Page 12 - IJA-Vol.3-No.19
Basic HTML Version
International Journal of Aquaculture, 2013, Vol.3, No.19, 105
-
114
112
October –December 2012. The collected shrimp were
kept in icebox, brought to lab, preserve in -20
℃
still
DNA isolation.
Figure 7 Collected site of penaeid population in South east
coast of India
3.2
DNA Isolation
Total genomic DNA was isolated from muscle tissue
by SDS–phenol/chloroform method described in
Williams et al
.
(1990)
with slight modifications.
Briefly, shrimp muscle (200 mg) was cut into small
pieces, crushed using a sterile porcelain mortar and
pestle with 1 ml of chilled TEN buffer (50 mM
Tris–HCl, pH 8.0, 50 mM EDTA and 100 mM NaCl),
and transferred to 2 ml Eppendorf tube. Proteinase K
8
ul (300 mg/mL), sucrose 20ul (2%), and 20ul
sodium
dodecyl
sulfate
SDS (2%) were added to the tube.
After overnight incubation at 60
℃
,
the lysate was
extracted once with phenol and twice with
chloroform/isoamyl alcohol. DNA was precipitated
with isopropanol, washed once with 70% ethyl
alcohol, and suspended in TE (Tris EDTA, pH 8.0)
buffer. DNA quality and quantity were determined by
Agarose gel electrophoresis and Biophotometer plus
(
Eppendorf, Germany).
3.3
RAPD–PCR Amplification and Data Analysis
Fifteen primers were used for RAPD analysis. DNA
amplification reactions were performed in 200 umol/L
each dNTP, 2 mmol/L MgCl
2
, 19
standard Taq
polymerase buffer, 0.2 umol/L random primer, 40 ng
genomic DNA and 0.75 U Taq polymerase in a final
volume of 25 µL. PCR conditions included initial
denaturation at 94
℃
for 5 min, followed by 45 cycles
of denaturation at 94
℃
for 30 seconds, annealing at
35
℃
for 1 min, extension at 72
℃
for 2 min and final
extension at 72
℃
for 10 min. The amplified DNA
was separated by electrophoresis through 2% agarose
gel containing Ethidium bromide in 1x TBE buffer at
a constant 80 V. To maintain consistency, only the
repeatable major bands ranging from 10000 to 1000
bp were scored. Molecular weights of amplified bands
were estimated by comparing with known molecular
weight marker (1Kbp DNA ladder, Bangalore Genie,
India). DNA profiles generated for all samples were
compared in a pairwise manner. RAPD bands were
recorded on spread sheets as binary matrix marking
alleles absent (0) and present (1). The distance matrix
between species (Jaccard, 1908) calculated and
subsequently the data used to construct a dendrogram
using the (UPGMA) algorithm of Rohlf (1993).
Acknowledgement
The authors express their special thanks to the Department of Science and
Technology, New Delhi, India, for financial assistance from DST–PURSE.
References
Aubert H., and Lightner D. V., 2000, Identification of genetic populations of
the pacific blue shrimp
Penaeus stylirostris
of the Gulf of California,
Mexico. Mar. Biol., 137: 875-885,
Allendorf F. W., and Ryman N., 1987, Genetic management of hatchery
stocks, In: Ryman N, Utter F (eds) Population genetics and fishery
management, University of Washington Press, Seattle, pp.141-159
Benzie J. A. H., Ballment E., and Frusher S., 1993, Genetic structure of
P.
monodon
in Austrialia: preliminary data form allozymes and mt DNA,
Aquaculture, 111: 89-93,
Benzie J. A. H., 2000, Population genetic structure in Penaeid prawn,
Aquacult. Res., 31: 95-119,
Benzie J. A. H., Ballment E., Forbes A. T., Demetriades N. T, Sugama K.
and Haryanti, Moria S., 2002, Mitochondrial DNA variation in
Indo-Pacific populations of the giant tiger prawn,
Penaeus monodon
,
Mol. Ecol., 11: 2553–2569,
.
2002.01638.
x
Brooker, A. L., Benzie J. A. H., Blair D., and Versini J. J, 2000, Population
structure of the black tiger shrimp
Penaeus monodon
in Australian
water, determined using microsatellite marker, Mar. Biotechnol.,
136(1): 149-157
Caetano-Anolles G., and Bassam B. J., 1993, DNA amplification
fingerprinting using arbitrary oligonucleotide primers, Appl. Biochem.
Biotechnol., 42: 189-200,
Chistiakov D. A., Hellemans B., and Volckaert F. A. M., 2006,
Microsatellites and their genomic distribution, evolution, function, and
applications: a review with special reference to fish genetics,
Aquaculture, 255: 1-29,
Dumas S., and Ramos R. C., 1999, Triploidy induction in the Pacific white
shrimp
Litopenaeus vannamei
(
Boone), Aquac. Res., 30(8): 621-624,
Dall W., 2007, Recent molecular research on
Penaeus sensu
lato,
J. Crust.
Biol., 27: 380-382,
De la Rosa-Velez J, Escobar R., Correa F., Maqueda M., and Torre J., 2000,
Genetic structure of two commercial Penaeids (
Penaeus californeinsis
and
P. styliristris
)
from the Gulf of California, as revealed by allozyme
variation, Fish. Bull., 98(4): 674-683
Duda Jr T. F., and Palumbi S. R., 1999, Population structure of the black
