high-myrcene plant is tall and skinny with no branching, reducing the yield. Crossing the two strains will produce F1 hybrid seeds. In some cases, F1 hybrids create unique desirable phenotypes (synergy) and the breeder’s work is completed. More often, traits act additively, thus we would expect the F1 to be of medium branching and medium myrcene production, a value between that of the values recorded for the parents (additive). Crossing F1 plants will produce an F2 population. An F2 population is comprised of the genes from both parents all mixed up. In this case we would expect the F2 progeny to have many different phenotypes. In our example, 25% of the plants would branch like parent A, and 25% of the F2 plants will have high myrcene like parent B. To get a plant with good branching and high myrcene, we predict that 6.25% (25% x 25%) of the F2 plants would have the correct combination.

The above-described scenario is how geneticists assign gene function, or generally called gene discovery. When the gene for height or branching is identified, it can now be tracked at the DNA level versus the phenotype level. In the above example, 93.5% of your F2 plants can be discarded, there is no need to grow them all to maturity and measure all of their phenotypes.

The most widely used method for gene discovery using natural genetic variation is by quantitative trait loci mapping (QTL). For these types of experiments, hundreds of plants are grown, phenotyped and genotyped and the data is statistically analyzed for correlations between genes (genotype) and traits (phenotype; figure). For example, all high-myrcene F2 plants will have one gene in common responsible for high myrcene, while all the other genes in those F2 plants will be randomly distributed, thus explaining the need for robust statistics. In this scenario, a gene conferring increased myrcene production has been discovered and can now be incorporated into an efficient marker-assisted breeding program to rapidly increase myrcene production in other desirable strains.

April 11, 2016

An Introduction to Cannabis Genetics, Part I

By Dr. CJ Schwartz

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What is DNA?

DNA stores information about how to build an organism. Just as a series of 0’s and 1’s represents digital data, DNA data is represented by four letters (A, C, G and T), which inherently allows DNA to store more information per unit (Figure 1).

The amount of DNA required to build a human is mind-boggling. The human genome has 3.2 billion A’s, C’s, G’s, or T’s, (called nucleotides). Cannabis has 820 million nucleotides. This is true for every cell in the organism. The DNA from a single human cell when spread out would stretch six feet long. A cell is not visible to the naked eye, yet it contains a microscopic thread of DNA six feet long! If you put all the DNA molecules in your body end to end, the DNA would reach from the Earth to the Sun.

DNA is common in all living things, and all living things are related through DNA. Humans and plants share 50% of their genes. In humans, 99.9% of the DNA is identical, thus just 0.1% of DNA differences accounts for all of the variation observed in humans. Cannabis, as a species, is more variable with approximately 1% of the DNA being different among strains. DNA is a super efficient and reliable information storage system. However, mistakes (mutations) do occur and while infrequent, these mutations account for all the differences observed within a species and is called natural genetic variation. Variation within the genomes of a species can help the species survive in unfavorable conditions (evolution) and is also the source of differences in traits, which is the material that is required for successful breeding.

Natural Genetic Variation

DNA mutations occur in every generation and these changes will be different in each individual creating natural genetic variation. Mutations (or more accurately referred to as DNA changes) will be inherited by offspring and will persist in the population if the offspring reproduce.

DNA differences maintain diversity in the gene pool, allowing organisms to respond to new environments (migration) or environmental changes (adaptation). The two most commonly described cannabis families are Indicas and Sativas. Indicas, being from cooler temperate regions, have wide leaves allowing the maximum capture of light during the shorter growing season. Sativas, being equatorial, have smaller leaves, which may be an advantage for such things as powdery mildew in a humid environment. Figure 2 shows the enormous amount of natural variation in leaves for one species with a worldwide population (Arabidopsis thaliana).

A DNA change that occurred a long time ago will be more useful to divide people/plants into different groups. For example, there are ancient DNA changes that differentiate humans originating from Europe or Asia. Other newer DNA changes allow us to further divide Europeans into those originating from Northern versus Southern Europe. Thus, different DNA changes have different values for determining relatedness or ancestry, yet every DNA change provides some information for determining heredity.

Family Trees

By comparing DNA changes among different strains, we can measure the relatedness between strains. For example, if strain A has a DNA change indicative of Kush ancestry and strain B has a DNA change indicative of hemp ancestry, we can assign strains to branches of the cannabis family tree comprised of strains that contain similar DNA changes. Figure 3 shows 184 strains that have been characterized for these changes, and the position of each strain is based on its shared DNA with neighboring strains. The two best-defined families of cannabis are hemp (blue) and kush (black). Strains within a family are more closely related. Strains in separate families, such as kush and hemp, are more distantly related.

Editor’s Note: This is the first installment in a series of articles focused on answering common questions regarding cannabis genetics. If you have questions regarding cannabis genetics, or wish to speak more about the topic please post in the comments section below. The next installment will delve into the THC synthase, gene discovery and manipulation and mapping chromosomes.