Protocol for identification of species and
hybrids within D. longispina complex
by restriction analysis of ITS region
following the method described in: Billiones R.,
Brehm M., Klee J., and Schwenk K. (2004): Genetic identification of Hyalodaphnia
species and interspecific hybrids. Hydrobiologia, 526: 43 – 53.
1) DNA isolation:
use any of your favourite protocol (e.g. using Chelex, proteinase K
solution, or their combination) to isolate DNA from a single specimen from field samples, or any number of individuals
from a clonal culture.
In case of ephippial females with sexual
eggs, it is advisable to remove the eggs from the ephippium (to avoid potential
contamination in case the eggs are hybrid).
2) Polymerase Chain Reaction
Gene region:
1500-2000 bp segment consisting of ITS2,
5.8S, ITS1 and a part of 18S
Primers:
ITS2-5.8S (forward): 5'-GGA AGT AAA
AGT CGT AAC AAG G-3'
ITS1-18S (reverse): 5'-CGG TGG TCG
ACA CTT CGA CAC GC-3'
Reaction mixture:
volume per sample: 25-35
µl
composition:
1x PCR buffer
3 mM MgCl2
0.2 mM of each dNTP
0.3 µM of each primer
1 unit of Taq polymerase
thermocycler program:
initial denaturation 94°C / 30 seconds
5x [94°C
/ 30 seconds
52°C / 30 seconds
72°C / 1 minute]
35x [92°C
/ 1 minute
52°C / 1 minute
72°C / 1.5
minutes]
final elongation 72°C / 5 minutes
pause at 4-10°C.
3) RFLP (Restriction Fragment Length Polymorphism) analysis
Cut the PCR product by the following two
restriction enzymes (1 unit enzyme per 10 µl PCR product) – Mwo I and Sau96 I – and incubate for the reaction mixture for
two hours at the enzyme-specific optimal temperature (reaction mix composition
and the incubation temperature may vary depending on the enzyme manufacturer).
4) RFLP pattern visualisation
Load the
whole content or restriction reaction mix on 2% agarose gel, and run agarose
electrophoresis (recommended settings: 100 V / 60 min, modify according to your
gel size and buffer used). We recommend using 100 bp and 25 bp
ladders as size markers.
Evaluate the
fragment presence and length, and follow the identification key in EDS.