Inbreeding for scientific purposes

FvG · Apr 5, 2016

Hi all,

I am a biologist interested in genome evolution in the Cannabaceae family (including hop but also tropical trees such as Celtis, Trema, Chaetachme). For my research I am looking into the possibilities of determining a genome sequence of Cannabis. First a bit of introduction:
:scripture:

Determining (assembling) a genome sequence is a hugely complicated puzzle. You can compare it with making a jigsaw puzzle with millions of pieces without knowing what is depicted. People have attempted to assemble Cannabis genomes but the results so far have not been great. I think this is because of the heterozygosity of Cannabis plants. Imagine that our metaphoric jigsaw puzzle contains each piece in two similar but slightly different forms - that would complicate solving the puzzle tremendously! :noway:

This is why I would like to have a completely homozygous Cannabis for research purposes. Normally, you can get homozygous plants through inbreeding. I am not a Cannabis breeder and have never grown a Cannabis plant myself. So I know virtually nothing about the practicalities. But what I have learned from various webpages and posts on this forum is that Cannabis plants suffer when they are inbred. They have reduced vigour: their performance deteriorates due to inbreeding depression. To avoid this problem, I understand breeders make backcrosses to maintain heterozygosity and 'hybrid vigour'. On the other hand, I see references to IBLs which are supposedly inbred. So perhaps it is possible to get to a homozygous line.

I am very interested in the advice and experiences of the expert breeders on this forum:

Are there any truly homozygous Cannabis strains/lines (not just homozygous for particular traits of interest but for all genes in the genome)?
How far can one go with inbreeding in terms of reduced vigour? (I would not mind having a poorly growing plant that requires a lot of care. But they should remain alive

Thanks in advance! :thank you:

MJPassion · Apr 5, 2016

Expert breeders that also study cannabis genome is equal to 1... maybe 2 here at IC Mag.
They are Chimera (a geneticist) & Sam Skunkman.

If there are more, theyre not making their expertise known.
At least not publicly.

I think finding an Homo plant at all loci will prove nearly impossible.

corky1968 · Apr 5, 2016

So to do this you must have a real laboratory.
Show us some pics please of your test tubes
and centrifuge.

Are you going to search for a rare Cannabis strain and amplify
it's genes with the PCR technique and implant that in another
plant like an orange to make THC Orange juice? I wish I had
some of that orange juice now.

On a serious note if only knew how to take plant tissue samples
and make those fancy phenotypic polymorphism bar tests.
The allelic stuff.

Note: I recommend that you learn how to grow some.

Get some seeds and get to it.

igrowone · Apr 5, 2016

not a breeder, except in the loosest sense, but very interesting topic
my understanding is if an initial cannabis population is isolated in the wild
then after some number of generations, you will see a population that will have a homozygous appearance
some disagreement here how long that takes
and the resulting landraces(term used here) tend to differ quite a bit, i.e. not homozygous from landrace to landrace
so subbing for the discussion, sure i will learn more than teach

rykus · Apr 5, 2016

I think because of the varied climates in a lot of the world that cannabis was cultivated,that only some of the areas where the strains where worked and bred by humans longer are closer to homo populations and breed more consistently. But only those areas with stable consistent weather. Some of those have been continually inbred for 30+ years now in Amsterdam as well in smaller populations(whether that is an asset or not you would have to prove out).but some seem a little weaker than less in bred lines.

Anyways in my tests with seeds, most populations express huge variances, and as often as not will express more pheno's when selfed or inbred. Some lines can become very uniform quickly though. I suspect this is usually dominant recessive and co dominant traits though and not true homo gene pool.

I think the wide variance is coming from the more wild populations that would have had to maintain wider expression to make it through thousands of years of storms and heat waves and any other obstacle that narrowed the gene pool...

Interesting stuff though, I'd love to hear what real breeders have to say now that they are getting more lab access!

Just hope we keep it GMO free for a while longer... Going to have to bury a few seed stashes for emergency repopulation ...

Lester Beans · Apr 5, 2016

I would bury a whole lot of seed for the future. Guys like the OP no nothing of cannabis but suddenly suddenly....

If you really want to do this work, work hops. They are the same family and your research will be much easier with ibl hops. Leave the cannabis to us pros.

iTsTimeNow · Apr 5, 2016

FvG said:
[*]Are there any truly homozygous Cannabis strains/lines (not just homozygous for particular traits of interest but for all genes in the genome)?

[*]How far can one go with inbreeding in terms of reduced vigour? (I would not mind having a poorly growing plant that requires a lot of care. But they should remain alive
[/LIST]

Thanks in advance!

A) No, currently there is not.. And assuming you speak of drug-type cannabis, there is also none homozygous even if only for traits of primary interest. The maths are seemingly insurmountable until science accomplishes feats such as successfully ceasing all crossovers during double haploid production (reverse breeding).

B) The question of duration a particular individual may sustain continued inbreeding will vary greatly from individual to individual starting material.

corky1968 · Apr 5, 2016

Op, seriously if you can't grow.

You better learn or your doomed.

FvG · Apr 5, 2016

Thanks everyone for the replies so far. Really appreciated! :tiphat:

MJPassion said:
Expert breeders that also study cannabis genome is equal to 1... maybe 2 here at IC Mag.
They are Chimera (a geneticist) & Sam Skunkman.

Thanks for the intel! Two breeders that know about genomics is not a lot but if they are willing to share some thoughts that would be of great help te me!

rykus said:
Some of those have been continually inbred for 30+ years now in Amsterdam as well in smaller populations(whether that is an asset or not you would have to prove out).but some seem a little weaker than less in bred lines.

That is very interesting! If they were truly inbred they should be more or less homozygous. But perhaps the breeders maintain genetic variation in their populations somehow? I would love to know some more specifics...
So I am curious about any experiences with inbreeding: how bad is the inbreeding depression: will plants die or become infertile after so many generations or will they simply grow a bit less rigorous which I guess would be a problem for breeders but OK for my purposes...

rykus said:
Just hope we keep it GMO free for a while longer... Going to have to bury a few seed stashes for emergency repopulation ...

I don't think GMO cannabis will be there anytime soon. But having a genome sequence available could help breeders understand better what is causing the phenotypes that they see (or feel ;-)

corky1968 said:
Note: I recommend that you learn how to grow some.
Get some seeds and get to it.

Yeah I may do that at some point but only if there is a chance in succeeding (getting a true homozygote I mean; growing a plant should be no problem ;-).

iTsTimeNow said:
A) No, currently there is not.. And assuming you speak of drug-type cannabis, there is also none homozygous even if only for traits of primary interest. The maths are seemingly insurmountable until science accomplishes feats such as successfully ceasing all crossovers during double haploid production (reverse breeding).

Hmmm. So even the IBL are heterozygous? I guess many people tried to get them homozygous.. what were the main problems?
My primary interest is not in the drug-type cannabis: I would be interested in any type of cannabis that I could get homozygous.
I should probably also note that homozygosity does not have to be 100%; 98% would be great.

iTsTimeNow said:
B) The question of duration a particular individual may sustain continued inbreeding will vary greatly from individual to individual starting material.

That makes sense: if the individual is very heterozygous it would take longer for the deleterious effects to start having an effect....

Thanks again! :thank you:

iTsTimeNow · Apr 5, 2016

Yes, any and all IBL (IL) you will currently find in drug type cannabis will be heterozygous at many integral sites. The main problem is the very nature of what makes a drug type desirable is very complex as it is a product of macro cannabinoids working in unison with over 100 lessor (though no less important) factors/compounds.

Inbreeding causes a deficit of heterozygosity but not necessarily deleterious effects.. This has been observed in many an outcrossing species thus far and will continue to remain exactly so. IE, some greatly inbred individuals/lines (though they are obligate outcrossers) will suffer no notable deleterious effects whatsoever. See many line of maize.

FvG · Apr 6, 2016

iTsTimeNow said:
Yes, any and all IBL (IL) you will currently find in drug type cannabis will be heterozygous at many integral sites. The main problem is the very nature of what makes a drug type desirable is very complex as it is a product of macro cannabinoids working in unison with over 100 lessor (though no less important) factors/compounds.

Inbreeding causes a deficit of heterozygosity but not necessarily deleterious effects.. This has been observed in many an outcrossing species thus far and will continue to remain exactly so. IE, some greatly inbred individuals/lines (though they are obligate outcrossers) will suffer no notable deleterious effects whatsoever. See many line of maize.

Ok so if I understand you correctly, doing rounds of inbreeding may be feasible for some individuals/lines but the plants will probably loose their desirable drug properties? I can perfectly live with that!

The most important question for me would then be: Does anyone know which individuals/lines have less deleterious effects?

bombadil.360 · Apr 6, 2016

You not only loose desireable drug properties, as you put it, but general desirable plant properties, which are vital if you were to try to classify as homozygous.

In other words, if you manage to inbreed to the point where genetically you can call a plant homozygous, is that plant worth growing at all? will it even have enough vigour to serve some purpose at all?

What's the point? What do you think this can accomplish? What applications do you think can come of that?

FvG · Apr 7, 2016

bombadil.360 said:
You not only loose desireable drug properties, as you put it, but general desirable plant properties, which are vital if you were to try to classify as homozygous.

In other words, if you manage to inbreed to the point where genetically you can call a plant homozygous, is that plant worth growing at all? will it even have enough vigour to serve some purpose at all?

What's the point? What do you think this can accomplish? What applications do you think can come of that?

As you say, the plant may have no purpose for breeding. But it would be very useful for reconstructing a genome, which is really difficult when a plant is highly heterozygous (see also my first post). So for my scientific purpose (reconstructing a genome) it would be very useful.

In the end, after the genome is reconstructed (based on the 'useless' homozygous plant) it becomes possible to see where the genomes of useful heterozygous plants are different. The genome of the homozygous plant is then used as a "reference" to compare other plants with. This could help find out which genes are important for traits that people care about. That would be the final purpose.

bombadil.360 · Apr 9, 2016

How do you "see where the genomes of useful heterozygous plants" from an inbred plant that has gone through unpredictable mutations?

Sounds like magic to me or am I an idiot?

FvG · Apr 11, 2016

bombadil.360 said:
How do you "see where the genomes of useful heterozygous plants" from an inbred plant that has gone through unpredictable mutations?
Sounds like magic to me or am I an idiot?

Yes, it is magic (kinda) so you are certainly not an idiot. The magic involves the nature of next-generation sequencing data and a computer. But let me try to simplify:

Let's say we look at two pieces of DNA sequence from a useful heterozygous plant. If they are similar but not really the same we cannot know if they come from two similar genes in the genome, or if they come from a single gene that is very heterozygous. This means we cannot know how many genes there are and which are heterozygous and which are not (which could be important to know for a breeder).

However, if we look at a homozygous plant we know that the sequences must come from two different genes - otherwise they could not be different. So we can then determine how many genes there are in a Cannabis genome and how they look like.

This is really useful because we can assume that the number of genes is more or less the same for all Cannabis plants. They only differ in what variants (alleles) they have of those genes. Therefore, if we know all the genes from the homozygous plant, we can compare the two pieces of DNA from the useful heterozygous plant with those genes and determine if they are two variants from one heterozygous gene or if they come from two different genes that are each homozygous.

I hope this demystifies things a little bit..

bombadil.360 · Apr 11, 2016

Thank you for that explanation FvG, it makes sense the way you explained it, but I may be a little idiotic nonetheless, as I don't really get how we can compare the homozygous with the heterozygous without accounting for mutations?

GrowerGoneWild · Apr 11, 2016

Would an isolated population eventually become homozygous, and this is how distinct landrace strains come about?..

Skinny Leaf · Apr 12, 2016

Again there is no point in mapping the genome of cannabis. What is your true objective? Never mind, the last company trying to do this came up with some bullshit answer also. You're the biologist figure it out. Crap they didn't teach y'all anything in a four year college except how to form a sentence with big words.

They're name is Phylos. Go work for them. You don't even smoke pot.

Rakosnicek · Apr 12, 2016

you can create a 100% homozygous cannabis plant, it's called dihaploid technology

you have to cultivate male anthers, pollen microspores or female ovum in very special in vitro conditions and if you're lucky enough you can get haploid plantlets. Next step is in vitro chromosome doubling (use of antimitotic agents like colchicine) in that haploid plant resulting in diploid with 2 identical sets of chromosomes (dihaploid, 100% homozygous)

corky1968 · Apr 12, 2016

Without Gel Electrophoresis your just playing a guessing game.

https://en.wikipedia.org/wiki/Gel_electrophoresis

Do watch the video in the following link for a much more information on what is gel electrophoresis and it's applications.
They video illustrates the basics of DNA extraction and gel electrophoresis in tomato.

http://articles.extension.org/pages/32366/gel-electrophoresis-principles-and-applications

With gel electrophoresis you can determine which plants are the homozygotes and which are heterozygous ones.

This would be a great asset to any cannabis breeders.

Inbreeding for scientific purposes

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