Microsatellites in Echinacea… they do exist

Hello all,
So this is my very first blog entry so it will be lacking all the neat links to pictures et al in that are in other people’s entries but it will talk about Echinacea.

Since this is my first blog I should probably spend a little time introducing myself. My name is Jennifer I am a in an inter-disciplinary PhD program, called LEAP, at the University of Illinois at Chicago in conjunction with the Chicago Botanic Garden. LEAP stands for Landscape Ecological and Anthropogenic Processes, it is an NSF funded IGERT program aimed at increasing biodiversity in human altered landscapes. For a much better description of LEAP see http://www.uic.edu/depts/bios/leap/. As for the Echinacea Project I have been involved with the project first as an intern back in 2003-2004 then as a graduate student (since summer 2005). My research mainly focuses on understanding how flowering phenology (when a plant flowers) shapes seed set, pollen movement, and ultimately genetic structure in a population. For more see my website at http://www2.uic.edu/~ison/.

To understand how flowering phenology shapes population structure we use a variety of methods. First we collect phenology data in the common garden. The current protocol has us counting anthers shedding pollen every other day. We then collect the seed heads in the fall and individually weigh a subset of the seeds to get an estimate of seed set. Why individually weigh seeds? Well it is the only non-destructive method of determining if an achene (the technical term for fruits in the Asteraceae) actually has a viable embryo. We know that 97% or seeds weighing greater than 2 mg will germinate and 91% of less weighing less than 2 mg will not germinate. As of this spring we have individually weighed (with the help of an amazing volunteer named Art) weighed 30,211 seeds. This June Art has embarked on weighing another 3,000 seeds from the 2006 flowering plants. So far we know that late flowering plants set much less seed than early or peak flowering plants. To get at the hereditability of flowering phenology we planted a second common garden (yes there is another common garden) on a site called Hegg Lake owned by the DNR. The site was planted with just about 4,000 seedlings in May 2006 and the plants will hopefully flower before I finish my PhD.

Finally, to understand how flowering phenology influences pollen movement we are using molecular genetic techniques, specifically microsatellites markers. Microsatellites are a molecular genetic marker that consist of repeating non-coding regions in the genome (eg GATGATGATGAT). Since they are repeating non-coding regions they mutate relatively rapidly so there are different number of repeats for the same microsatellite in a population–alleles. With these microsatellites we will be able to, eventually, take a seed from a known maternal plant and find out who the dad is. I developed microsatellites specifically for Echinacea last fall at the Field Museum of Natural History in Chicago. I now need to determine of these microsatellites I found do they actually have enough alleles to conduct paternity analysis. While everyone else has been up in Minnesota flying kites I have been spending time in the genetics lab trying to get the microsatellites to work. After spending too long figuring out the optimal number of cycles and temperature in the PCR, plus how much, if any, Mg to add I finally have been having success with about 5 microsatellites.

Today I ran four out of the five primers on 16 plants (8 from the preserve and 8 from Steven’s approach) and had multiple alleles!!! I had between 4 to 6 alleles just in these 16 plants. It was very exciting after spending so long playing with PCR conditions. It was very rewarding to run samples that actually worked and even better that all the microsatellites were at least moderately variable. My goal is to get 8 primers with all with around 6 alleles, which should be enough to do figure out who the dad is. For my next blog entry I’ll see if I can figure out how to add pictures and I’ll insert some images of my microsatellite alleles.

I think that is more than enough for my first entry. I will hopefully have more exciting news regarding the microsatellites before I come up to Minnesota (which is on July 15th).
Night!
Jennifer

Notes to self:
Equipment for MN
-2 meter sticks
-camera
-data logger (?)–talk to JF
Finish up at CBG
-put seeds into freezer–talk to AS?
-data entry for Theresa
-get tissue samples into fresh silica gel
-molecular work for John and Eric