How to collect for "Sampling protocol for population genetics" protocol

mardi 2 juin 2015
par  Dorian GUILLEMAIN, Romain DAVID
popularité : 12%

Index of "Sampling protocol for population genetics?"
1. Principle
2. Method
3. Sampling
4. Deliverables
5. To summarize
Back to protocols Index
How to cite this content :

David R., Arvanitidis C., Çinar M.E., Dubar J., Dubois S., Erga Z., Guillemain D., Sartoretto S., Thierry de Ville d’Avray L., Zuberer F., Chenuil A., Féral J.-P., (2014), with contributors : Açik Çinar S., Andral B., Aurelle D., Aysel V., Bakir K., Bellan G., Bellan-Santini D., Bouchoucha M., Celik C., Chatzigeorgiou G., Chatzinikolaou E., Chenesseau S., Dağli E., Dailianis T., Dimitriadis C., D’Iribarne C., Doğan A., Dounas C., Egea E., Elguerrabi W., Emery E., Evcen A., Faulwetter S., Gatti G., Gerovasileiou V., Güçver S.M., Issaris Y., Katağan T., Keklikoglou K., Kirkim F., Koçak F., Koutsoubas D., Marschal C., Önen M., Önen S., Öztürk B., Panayiotidis P., Pavloudi C., Pergent G., Pergent-Martini C., Poursanidis D., Ravel C., Reizopoulou S., Rocher C., Ruiton S., Sakher S., Salomidi M., Sarropoulou E., Selva M., Sini M., Sourbes L., Simboura N., Taşkin E., Vacelet J., Valavanis V., Vasileiadou A., Verlaque M. Protocols for monitoring of coralligenous habitats of mediterannean (Coralligenous based Indicators to Evaluate and Monitor the "good ecological status" of the MEDiterranean coastal waters) Protocoles de suivi du coralligène en méditerranée (Coralligenous
based Indicators to Evaluate and Monitor the "good ecological status" of the MEDiterranean coastal waters).

a. Operations
[§ 1] 32 zip bags should be numbered with an indelible marker as showed in Figure 1(1). Numbers will correspond to the segments used for the profile? characterization.

The preparation of the bags should preserve the samples without any hesitation during the dive. Dangle an elastic on a snap hook in order to obtain a loop (Figure 1(2)) ;pass on the elastic loop through the hole of each bag, starting from the last one (Figure 1(3)). Secure the loop into the snap hook in order to avoid loosing the bags (Figure 1(4)). During the dives, unhook the loop and wear it around the wrist(Figure 1(5)). Releasing the elastic loop from the wrist,the diver can take the first bag,put the samples inside and then place it in its mesh bag.

IT IS IMPERATIVE TO MAINTAIN CORRISPONDENCE BETWEEN THE NUMBER OF THE BAG AND THE NUMBER OF THE TRANSECT?’S SEGMENT.
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Figure 1 : methodology for the samples’ collection

[§ 2] Along each segment, they should be collected : 2-3 fragments of Myriapora truncata (Pallas, 1766) about 3 cm long, by carefully braking a piece from the top of the colony (becareful not to break all the colony), and 3-4 fragments of Lithophyllum stictaeforme/cabiochiae (in order to compensate a possible confusions with Mesophyllum spp.), sized about 3 cm. It is better to collect bigger fragments than the minimum size every time it is possible because it will make the genetic analysis easier (more DNA available) and allow morphological analysis (using microscopy).16 samples of each species should be collected from each side of the site? at 28 m (+/-1m) depth, thus totally 32 samples from each site.The same protocol will be applied at 45 m depth, or any operational depth between 40 m and 50 m. Anyway, it is essential to have at least 10 m between the two sampling depths.

b. Samples conditioning
[§ 3] Samples should be placed into a portable fridge immediately after the dive, to tansfer them to the laboratory. In case of impossible immediate samples treatment, they can be conserved for no more than 48 h in aquarium.

Myriapora truncata
[§ 4] Sample manipulation needs pliers and a proper surface. Clear samples of epibionts with the help, whether necessary, of a binocular microscope, to remove any DNA which could distort genetic results. Then, conserve the samples in 20 ml or 50 ml Falcon tubes, filled to the ¾ of their volume with 95% ethanol. Change the ethanol after 24 hours to remove water and pigments. If it is not possible, rinse the samples with ethanol in a beaker before putting them into their individual tube. If samples are too large for the tube, they can be breaked.

[§5] Each sample should be identified with an individual code (see below), marked with a permanent marker on the tube, and with a pencil on a tracing paper placed inside the tube (Figure 2) :

[program]_[site]_[date] _D[depth]_[species]_T[n°transect]_S[n°segment]_R[n°replicate]_[observer?] Exemple :CIGESMED _CAS_20140123 _D1_truncata_T02_S07_R01_AA01.

Store in a cold room at 4°C.

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Figure 2 : M. truncata sample in a 20 ml tube

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Figure 3 : samples stored in a cold room

Lithophyllum stictaeforme/cabiochiae
[§ 6] Clear epibionts on specimens, in order to make the morphological traits useful for identification visible. Once confirmed the identification of Lithophyllum stictaeforme/cabiochiae (see ”Identification of Lithophyllum stictaeforme/cabiochiae” section), dry the sample with laboratory paper. Conserve samples in plastic bags filled silicagel? , enough to totally cover them (quantity vary with samples’ size) (Figure 4), stored in a dry and dark room to best preserve DNA (Broom et al.,2008).

Note with permanent marker on the bag, and with a pencil on tracing paper, which should be place into the bag, the sample code as follows :
[program]_[site]_[date]_

D[depth]_[species]_T[n°transect]_S[n°segment]_R[n°replicate]_[doer] Example : CIGESMED _CAS_20140123 _D1_cabiochiae _T02_S07_R01_AA01
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Figure 4 : Lithophyllum stictaeforme/cabiochiae conditioning.

Be very careful not to consider a broken sample as two different ones.

[§ 7] All samples should be listed in an excel file according to their code. Samples and files should be sent to the project responsible (anne.chenuil@imbe.fr) or to the responsible of the project database (romain.david@imbe.fr).

c. Material list
[§ 8]
• 16 zipped plastic bags
• 1 elastic
• 1 snap hook
• 1 picnic transport fridge

d. Work time quantification
[§ 9] For each site, 2 divers can carry on all sampling activities during 2 dives : totally, 4 man’s dives.

WE EXPECT YOUR SUGGESTIONS OF CORRECTIONS !

- Deadline : 14th December 2015
- Please add comments for each paragraph (§) by means of a corrected new version (one form per §)
- It is recommended to add scientific references if necessary

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