The determinator of sex

[Sam_Skunkman]

Chimera,
If you test a female that has been STS treated to turn male, with a RAPD or other marker DNA test what sex does it show?
Same with a male plant treated with ethephon to turn female, test it and what sex does it show?
If you are testing with RAPD or other markers to find males do you also test with RAPD or other markers to confirm they are female? Have you ever done female RAPD or other DNA testing?
If you test a full blown inter-sexed plant what is the results? With which tests? Does it make a difference if you use plant DNA from the male or female parts of an inter-sex plant?
-SamS

For everyone else here are some references for DNA based sex determination:
If you know of more please post a reference so I can get them. I am looking for an early Chinese and even earlier Hungarian sexing via DNA article. Anything about MADC1 or MADC2 or MADC 3, MADC4, MADC5, MADC6, SCAR332 or any others.

Theoretical and Applied Genetics
January 1999, Volume 98, Issue 1, pp 86-92
Identification of DNA markers linked to the male sex in dioecious hemp (Cannabis sativa)
G. Mandolino, A. Carboni, S. Forapani, V. Faeti, P. Ranalli

A male-associated DNA sequence in a dioecious plant, Cannabis sativa L.
Sakamoto K, Shimomura K, Komeda Y, Kamada H, Satoh S.
Plant Cell Physiol. 1995 Dec;36(8):1549-54.

Oxford Journals Life Sciences Plant and Cell Physiology Volume 36, Issue 8Pp. 1549-1554.
1995, A Male-Associated DNA Sequence in a Dioecious Plant, Cannabis sativa L.
Koichi Sakamoto, Koichiro Shimomura, Yoshibumi Komeda, Hiroshi Kamada and Shinobu Satoh

Maybe this one:
Sakamoto K, Akiyama Y, Fukui K, Kamada H, Satoh S (1998) Characterization; genome sizes and morphology of sex chromosomes in hemp (Cannabis sativa L.). Cytologia 63: 459–464. doi: 10.1508/cytologia.63.459

Identification of DNA markers linked to the male sex in dioecious hemp (Cannabis sativa L.)
G. Mandolino, A. Carboni, S. Forapani, V. Faeti, P. Ranalli
Theoretical and Applied Genetics (Impact Factor: 3.66). 12/1998; 98(1):86-92.

Maybe this one:
Comparison of Hemp Varieties Using Random Amplified Polymorphic DNA Markers
Crop Science Vol 41 nov-Dec 2000 pg 1682-1689
Silvia Forapani, Andrea Carboni, Claudia Paoletti, V. M. Cristiana Moliterni, Paolo Ranalli,
and Giuseppe Mandolino

Maybe this one:
Sakamoto K, Ohmido N, Fukui K, Kamada H, Satoh S (2000) Site-specific accumulation of a LINE-like retrotransposon in a sex chromosome of the dioecious plant Cannabis sativa. Plant Mol Boil 44: 723–732.

Euphytica
07-2002, Volume 126, Issue 2, pp 211-218
Occurrence and frequency of putatively Y chromosome linked DNA markers in Cannabis sativa L.
Giuseppe Mandolino, Andrea Carboni, Manuela Bagatta, V.M. Cristiana Moliterni, Paolo Ranalli

Törjék O, Bucherna N, Kiss E, Homoki H, Finta-Korpelová Z, et al. (2002) Novel male-specific molecular markers (MADC5, MADC6) in hemp. Euphytica 127: 209–218. doi: 10.1023/a:1020204729122

Törjék O, Bucherna N, Kiss E, Homoki H, Finta-Korpelová Z, et al. (2002) Novel male-specific molecular markers (MADC5, MADC6) in hemp. Euphytica 127: 209–218.

Theoretical and Applied Genetics
June 2003, Volume 107, Issue 1, pp 102-109
Sex-linked AFLP markers indicate a pseudoautosomal region in hemp (Cannabis sativa L.)
A. Peil, H. Flachowsky, E. Schumann, W. E. Weber

Journal of Industrial Hemp Vol 8 Number 1, 2003, Female-Associated DNA Polymorphisms of Hemp (Cannabis sativa L.) Hong Shao, Shu-Juan Song, Robert C Clarke

Plant Breeding
Volume 124, Issue 2, pages 167–170, April 2005
Sex-linked SSR markers in hemp
J. Rode, K. In-Chol, B. Saal, H. Flachowsky, U. Kriese andW. E. Weber

Sakamoto K, Abe T, Matsuyama T, Yoshida S, Ohmido N, et al. (2005) RAPD markers encoding retrotransposable elements are linked to the male sex in Cannabis sativa L. Genome. 48: 931–936.
RAPD markers encoding retrotransposable elements are linked to the male sex in Cannabis sativa L.
Koichi Sakamoto, Tomoko Abe, Tomoki Matsuyama, Shigeo Yoshida, Nobuko Ohmido, Kiichi Fukui, Shinobu Satoh
Genome, 2005, 48(5): 931-936

Plant Biotechnology
Electronic Journal of Biotechnology ISSN: 0717-3458 Vol. 10 No. 4, Issue of October 15, 2007
DOI: 10.2225/vol10-issue4-fulltext-4
RESEARCH ARTICLE
Inter simple sequence repeats separate efficiently hemp from marijuana (Cannabis sativa L.)
Erdogan E. Hakki, Seyit A. Kayis, Emine Pinarkara, Ayla Sag

Maybe this one:
Yang MQ, van Velzen R, Bakker FT, Sattarian A, Li DZ, et al. (2013) Molecular phylogenetics and character evolution of Cannabaceae.Taxon. 62: 473–485.

Maybe this one:
Molecular Cytogenetic Characterization of the Dioecious Cannabis sativa with an XY Chromosome Sex Determination System
Mikhail G. Divashuk, Oleg S. Alexandrov, Olga V. Razumova, Ilya V. Kirov, Gennady I. Karlov
Published: January 21, 2014 at PLOS One

 

[Only Ornamental]

Chimera,
If you test a female that has been STS treated to turn male, with a RAPD or other marker DNA test what sex does it show?
Same with a male plant treated with ethephon to turn female, test it and what sex does it show?
If you are testing with RAPD or other markers to find males do you also test with RAPD or other markers to confirm they are female? Have you ever done female RAPD or other DNA testing?
If you test a full blown inter-sexed plant what is the results? With which tests? Does it make a difference if you use plant DNA from the male or female parts of an inter-sex plant?
-SamS.....

Hi Sam,
Principally, RAPD 'randomly' amplifies DNA (you don't know by default which genes you see and if there's truly a connection to, lets say, gender or heterosomes). That means, a genetic male should test as male no matter it's sexual phenotype and chemical treatment. Obviously, the same applies to females... Using reverse transcriptase PCR where you look at RNA or protein sequencing may and should be different stories and I'd expect (hypothetically) them to reveal the phenotype rather then the genotype.
I read one of those RAPD cannabis papers and I think to remember (would have to look it up again to be sure) that they linked male/female RAPD results to the karyotyping results. Can't remember if they also looked at the expressed sex too (but it seems obvious). Which would leave me with the conclusion that they hadn't any inter-sex or hermaphroditic plants in their test lot...

 

[Sam_Skunkman]

That is pretty much as I suspected, I just wanted to hear from someone that did it and which method they used to get the results.
Have you heard of anyone that uses DNA markers to ID females or just for males?
-SamS

Hi Sam,
Principally, RAPD 'randomly' amplifies DNA (you don't know by default which genes you see and if there's truly a connection to, lets say, gender or heterosomes). That means, a genetic male should test as male no matter it's sexual phenotype and chemical treatment. Obviously, the same applies to females... Using reverse transcriptase PCR where you look at RNA or protein sequencing may and should be different stories and I'd expect (hypothetically) them to reveal the phenotype rather then the genotype.
I read one of those RAPD cannabis papers and I think to remember (would have to look it up again to be sure) that they linked male/female RAPD results to the karyotyping results. Can't remember if they also looked at the expressed sex too (but it seems obvious). Which would leave me with the conclusion that they hadn't any inter-sex or hermaphroditic plants in their test lot...

 

[Sam_Skunkman]

Here are a few recent for Cannabis Karyotyping.
-SamS


https://www.facebook.com/permalink.php?id=206098782849331&story_fbid=597734820301089
http://www.cibtech.org/J LIFE SCIENCES/PUBLICATIONS/2012/Vol 2 No 1/41 Suman Kaushal Kangra.pdf
CYTOLOGICAL STUDIES OF CANNABIS SATIVA IN SHIMLA HILLS 
OF HIMACHAL PRADESH
ABSTRACT 
Chromosome counts made in Cannabis sativa, clearly depict that the genus is diploid with 2n= 20. On the basis of the position of centromere, they can be represented as 10M + 6SM + 4ST i.e., 10 median, 6 sub-median and 4 sub-terminal. Polyploidy were also observed in some of the tetraploid cells with 4n=40. The root-tips showing polyploidy were thicker in appearance than those showing diploid number.
Suman Kaushal - Department of Botany, Govt. Degree College, Dharmshala, Distt. Kangra (H.P) - Journal...Life Sciences (jls) > Volume 2, No. 1
Indian Journal of Fundamental and applied Life Science




http://en.cnki.com.cn/Article_en/CJFDTOTAL-ZGMZ902.004.htm
KARYOTYPE ANALYSIS OF HEMP (Cannabis stativa L.)
Guo Yunling Xiong Heping Tang Suwei Yu Chunming (Institute of Bast Fibre Crops,CAAS,Yuanjiang,Hunan 413100)
The present paper describes the chromosome number and the karyotype of hemp (Cannabis stativa L.)from Liuan Anhui province, China. The results of observation showed that the somatic chromosome number is 2n=20,and the karyotype is 2n=7(L)+3(S)M,belongs to 1B type,and its N.F is 40.somatic chromosome is 2n=20=7(L)M+3(S)M.Its N.F is 40.




http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085118
Molecular Cytogenetic Characterization of the Dioecious Cannabis sativa with an XY Chromosome Sex Determination System

Mikhail G. Divashuk,X
Oleg S. Alexandrov,Russia
Olga V. Razumova,X
Ilya V. Kirov,
Gennady I. Karlov
Published: January 21, 2014
PLOS one

 

[Only Ornamental]

Have you heard of anyone that uses DNA markers to ID females or just for males?
-SamS

Absence can be as good as presence to prove something .
Couldn't find the one publi I remember but the one by Sakamoto et al. you mentioned earlier identified the genes found by RAPD; by chance they were male markers. For further gender testing, the 'real' approach would now be to test only for one or both of these DNA sequences or respective proteins. No need to run a RAPD (or similar method) each time. That one's pretty handy when you don't know what to look for but as soon as you do, other more specific methods (p.ex. ELISA) are preferred.

And I have no idea who does what especially when it comes to commercial providers.
If you need to run such a test and can't find an official source you could do a RAPD (whole primers/probe mix or a selection based on said publication) with plants you know and re-discover the distinctive bands. Then, you simply ignore what they really are as long as you can find the bands again in unknown samples. If that's what you're really after .

 

[browntrout]

Having not paid attention in bio class, I often have wondered if it is possible to see true males and true females in humans?

"The way I understand it is that, while the sex of the fetus is determined by
the xx/xy chromosome combination, the sex organs start out looking female and
then later the penis/testicles develope in the xy (male) fetus. This is
different from having the fetus begin as female and then become male."

"For the first 7 weeks the fetus is undifferentiated - and only an
examination of the chromosones can indicate the sex."

So it is pre determined before the fetus is formed?

Off topic, but would like to hear what you guys think.

 

[GreenintheThumb]

I hope they find/develop DNA tests for inter-sex plants, maybe for both genotype and phenotype inter-sex like they already have for Male and Female genotype plants, the sooner the better.
Any ideas?

Since we can induce different sexual phenotypes this way I think what' we're really looking for is the genes that control ethylene production or that block ethylene somehow. And I don't know about you but I don't think it's a simply inherited trait. I've seen inter sex plants come from non intersex parents and vice versa. I wish it was as simple as Bt vs Bd but I doubt it.

 

[Chimera]

Sam I use a PCR based method for determining the presence of the Y, or MADC's... it is a slightly modified PCR reaction based on Sakamoto's 1995 protocol. Females lack the marker, males show it with 100% concordance in my experience.

STS treated females do not show the marker (neither will the selfed progeny); the STS is an ethylene blocker which leads to the altered phenotype but does not alter the DNA of the plant or that of subsequent generations. Ethephon works through similar but opposite methods, and also does not alter DNA... this is why the reversal effects are transient.

The chinese paper Rob published in the JIH is a bit of an anomaly as it offers certain implications to the model which don't fit with the puzzle as I understand it, I keep hoping to run into Rob to discuss it but never seem to be over there at a time that coincides with one of his visits... when you see him please pass on that I'd like to meet with him about this at some point. The troubling part of that paper are the very low N's used and no replicability with other individuals of the same population - we should see that marker repeatedly in other females if their results are correct. Alternatively it could possibly suggest active TE's in the species which would not be surprising, however it would be a novel discovery at this point as it has not yet been reported in the literature afaik.

The monoecious plants I tested came from a monoecious X female cross. They all showed as females (no male marker), essentially indicating a female only "base" population with a 'floating' intersex locus presenting the appearance of a sex ratio. Really, it is a separate trait from gender; and I consider it as a second trait in a dyhybrid cross a la Mendel's second law - at least in the population we were investigating. I suspect there are multiple inducer loci for 'intersex induction', based on the type of intersex morphology from full blown intersex plants with both floral types on each bud (monoecious), to single branch intersex individuals, late-florescence intersex individuals with single staminate flowers appearing only on apical pistillate clusters, single staminate flowers that hang at the base of otherwise pistillate inflorescences, etc etc.

Hope that helps,
-Chimera

 

[Chimera]

Sense we can induce different sexual phenotypes this way I think what' we're really looking for is the genes that control ethylene production or that block ethylene somehow. And I don't know about you but I don't think it's a simply inherited trait. I've seen inter sex plants come from non intersex parents and vice versa. I wish it was as simple as Bt vs Bd but I doubt it.

I'd have to agree with this; as I've stated before ethylene alterations occur throughout the lifecycle in response to regulatory and environmental cues, but also fluctuate in response to environmental stress.

Considering there are only 5 major hormones in plants that control a HUGE variety of functions, metabolic and gene regulatory 'check-points', and the well agreed upon fact that modifications in 2 of those pathways are known to induce sexual phenotype changes (through both gibberelic and ethylene pathway manipulation), I think that the appearance of 'intersexism' has multiple possible causes in some cases, and there will be multiple underlying genetic contributors and possibly QTL's involved, depending on how we and what we define as 'intersex'.

As per the (monoecious) X (female) cross discussed above, it does seem to indicate a single locus is possible to induce intersexuality - however my contention is that there are multiple possible causes for the appearance of staminate flowers on otherwise female plants, and all of those different types of intersexuality can be lumped together by their shared phenotype, whereas they may share divergent, unlinked, underlying genetic causes.

-Chimera

 

[Sam_Skunkman]

I'll guess that if an inter-sex plant easily show inter-sex it maybe has copies for that from both parents, while if only from one parent gave it maybe the plant does not express it so easy? But cross the plant with a single set again with another plant that does carry inter-sex maybe both copies, and the progeny do all express inter-sex. I am guessing there could be many different control mechanisms depending if it is a true inter-sex genes or something that alters ethylene expression for example.
We will see....
-SamS

Since we can induce different sexual phenotypes this way I think what' we're really looking for is the genes that control ethylene production or that block ethylene somehow. And I don't know about you but I don't think it's a simply inherited trait. I've seen inter sex plants come from non intersex parents and vice versa. I wish it was as simple as Bt vs Bd but I doubt it.

 

[mofeta]

I think what' we're really looking for is the genes that control ethylene production or that block ethylene somehow.

I think along the same lines. I don't go along with it being something to do with "production" of the hormone, or the amount of it- there are too many other effects that would be obvious in a plant with increased global levels of ethylene (epinasty, auxin dysregulation, etc.). When you say block, though, I think that is heading in the right direction.

Remember that although hormones are powerful players in determining the form of an organism, it is the target receptors that actually do the work. Many chronic human diseases are probably the result of not receptor modulation, but of modification of the receptor itself. Here is a link to a post I made with an example of a genetically controlled human disorder that fleshes this idea out some:

POST ON COMPLETE ANDROGEN INSENSITIVITY

The example in the post linked above is genetic, but it turns out that lots of these "receptor diseases" (diabetes, mental illnesses, etc.) are caused by epigenetic mechanisms. Interestingly, it has been shown that sex chromosomes can regulate autosomal expression epigenetically, in ways that have nothing to do with hormones.

So, I think it is genes regulated by epigenetic factors that are probably regulated by genes that are controlled epigenetically......

mofeta

 

[GreenintheThumb]

I'll guess that if an inter-sex plant easily show inter-sex it maybe has copies for that from both parents, while if only from one parent gave it maybe the plant does not express it so easy? But cross the plant with a single set again with another plant that does carry inter-sex maybe both copies, and the progeny do all express inter-sex. I am guessing there could be many different control mechanisms depending if it is a true inter-sex genes or something that alters ethylene expression for example.
We will see....
-SamS

I don't know. What you're describing is codominance and if it were truly that simple I feel like we would have eliminated this trait from the drug pool by now. In the same way that western bred drug cultivars have eliminated Bd from our populations. And they did that pretty much on accident. You'd think intersex would be eliminated faster if it was one locus with codominance. Everyone selects against these traits (except Soma I guess).

But who knows, this could be the first time I could be accused of giving TOO much credit to cannabis breeders. It feels weird.

It took you many generations to clean up Durban, right?


Has anyone studied stress hormones in cannabis? I'm not very familiar with the subject honestly but what does abscisic acid do in our favorite plant? How about nitric oxide?

A few interesting bits in here, Plant hormone interactions
http://jxb.oxfordjournals.org/content/63/9/3499.full

 

[Sam_Skunkman]

It could codominance or something else.
Allelic dominance always depends on the relative influence of each allele for a specific phenotype under certain environmental conditions, so if there are multiple alleles at the locus controlling inter-sex or multiple alleles on other locus that promote inter-sex, the results can be all over the board.
I have to think about Partial dominance or Over dominance to see if they might be possible to be part of the picture. My brain hurts when I think to hard or long, getting old.
I will be happy when most of it is all figured out.
-SamS

I don't know. What you're describing is codominance and if it were truly that simple I feel like we would have eliminated this trait from the drug pool by now. In the same way that western bred drug cultivars have eliminated Bd from our populations. And they did that pretty much on accident. You'd think intersex would be eliminated faster if it was one locus with codominance. Everyone selects against these traits (except Soma I guess).

But who knows, this could be the first time I could be accused of giving TOO much credit to cannabis breeders. It feels weird.

It took you many generations to clean up Durban, right?


Has anyone studied stress hormones in cannabis? I'm not very familiar with the subject honestly but what does abscisic acid do in our favorite plant? How about nitric oxide?

A few interesting bits in here, Plant hormone interactions
http://jxb.oxfordjournals.org/content/63/9/3499.full

 

[Sativied]

Great thread, nice to see a proper discussion on this topic. Still got a few pages of the thread to read but a search didn't show this one, so figured I add the following reference for completeness

The sexual differentiation of Cannabis sativa L.: A morphological and molecular study
V. M. Cristiana Moliterni, Luigi Cattivelli, P. Ranalli, Giuseppe Mandolino
2004
Summary

Cannabis sativa L. is a dioecious species with sexual dimorphism occurring in a late stage of plant development. Sex is determined by heteromorphic chromosomes (X and Y): male is the heterogametic sex (XY) and female is the homogametic one (XX). The sexual phenotype of Cannabis often shows some flexibility leading to the differentiation of hermaphrodite flowers or bisexual inflorescences (monoecious phenotype). Sex is considered an important trait for hemp genetic improvement; therefore, the study of the mechanism of sexual differentiation is of paramount interest in hemp research. A morphological and molecular study of Cannabis sativa sexual differentiation has been carried out in the Italian dioecious cultivar Fibranova.


Microscopic analysis of male and female apices revealed that their reproductive commitment may occur as soon as the leaves of the fourth node emerge; the genetic expression of male and female apices at this stage has been compared by cDNA-AFLP. A rapid method for the early sex discrimination has been developed, based on the PCR amplification of a male-specific SCAR marker directly from a tissue fragment.


Five of the several cDNA-AFLP polymorphic fragments identified have been confirmed to be differentially expressed in male and female apices at the fourth node. Cloning and sequencing revealed that they belong to nine different mRNAs that were all induced in the female apices at this stage. Four out of them showed a high degree of similarity with known sequences: a putative permease, a SMT3-like protein, a putative kinesin and a RAC-GTP binding protein.


http://link.springer.com/article/10.1007%2Fs10681-004-4758-7