RAPD 40 RAPD primers that is 2 set of

RAPD and
ISSR analysis details

              In present study, initially 40
RAPD primers that is 2 set of Operon primer kits OPG and OPR (20 primer from
each kits) were used to detect genetic polymorphism of S. oblonga, S. fruticosa, S. chinensis and S. macrosperma. Out of the 40 RAPD primers, 10 primers i.e. OPG-02,
14, -16, -17, -18, -19 and OPR-02, -03, -07, -08 showed reproducible amplified
DNA polymorphism. All the chosen primers amplified fragments across the 21
samples, with the number of amplified fragments ranging from 4 to 12. Minimum number
of loci were seen in the primer OPG18 (4 bands) and maximum bands were observed
in primer OPG17-12 bands. From the ten primers, a total of 76 loci were
generated of which 70 were polymorphic, making polymorphism generated by RAPD
makers to be 92.11%. Multiplex ratio of RAPD analysis was calculated to be 7.6.
Cumulative resolving power of 10 RAPD primer was 54.67.

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              While in ISSR analysis 10 primers
produced 67 loci of which 61 bands were polymorphic, accounting for 91.04% of
polymorphism. Number of loci varied from minimum of four in primer ISSR 5 to
maximum of nine in ISSR 10. Multiplex ratio of ISSR analysis is calculated to
be 6.8. Cumulative resolving power of 10 ISSR primer was 58.48. The marker
index for RAPD and ISSR was 6.54 and 5.45 respectively.

              Observed number of alleles,
effective number of alleles, Nei’s genetic diversity, Shannon’s information
index, for 21 samples of Salacia
species analyzed using ten each of RAPD
and ISSR primer were found to be 1.9211, 1.4537, 0.2785, 0.4294 and 1.9104,
1.5108, 0.2988, 0.4509 respectively. Total genotype diversity among population (Ht) was estimated to be 0.2713 while within population
diversity (Hs) was estimated to be 0.1514 for RAPD and for ISSR Ht was 0.3055
and Hs was 0.1222. Mean coefficient of gene differentiation (Gst) value for
RAPD was 0.4418 and ISSR was 0.5999. Suggesting that 55.8% and 40.1 % of the
genetic diversity resided within the population as per RAPD and ISSR markers.
Estimates of gene flow in the population for RAPD and ISSR were 0.6318 and
0.3334 respectively. (Table 3).




Dendrogram and PCoA of RAPD and ISSR

In RAPD dendrogram, 21
samples of Salacia grouped into two clusters
(Cluster 1 and 2). Cluster 1 contained S.
chinensis SC1 to SC5 and cluster 2 was further divided into two
sub-clusters (sub-cluster 1 & 2). In cluster 2, sub-cluster 1 contained all
samples of S. macrosperma along with
two samples of S. fruticosa SF1 &
SF3 (Fig 1) and sub-cluster 2 contained three remaining samples of S. fruticosa along with S. oblonga samples. The cumulative total
variation of three principle components accounted for 65.68 % of variation.

Dendrogram of ISSR data
showed that the samples clearly grouped into four clusters (I, II III and IV)
of its respective species S. chinensis,
S. macrosperma, S. fruticosa, S. oblonga. For ISSR analysis cumulative
total variation of three principle components accounted for 74.05% of the
variation. The results of RAPD and ISSR PCoA analysis were comparable to the
cluster analysis (Fig 2).

ITS analysis

              For ITS analysis, 19 samples of
current study and an outgroup Pristimera
preussii belonging to sub-family Hippocrateoideae was used to construct
phylogenetic tree. Two samples SM1 and SM14 produced faint bands and could not
be sequenced. Sequence alignment of 20 samples resulted in overall sequence
length of 752 bp, of which 221 bp (29.38%) were conserved, 503 bp (66.88%) were
variable sites and 103 bp (13.69%) were parsimony informative sites. Three
major clades were observed from ML tree. Clade 1 contained all the samples of S. macrosperma along with samples of S. oblonga which were nested with-in the
clade. Clade 2 and 3 contained S.
chinensis and S. fruticosa
samples respectively (Fig 3).


Comparative analysis of population

              Values of observed number of
alleles, effective number of alleles, Nei’s genetic diversity, Shannon’s
information index of each population were compared to observed diversity and
degree of polymorphism with-in the population (Table 3 and 4). In comparison,
the RAPD values were marginally higher than the ISSR except in the S. fruticosa population. Significant
differences were observed in all the parameters. Highest percentage of
polymorphism and highest polymorphic loci were seen in S. fruticosa population in RAPD analysis. In RAPD, ISSR and ITS analysis
high degree of polymorphism was seen in S.
macrosperma and S. fruticosa population
followed by S. chinensis population. Although
only two samples are in S. oblonga
population, RAPD, ISSR and ITS analysis detected polymorphism of 15.79%,16.42%,23.36%
respectively. Also, in parameters such as Ht, Hs, Gst and Nm significant
differences in value were observed (Table 4). The level of polymorphism
revealed by RAPD was (41.45%±10%) which was higher than ISSR (33.58%±6.52%) and
ITS (25.50%±17.25%). The polymorphism of each population of S. chinensis, S. macrosperma, S. fruticosa and S. oblonga from RAPD was35.53%,
55.26%, 59.21%, 15.79%and ISSR was32.84%,
47.76%,37.31%,16.42% respectively.

              For comparative analysis of ITS
with the RAPD and ISSR, sequences data of ITS was analyzed in GenAlEx. Before
exporting the data, the outgroup sequence and sequence SF5 were removed. Only
polymorphic nucleotide positions were converted to numeric codes (A=1, C=2,
G=3, T=4, hypen/colon=5) and 137 sites showed the polymorphism which were used
for the further analysis. The polymorphism of each population of S. chinensis, S. fruticosa, S. macrosperma
and S. oblonga from ITS analysis was
6.57%,19.71%,24.82%,23.36% and overall polymorphism was 18.61%±4.16%. For ITS coefficient
of evolutionary differentiation was 0.797which indicated that 20.3% of the
genetic diversity resided within the population. Tajima’s D neutrality tests were
performed to check whether genus Salacia
populations followed a neutral model of evolution with constant population size
over time. The observed values of Tajima’s D neutrality tests were -1.089757
for S. macroperma and S. oblonga population, -1.105205 for S. fruticosa and -0.174749 for S. chinensis and-1. 181277 for all the
19 samples. After removing sample SF5 since it showed high divergence,
neutrality test was performed for 18 sample of Salacia which gave observed value of 0.606285.

AMOVA, which helps in
partitioning of the overall variations among groups and among populations
within the group were performed for RAPD, ISSR and ITS data matrices. From RAPD,
39% of molecular variance was found among population while, within the population
this value was found to be 61% indicating that there were more variations
within the population. While in ISSR, 55% molecular variance was found among
population and 45%within the population. For ITS sequence analysis 80%
variances was among the population and 20% variance was within population which
was similar to coefficient of evolutionary differentiation. (Table5).

Nei genetic pairwise
distance of Salacia species was found
to be > 0.5 for RAPD, ISSR and ITS sequence. But in ITS sequence analysis,
the pair-wise distance between the S. oblonga
and S. macrosperm was 0.061
suggesting that they are very closely related. In addition, the pair-wise
distance and identity of S. oblonga
and S. fruticosa was 0.915 and 0.088
indicating that they are highly dissimilar. (Table 6).

Statistical comparative analysis

              Mantel test was employed to
determine the coefficient of correlation between the genetic distance matrices
generated by RAPD and ISSR markers. The coefficient of correlation between RAPD
and ISSR marker was R2=0.3781, r=0.614 which is high. This value signifies
that there was considerable correlation between RAPD and ISSR genetic distances
matrices. Twenty-one samples grouped into two clusters in RAPD dendrogram
whereas in ISSR dendrogram four cluster were observed. Comparing RAPD
dendrogram with ISSR dendrograms we can notice that S. oblonga was an Operational Taxonomic Units (OTU). In all
analysis, results of cluster analysis were comparable to PCoA.

              Mantel test was also employed to
analyze the ‘goodness of fit’ for each marker system. This was done by
comparing cophenetic similarity matrices of genetic distance with cophenetic
similarity matrices with the Nei’s Genetic Distance for each marker technique.
It revealed values higher than 0.80 for all the markers used RAPD (r = 0.827,
P = 0.01), ISSR (r = 0.816, P = 0.01) thus confirming their authenticity and
very good fit of PCA clustering.