On epigenetic heredity

[Ruosk]

Ok, this has been bothering me for a while, even though I don't know that much on the subject. I would just like to hear breeders take on whether or not epigenetics hold any significance on plant (cannabis) breeding?

Could some traits be due to these mechanisms since the plants have been generation after generation bred under certain conditions? Could this mechanism allow certain line to later become more accustomed to conditions on genetic level too, guided by epigenetics?

Recent studies have shown that on humans this system of heredity may have huge impact on how we cope with changing world, though facts are still quite unclear. In short, epigenetics allow exactly same genotypes have different phenotypes: the genes don't change, but epigenetics allow the activation or passivation of certain genes to be passed on to next generation.

http://en.wikipedia.org/wiki/Epigenetics

This is propably kind of a stupid newbie question, I'm not that much into heredity nor breeding in general.. just bugged me after I read a couple of articles on it.

 

[Growdoc]

This is a vaild point and yes it is true to some degree. All Cannabis plants come from the great outdoors. It is us humans that have brought them inside. Some visable genophysical experessions like height shape and width are effected to some degree when plants are brought inside for many generations. This effect can also been seen again when the same family is taken back outside.

misinformation- most people think that outdoor sativa is the tallest species of Cannabis but this is not true, Indica is much taller outdoor.

GrowDoc

 

[drrico]

Ok, this has been bothering me for a while, even though I don't know that much on the subject. I would just like to hear breeders take on whether or not epigenetics hold any significance on plant (cannabis) breeding?

Could some traits be due to these mechanisms since the plants have been generation after generation bred under certain conditions? Could this mechanism allow certain line to later become more accustomed to conditions on genetic level too, guided by epigenetics?

Yah sure, you betcha!

There is a vast literature about this, but not from Cannabis per se. But, yeah, back in the 70s I messed with this rather a lot, using methylation inhibitors, histone modification inhibitors, folate/methionine supplements to enhance/support methylation, etc, etc..and Heck Yeah it matters!

Should I write this stuff up? I'm wicked bizzzeee...but if the community wants to collectively wrap their brain about this, I'm game.

Da Good Doc

 

[Growdoc]

please write this up...!!! i am game

GrowDoc

 

[zamalito]

Hell yeah, Dr. type it up! Mi nombre Jorgé Curioso y soy muy curioso!

 

[drrico]

Hola mi amigos,

Okey-doke, I'll get on it and report back. Sorry for the delay, I am freshly back from the rainforest in El Yunque...mebbe a couple daze, max. Deal?

Hasta -> Rico

P.S. As a taster, chromosomes carry two types of information: 1) Dat stuff encoded in the DNA sequence and 2) dat stuff encoded in da pattern of DNA methylation and histone (protein) modification (acetylation, methylation, etc). Epigenetic phenomena arise, in part, from numero dos. These modifications to DNA and the proteins that give chromosomes their shape, alter the ability of the information encoded in DNA to be expressed. In general, some parts of chromosomes acquire modifications to DNA and histones that shut off expression of the DNA sequences. This tendency to be silent is heritable through multiple rounds of cell division. These patterns are STRIPPED AND RE-ESTABLISHED during the sexual process that gives rise to seeds. This is one reason why propagation through seeds yields variability in the traits we see. On the other hand, sexual reproduction rejuvinates a line and explains why clones fade through multiple rounds of propagation...this is a topic worth exploring in a different thread ifn there is any interest in this sorta thang.

(I could go on for daze like this if there is a "market" for the info. Otherwise I'll channel it into my books and teaching material for my gradual students.).

 

[zamalito]

I can't speak for anyone else but I for one am intrigued. Thanks in advance for typing this up.

So if I understand this right. The use of methylation inhibitors allow you to identify which traits are the result of epigenetic phenomena. Then you can adjust your breeding techniques accordingly.

I'd love to hear more about not only your techniques and the the results but the hows and why's of both the results and the development of your experimental techniques.

 

[drrico]

I can't speak for anyone else but I for one am intrigued. Thanks in advance for typing this up.

Gracias Jorge, it is truly my pleasure. I live to serve, but recognize that sometimes my services are irrelevant (hence the query RE: interest).

So if I understand this right. The use of methylation inhibitors allow you to identify which traits are the result of epigenetic phenomena. Then you can adjust your breeding techniques accordingly.

Indeed, but with some caveats. Many traits are the result of complex interactions between alleles of genes (DNA sequence variation) and metastable gene expression states (epigenetic phenomena). The use of 5-azacytidine, for example, leads to depletion in DNA methylation which reverses (in part) the portion of the genome that is tightly packed (heterochromatic)...leading to expression of previously silent genes. If a trait is reproducibly lost when methylation is relieved, you might reasonably propose that the trait resulted from inactivation of some previously silent region. That region could be identified using modern molecular techniques, including cloning the methylated portion of the genome (using methyl-CpG binding proteins to select this portion of the genome), sequencing of the methylated sites (using hydroxylamine to identify the regions where "C" has become "5-methyl-C"), and whole genome scanning on chips. Lots of options...if only the funding were generally available to pursue this further. Thus sorta of study is in full force in other plants, though.

I'd love to hear more about not only your techniques and the the results but the hows and why's of both the results and the development of your experimental techniques.

I will dig up my notebooks from those hoary old studies and supplement them with anecdotes, when appropriate. Much of this work was done by me and my fellow inner space fellows in the late 70s-early 80s and again (a bit) in the early 90s.

Science is very cool....

 

[drrico]

Hmm...looks like I don't have permission to start a thread here, alas.

I suppose I could use this thread to start recording my experiments in the 70s and 80s with inhibitors of DNA and chromatin modification in my breeding program.

To start off, I think I'll dump what I recall from my lysergically challenged synapses and reorganize it as I locate what remains of my notes and data (most is long lost).

IN DA BEGINNING...
Short-chain organic acids (sodium butyrate, mostly). We did not know what this was doing back then, except leading to heritable changes in phenotypic expression that was maintained during cloning but often lost when using seeds to propagate traits. Now we know that this is a weak inhibitor of HDACs (histone deacetylatases) and promotes re-activation of silenced genes by opening up chromatin. Active in the millimolar range...rather alot when compared to modern HDAC inhibitors (e.g. Trichostatin A which is active in nanomolar concentrations... ~ a million fold more potent!). These were used to treat seeds and in sterile propagation media when cloning tissue. We isolated many morphological variants (leaf size and shape, colour, flower morphology, seed morphology) and variants with different tastes and smells. There were qualitiative differences in psychoactive properties. We did not use objective criteria to evaluate potency.

5-Azacytidine (5-AC). 5-AC is a deoxycytidine nucleoside analogue that gets incorporated into DNA during DNA synthesis, replacing some of the "C" residues oin the genome with "5-AC". When incoporated at targets for cytidine DNA methyltransferases (DNA M-ases), mostly at 5'-CpG-3' sites but also some others, these create suicide substrates for DNA methylation. What happens is that the DNA M-ases try to transfer a methyl group from S-adenosylmethionine to the 5-AC and get stuck halfway through the reaction (the aza group is an electron sink and traps the intermediate in the transfer). This leads to formation of covalent complexes between the M-ases and the DNA, welding the M-ase to ther DNA and blocking subsequent methylation of other sites in the genome, thereby depleting the nucleus of M-ase activity. As a consequence, cytidine methylation is lost (mostly through DNA replication, but there is some evidence for DNA methylation removal by enzyme action as well in some tissues and in some developmental stages). A previously unrecognized secondary consequence of trapping the M-ase on the DNA is that it creates a non-replicatable DNA lesion (like welding a cow to some train tracks, thereby blocking a train from translocating down the tracks) that causes replication forks to stall and eventually break. These DNA breaks lead to mutations and genome rearrangements with profound GENETIC (not epigenetic) consequences...The take home point: 5_AC is a potent inhibitor of DNA methylation, but the experimental breeder muts recognize that it can also cause genetic changes.

We used 5-AC and other analogues to create phenotypes that were shockingly similar in morphology and heritability to those caused by sodium butyrate (and, later, to Trichostatin A). An early conclusion from these studies is taht whatever these inhibtors did to alter patters of gene expression, they were acting in the same overall "pathway". We now know atht this pathway relates to heritable chromatin structures that establish stable patterns of gene expression.

To be continued...

 

[Evolution]

extremely interesting info....keep it coming...thanks in advance...

 

[drrico]

Thanks, Evolution! I've updated the entry again to add some info about methyltransferase inhibitors.

 

[drrico]

Ok, since my post count is too low to initiate a thread here, I moved the description of my work on epigenetics to here:

http://www.icmag.com/ic/showthread.php?t=60733

Please feel free to drop by there if you enjoy this stuff. I will start up something more nitty-gritty here after I get street legal.

 

[drrico]

I've added a new installment in the thread about the role of sexual reproduction in resetting patterns of epigentic inheritance. See the link I previously posted for the redirect (until I get street legal over here...).

Doc Snock

 

[GMT]

Personally, and I'm not where near the Dr league, I tend to think of epigenetics as a pre-evolution stage. Children born after a sustained food shortage, are born slightly smaller and will grow into smaller adults than their parents did. This is not because their dna has changed but because certain genes have been turned on/off that alreadys existed. It is a coping mechanism that allows adaptation to the environment the genetic entity finds itself in. I have found that in trifoliar cannabis, restricting the root space a plant has grown in soil, will increase the chance of a reversion to bifoliar growth structure. And suspect that carries over into their kids. But I'm now going to read the good doctors work.

 

[OG bub]

awesome stuff!
I agree with this theory.

one thing that has me thinkin tho.. It would be difficult to recognize epigenetic effect apart from recessive traits, in generational offspring.. especially if poly parents are used.



Peace, bub.

 

[drrico]

Thank you all for reading my notes from the lab.

Epigenetic phenomena can generate metastable phenotypes that resemble many genetic changes. This can lead to changes in development, morphology, chemical composition, time to maturation, node spacing, number of floral meristems (producing buds...1 cola or several...), etc. Interestingly, one can recover dominant traits just as readily (even more readily) than recessive traits.

In a plant that has two copies of a silenced gene, reactivation of one copy creates a different phenotype. Since expression of this gene copy is visible phenotypically, it can be thought of as dominant to the silenced allele.

In a plant that has silenced a transcriptional repressor, reactivation of one copy of the repressor gene can create a different phenotype due to shutting down of gene expression at the target of the repressor. In this case, expression of the gene has a negative effect on phenotypic expression by repression of the genes targetted by the repressor....and this effect is also dominant, though its is "negative". In other words, dominance can be "positive" or "negative" depending on the type of gene that was regulated by epigenetic gene silencing.

I've seen many traits arise through mutagenesis that are stable genetic changes. I have recovered similar phenotypes through metastable (stable but reversible) epigenetic modifications. The distinction I have made is through susceptibility to inhibitors of DNA or histone modification...though this can be a bit dicey as well. I wish I could have isolated the alleles to determine if they resulted from genetic or epigenetic changes (determined by DNA sequencing and by sequencing DNA that was previously treated with a chemical that allows the investigator to distinguish methylated C residues from unmethylated C residues. Pretty cool tool, that...).

I have been super busy running herd in my lab (I have 13 folks working there now). After the dust settles, I'll drag out more information from my notebooks and memory.

I appreciate your interest. Science is fun.

 

[Evolution]

I appreciate your knowledge and narrative writings. Your work is extremely interesting...can you elaborate on your lab? Science is the best way to discern and quantify the universe, I love it also...


Peace my brother, I can't wait to learn more...

 

[drrico]

Thank you for the kind words, Evolution! Science rawks.

My lab studies the mechanics of inheritence, the forces of genome conservation and change, and the machines that make it all happen. We use genetics ("classical" and "molecular"), enzymology, biophysical chemistry (lasers and lots of voltage and loud dangerous things that go fast), bioinformatics and mathematical modeling in computer simulations.

That's about all I care to say about the details, the WOD being what it is and all...

I've been at the bench and in the field for 30 years. My interest in plant genetics and manipulations of the noble herb go back to the very first days and are largely responsible for me havibg this career now. Just hoping to give back some of the wonder and knowledge I've picked up on this trip. I'm lucky!

 

[Sam_Skunkman]

drrico,
What if any work have you done on Cannabis? Have you used your knowledge to solve problems or create anything special in the Cannabis world? You seem to understand the field much better then me. But I do have 40 years hands on experience, and have quite a few firsts in the Cannabis science world. Do you work with DNA markers, like the ones found for Cannabis that are found with male plants? Or the markers for female plants? I am interested in finding intersexed markers. PM me if you prefer.

-SamS

 

[drrico]

Hi Sam,

I may have answered some of your questions at the RKS thread. I hope so at least!

My work with C. sativa started in the 70s and has been an on-again, off-again effort ever since. Some of this is described in that other thread. I do not think anything we isolated was particularly special, and our interests were scientific, not commercial. I guess it depends on what "special" means! We isolated some interesting strains that exhibited traits that amused us. Some of these traits appear to be genetic, and others epigenetic and others still are complex interactions of both.

The markers we worked with were identified in our labs. I would like to return and mess around with male-specific markers like the MADC RAPD markers reported recently. They look neat. If I come upon anything I think would be useful to you, I'll let you know.

Thank you for your interest. Science rawks.