Animal Molecular Breeding 2015, Vol. 5, No. 1, 1-8
3
reliably distinguished using morphometric methods on
the basis of the striking polymorphisms exhibited by
their shells.
1.2
DNA extraction and quantification
DNA was extracted from the meat of each sample
according to the CTAB method of Saghai-Maroof et al.
(1984), with minor modifications containing 2% (v/v)
β
-mercaptoethanol (added just before use) and 2% (w/v)
polyvinylpyrrolidone, followed by RNase treatment for
the removal of contaminating RNA. The purity of the
extracted DNA was examined by agarose (0.8%) gel
electrophoresis while its concentration was determined
by UV-visible spectrophotometer (UV-1601, Shimadzu,
Japan).
1.3
Screening of RAPD primers and polymerase
chain reaction (PCR) amplification
Twenty of the 50 RAPD primers ordered from
Eurofins Genomics
TM
.
eu/media/962761/rapd_10mer_kits_sequences.pdf)
were randomly selected and screened for
polymorphism on template DNA from land snail
accessions. Three of the screened primers (OPB03,
OPB04 and OPB08), developed by Hadris et al. (1992),
showed significant polymorphism (as outlined in Table 1)
and were used for further analysis (Figure 1).
Figure 1 RAPD banding pattern of the sixteen (16) accessions of
the snails
Table 1 Oligonucleotide primers selected for the study
Serial number
Primer
Sequence
1
OPB-03
5′ AGACGTCCAC 3′
2
OPB-04
5′ GGAAGTCGCC 3′
3
OPB-08
5′ GTCCACACGG 3′
Polymerase chain reaction amplification was
performed in 25
μ
l reaction mixtures containing 1x
Taq
,
100 ng genomic DNA, 3mm MgCl
2
, 250
μ
M of each
dNTPs, 0.2 nm primer and 2 units of Taq polymerase
(BioGene, USA) using MJ Research PTC-200
Thermal Cycler (GMI Inc., Ramsey, Minnesota, USA).
The cycling conditions consisted of 1 cycle of 94
o
C for
5 min (initial denaturation), followed by 45 cycles of
94
o
C for 30 sec (denaturation), 35
o
C for 1 min
(annealing), and 72
o
C for 2 min (polymerization), with
a final extension of 7 min at 72
o
C. A negative control
without the snail genomic DNA was kept for
amplification along with each primer to check the
quality of the primer and to avoid the possibilities of
contaminations and primer dimers. The amplification
products (10μl) were loaded in an ethidium
bromide-added agarose gel (1.2%) for electrophoresis
(at 125V) in 1x Tris-boric acid-EDTA buffer. The
electropherograms were documented using gel Alpha
Imager documentation analysis system (Alpha
Innotech, USA). DNA bands were compared with 100
and 1500 bp DNA standard markers (BioGene, USA).
The amplifications were performed twice with
genomic DNA isolated independently to confirm the
reproducibility.
1.4
Data Analysis
Basic statistical analyses method for morphological
variations including the range, mean and their standard
deviation were calculated to assess morphometric
variation using the standard statistical procedures as
described by Snedecor and Cochran (1994).
For the PCR-RAPD fingerprinting, all positive
amplicons were treated as separate characters and
scored for the presence (1) or absence (0) of bands.
Polymorphism Information Content (PIC) was
calculated based on the number of bands/primer, using
the formula PIC
=
1
− Pi
2, where
Pi
is the frequency of
the
i
th band.
Cluster analysis and Principal Coordinate Analysis
(PCA) were carried out in NTSYS (Numerical
Taxonomy and Multivariate Analysis System) version
2.01i. Genetic similarities based on Dice coefficient
were calculated among all possible pairs using the
SIMQUAL option and ordered in a similarity matrix. A
