Protocols for Micropropagation of Woody Trees and Fruits

452
M.G. OSTROLUCKÁ ET AL.
2.6.3. PCR–ISSR
For ISSR technique 3´OH anchored primers [(CA)6GT, (GAG)3GC, (GT)6CC,
(GA)9C] and 5´OH anchored primer GT(CA)4 were used. Amplification reactions
were performed in 25 µl of reaction volume containing 20 mmol × dm –3 Tris-HCl
(pH 8.0), 50 mmol × dm –3 KCl, 3 mmol × dm –3 MgCl2, 0.1 mmol × dm –3 dNTPs, 0.4
µmol × dm –3 primer, 1U Taq polymerase and 30 ng of DNA. All reactions were
heated to 94°C for 2 min, followed by 45 cycles of 94°C for 1 min, 55°C for 1 min
and 72°C for 3 min. Polymerisation was followed by final extension cycle of 72°C
for 10 min.
2.6.4. Electrophoresis and PCR Products Evaluation
The amplification PCR–ISSR products were separated by electrophoresis on 2%
(w/v) agarose gel (120 V, 80 mA). Electrophoregrams were evaluated by KODAK
EDAS 190 1D software (Figure 4) where the size (in bp) of individual DNA bands
was recorded. DNA bands were retrieved based on their size and these information
was transferred into Microsoft Excel Application where the size was replaced by
binary 1 (for present band on specific position) or 0 (for absent band). Consequently,
Nei and Li (1979) similarity index (SINL) based on ISSR profiles was calculated in
the MS Excel according to the SINL = 2 × sum of common bands of lane A and B/
(sum of the bands in the lane A + sum of the bands in the lane B).
Figure 4. ISSR profile using primer (GT) 6CC evaluated by KODAK EDAS 190 1D software.
M-250 bp ladder, 1–4 Berkeley, 5–7 ‘Bluecrop’, 8 ‘Blueray’, 9 ‘Darrow’, 10 ‘Linnea’.
Distance index (DINL) values were calculated according to the DINL=1 – SINL.
Dendrograms were constructed using UPGMA (Unweighted Pair-Group Method
Using Arithmetic Averages) analysis for the objective position of ISSR in the
statistical program SYNTAX. Dissimilarity of every single cluster is showed by the
Euclidian Distance Averages of Clusters (EDAC).
The group of tested genotypes was divided into two main clusters (Figure 5).
First group consists of ‘Bluecrop’ and ‘Berkeley’ genotypes with an average GINL
0.308. Second group consist of three individual genotypes, two of species V.
corymbosum (‘Blueray’ and ‘Darrow’) and one of species V. vitis-idaea (‘Linnea’)
with an average GINL 0.552. The polymorphism of analyzed samples varied from
96% to 99% depending on the used primer. Unique bands appeared in very low