Feminized Breeding in Cannabis and a Y Chromosome Bottleneck

OG growers outsmart "RFKjr BroScience" accusations from Ivory Tower Propeller heads

Figure 1. The homology between Cannabis X and Y chromosomes. The blue region is the PAR locus on Y that has homology to X and recombines with X during Male meiosis.

This week a spectacular thread unrolled on LinkedIn in regards to Cannabis breeding.. Yes, that CV cess pool where degreed people like to show off their virtue and adherence to the Zeitgeist on a platform owned and operated by a frequent flyer on the Lolitta Express. I don’t traffic LinkedIn much as it was Orwellian during the pandemic and much of that tyranny is a reflection of its founder who viciously defended the Biden administration and Sam Altman’s redirection of OpenAI.

Cannabis is a fun market because most of the intel resides in the clandestine growers who refrained from publishing their work as to avoid a DEA visit. Once Canada federally legalized we saw an influx of PhD’d people coming in to lecture everyone on how their ‘broscience’ wasn’t validated and required their omniscience. It has many parallels to the Covid debacle where universitied ‘Experts’ were routinely panted and punched in the mouth by anonymous cats and mice on twitter who have more than a few neurons to spare to dissect their delusional advice.

One well meaning but also RFKjr ridiculing PhD tried to lecture some breeders on the disposable nature of the plants largest chromosome: The Y chromosome.

In Cannabis, male plants are a nuisance as they pollinate female flowers and seed them up. Once the plants energy is focused on seeds, the cannabinoid content in the flowers drops and the quality and market value of the flower decays precipitously.

Back in the 1990’s growers began experimenting with using silver nitrate or silver thiosulfate to arrest the plants Ethylene pathway. This triggers the female plants to hermaphrodite and self pollinate. If female plants self pollinate, they can only make female XX seeds. Clever approach to make all your seeds be females and you don’t have to walk your farm yanking out pollinating males.

Figure2. From McKernan et al. Pictures of male and female flowers are only distinguishable once in flowering. They can be segregated with qPCR in seedling stage.

The so sure of themselves PhDs even wrote a whole blog post about how paranoid growers don’t need to keep males around as females had all the diversity required from their single X chromosome.

For anyone actually paying attention in a university genetics class, you likely knew this was off as breeding with a population that is missing an entire chromosome will create a genetic bottleneck.

What this PhD was missing was the understanding that X and Y sex chromosomes often engineer a safeguard against Muller’s Ratchet so deleterious variation on the mostly haploid Y chromosome doesn’t become homozygous in one mutation. For this reason the X and Y chromosomes often share large regions of the chromosome that recombine with each other. This is know as the 30 million base region of Y called the PAR locus (Pseudo Autosomal Region). This recombination can only happen during male meiosis (between PAR on Y and X- See Figure 1 above) so when you eliminate males, these 1300 genes are removed from the card deck and you’re not shuffling the cards with a full deck. A Thelma and Louise breeding strategy if you will.

The retention of male genetics was referred to as “RFKjr BroScience”. The elite being so sure of themselves, had learned nothing from the pandemic and how this elitest attitude leads to despair and is anti-science.

When ChatGPT4.o agreed with this overt breeding bottleneck , it was summarily dismissed as a AI hallucination. But we don’t need to hypothesize about this bottleneck when we have sequenced over 1000 cannabis genomes and can flat out measure it.

So we took 2 trillion bases of cannabis sequence from males and females and charted it out. Figure 3 has the heterozygosity of cannabis genomes in Blue and the heterozygosity in the PAR locus in Orange. A heterozygosity of 1% = 1 SNP every 100 bases in the genome. 2% is every 50 bases.

Figure 3. Heterozygosity of 90 cannabis genomes measured genome wide (Blue) and in the PAR locus (Orange).

You can see from this chart that the males on the left (in blue text) have higher overall genome wide diversity (heterozygosity) and higher diversity in the PAR locus than females. We used only TypeI (THC dominant) cannabis plants as these are most commonly grown in door with feminized seeds or cloning. Very few indoor THC grows are popping Male/Females seeds and culling the males. The real estate costs for indoor growing are substantial as you need camera’s, barcodes and seed to sale tracking. The security for indoor THC grows is prison grade. If Jeffrey Epstein was detained in a Cannabis grow, we’d know that he didn’t kill himself.

You can find several plants where the Orange is less than the Blue (PAR diversity less than Genome wide). One such plant is Gary Payton (RSP12898). This plant was in the pipeline as it was infected with HLVd and even transferred HLVd to leaf Hoppers.

So lets chart out the coverage over the whole Y chromosome to genetically verify the Males vs Female genomes and compare that to the PAR diversity minus the Genome wide diversity.

Figure 4. PAR diversity - Genome-wide diversity on the Y axis. Very few males are below zero in this chart but many females are. The X axis is the Y ratio in Kannapedia. This is a measurement of the sequence coverage over the entire ~100Mb Y chromosome and all Males are clearly over 0.05.

Figure 5. A summary of the heterozygosity rates in Males and Females. Het_rate_all is genome-wide diversity and het_rate_PAR is diversity in the PAR locus (30mb on Y that crosses over with X).

From this data, we can clearly see this Y bottleneck in play and the OG growers were instinctively correct and also theoretically correct. Make no mistake, I was once one of these academic newbies in the cannabis field and have learned lot from OG growers in the 15 years I’ve been in the field. But this particular topic required numbers to back it as we have always been accused of having a bias in this debate. Our company sells qPCR kits that detect the Y chromosome for breeders who still prefer non-feminized breeding approaches. There isn’t one answer on how to breed. If you are looking to introduce new traits, bringing in more diversity from males is a good idea as this PAR locus has 1300 genes and one in particular everyone should pay attention to.

Cannabis sativa ent-copalyl diphosphate synthase 1 (LOC115703738), transcript variant X1, mRNA. Gibberellins.

What does this gene do?

The gene **Cannabis sativa ent-copalyl diphosphate synthase 1 (LOC115703738)** encodes an enzyme that catalyzes the **first committed step in gibberellin (GA) biosynthesis**.

As you can see this is an important gene for flowering and trichome development.

According to a GO term search, there are ~110 gene related to pathogen response as well. This might explain the HLVd susceptibility of the Gary Payton plant.

For people trying to breed stabilized seed lines where each sibling is more like one another than different, backcrossing to drive the heterozygosity down is a smart plan. It just may need to be done with careful attention to the microbiome. You don’t want to breed the perfect plant only to find it cant tolerate pathogens.

You also don’t want to breed yourself into a non-reproductive corner and be stuck with only clonal propagation. Some feminized breeding programs are backcrossing to get stabilized seeds (low heterozygosity so sibling seeds are more related). Some of these programs have run into plants that make sterile pollen. If your plants have great traits but can’t be reversed with silver to make any feminized pollen, then you can only propagate that plant through cloning and tissue culture.

Overtime, serial cloning can lead to telomere shortening and epigenetic drift. While this has not been ‘genomically’ measured in Cannabis, it has been seen in other plants and is suspected to occur in cannabis.

Ever since we used Pacific Biosciences to sequence the Jamaican Lion Trio we have been fascinated with Cannabis telomere length. These long read sequencers found the telomeres on Jamaican Lion chromosomes to vary substantially. Some were just a few thousand bases while others were longer than 40,000 bases.

We even performed methylation based sequencing to understand how methylated these telomeres are. PacBio and ONT plant methylation tools we not as mature back in 2018 so we resorted to EM-Seq on Illumina which converts all Non-methylated Cytosines into Thymine. The Red bars on the chart below are unmethylated Cs that were converted to Ts. The blue bars represent Cs which were methylated and resistant to this conversion chemistry.

We are engineering tools to monitor this as we believe grows that rely on serial multi-generational Cannabis cloning will experience telomere decay and this can be quantitated and measured to help inform growers of when they have reached the end of the line and need to regenerate the telomeres through tissue culture or pollination.

The moral of the story: Stay humble and let the data speak for itself. There is much knowledge in the trenches that escapes the white lab coat class and the cannabis community has never been one to appeal to authority. Nature is the authority and while the academic class may have their scientific terminology perfected which they love to lord over the colloquials, don’t subscribe to it without replicable open source data. Try to keep your politics out of Cannabis Science. It has never mixes well.

Its very obvious the pharma funded mainstream media loves to demonize RFKjr “Broscience” but the more you dig into his stance on topics, you will find he is incredibly well versed and often runs circles around the people who parrot the mainstream media Overton window.

This is the headline they chose to run when RFKJr announced his plans to keep drug pricing in the US the same as it is in other countries. Drugs that cost 1% of what they cost in the US will now be normalized country to country. A earth shattering event for Pharma but him swimming in Rock Creek is what you need to worry about.

These Cannabis genomes are all open source on Kannapedia.net.