Peas in the lab – results

Principal coordinate analysis of old Danish peas
(Click on picture to see more clearly)

Finally I finished writing a small report (in danish) about the old peas in the laboratory.
The report is located here: 2010 Resultat af ærteforsøg (DNA)
I had google translate this version, with a bit of my help: 2010 Pea results

When reading the results, so it is vital to understand that this investigation can determine whether some of the peas are genetically different, but can not determine whether they are genetically identical! We have investigated only some small bits of the total DNA.
The peas that look similar, are either identical or different, we do not know!

At the top is shown the principal coordinate analysis. It gives a good visual picture of the distance between our varieties. Some old Swedish varieties was blended in, as they have been studied previously. It gives some reference points to past studies.

The principal coordinate analysis is based of course, on some very concrete data, namely the length of the micro-satellites we tested.

Table. Displays length of each micro-satellite.

Micro-satellites we have measured have letter names. A9 – D21 – AC58 – C20 – AA5 – AC75.
We investigated 6 different, but one would not work, the second was identical in all varieties, and therefore uninteresting. The last four showed differences. Each micro-satellite was measured twice.

I end up concluding:

So there are varieties that we now know is different from all others in this sample.
There are also groups of varieties we should check for phenotype differences. It is a great advantage that it is now clearly delineated the varieties to be compared by cultivation experiments. There is probably also varieties that are identical with each other. It is not something that can be resolved by this trial, which is only suitable to detect differences in the 4 areas (micro-satellites), we got useful results from. Whether they are identical in many other micro-satellites, we can not know before we make a new attempt!

Read in the google translated report: 2010 Pea results

If you can take a bit more, this is the excel file we were sent after the course: Diversity analysis Pisum

 

Heirloom peas in the laboratory

Pee shoots of heirloom varieties in the laboratory at Department of Agriculture and Ecology, Copenhagen University (Copyright Ingrid Nolde)

Last weekend I joined the Danish seed savers at the laboratory at the Department of Agriculture and Ecology (Copenhagen University). Gunter Backes and Jihad Orabi led us through a study of our heirloom pea collection. It was a fantastic and educational weekend!

In preparation, we had grown peas for 3-4 weeks, so they were ready for analysis. We had to use leaves that had not been given too much light. Chlorophyll can make it difficult to extract DNA material from the rest of the leaf tissue. For safety’s sake, we were two cultivating the same peas. It went well and we only used one set.

Test tubes on ice (Copyright Ingrid Nolde)

We were divided into four groups. That way we could manage to work with all the varieties we had brought. After a brief introduction to laboratory work, to use gloves and gowns – take them off when you leave the lab, we started slicing the pea leafes.
Once cut, the natural enzymes in the leaves activates. To inhibit them, we set all tubes in a tray of ice. Millimetre small pieces were placed in the tubes, a solution of caustic soda were added and the tubes were boiled for 1 minute to denature the proteins. Then we blended the boiled leaves with the tip of a plastic stick and added a resolution that brought the pH down to a desired level. To separate DNA from the rest of the leaf broth, test tubes were centrifuged.

Pea leaf broth bottom, DNA located at the top in the clear part of the liquid (Copyright Ingrid Nolde)

Now we could move the purified DNA into new tubes, still set in ice.
Then it was time to mix liquids with primer for the DNA fragments we searched for. We looked at 6 different micro satellites. Micro satellites are good to tell the difference between varieties of a species. A primer is the DNA sequence immediately before the micro satellite, leading the DNA strand to be cut at the desired location. We used a primer for each end of the micro satellite, because it must be “cut” at both ends. To the liquid is also added Taq DNA polymerase, an enzyme that replicates DNA at specific temperatures.

We used pipettes for several hours. Even with multi pipettes there was plenty to do (Copyright Ingrid Nolde)

Next the test tubes are undergoing repeated temperature changes between
94 °C, where DNA strand separates
64-55 °C, where the primer binds to the DNA strand, and
72 °C, where Taq DNA polymerase copies the DNA strand from the primer and forward

By the repeated changes in temperature, we multiplied the selected micro satellite exponentially. The first cycle gave very little DNA material, but for each cycle, there was more copies. Just like reproduction of yeast in a bread dough.

Finally, we let an electric field pull micro satellites through a viscous liquid in capillary thin tubes. A dye was coupled to micro satellites, for a photocell at the end of capillaries to detect how quickly they passed by. Small micro satellites migrate faster through capillary tubes than larger.

First presentation results

The photo shows the horizontal bands, each representing a pea variety. The small vertical bands are micro satellites. The red vertical band marks a scale, so we know how long the micro satellite strands are.
Jihad and Gunter did the analysis work Sunday morning, before we all met up – thank you!

It was best to group the peas in 7 groups

The results will soon arrive in my Inbox. I look forward to delve deeper into the results. I might have a more to write about then.

Big thanks to both Gunter and Jihad for a learning and rewarding weekend 🙂

PS: I’m just a happy amateur. Feel free to comment if I’m wrong in some of the techicalities 🙂