[Annelida] Problems with sequencing CO1 in polychaetes

Dirk Steinke via annelida%40net.bio.net (by dsteinke from uoguelph.ca)
Tue Jun 7 11:05:11 EST 2011


Dear Elena,

We did a fair bit of testing with polychaetes at the Biodiversity 
Institute in Guelph . Mainly because we had exactly the same problems 
you've described in your post: Folmer primers did not work, design of 
alternative primers was an issue.
I won't say we have solved the problems 100% but we were able to make 
some good progress on Polychaete barcoding. It turns out that often it 
is the DNA extraction that represents the problem for further lab work 
not so much the PCR itself.

The best general protocol we currently have is the following:

DNA extraction with CTAB. An example protocol utilizing 96-well plates 
you can find here: 
http://www.ccdb.ca/pa/ge/research/protocols/extraction (pick the plant 
etc. protocol). There are also plenty of protocols on the web for 
smaller scale extractions - not everyone has 96 samples available at once.
CTAB has been widely used for plants but many researchers have 
discovered that it works nicely with all sort of "slimy" animals. The 
idea is that this removes potential PCR inhibitors and looking at our 
results in the last years it might well be true.
There is also another method to remove the suspected polysaccharides 
from the extract described in this paper: 
http://www.springerlink.com/content/j25wux7m21760q20/

As for the PCR we almost completely moved away from standard Folmer 
primers and use Polychaete specific variants (see 
http://connect.barcodeoflife.net/group/marinebarcoding/forum/topics/primer-combinations-for-marine) 
and even a primer cocktail (mammal cocktail in Ivanova NV, Zemlak TS, 
Hanner RH, Hebert PDN (2007) Universal primer cocktails for fish DNA 
barcoding. Molecular Ecology Notes, 7, 544-548.) that initially has been 
designed for vertebrates. Sometimes re-PCRing of templates that 
initially did not work helped too although I see that as the very last 
resort.


There is still quite some room from improvement in some groups (e.g. 
some serpulids). However, recent papers have shown that there is hope :-)

Barroso R, Klautau M, Sole-Cava AM, Paiva PC (2010) Eurythoe complanata 
(Polychaeta: Amphinomidae), the 'cosmopolitan' fireworm, consists of at 
least three cryptic species. Marine Biology 157: 69-80
Carrera-Parra LF, Salazar-Vallejo SI (2011) Redescriptions of Eunice 
filamentosa and E. denticulata and description of E. tovarae n. sp. 
(Polychaeta: Eunicidae), highlighted with morphological and molecular 
data. Zootaxa: 51-64
Heimeier D, Lavery S, Sewell MA (2010) Using DNA barcoding and 
phylogenetics to identify Antarctic invertebrate larvae: Lessons from a 
large scale study. Marine Genomics 3: 165-177
Hilário A, Johnson SB, Cunha MR, Vrijenhoek RC (2010) High diversity of 
frenulates (Polychaeta: Siboglinidae) in the Gulf of Cadiz mud 
volcanoes: A DNA taxonomy analysis. Deep Sea Research Part I: 
Oceanographic Research Papers 57: 143-150
Luttikhuizen PC, Dekker R (2010) Pseudo-cryptic species Arenicola 
defodiens and Arenicola marina (Polychaeta: Arenicolidae) in Wadden Sea, 
North Sea and Skagerrak: Morphological and molecular variation. Journal 
of Sea Research 63: 17-23
Schueller M (2010) Evidence for a role of bathymetry and emergence in 
speciation in the genus Glycera (Glyceridae, Polychaeta) from the deep 
Eastern Weddell Sea Polar Biology. Springer Berlin / Heidelberg, pp 1-16
Tovar-Hernandez MA, Carrera-Parra LF (2011) Megalomma Johansson, 1925 
(Polychaeta: Sabellidae) from America and other world-wide localities, 
and phylogenetic relationships within the genus. Zootaxa: 1-71

Hope that helps a bit.

Best,

Dirk

-- 
_______________________________________________
Dr. Dirk Steinke

Lead Scientist Marine Barcoding of Life

Biodiversity Institute of Ontario
University of Guelph
50 Stone Road West
Guelph, ON, N1G2W1

phone 1-519-824 4120 ext. 53759	
fax 1-519-824-5703
email dsteinke from uoguelph.ca




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