Hi!

First of all, your question is not stupid. Second, I believe your problem is technical.

You have to make sure that the DNA you isolated from your material is suitable for PCR. It is not trivial, actually.

I would try a set of primers - specific primers for very conserved nuclear gene. Only after you are absolutely sure that your DNA is good, high quality for further research, try the primers for ITS.

It is possible that you are having difficulty amplifying ITS from herbarium specimens because the nuclear DNA is degraded.

The Taberlet primers are a good place to start: you may have the most success with primers "e" and "f" - these amplify a usually variable integenic spacer. Since trnLF is a chloroplast marker, you should have better success than with a nuclear marker such as ITS. I would recommend trying it with your samples to see if they amplify.

As for any phylogenetic inquiry, it is important to consider your question before pursuing markers to address it.

How many species are in the group that you are studying?

How closely related?

Is hybridization or allopolyploidy a potential factor?

Of course, it is also important to make sure that you have optimized your PCR conditions. Start with those suggested in recent work on the group or related groups, and go from there.

This paper: Phylogenetic Analysis and Evolutionary Diversification of Heliotropium Sect. Cochranea (Heliotropiaceae) in the Atacama Desert

would be a great start. They use several markers, including ITS and trnLF - you can look at their amplification protocols and get some understanding of which regions may be the most informative for your study.

Regarding ITScomplete, we used P5 (forward primer: GGA AGT AAA AGT CGT AAC AAG G) and P4 (reverse primer: TCC TCC GCT TAT TGA TAT GC) ex White et al. (1990). Regarding trnLF we used c (forward primer: CGA AAT CGG TAG ACG CTA CG) and f (reverse primer: ATT TGA ACT GGT GAC ACG AG) ex Taberlet et al. 1991. This set represents a good starting kit. However, ITS1 and trnLUAA probably will not provide sufficient information in order to obtain a nicely resolved phylogeny. Especially ITS sometimes is difficult to amplify and silica dried material is highly recommended. What species/section you are working on?

my species belong to 7 different sections (sensu Forther 1998); all from middle eastern localities (Egypt-Yemen-Saudi Arabia)

- Orthostachys (now Euploca)

- Heliotropium

- Pleurolasia

- Rutidotheca

- Pterocarpum

- Pseudocoeloma

- Zeylanica

Please have a look at what already has been published on this topic (publication list "Boraginales-Publikationen ab dem Jahre 2000": http://www.bcp.fu-berlin.de/biologie/arbeitsgruppen/botanik/ag_hilger/publikationen/index.html

You might like to get in touch with Dr. Federico Luebert who is a specialist in this area of research ([email protected]).

Have a look here to see who also works in this area: http://www.cmep.org.uk/cmep-team/alan-forrest/

Hope that helps!

Hi Asma'a,

I know we had been in contact before about Heliotropium. I can answer some of your questions.

First, it is true that you need very good quality DNA from herbarium specimens to amplify ITS. I always use Qiagen DNA extraction protocols as they give high quality DNA. I also dilute the DNA so any impurities are also diluted. I still sometimes have trouble: it depends on how the herbarium sample was collected. However, you can add some chemicals to your PCR to help - betaine is particularly effective.

Second, as all your species are in different sections, it should be easy to tell them apart with ITS and an appropriate chloroplast marker. I have designed Heliotropium specific primers for the rpl32-trnL spacer that work very well, and give good resolution, even among some species and within the H. bacciferum complex. In fact, your sections are so different, especially Euploca, that ITS sequences might be difficult to align. The trnL markers will work quite nicely, but the resolution will be lower. ITS resolution is quite good for closely related species in Heliotropium.

If you need more advice or would like to try some primers, please contact me directly through the CMEP website.

Best wishes,

Alan