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International Journal of Aquaculture, 2013, Vol.3, No.24, 138
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The diversity was comparable with those obtained
earlier based on RFLP analysis of mtDNA (Oleinik
and Polyakova, 1994).
The statistical analyses using one way ANOVA also
depict 100% variations between the species. Also in
our study the value of H is not too high; hence do not
represent much diversity. In contrast to the results
reported by Hansen and Loeschke (1996) polymorphism
was found neither in Danish brown trout (
Salmo trutta
L.)
strains by digestion with 18 restriction enzymes,
nor in rainbow trout (
Onchorhyncuhus mykiss
) using
12 endonucleases (Sajedi et al., 2003). Thus mtDNA
is not appropriate for reconstruction of relationships
among populations, subspecies that diverged > 10
million years ago (Peacock et al
.
, 2001). Interestingly,
the dendrograms generated by RAPD and RFLP
resulted showed clustering of
C.c. communis
and
C.c.
specularis
in one major group and
C. carassius
into
separate group. These results suggested that
C.C.
communis
and
C.C. specularis
have a common
ancestor and are genetically closer to each other than
to
C. carassius.
These results above clarified the
utility of RAPD (in terms of degree of polymorphism,
precision of inter- and intra-specific genetic variations)
in characterizing the fish species. However,
PCR-RFLP of Cyt b
gene has been found an important
marker for inter- specific study of cyprinids. The
RAPD assay has been used to construct Phylogenetic
trees for resolving taxonomic problems in many
organisms (Bardakci and Skibinski, 1994; Barman et
al., 2003).
PCR–RFLP of Cyt b cannot help resolve the
interspecific relations but Phylogenetic relations
among the species under consideration. Mitochondrial
DNA variation can resolve relationships of species
that have diverged as long as 8-10 million years
before present (Peacock et al., 2001). After about 8-10
million years, sequence divergence is too slow to
allow sufficient resolution of divergence times. Thus
mtDNA is not appropriate for reconstruction of
relationships among populations, subspecies and
species that diverged>10 million years ago (Peacock
et al., 2001). Further the use of more restriction
enzymes might offer alternative marker and could
make the analysis more explanatory. We also observed
intense extra restriction fragments that yield total Cyt
b
length greater than 680 bp. This is in conformity
with the patterns obtained for several restriction
enzymes by Wen et al (2005) and Durand et al (2002)
in their studies. The intensity of these bands suggests
that they occur at the same frequency as those bands
of the ‘standard’ repeats. The fact that the fragment
size is constant for each restriction enzyme used and
that such bands were repeatedly obtained in replicate
digestions indicates that they are not the result of
partial digestions. There may be possibility that the
extra bands observed are a consequence of interlocus
length heterogeneity due to low rates of concerted
evolution in cyprinid species. Out of the four
restriction enzymes MboI and MspI showed higher
Cyt b
variations than that by Hin6I
and
BsuRI. In our
study Mitochondrial DNA analysis has proven a
powerful tool for assessing intraspecific Phylogenetic
patterns in cyprinid species as also reported by
(Bernatchez et al., 1992; Avise, 1994). Data confirmed
occurrence of variability in the mitochondrial
Cyt b
is
an appropriate tool for studying intraspecific genetic
variability among Cyprinidae
species.
3 Material and Methods
3.1 Fish material
Twenty five specimens for each of the
Cyprinidae
species viz
Cyprinus carpio var. Communis
,
Cyprinus
carpio
var
. Specularis
and
Carassius carassius
collected from different water bodies of Kashmir,
India
were
analyzed for present study (Table 2 and
Figure 4). The fishes were identifies using the
taxonomies of Talwar and Jhingran (1991). Muscle
tissues samples were preserved in 95% ethanol and
subjected for DNA isolation. Voucher specimens
were fixed in 10% formalin and preserved in the
Museum of Faculty Fisheries Sher-e-Kashmir
University of Agricultural Sciences and Technology of
Kashmir, India.
Figure 4 Map showing collection sites for 75 specimens of two
Cyprinidae species.