RE: [DIYbio] Barcoding of wheat

[Sebastian Cocioba]

DNA barcode for plants may give some insight to the evolutionary origin of said grain. I'm not sure how varied the sites are in the same species across time.

Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D

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From: Mega [Andreas Stuermer]
Sent: 10/14/2013 3:42 PM
To: diy...@googlegroups.com
Subject: [DIYbio] Barcoding of wheat

Hi everyone,

Near my university hundreds of kilograms of old grains were found in holes below the ground. They were dated to the 1st century after Christ, and Roman origin (Roman settlement).
It seems to have been some kind of a reserve, or storage facility. Strangely, the grains are black.

Well, as a project we have to do some analysis. Regarding protein content, nitrogen content, etc etc.

I was wondering, wheter we could use PCR to identify the strain of wheat used / or relationship to modern strains...

A google research, however, showed no viable results.

Would be nice if someone had an idea ;)

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[Cathal Garvey]

Does barcoding have any relevance to phylogeny? I thought it was like a
"bio-hash", with the output guaranteed to be unique but not necessarily
have any other useful purpose.. :)

Certainly 16S rRNA and other medium-high conserved genes are useful for
inferring relationships between species, but for intra-species
relationships there's probably a different set of more medium-variable
genes that vary more within centuries.

On Mon, 14 Oct 2013 13:06:09 -0700
Sebastian Cocioba <scoc...@gmail.com> wrote:

> DNA barcode for plants may give some insight to the evolutionary
> origin of said grain. I'm not sure how varied the sites are in the
> same species across time.
>
> Sebastian S. Cocioba
> CEO & Founder
> New York Botanics, LLC
> Plant Biotech R&D

> ------------------------------
> From: Mega [Andreas Stuermer] <masters...@gmail.com>

[Dakota Hamill]

http://www.ccdb.ca/docs/CCDB_PrimerSets-Plants.pdf

I use rbclA-F/R for plants.  For plants, they also tend to use 2 different primer pairs, but I forgot the 2nd pair, and multiplex them.  For bacteria/fungi it seems "acceptable" by barcode people to use just 16s or ITS, but for plants and animals I see a lot of double pair multiplexing.  Kind of fell off my barcoding obsession and been doing other things so I can't remember the primer pairs anymore off the top of my head.

[Patrik D'haeseleer]

On Monday, October 14, 2013 2:12:02 PM UTC-7, Cathal Garvey wrote:

Does barcoding have any relevance to phylogeny? I thought it was like a
"bio-hash", with the output guaranteed to be unique but not necessarily
have any other useful purpose.. :)

Yes, the genes selected for barcoding are definitely useful for phylogeny. With the exception of things like V(D)J recombination in immune receptors, there's no such thing as a "bio-hash" - just genes that evolve at different rates. Pick a slow evolving gene, and you won't be able to tell species apart, but you might be able to reconstruct deeper branchings between phyla. Pick a very fast evolving gene, and you might be able to tell strains or sub-strains apart, but a fast evolving gene will typically only be useful within a specific taxonomic range (and may not have the same rate of mutation across that entire range). The genes picked for the Barcodes of Life project are typically picked to give enough separation between species and genera, but may not have enough resolution to tell different strains apart - or to reconstruct the evolution of a new species, such as modern wheat from its ancestral strains.

To quote barcodeoflife.org:

The gene region that is being used as the standard barcode for almost all animal groups is a 648 base-pair region in the mitochondrial cytochrome c oxidase 1 gene (“CO1”). COI is proving highly effective in identifying birds, butterflies, fish, flies and many other animal groups. COI is not an effective barcode region in plants because it evolves too slowly, but two gene regions in the chloroplast, matK and rbcL, have been approved as the barcode regions for plants.

This Plos One paper has a lot more info:

The outcome of these trials was that although some markers could be eliminated from consideration (e.g. rpoC1 and rpoB showed markedly lower discriminatory power), there was no straightforward solution as to which should form the plant barcode, as each of the candidate markers had different strengths and weaknesses. The majority preference of the CBOL Plant Working group was to recommend a core-barcode consisting of portions of two plastid coding regions, rbcL+matK, to be supplemented with additional markers as required [11]. The rbcL barcode consists of a 599 bp region at the 5′ end of the gene, located at bp 1–599 (including primer sites) in the complete Arabidopsis thaliana plastid genome sequence (gi 7525012:54958–56397). The matK barcode region consists of a ca. 841 bp region at the center of the gene, located between bp 205–1046 (including primer sites) in the complete A. thaliana plastid genome sequence (gi 7525012:2056–3570).

To tease apart the evolution of a specific species, you may need to look at features that mutate much faster, such as microsattelites or transposable elements:

Microsatellite markers: an overview of the recent progress in plants

Marker utility of miniature inverted-repeat transposable elements for wheat biodiversity and evolution

See also:

Genetic Diversity, Evolution and Domestication of Wheat and Barley in the Fertile Crescent

Genetic diversity, evolution and domestication of Triticeae in the Fertile Crescent

Patrik

[Mega [Andreas Stuermer]]

Ok, thanks a lot!!

[Mega [Andreas Stuermer]]

Btw, any other ideas what research to do on 2000 year old grains? :D

we want to put some on soil and grow them :D

[Simon Quellen Field]

It might be interesting to look at commensal fungi and other organisms that might have aided storage, or selectively degraded some of the grains. There might also be links between commensals and the germination rates of seeds that old (if you get any germination at all).

Measuring the amino acid composition, fatty acid composition, and other nutritional aspects might be interesting. What has been gained over the years, and what has been lost?

Measuring mineral content such as arsenic might tell you about groundwater depletion as a result of agriculture, or changes in local heavy metal concentration as a result of local mining and smelting.

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On Tue, Oct 15, 2013 at 10:19 AM, Mega [Andreas Stuermer] <masters...@gmail.com> wrote:

Btw, any other ideas what research to do on 2000 year old grains? :D

we want to put some on soil and grow them :D

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[Mac Cowell]

If you are barcoding the wheat anyway, you could also get a fungal genome isolation kit and try to barcode any fungi present. You would probably have to use next-gen sequencing since you'll have isolated a heterologous mixture of fungal genomes.

uBiome is offering some kind of barcoding service on scienceexchange for $69, but I don't think it's next-gen based. But they might be open to an affordable collaboration. http://widgets.scienceexchange.com/facilities/ubiome/services/16s-rdna-sequencing-analysis

Mac

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[Andreas Stuermer]

Ok thanks!

We'll also do mycotoxins.

But heavy metal contamination won't say much, because it was under some kind of factory... The heavy metals may come from there - if there are any...  

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[Patrik D'haeseleer]

You might also want to check whether the black color of the grains is due to fungal melanin. That's the first thing that came to my mind.

And keep in mind that fungal melanin is often associated with pathogenicity as well - such as in the human pathogenic fungus Aspergillus fumigatus. I don't know whether fungal spores remain viable after a couple thousand years, but I wouldn't be handling any of that grain without a face mask...

Patrik

[Cathal Garvey (Phone)]

Do, and then give some to heritage seed keepers/sellers. Even though that's kinda illegal now. Civil disobedience is only way to kill that.

"Mega [Andreas Stuermer]" <masters...@gmail.com> wrote:

Btw, any other ideas what research to do on 2000 year old grains? :D

we want to put some on soil and grow them :D

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Sent from my Android device with K-9 Mail. Please excuse my brevity.

[Patrik D'haeseleer]

On Tuesday, October 15, 2013 11:16:02 AM UTC-7, Mackenzie Cowell wrote:

uBiome is offering some kind of barcoding service on scienceexchange for $69, but I don't think it's next-gen based. But they might be open to an affordable collaboration. http://widgets.scienceexchange.com/facilities/ubiome/services/16s-rdna-sequencing-analysis

I would assume uBiome is using iTag sequencing, multiplexing a couple hundred samples on a single Illumina run. There's no way they can get down to $69 for a mixed sample without doing next-gen.

Of course, uBiome's $69 offering is for 16S - they'd have to target 18S or ITS for fungi. Not sure how much optimization that would require beyond merely swapping out a different set of primers.

Patrik

[Patrik D'haeseleer]

On Tuesday, October 15, 2013 10:55:45 AM UTC-7, Simon Field wrote:

Measuring mineral content such as arsenic might tell you about groundwater depletion as a result of agriculture, or changes in local heavy metal concentration as a result of local mining and smelting.

I wonder if it's possible to tell something about fertilization practices by looking at nitrogen isotopes. 15N/14N ratio tends to increase with trophic level, so you may be able to see a slight increase in 15N in plants fertilized with human feces, vs animal dung, vs unfertilized crops.

The same approach has been used with carbon isotope ratios in human remains, to see if their diet was based primarily on C4 or C3 plants, or based on marine vs freshwater fish.

Patrik

[Patrik D'haeseleer]

On Tuesday, October 15, 2013 11:49:18 AM UTC-7, Patrik D'haeseleer wrote:

On Tuesday, October 15, 2013 10:55:45 AM UTC-7, Simon Field wrote:

Measuring mineral content such as arsenic might tell you about groundwater depletion as a result of agriculture, or changes in local heavy metal concentration as a result of local mining and smelting.

I wonder if it's possible to tell something about fertilization practices by looking at nitrogen isotopes. 15N/14N ratio tends to increase with trophic level, so you may be able to see a slight increase in 15N in plants fertilized with human feces, vs animal dung, vs unfertilized crops.

Aha - let me just answer my own question... Here's a Plos One paper talking about nitrogen isotope ratios in plants, and fertilization sources:

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0053763
"The second potentially significant source-related cause of plant δ¹⁵N variation is the uptake of fertilizer-derived N by plants. Animal fertilizers are characterized by extremely variable δ¹⁵N values depending on the relative proportions of N-bearing species in the fertilizer (e.g. urea, uric acid, ammonium, organic matter) [143]. Manures consisting primarily of solid waste derived from terrestrial herbivores tend to have δ¹⁵N values between 2 and 8 ‰ [144], while those that contain a mix of solid and liquid waste (slurry fertilizers) tend to have higher δ¹⁵N values, often between 6 and 15 ‰ [145], [146]. The highest δ¹⁵N values for animal fertilizers (>25 ‰) have been recorded for seabird guano [143], [147], which consists primarily of uric acid and is subject to significant NH₄⁺ volatilization. The addition of animal fertilizer N to the soil therefore adds an N source with an isotopic composition that is usually enriched in ¹⁵N relative to endogenous soil N. This results in higher δ¹⁵N values for plants growing in soils fertilized with animal waste than those plants growing in unfertilized soil or soils fertilized with chemical fertilizers [143], [145]–[147]."

You might be able to identify some potential collaborators with the expertise and resources to do this kind of analysis, by tracking down those references...

Patrik

[Eugen Leitl]

On Tue, Oct 15, 2013 at 11:24:03AM -0700, Patrik D'haeseleer wrote:
> You might also want to check whether the black color of the grains is due
> to fungal melanin. That's the first thing that came to my mind.

I would just expect plain old oxidation.

[Eugen Leitl]

On Tue, Oct 15, 2013 at 10:55:45AM -0700, Simon Quellen Field wrote:

> Measuring mineral content such as arsenic might tell you about groundwater
> depletion as a result of agriculture, or changes in local heavy metal
> concentration as a result of local mining and smelting.

Arsenic typically starts moving when oxidative milieu turns reductive.
This can be associated with agriculture.