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International Journal of Aquaculture, 2013, Vol.3, No.24, 138
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with BsuI varied from 170 bp to 308 bp in
C.c.
communis
, 170 bp to 308 bp in
C.c. specularis
and
296 bp to 493 bp in
C. carassius.
Hin6I showed the
varied size of restriction fragments in
C.c. specularis
and
C. carassius
varying in size from 105 bp to 392
bp and 204 bp to 523 bp respectively. However, no
restriction site of Hin6I was found in
C.c. communis.
Furthermore, MspI showed the varied size of
restriction fragments in
C.c.communis
and
C. carassius
varying in size from 178 bp to 317 bp and 178 bp to
317 bp respectively, no restriction site of MspI was
found in
C.c. specularis
.
Based on Shannon’s index (H) of combined fragment
size patterns averaged with four restriction enzymes
was 0.58 with values ranging from 0.23 to 0.29 which
attributes to differences among the populations shown
in Table 3. Further genetic differentiation for haplotype
diversity between and within populations of two
species were studies ARLEQUIN version 3.1 software
package (Tajima, 1993; Nei, 1987). The mean nucleotide
diversity of all the species was 1.0000±0.0113 and the
value of mean haplotype diversity was 0.0200. As
expected the value for nucleotide diversity were similar
(1.0000±0.0113) for all the species. It is evident that
the low number of nucleotide substitutions between
the haplotypes determined the low indices of genetic
diversity (1.0000±0.0113). Hierarchic analysis of
interspecies haplotype differences was performed by
one way ANOVA (Tajima, 1993) shown in Table 4.
Table 4 AMOVA design and results Hierarchical analysis of inter-population haplotype differences in two Cyprinidae species.
Source of variation
DF
Sum of squares
Variance components
Percentage of variation
Among population
2
316.667
6.33333Va
100.00
Within population
72
0.000
0.00000Vb
0.00
Total
74
316.667
6.33333
Note: Fst =1.00000; P-value=0.00000±0.00000
Further to estimate the pairwise genetic distance
between 75 individuals Jaccard’s similarity coefficient
was used. From cluster analysis UPGMA dendrogram
revealed that Crucian carp i.e. Cc-1 to Cc-25
distributed in III cluster displayed maximum
dissimilarity coefficient of 0.38 with other individuals,
while other two species i.e. Scale carp and Mirror carp
clearly distributed in two clusters I and II resp. shown
in Figure 2. The distribution of different individuals
revealed that there were 25 individuals in cluster I i.e.,
Sc-1 to Sc-25 in cluster II i.e., Mc-1 to Mc-25. The
PCR-RFLP results showed no polymorphism within
the species. However, the marker clearly helps in
diversifying the species.
In case of RAPD, among all the primers S-145, S-71,
S-159 and S-161 generated robust banding pattern and
was found suitable for the species characterization.
However, in RFLP MboI and BsuRI was best suited for
species discrimination. In contrast S-131 and Hin6I and
MspI were least informative for polymorphism. Cluster
analysis (UPMGA) was used to generate two
independent dendrograms for the two types of markers
(Figure 2). As expected in the dendrogram generated
by the RAPD and PCR-RFLP, the individuals were
grouped according to the species. The cluster I consists
of
C.c. communis
, whereas cluster II and III represents
C.c specularis
and
C. carassius
respectively in both
RAPD and RFLP. As expected the high value of cluster
information was found in case of RAPD and RFLP
helped in species discrimination. Further, the result
obtained by two markers showed that
C.c. communis
and C.c. specularis are
closely related with each other
than
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.
2 Discussion
PCR based molecular markers are the ideal means of
identifying genotypes and following inheritance of
economically important characters. Phylogenetic
studies specifically attempt to show relationships
based on reconstructing the evolutionary history of
groups or unique genomic lineages. Phylogenetic
analysis of related species can be undertaken in
various ways and with various DNA markers.
However for closely related species, “gene trees” do
not always reflect “species trees”, because of
intraspecific heterogeneity (Nei, 1987). Therefore, it is
important to compare many Phylogenetic trees, using