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:D Genetic Preservation :D - Breeding

acespicoli

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Proc Natl Acad Sci U S A. 2018 Nov 6; 115(45): 11354–11356.
Published online 2018 Oct 26. doi: 10.1073/pnas.1816187115
PMCID: PMC6233102
PMID: 30366950

Outbreeders pull harder in a parental tug-of-war​

Yaniv Brandvaina and David Haigb,1
Author information Copyright and License information Disclaimer

See the article "Intersexual conflict over seed size is stronger in more outcrossed populations of a mixed-mating plant" in volume 115 on page 11561.
This article has been corrected. See Proc Natl Acad Sci U S A. 2019 October 22; 116(43): 21947.


During development, seeds compete, directly or indirectly, for access to resources supplied by the maternal plant. This competition is expected to be more intense when embryos (and associated endosperms) are less related to each other because they are fertilized by less-related pollen (1). Imprinted genes of paternal origin in offspring tissues (embryos and endosperms) are predicted to engage in more intense competition for maternal resources than imprinted genes of maternal origin because offspring tissues share the same mother but may have different fathers (2, 3). Plant species vary tremendously in the extent to which they produce selfed siblings (the progeny of self-pollination), half-siblings, and full-siblings (the progeny of outcrossing). The “weak inbreeder/strong outbreeder” (WISO; pronounced “why so”) hypothesis predicts that this maternal–paternal conflict will escalate to higher levels as the degree of outcrossing increases (4). In PNAS, Raunsgard et al. (5) test this hypothesis using crosses among Mexican populations of Dalechampia scandens.
The conflict over resource allocation in seeds largely takes place in the endosperm, an ephemeral organism that acquires nutrients from the diploid mother and passes them on to the diploid embryo. Endosperm is triploid in most flowering plants: two maternal genomes for each paternal genome (2m:1p). The antagonistic effects of maternal and paternal genomes on endosperm development are revealed in reciprocal crosses between diploids and their autotetraploids. When the seed parent is diploid, the resulting endosperm is tetraploid (2m:2p) and can be described as exhibiting “paternal excess.” When the seed parent is tetraploid, the resulting endosperm is pentaploid (4m:1p) and can be described as exhibiting “maternal excess.” In species in which the earliest phases of endosperm development occur without cell division, paternal excess is associated with prolonged proliferation of endosperm nuclei, with delayed or absent cellularization of endosperm. In contrast, maternal excess is associated with reduced proliferation of endosperm nuclei and precocious cellularization (2). Of particular interest, some interspecific crosses between diploid species exhibit features of maternal excess in one direction of the cross and paternal excess in the reciprocal cross despite triploid (2m:1p) endosperms in both directions of the cross. We have proposed that these hybridization barriers could be explained by differences in mating system between the two species, with genomes from the more outbred species having greater strength or “effective ploidy” (i.e., features of paternal excess were predicted when the pollen parent was more outbred, but features of maternal excess were predicted when the seed parent was more outbred). We found support for these predictions in a review of earlier literature (4).
Subsequent studies have provided further support for the WISO hypothesis. For example, a recent study by Lafon-Placette et al. (6) intercrossed three diploid species of Capsella and assigned effective ploidies to their genomes based on each genome’s effects in hybrid seeds. Capsella grandiflora (an obligate outcrosser) was assigned higher effective ploidy than Capsella rubella (a recent selfer), which, in turn, was assigned higher effective ploidy than Capsella orientalis (an ancient selfer). Endosperms from interspecific crosses exhibited symptoms of paternal excess when the pollen parent had higher effective ploidy but exhibited symptoms of maternal excess when the seed parent had higher effective ploidy. The maternal-excess seeds were very small and underwent precocious cellularization of endosperm, whereas the paternal-excess seeds did not undergo cellularization of endosperm and were shriveled. These results are broadly consistent with the WISO hypothesis, because effective ploidy decreases with an increased evolutionary history of selfing. However, the paternal-excess seeds were shriveled and failed to extract extra maternal resources. This failure can be ascribed to an imbalance in the effective ploidies of the maternal and paternal parents sufficiently great to be incompatible with seed development. One of the crosses that failed, between C. rubella as seed parent and C. grandiflora as pollen parent, succeeded when a tetraploid C. rubella was used as seed parent. Thus, an increase in actual ploidy of the self-fertilizing parent was able to correct the imbalance in effective ploidies at the diploid level.
Interspecific crosses test the WISO hypothesis in circumstances in which there has been substantial evolutionary divergence between the parents. It is also desirable to test the hypothesis in crosses in which the parents are more recently diverged and have more subtle differences in mating system. Raunsgard et al. (5) provide such a test in crosses between geographically separated populations of D. scandens, a vine with a mixed mating system. The relative frequency of outcrossing and selfing in Dalechampia varies geographically and is reflected in structural features of the inflorescence—in particular, the distance between male and female flowers (herkogamy). Raunsgard et al. (5) found that seed size tended to be larger when the pollen parent came from a population with a higher frequency of outcrossing but found that it was smaller in the reciprocal cross. They also found a stronger relationship between seed size and the difference between maternal and paternal herkogamy than between seed size and paternal herkogamy. This was interpreted as evidence in favor of a “tug-of-war” mechanism rather than a “recognition-avoidance” mechanism. Under the tug-of-war mechanism, seed size is determined by the quantitative balance between maternally expressed growth suppressors and paternally expressed growth enhancers, with levels of both suppressors and enhancers escalated in more-outcrossed populations. Under the recognition-avoidance mechanism, the expression of paternally expressed growth enhancers is elevated in more-outcrossed populations, but these are blocked by maternal countermeasures that are specific to each population. We think both mechanisms can be subsumed under the broader WISO hypothesis.
We know of two other studies that found an association between differences in seed size in reciprocal crosses and differences in mating system between the parental populations. Willi (7) intercrossed Arabidopsis lyrata from populations with different frequencies of selfing and found that seed size was larger when mothers from outcrossed populations were pollinated by fathers from selfing populations but was smaller when the parental roles were reversed. Similar data can be extracted from an old study of Leavenworthia by Lloyd (8), in which the difference in seed size in reciprocal crosses also increased as the difference in mating system between the parental populations increased (Fig. 1). Thus, studies in Dalechampia, Arabidopsis, and Leavenworthia provide evidence that variation in mating system within species can generate subtle incompatibilities between maternal and paternal genomes in seed development (68). An important direction for future research is to connect these phenotypic observations with mechanisms, including changes in the expression of imprinted genes.
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Fig. 1.
Relationship between relative seed size and relative mating system in Leavenworthia (data from ref. 8). Relative seed size = log(seed weight) − log(maternal seed weight). Mating systems are classified as self-compatible (sc), weakly self-incompatible (wsi), and strongly self-incompatible (ssi). Relative mating systems are as follows (mother × father): −2 (ssi × sc), −1 (wsi × sc, ssi × wsi), 0 (ssi × ssi, sc × sc), 1 (sc × wsi, wsi × sci), and 2 (sc × ssi).
Most research comparing imprinted expression in species with different mating systems have used Capsella and Arabidopsis, two genera of Brassicaceae. In Capsella, the number of paternally expressed genes (PEGs) and their levels of expression were both higher in outcrossing C. grandiflora than in the self-fertilizing species, but the number and expression of levels of maternally expressed genes (MEGs) did not differ (6). Similarly, the number and expression of PEGs, but not MEGs, was elevated in outcrossing A. lyrata compared with self-fertilizing Arabidopsis thaliana (9). PEGs are also more frequently flanked by transposable elements (TEs) than are MEGs (10). Why PEGs appear more responsive to variation in the intensity of parental conflict, why PEGs are preferentially associated with TEs, and how this apparent one-sided molecular response results in seed phenotypes that are consistent with the tug-of-war model require further study.
There is substantial evidence of coevolution between TEs and imprinted gene expression, including genomic colocalization of TEs and PEGs (10) and the role of Polycomb group-mediated histone methylation in the control of both imprinted expression and transposition (11). Therefore, a more complete picture of the genomic effects of mating system on imprinted expression needs to take account of the distinctive selective forces acting on TEs. New mutations of TEs, at each and every location in the genome where a TE is inserted, are under selection to reduce rates of transposition. This includes selection on the TE sequence for the production of small interfering RNAs (siRNAs) that inhibit transposition. On the other hand, actively proliferating TEs are also under selection for their ability to transpose, because insertions at new sites select for elements that are still able to transpose. For a TE lineage to remain active, it must continually move to new sites faster than inactivating mutations occur at established sites (12). If a TE is differentially expressed in maternal and paternal germ tracks, then paternally expressed transcripts are subject to the same selective forces as PEGs, and maternally expressed transcripts are subject to the same selective forces as MEGs. By this process, TEs may have been coopted into roles in the control of nutrient transfer in seeds.
The evolutionary dynamics of TE-derived sequences vary with mating system in complex ways (13), although the general expectation is that TEs will be less abundant and less active in species with a long history of self-fertilization, because self-fertilization prevents active TEs segregating away from the mutational costs of transposition. Consistent with this expectation, TEs are much more abundant in the outcrossing C. grandiflora and the recent selfer C. rubella than in the ancient selfer C. orientalis (14). The greater activity of TEs and silencing siRNAs could partially account for the greater effective ploidy of outcrossing species in intraspecific crosses. Thus, a relative excess of maternally expressed siRNAs derived from TEs has been associated with precocious cellularization in maternal excess endosperms, whereas a relative deficiency is associated with increased nuclear proliferation in paternal excess endosperms (15).
Because of coevolution between maternal and paternal genomes in species subject to genomic imprinting, normal development comes to depend on the correct balance of antagonistic forces. The strength of these antagonistic forces is predicted to be responsive to changes in mating system. Therefore, divergence in mating systems between reproductively isolated populations can be a source of barriers to hybridization. The work of Raunsgard et al. (5) suggests that these barriers can evolve rapidly and presents D. scandens as a model for studying early stages in this process. A combination of genomic and transcriptomic studies in this group could help unravel the molecular underpinning of the coevolution of imprinting, transposition, and mating system.
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Footnotes​


The authors declare no conflict of interest.


See companion article on page 11561.

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References​

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acespicoli

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Cannabinoid potency​

It is known from other systems that increases in copy number of biosynthetic gene clusters can elevate secondary metabolite production (Manderscheid et al., 2016) and it has been suggested that cannabinoid synthase gene copy numbers might play a role in determining overall cannabinoid content (Vergara et al., 2019). Although multiple copies are present in tandemly repeated arrays in CBDRx, only a single copy of CBDAS was expressed. Also, contrary to the prediction of the copy number hypothesis, none of the six significant QTLs for total cannabinoid content (potency) in a segregating population was associated with the cannabinoid synthase arrays on chromosome 7 (Fig. 5; Table S11). However, a potential marker association near the cannabinoid ratio QTL, at 38.89 cM, did not reach the experiment‐wise threshold for statistical significance (max LR = 13.9, LOD = 3.02, at 31.65 cM). Repeating the potency analysis with additional marker cofactors on chromosome 7 increased the likelihood ratio to 17.95 (LOD = 3.9) but still fell just short of significance.
We might expect genes expressing other metabolic enzymes upstream of THCAS and CBDAS in the cannabinoid pathway to be associated with potency. The hexanoate pathway, the methylerythritol phosphate (MEP) pathway, and the geranyl diphosphate pathway produce essential substrates for cannabinoid synthesis. Previous experimental work identified each of the enzymes involved in these pathways (Laverty et al., 2019). We located these genes in our assemblies and verified their expression with full‐length cDNA libraries (Table S12). We then compared their physical map positions with our genetic map and found two candidate genes proximal to potency QTLs. The gene coding Acyl‐activating enzyme 1 (AAE1), the last enzyme of the hexanoate pathway (Gagne et al., 2012), is located at 39.7 cM on chromosome 3 and a QTL (LOD peak 40.2 cM) associated with 17% of the variance in potency. The gene coding for 4‐hydroxy‐3‐methylbut‐2‐enyl diphosphate reductase (HDR), the last enzyme in the MEP pathway, is located 1.61 cM from a QTL (LOD peak at 40.59 cM) on the X chromosome that is associated with 9% of potency variance. These associations point to potential directions for future studies of mechanisms enhancing cannabinoid expression and selection of this economically important trait.

CBDRx ancestry and the origin of CBD‐type Cannabis

Weiblen et al. (2015) argued that marijuana breeding would favor plants lacking CBDAS. Without the enzyme that competes with THCAS for the same precursor, CBG, the cannabinoid ratio is skewed in favor of intoxicating THC (Onofri et al., 2015). Evidence from dN : dS ratios suggests strong, positive selection for nonfunctional variants of CBDAS in marijuana (Weiblen et al., 2015). Marijuana breeders also possibly selected other independently inherited traits affecting cannabinoid content (potency) such as inflorescence architecture or trichome size (Small & Naraine, 2016). Once highly potent marijuana cultivars were developed (ElSohly et al., 2000) they could be crossed with hemp‐type Cannabis, decoupling the THC : CBD ratio from overall cannabinoid content so that highly potent CBD‐type Cannabis could be selected by introgressing functional CBDAS into marijuana.
Multiple lines of evidence from CBDRx support this scenario. The CBDRx genome is predominantly of marijuana ancestry (Figs 1, S2) and much of the hemp‐derived ancestry in the CBDRx genome is found on chromosome 7 where CBDAS is located (Fig. 2). The lone QTL for the cannabinoid ratio maps to the 31 Mb position of CBDAS on this chromosome (Fig. 2d) and there is population genetic evidence of recent, positive selection in the vicinity (PBS; Figs 2b, S3). These observations are consistent with the interpretation that a CBD‐type cannabinoid profile is the result of introgression of hemp‐like alleles into a drug‐type genetic background to elevate CBD production at the expense of THC. Further evaluation of this hypothesis will require additional genome assemblies from THC‐type C. sativa.
It appears that introgression followed by artificial selection has yielded new types of Cannabis like CBDRx with unprecedented combinations of phenotypic traits, as has been observed in other domesticated plants, including sunflower (Rieseberg et al., 2003). Marijuana and hemp cultivars have a history of independent breeding and reduced gene flow between domesticated populations selected for divergent traits. We suggest that breeders have responded to recent interest in CBD with targeted introgression to produce marijuana cultivars with exceptionally high concentrations of CBD.

Conclusion​

We trace the origin of a Cannabis cultivar with elevated CBD content to chromosome 7 where the introgression of CBDAS from hemp into a marijuana background has shifted the predominant cannabinoid from THC to CBD while maintaining an overall quantity of cannabinoids that is typical of drug‐type Cannabis. Cannabinoid synthase gene clusters could be further manipulated, but QTL analysis suggests that other genetic regions on different chromosomes can be targeted to either enhance or reduce potency. It is highly abnormal for a plant to allocate 20–30% of its flowering biomass to one or two specialized metabolites, as do modern Cannabis cultivars. Dissecting this trait will require much additional study. We speculate that integrating cell biology and developmental genetics with existing knowledge of the relevant metabolic pathways will be necessary. Although controlled substance regulations have hindered Cannabis science for decades, economic trends, recent changes in law and the genomic results described here have the potential to accelerate the study of a plant that has coevolved with human culture since the origins of agriculture.
 

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The seedless Triploid cannabis genetics revolution​


Triploid cannabis seeds may not be too far away from being a commercial reality. Triploid genetics have been used extensively in farming, bringing stabilised increases in both yield and quality. The same could also prove true for cannabis and that has got everyone in the cannabis industry very excited. One other benefit for the home grower is the fact that plants grown from cannabis seeds containing triploid genetics won’t ever produce seeds. Even if you have a rogue hermaphrodite (or male) nearby.


A brief history of cannabis genetics breeding​

Traditionally cannabis genetics contain two sets of chromosomes making it diploid. In regular cannabis seeds, one comes from the male parent (the ‘Y’ chromosome) and one from the female parent (the ‘X’ chromosome). Regular cannabis seeds therefore have ‘XY’ chromosome pairs.

In feminised cannabis seeds only two ‘X’ chromosomes from the female are present, giving rise to (generally) all female offspring. Photoperiod feminised cannabis seeds (and feminised autoflower seeds) have an ‘XX’ chromosome pair.

With triploid seeds there are three sets of chromosomes. The extra set comes from either the male or the female parent. Because there is an odd number of chromosomes fertilisation of a triploid plant can’t take place - the plants are sterile and therefore triploids are always completely seedless.

There are many industries that take advantage of triploid genetics, the best example of a mainstream agricultural crop that uses triploid genetics is the global banana industry. In order to guarantee commercial-sellable bananas, without hard black seeds inside, triploid banana genetics are used from the ‘Cavendish’ strain.

Triploids bananas are seedless. Since triploid plants can’t be pollinated, triploid banana plants can only be propagated by taking cuttings (clones).

Feminised cannabis seeds vs triploid cannabis seeds​

Feminised cannabis seeds are now the industry standard following their 1990’s introduction by Dutch Passion. Even Dutch Passion, which still boasts one of the largest remaining collection of regular cannabis seeds in the industry only sells around 2% of their cannabis seeds in regular form. Cannabis breeding, both for autoflower seeds and photoperiod feminised seeds, has until now focussed on traditional ‘diploid’ (2 sets of chromosomes) genetics. But the coming years may see the first triploid cannabis seeds becoming available. And with them could come a step-change in yields and quality.

Cannabinoid levels could be stable in triploid cannabis even at saturation levels and you would never need to worry about accidentally seeded crops. What’s more, if you wanted to give your friends cuttings of your favourite triploid cannabis plants you can. They will root easily and grow into healthy new triploid plants with the same elite genetics. You can take as many cuttings from a triploid cannabis plant as you could from a non-triploid.

Triploid cannabis genetics wouldn’t be the same as GMO, triploidy is genetic manipulation rather than genetic modification. So if it meant that you could grow heavier yields of more potent buds would you grow seedless triploid cannabis genetics?

Sinsemilla and the problem with cannabis pollen​

Cannabis pollen is one of the biggest threats to a crop, both for home growers as well as professional licensed growers. It’s a source of anxiety and worry. Allow a crop to become seeded and you will inevitably feel bitterly disappointed.

Feedback from Dutch Passion customers over many decades has shown that the top-3 items on the wish-list for new seeds are:

• Cannabis seeds that are easier & faster to grow
• Cannabis seeds that produce more potent buds in heavier quantities
• Strains that wouldn’t form their own cannabis seeds if accidentally exposed to pollen

Seedless cannabis, or sinsemilla, is the goal of all growers (apart from breeders). Feminised triploid cannabis seeds offer growers the option to guarantee seedless crops and at the same time produce highly consistent harvests. If cannabis breeders can combine killer genetics and deliver them in triploid form, the benefits for cannabis growers may quite easily surpass all current expectations.

Without wanting to over-state the potential, triploid cannabis seeds could well be an even more significant event than the introduction of feminised seeds.

Some of the current research, whilst still awaiting confirmation, hints at some possibly revolutionary improvements. Watch this space for more.


What is Triploid cannabis?​

What-is-Triploid-cannabis

Cannabis is normally diploid, with two sets of chromosomes. But triploidy (3 sets of chromosomes) and even tetraploid (4 sets of chromosomes, 2 female & 2 male) are possible, more so in plants than animals. Triploid cannabis could be created by crossing diploid and tetraploid parents. This was the approach taken to create the modern banana.

It’s important to add that the triploid product being grown (bananas, fruits or potentially cannabis) is fundamentally the same product. A triploid banana tastes the same (or better) than a seeded banana and the final product convenience/value is significantly higher. The plant structure is similar. The only change is the chromosome count.

As well as conferring sexual sterility (meaning no seeded crops ever), triploid genetics tend to be very stable with yield enhancements and quality improvements. Therefore, for any cannabis seed breeder considering creating triploid cannabis genetics it makes most sense to combine the triploid breeding process with truly elite genetics, maximising the inherent yield/quality enhancements that triploid can deliver.

Remember that use of triploid breeding to deliver superior yields & quality is common practice in agriculture and has been so for decades. As well as bananas, seedless watermelons and other fruits have also been created.
 

acespicoli

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While male plants produce small amounts of cannabinoids, in cannabis cultivation, the primary products are the female flowers clustered in inflorescences (Ohlsson et al., 1971). Stalked glandular trichomes are primarily concentrated on the calyces and bracts (Figure 1A; Spitzer-Rimon et al., 2019; Leme et al., 2020) with populations extending to the inflorescence “sugar leaves”; these are the sites of accumulation for secreted metabolic products. These valuable secretions include tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA), terpenes, and flavonoids (ElSohly and Slade, 2005; Flores-Sanchez and Verpoorte, 2008). Cannabis plant morphology and cannabinoid profiles are influenced by genetics and the cultivation environment, highlighting the importance of controlled conditions for cannabis cultivation (Magagnini et al., 2018; Danziger and Bernstein, 2021a, b).

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One hundred ten whole genomes of cannabis cultivars, from wild plants and historical varieties to modern hybrids, with a focus on Asian sources to account for the likely domestication origin, were recently sequenced and analyzed to provide an invaluable genetic framework for the history of the plant; the resulting information can be applied to secondary metabolite investigations (Ren et al., 2021). With time, the validity of these hypotheses is sure to be determined thanks to this new genomic information, along with valuable insight into the impressive complexity seen within them.
 

acespicoli

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Tetraploids displayed reduced female fertility as compared to diploids, but were still fertile. When pollinated by diploid plants, the tetraploid seed parents showed an average of 78% fewer filled seeds than the diploid × diploid cross (Table 2). A similar phenomenon was observed when using the tetraploids as pollen donors. When pollinated by tetraploid plants, the tetraploid seed parents showed 42% fewer total seeds than the diploid × tetraploid combination. These results, taken together, indicate that the tetraploid plants assessed in this study have reduced female fertility, but are still able to produce viable seeds.
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Several methods of ploidy manipulation in C. sativa were published prior to this study; however, this is the first report that tracks phenotypic differences between diploids, triploids, and tetraploids in relation to seed production, biomass, and cannabinoid yield.
 

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  • Methods

    • Morphological characters

    • Phytochemical characters

    • Genetic characters


  • Results

    • Taxonomic analysis

    • Key to four varieties of C. sativa subsp. indica1

    • Taxonomic treatment

    • Variety 1: South Asian domesticate

    • Variety 2: South Asian wild-type

    • Variety 3: Central Asian domesticate

    • Variety 4: Central Asian wild-type

1. General Introduction to Cannabis spp.: Taxonomy and History of Cultivated Varieties​

Cannabis sativa L. is an agricultural plant species that today enjoys great interest because of its multiple uses in the recreational, medicinal, and industrial areas (Kovalchuk et al., 2020). This plant can be cultivated for the production of fibers (used to make different textiles), seeds (rich in unsaturated fatty acids for edible oils), and drugs from its female inflorescences that contain cannabinoids (compounds with psychotropic or psychopharmaceutical effects). Among these latter, the principal psychoactive constituent of cannabis is THC (tetrahydrocannabinol), and the concentration of this metabolite is at the basis of the distinction between hemp and drug (marijuana) types, with hemp considered low in concentration, 0.3% or less THC content (non-psychoactive), and marijuana, on the other hand, containing up to 30% THC by dry weight. In the present review, we will mainly focus on drug type cannabis.
The genus Cannabis belongs to the family of Cannabaceae (order Rosales). Its botanical classification had a very troubled genesis since the times of Linnaeus considering it was not clear whether the genus was mono- or polytypic (Schultes, 1970; Small and Cronquist, 1976; Schultes and Hofmann, 1980). In 1597, John Gerarde (Gerarde, 1597) first defined the plant species as dioecious, but the question remained open because monoecious plants can occur and hermaphroditism is also possible with plants that show reproductive organs within the same flower (Small and Cronquist, 1976; Clarke, 1981; Ming et al., 2011). All these biological variants are known to be very frequent in fiber varieties (Small and Cronquist, 1976). Plants also manifest sexual dimorphism, with male individuals being often characterized by a shorter crop cycle and a taller stature than female ones. Lamarck originally recognized two interfertile species C. sativa (from Persia) and C. indica (from India) (Lamarck, 1785). Based on this old taxonomy, many varieties available on the market are still classified as C. sativa × C. indica hybrids. As a matter of fact, the reproductive system of cannabis plants is characterized by allogamy and anemophily, and therefore open pollination is necessarily responsible for a certain degree of hybridization between improved and wild populations. This is why, according to Schultes, landraces of cannabis should no longer exist since several decades (Schultes, 1970). Later on, Small and Cronquist (Small and Cronquist, 1976) proposed a unique species system that is still widely accepted and that is based on two subspecies of C. sativa: C. sativa subsp. sativa and C. sativa subsp. indica. Although several authors, supporting the one-species system for cannabis, recommend to classify its varieties based on the cannabinoids and terpenoids profile (Hazekamp et al., 2016; Piomelli and Russo, 2016), a molecular system based on DNA barcoding could represent a cost- and time-effective technique of great help in clarifying some of the taxonomic issues related to the genus Cannabis. DNA barcoding could also play a crucial role in the identification and characterization of those uncertified cannabis strains, which are mainly derived from black market. Section 2 reviews the DNA barcoding data available for this genus and explores the potential use of this technique for taxonomic identity surveys.
According to Charlesworth et al. (2005), the dioecious species evolved from a common monoecious ancestor shared by Cannabis and Humulus (Kovalchuk et al., 2020) both characterized by having sex chromosomes (Renner, 2014). In particular, C. sativa possesses nine pairs of autosomes and a pair of X and Y sex chromosomes. The male sex is heterogametic (XY), while the female is homogametic (XX), and different authors reported distinct mechanisms involved in the determination of sex (Sakamoto et al., 1998; Faux et al., 2016). This uncertainty could derive from the fact that environmental conditions, and in particular abiotic stress factors, can influence the expression and the determination of sex (Vergara et al., 2016a). Although the structure of sex chromosomes is poorly understood in Cannabis spp., since it is not detectable with standard microscopic techniques (Sakamoto et al., 1998; Peil et al., 2003), the Y chromosome was shown to have larger dimensions than the X chromosome (Sakamoto et al., 1998; van Bakel et al., 2011). More recently, both male and female karyotypes of C. sativa L. were extensively characterized by DAPI banding procedures and FISH analyses using rDNA probes (Divashuk et al., 2014). Sex determination represents one of the main problems when breeding new cannabis varieties since it can only be assessed at the beginning of flowering, when male and female flowers are visible and distinguishable. The genetic control of dioecy seems to be determined by two specific genes at linked loci acting as sex determinants (Bergero and Charlesworth, 2008; Divashuk et al., 2014; Henry et al., 2018): Male plants would require a dominant suppressor of female organs (SuF) and a dominant activator of maleness (M), while female plants would share homozygosity for their recessive alleles at both loci (suFsuF mm), as illustrated in Figure 1. For breeding purposes, male and female plants can then be identified in the early stages of development through the use of Y-specific DNA markers (Mandolino et al., 1999; Törjék et al., 2002). Apart from that, the molecular mechanisms underlying dioecy are essentially unknown but, considering that this condition is fully reversible (e.g., through chemical products treatment), the hypothesis that those genic regions involved in both sexes development remain potentially functional throughout the entire life cycle cannot be excluded (Di Stilio et al., 2005; Khadka et al., 2019). Given the role of homeotic genes in flower whorls identity (including anthers, pistils, and ovary), the hypothesis for their involvement in sex determination (Pfent et al., 2005; Sather et al., 2010; LaRue et al., 2013) and the lack of any information on the ABCDE model in the Cannabis genus, we screened all cannabis genomic and transcriptomic data available for homeotic genes and summarized them in Section 3. Traditionally, hemp-type and drug-type varieties have been bred mainly through mass selection. This method has been effectively used for the selection of cannabis showing improved quality traits such as fiber, oil, and cannabinoid content (Hennink, 1994). Nevertheless, one of the main problems associated with the first attempts of cannabis genetic improvement was, on the one hand, the need to avoid hemp genotypes with high THC contents, on the other hand, the availability of uniform medical genotypes, which was often linked to clandestine growers. More recently, cannabis cultivars were obtained from controlled mating using selected individuals from different landraces and cultivars. Usually, several selected individuals were used for open-pollination so that each of the female plants could be fertilized by each of the male plants (i.e., intercrosses). Synthetic varieties were also obtained by open-pollination using many female and male plants vegetatively propagated via cuttings (i.e., polycrosses).
FIGURE 1
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Figure 1 Information on sex determinants (A) and sex chromosomes (B) in cannabis [adapted from (Bergero and Charlesworth, 2008; Divashuk et al., 2014)].

 

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The morphology of Cannabis sativa L. Achenes

Toronto Metropolitan University
https://rshare.library.torontomu.ca › articles › thesis
May 24, 2021 — Marijuana achenes, in comparison with hemp achenes, are shorter and darker. Achenes of fibre cultivars are larger than the achenes of oilseed ...

Analysis of Morphological Traits, Cannabinoid Profiles ...

Frontiers
https://www.frontiersin.org › fpls.2022.786161 › full
by J Murovec · 2022 · Cited by 3 — In this work, we investigated leaf morphology, plant growth characteristics, cannabinoid profiles, THCAS gene sequences, and plant ...

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Tetraploid clones were assessed for changes in morphology and chemical profile compared to diploid control plants. Tetraploid fan leaves were larger, with stomata about 30% larger and about half as dense compared to diploids. Trichome density was increased by about 40% on tetraploid sugar leaves, coupled with significant changes in the terpene profile and a 9% increase in CBD that was significant in buds.
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Master Kush, also known as "High Rise," "Grandmaster Kush," and "Purple SoCal Master Kush" is a popular indica marijuana strain crossed from two landrace strains from different parts of the Hindu Kush region by the Dutch White Label Seed Company in Amsterdam. The plant produces a subtle earthy, citrus smell with a hint of incense, which is often described as a vintage flavor. The taste of Master Kush is reminiscent of the famous hard-rubbed charas hash. This strain holds a superb balance of full-body relaxation without mind-numbing effects.

Instead, Master Kush offers a sharpened sensory awareness that can bring out the best of any activity.
 
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Below is an example of drift. Imagine a rare species kept in a zoo with a population of only six diploid individuals.


There are a total of 12 alleles (numbered 1-12 in generation 0). All alleles are assumed equally fit, so that evolution is neutral. The alleles may also be genetically distinguishable, or "different in state" (represented by colours).
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how to calculate inbreeding coefficient
In general, for autosomal loci, the inbreeding coefficient for an individual is F = (½)(n1+n2+1), where n1 and n2 are the numbers of generations separating the individuals in the consanguineous mating from their common ancestor. (This formula assumes that the common ancestor is not inbred.)

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What is the genome of the cannabis plant?



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The plant is annual, wind-pollinated, and predominantly dioecious. It is diploid, with 10 pairs of chromosomes (2n = 20) and is characterized by an XY/XX chromosomal sex-determining system, with a genome size of about 830 Mb

Taxonomy​

Relation to Cannabis sativa

The hop is within the same family of plants such as hemp and marijuana, called Cannabaceae.[13] The hop plant diverged from Cannabis sativa over 20 million years ago and has evolved to be three times the physical size.[20][21][12][22] The hop and C. sativa are estimated to have approximately a 73% overlap in genomic content.[23] The overlap between enzymes includes polyketide synthases and prenyltransferases.[24] The hop and C. sativa also have significant overlap in the cannabidiolic acid synthase gene, which is expressed in the tissues of the leaves in both plants.[13]



The hop and C. sativa also have significant overlap in the cannabidiolic acid synthase gene, which is expressed in the tissues of the leaves in both plants.[13]

Because these products are toxic to the plant, THCA synthase is secreted into the trichome storage cavity.[7]

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Molecules | Free Full-Text | Biosynthesis of Nature-Inspired Unnatural  Cannabinoids

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Discussion​

Here, we show that inheritance of cannabinoids is far more complex than previously appreciated.2,4,5,28,29 In fact, not only are cannabinoid concentrations polygenic but we have also documented that they are influenced by maternal and cytoplasmic genetic effects. These complex genetic models alter the variance components of the one-locus, two-allele model in fundamental and enlightening ways, as detailed below. By using an I-T approach to identify the best genetic models, rather than a typical LCA approach to elucidate cannabinoid inheritance, we have had more power to predict variation in cannabinoids. However, it is difficult to disentangle the differences in these results because the genetic algorithms of SAGA are unclear in the original article and R documentation. The role of genetic variation on the inheritance of THC:CBD ratio is well described (and our modeling efforts confirmed the general consensus developed in the literature that the ratio is largely an additive inheritance process in C. sativa).2,5 Importantly, it appears as though there is a trade-off between the production of THCA and CBDA, which influences the relative cannabinoid abundance. Nevertheless, how absolute cannabinoid abundance is controlled by genetic factors still requires significant research. Our model predicts that the absolute abundance of cannabinoids, especially THC and CBD, is controlled by different genetic architectures. This discrepancy between the models of relative and absolute abundance hinders careful and predictable breeding efforts of cannabis cultivars, and experimentation is required to reconcile these two conclusions. Because we did not have access to data from unselected parental populations, reciprocal crosses for F1 generations, or backcrossed offspring,2,33 the available data were less likely to yield complex genetic effects. Yet, despite the reduced probability of detecting complex genetic effects and nonadditivity, we found evidence for complexity in the inheritance of all three cannabinoids. Therefore, the elucidated patterns are likely important and understudied aspects of cannabinoid inheritance. For example, altered CBD and THC concentration appears to result from cytoplasmic effects; maternal effects may explain CBC concentrations; and allelic dominance likely influences THC and CBC concentrations. Earlier studies, using traditional analytical approaches, did not explore phenotypic variance explained by the polygenic nature of these traits and nonadditive genetic effects and so their conclusions were limited to additive genetic effects.2–4,28 By identifying genetic effects on cannabinoid concentrations, we expect our results will make it easier to predict and control cannabinoid production.

Population-level studies involve limited parental germplasm and thus provide information of CGEs acting on that population alone (i.e., results are not globally generalizable). Our use of two complementary data sets with unique parental genotypes, and the comparison of our results with a third study using unique parental genotypes,5 highlights those CGEs common to multiple breeding populations. This confirms that cannabinoid abundance is, in part, controlled by a nonfunctional CBDA synthase, in contrast to a single-locus additive gene, as originally proposed.2,29 Furthermore, we detected the presence of multiple genes acting on the concentration of THC in both 20032 and 2014,29 consistent with de Meijer's original quantitative model2 and the mapped “cannabinoid quantity” QTL.5 However, we present only summarized data because the raw data set was a gift from de Meijer et al.2,29 We encourage readers who want to explore our approach to contact the authors of these studies directly.

CBD is likely controlled by at least one major gene and a series of smaller genetic effects. In contrast to previous research, cytogenetic effects appear to contribute to the inheritance of CBD in both populations, suggesting a role of mitochondrial or plastid genomes in its expression (Fig. 3; Appendix Table A2). Comparative sequencing of chloroplast and mitochondrial DNA noted the utility of extranuclear genomes in differentiating among fiber and drug cultivars,41 which are differentiated by THC concentrations but often also differ in CBD concentration.23 However, this remains understudied because other studies have removed “sequences obviously originating from organelles or ribosomal RNA.”42 Finally, CBC concentrations are likely the product of two major genes, one additive and one dominant, given the consistent role of these two CGEs across both data sets.

Our analyses also detected differences in the role of CGEs among interbreeding populations, suggesting that convergent evolution is responsible for some of the chemical diversity within C. sativa populations. For instance, where cytotype and epistatic additive effects appear to play a role in the expression of THC concentration in the 2014 populations,29 they play more minor roles in the 2003 population.2 Maternal effects influenced CBC and chemotype expression in the 2003 data set,2 whereas they were not included in the model in the 2014 data set.29 These differences in genetic effects may help explain the broad difficulties for hemp breeders when attempting to limit the production of THC, and for cannabis growers in the production of consistent concentrations of cannabinoids between and within crop varieties. Furthermore, more general epistatic effects have not been considered and may contribute to fluctuating chemotype expression.

The Castle–Wright estimator is commonly used to explore the genetic basis of trait evolution and has been commonly used to evaluate crop traits that have likely involved adaptation through altered expression of second metabolites (e.g., resistance to fungal infections, insect resistance, and heat tolerance43–45). Previous QTL and modeling approaches suggest that the Castle–Wright estimator may be robust for traits with few loci, even if assumptions are not met.40,46,47 Our Castle–Wright estimator results for THC, CBD, and CBC in 20032 are consistent with the independent CGE analyses run in SAGA, in terms of our expectations of the number of genes involved in a trait. However, the results differ dramatically between the two data sets, suggesting that populations may differ dramatically in the mechanisms of inheritance and the number of genes involved.


Our results suggest that variation in cannabinoid expression was not previously fully described. We hypothesize that nonadditive genetic effects play prominent roles in cannabinoid genetics. A formal test of integration, whether phenotypic,48 genetic,49,50 or developmental,51 would make significant advances in our understanding of cannabinoid inheritance. Thus, new crosses that map loci relative to psychoactive chemicals and pathways in the F1 and F2 generations of diverse chemotypes52 will complement ongoing efforts to characterize the genome of this plant.53,54

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