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International Journal of Marine Science 2015, Vol.5, No.5: 1-8
http://ijms.biopublisher.ca
3
Function Analysis (DFA) was performed to determine
the most reliable morphological characters that are
important in distinguishing two species. Wilks' lamda
was used to test the significance of the discriminant
function as a whole. Canonical scores derived from
DFA analysis were plotted in two dimensional spaces
for visual detection of the separation of two
populations. Analysis was performed using MINITAB
V. 14 (2004) and SPSS V. 20 (IBM corp. 2011).
Number of individuals analyzed from each population
for each gene region is given in the Table 1. For genetic
analyses individuals from each population were
selected randomly. Total DNA was extracted from
small muscle sample using DNEasy Extraction Kit
(QIAGEN) following manufacturer's protocols. Four
mtDNA gene regions and two nucDNA gene regions
were partially amplified using primers published in the
past studies (Table 2). Initially, amplification of PCR
reactions for all six regions were conducted in 25 µL
reaction mixture contained 1xTaq polymerase buffer,
1.5 mM MgCl
2
, 0.4 mM each dNTP, 0.2 µm of each
primer, 100 ng of DNA template and 0.5 U of Taq DNA
polymerase. When optimize the conditions, less primer
amounts were added (0.15 µm) to 18S and H3 nuclear
gene regions and additional MgCl
2
was (final
concentration 2 mM) added to amplify the control gene
region. PCR conditions of the initial denaturation and
final extension stages are common for all six regions
which were used as 2 min at 95
℃
and 72
℃
for 7 min.
respectively. PCR cycle conditions optimized for each
gene region are given in the Table 2. PCR products
were purified using PrepEaseTM PCR Purification
96
-
well Plate Kit (USB Corporation). Purified samples
were sequenced at the DNA sequencing laboratory,
Brigham Young University Utah, USA, using next
generation sequencing method. Sequences emerged
from this study were deposited in the Genbank
under the given accession numbers (Table 1). Nucleotide
divergence levels and phylogenetic relationships
among derived sequences were analyzed using MEGA
(V 5.1) program.
Table 1 Gene regions sequenced, number of individuals used, results reveled from the of the sequencing process and Genbank
Accession numbers
Gene region
Sample
number
per
population
Length of
the seq.
(bp)
Mean nucleotide
composition (%)
No. of haplotypes
produced from each
population
No of
shared
haplotypes
Genbank
Accession
numbers
A
C
G
T
Northern Southern
Mitochondrial
16S rRNA
08
550
33.27 13.09 20.36 33.27
01
01
01
KM486609
Mitochondrial
12S rRNA
08
407
35.63 14.25 18.18 31.94
01
01
01
KM486608
Mitochondrial
COI
10
690
34.97 17.87 19.86 27.29
02
02
01
KM528139
-
KM528141
Mitochondrial
Control
15
522
42.45
9.24 6.78 41.52
07
07
07
KM486612
-
KM486618
Nuclear
18S RNA
08
735
22.86 23.13 29.39 24.63
01
01
01
KM486610
Nuclear
H3
08
376
21.01 34.04 25.53 19.41
01
01
01
KM486611