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Research Article
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An Identification in Fish of the Genus Puntius Hamilton
1822 (Cypriniformes:Cyprinidae) of Some Wetlands in Northeast Thailand
With the Use of Random Amplified Polymorphic DNA Technique |
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T. Champasri,
R. Rapley,
M. Duangjinda
and
A. Suksri
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ABSTRACT
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The experiment was carried out during the 2003 to
2006 at the Department of Fisheries, Khon Kaen University, Khon Kaen,
Thailand in collaboration with the Department of Biosciences, the University
of Hertfordshire, College Land, Hatfield, Herts, UK. Molecular RAPD technique
was used for the determinations of DNA patterns of the fish genus Puntius
Hamilton 1822. The fish samples of 1,500 individual fish were collected
from fifteen wetlands in Northeast Thailand and they were used for DNA
extraction. Before the experiment was carried out the fish samples were
morphologically identified and it was found that the collected fish consisted
of 9 species i.e., Puntius altus, P. aurotaeniatus, P. binotatus, P.
gonionotus, (e) P. leiacanthus, P. orphoides, P. partipentazona,
P. schwanenfeldi and P. wetmorei. Genomic DNAs were extracted
from 5 mg of muscle tissues (skeleton muscles) with the use of PUREGENETM
DNA Isolation Kit for Laboratory Use, Gentra Systems, USA. Eighty decamer
primers from four kits were subjected to a preliminary test. It was found
that only 10 decamer primers were most suited for this PCR amplification.
The results showed that genetic distant values being established among
and between pairs of the fishes of the 9 fish species ranged from 0.191
to 0.456 for a pair between Puntius gonionotus and Puntius altus
and a pair between Puntius schwanenfeldi and Puntius leiacanthus,
respectively. Similarity coefficient values within the 9 fish species
ranged from 0.109 to 0.231. The results on a Dendrogram of clusters showed
that there were 5 minor groups of the 9 fish species but the 9 species
could not be split or shifted into other genera of the fish due to small
differences found within the values of similarity coefficients.
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How
to cite this article:
T. Champasri, R. Rapley, M. Duangjinda and A. Suksri, 2008. An Identification in Fish of the Genus Puntius Hamilton
1822 (Cypriniformes:Cyprinidae) of Some Wetlands in Northeast Thailand
With the Use of Random Amplified Polymorphic DNA Technique. Pakistan Journal of Biological Sciences, 11: 525-531. DOI: 10.3923/pjbs.2008.525.531 URL: https://scialert.net/abstract/?doi=pjbs.2008.525.531
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INTRODUCTION
With the work of Champasri et al. (2007) on
fish of the genus Puntius Hamilton 1822 where the fish were morphologically
identified and the results revealed that the fish of this genus could
not be split or shifted into other genera as previously stated by Rainboth
(1996a, b) where he did give out another three genera for the fish of
the genus Puntius Hamilton 1822. In order to justify the results
of the work being carried out by Rainboth (1996a, b) and Champasri et
al. (2007), it may be of tangible value to identify the fish of this
genus again with the use of a molecular technique (Random Amplified Polymorphic
DNA Technique or RAPD) where DNAs of the fish are used in the laboratory
in order to search for their identities in terms of similarities where
the results could provide adequate information if the results found with
the use of molecular technique could confirm the results being carried
out by morphological technique as reported by Rainboth (1996a, b) and
Champasri et al. (2007). Thus there is an urgent need to carry
out more works on this particular respect. Long before RAPD technique
was used, fish taxonomists have morphologically classified the various
types of fish into their respective genera, families and species, i.e.
they used, more or less, morphological characteristics of fishes as tools
to identify fishes for their systematic identification, hence such information
with respect to fish taxonomic identifications were accumulated.
For the past 30 years, it has been advocated that molecular
technologies have contributed largely on a large number of DNA-based markers
capable of revealing genetic variation in many species (Isabel et al.,
1999). It has been advocated that RAPD-PCR technique is a simple and straightforward
method where the work based on amplification of discrete regions of genome
by using arbitrary primers (Williams et al., 1990). Several authors
have used RAPD markers to evaluate genetic variability and structure of
a variety of species of Neotropical fish in the rivers of South America
e.g., Almeida et al. (2001), Wasco and Galetti (2002), Leuzzi et
al. (2004) and Matoso et al. (2004). Molecular markers could
also be applied for use in identifying different types of populations
of fish such as species, hybrid identification, phylogeny and many others
(Almeida and Sodre, 2002). Other workers have also used Random Amplified
Polymorphic DNA (RAPD) technique for their investigations such as Welsh
and McClelland (1990), Williams et al. (1990), Hassanien et
al. (2004) and Toth et al. (2005). Therefore, it may be of
important value to investigate further if the results found with the use
of RAPD technique could profoundly confirm the results being carried out
with the use of morphological technique of Champasri et al. (2007)
where they stated that the fish of the genus Puntius Hamilton 1822
could not be classified into other different genera as stated by Rainboth
(1996a, b). MATERIALS AND METHODS The
experiment was carried out at the Department of Fisheries, Faculty of
Agriculture, Khon Kaen University, Khon Kaen, Thailand in collaboration
with the Department of Biosciences, University of Hertfordshire, UK during
the 2003 to 2006. The fish samples were collected from fifteen different
wetlands in Northeast Thailand. The fifteen locations include provinces
of (1) Kalasin, (2) Yasothon, (3) Udon Thani, (4) Chaiya Phume, (5) Sisaket,
(6), Mahasarakham, (7) Sakon Nakhon, (8) Roi-Et, (9) Nong Khai, (10) Nakhon
Phanom, (11) Mukdaharn, (12) Nakhon Ratchasima, (13) Khon Kaen, (14) Nong
Bua Lampoo and (15) Loei. The individual fish samples were collected with
the use of trawl, gillnets, seine nets, hand-line and bamboo trap. The
fish samples were morphologically identified and they consisted of nine
species i.e., P. altus, P. aurotaeniatus, P. binotatus, P. gonionotus,
P. leiacanthus, P. orphoides, P. partipentazona, P. schwanenfeldi
and P. wetmorei. The collected fish samples were kept in liquid
nitrogen containers with the use of the method of Champasri et al.
(2007) and then the fish samples were used for DNA extraction. The laboratory
works on DNA analysis were carried out at the Department of Animal Science,
Faculty of Agriculture, Khon Kaen University, Khon Kaen, 40002 Thailand.
Genomic DNAs were extracted from 5 mg of muscle tissues (skeleton muscles)
with the use of the method of PUREGENETM DNA Isolation Kit
for Laboratory Use, Gentra systems, USA (Gentra Systems Inc., 2000). DNAs
were quantified using a spectrophotometer and then stored in a deep freezer
at -20°C.
Polymerase Chain Reaction (PCR) amplification and gel electrophoresis:
Initially, 80 decamer primers from four kits i.e., 20 decamer primers
from each kit of A, C, E and Y of random sequences (Operon Technologies,
Inc., Alameda, USA) were used. The work started from the screening of
samples i.e., the searching for suitable primers where two randomly chosen
fish samples from each population were subjected to PCR amplification.
This was carried out in order to attain suitable polymorphic bands of
DNAs for the nine fish species being used for this investigation (Bardakci
and Skibinski, 1994; Barman et al., 2003; Liu and Cordes, 2004).
It was found that only 10 decamer primers were suited most, thus the ten
primers were used for this work. They include OPA-03, OPA-04, OPA-13,
OPC-02, OPC-05, OPC-09, OPE-03, OPY-01, OPY-02 and OPY-11. RAPD amplification
reactions were maintained followed the method of Williams et al.
(1993). The RAPD profiles were generated from total genomic DNAs where
PCR protocol for RAPD analysis was mixed with 1.0 unit of Ampli Taq DNA
polymerase, 2.5 µL of 10x Taq buffer, 2.0 mM each of dNTPs (dATP, dCTP,
dGTP and dGTP), 100 pmol (0.1 mM) of primer, 2.0 mM of MgCl2
and 50 ng (1 µL) of genomic DNA to form a final volume of 25 µL, i.e.,
the samples were added with a high purification of distilled water (three
times in distilling distilled water to attain the utmost water purification).
PCR amplification was carried out in a Thermal Cycler machine (Corbett
Research, Netherlands). Firstly preheated at 94°C for 2 min (step 1, denature)
followed by 43 cycles of 1 min denaturation at 94°C and another 1 min
at 36°C and then elongation or extension for 2 min at 72°C (step 2, annealing)
and finally 7 min at 72°C was carried out to allow a complete extension
of all amplified fragments (step 3, additional extension). For every PCR
cycle, one PCR tube of negative control must not bear any DNA content
(not being contaminated). This was carried out to avoid any possible contamination
of other DNAs in each set of the PCR amplification.
Approximately 10 µL of amplification product plus 3 µL of
loading dye were resolved in 2.0% agarose gel and then the sample was
assayed by electrophoresis in 1x TBE buffer (89 mM Tris-HCl, 89 mM boric
acid and 2.5 mM EDTA) with pH value of 8.0 at 100 volt for 40 min. Finally,
the gels were stained with ehtidium bromide (0.5 µg mL-1) for
10 min and then viewed under UV light gel-document (SYNGENE, The United
Kingdom) where the GeneSnap software (installed within the UV light gel-document)
was used to identify photographs of gels and then the software gave out
scores of DNA bands. DNA bands were recorded (the score of 1 was used
for the presence of DNA band and the score of 0 was used for the absence)
and then transformed the reading scores of DNA bands from GeneSnap to
XLS file and finally the file was subjected to NTSYS-pc 2.10 programme
(Rohlf, 2000) for further calculations to attain similarity index values
and genetic distances. At the final stage of calculations, both similarity
index values and genetic distances were calculated further through a SAS
Computer Programme (Littell et al., 2002) for multivariate analysis
of variance (MANOVA) where the attained statistical values were used for
discriminatory effectiveness of the analysis. RESULTS
The nine collected species of fish of the genus Puntius Hamilton
1822: The collected species of fish from 15 different sites (provinces)
in Northeast Thailand consisted of 9 nominal species. The 9 nominal species
were identified into their respective species along with names of the
authors of the published works and their respective numbers of the harvested
locations
(Fig. 1-9), i.e., 1 = Puntius altus, 2 = P.
aurotaeniatus, 3 = P. binotatus, 4 = P. gonionotus,
5 = P. leiacanthus
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Fig. 1-9: |
Photographs of the nine species
of fish, genus Puntius Hamilton 1822 with their respective
numbers of the locations of the 15 wetlands (provinces) in Northeast
Thailand: 1 = Puntius altus (Gunther, 1868), harvested
from locations 3, 9, 10, 11 and 13; 2 = Puntius aurotaeniatus
(Tirant, 1885) harvested from locations 1, 3, 6, 11 and
15; 3 = Puntius binotatus (Valenciennes, 1842) harvested
from locations 3, 7, 9, 11 and 14; 4 = Puntius gonionotus
(Bleeker, 1850) harvested from locations 1, 3, 6, 9 and
13; 5 = Puntius leiacanthus (Bleeker, 1860) harvested
from locations 2, 4, 5, 7 and 9; 6 = Puntius orphoides
(Valenciennes, 1842) harvested from locations 6, 8, 12, 14 and
15; 7 = Puntius partipentazona. (Fowler, 1934) harvested
from locations 1, 3, 4, 8 and 13; 8 = Puntius schwanenfeldi
(Bleeker, 1853) harvested from locations 3, 9, 10, 11 and 13;
9 = Puntius wetmorei (Smith, 1931) harvested from locations
1, 9, 10, 11 and 13. Numbers of locations: 1 = Kalasin; 2 =
Yasothorn; 3 = Udonthani, 4 = Chaiyaphum, 5 = Srisaket, 6 =
Mahasarakham, 7 = Sakonnakhon, 8 = Roi-Et, 9 = Nong Khai, 10
= Nakhonphanom, 11 = Mukdahan, 12 = Nakhonratchasima, 13 = Khon
Kaen, 14 = Nongbualumphu and 15 = Loei |
Table 1: |
Genetic distances among the
nine fish species of the genus Puntius Hamilton 1822
being harvested from fifteen wetlands in Northeast Thailand
during the years 2003 to 2006 |
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Where 1 = Puntius wetmorei, 2 =
P. altus, 3 = P. aurotaeniatus, 4 = P. binotatus,
5 = P. gonionotus, 6 = P. leiacanthus, 7 = P.
orphoides, 8 = P. partipentazona and 9 = P. schwanenfeldi
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6 = P. orphoides, 7 = P. partipentazona, 8
= P. schwanenfeldi and 9 = P. wetmorei. These nine nominal
fish species possess a similar body structure with respect to their morphological
identifications where the identification followed the method of Smith
(1945), Rainboth (1996a, b) and Champasri et al. (2007) although
the sizes of the fish were not of the same sizes.
Genetic distances of the nine fish species:With the results on
genetic distances (Nei and Li, 1979), the nine fish species possessed
genetic distant values ranged from 0.191 to 0.456 for two pairs of fishes
i.e., between Puntius gonionotus and Puntius altus and between
Puntius schwanenfeldi and Puntius leiacanthus, respectively
(Table 1). The differences among the nine species were
relatively small and not statistically significant.
Distribution of similarity coefficients of the nine fish species:Amongst
the nine fish species of the genus Puntius Hamilton 1822, when
displayed separately into species, it was found that the highest value
of similarity coefficient was found with Puntius orphoides followed
by Puntius aurotaeniatus, Puntius wetmorei, Puntius gonionotus, Puntius
schwanenfeldi, Puntius binotatus, Puntius leiacanthus, Puntius altus
and Puntius partipentazona with values of 0.231, 0.214, 0.188,
0.175, 0.170, 0.152, 0.143, 0.132 and 0.109, respectively (Fig.
10). Similarity coefficients of individual fish species were relatively
small and not statistically significant.
Dendrogram structure of clusters of the nine fish species:The
results of UPGMA (un-weighted pair-group method) revealed that there were
five different groups of clusters of the nine fish species i.e., group
1 consisted of Puntius gonionotus, group 2 include Puntius leiacanthus
and Puntius orphoides, group 3 include Puntius partipentazona
and Puntius schwanenfeldi, group 4 consisted of Puntius altus
and Puntius binotatus and finally group 5 include Puntius wetmorei
and Puntius aurotaeniatus. Nonetheless, similarity coefficient
values
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Fig. 10: |
The distribution of similarity
coefficients of the nine fish species of the genus Puntius
Hamilton 1822 being harvested from fifteen wetlands (provinces)
in Northeast Thailand. Where 1 = Puntius wetmorei, 2
= P. altus, 3 = P. aurotaeniatus, 4 = P. binotatus,
5 = P. gonionotus, 6 = P. leiacanthus, 7 = P.
orphoides, 8 = P. partipentazona and 9 = P. schwanenfeldi
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Fig. 11: |
A dendrogram structure showing
five fish clusters and correlation coefficients of the nine
fish species being harvested from fifteen wetlands (provinces)
in Northeast Thailand. Where 1 = Puntius wetmorei, 2
= P. altus, 3 = P. aurotaeniatus, 4 = P. binotatus,
5 = P. gonionotus, 6 = P. leiacanthus, 7 = P.
orphoides, 8 = P. partipentazona and 9 = P. schwanenfeldi
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attained for all of the nine fish species were relatively
small and not statistically significant where the values ranged from 0.109
to 0.231 for Puntius partipentazona and Puntius orphoides,
respectively (Fig. 11). DISCUSSION
It has been advocated that in Thailand the fish of the genus
Puntius Hamilton 1822 comprises of 36 nominal species (Smith, 1945).
However, with the results of Champasri et al. (2007) they did carry
out research on this genus of fish of fifteen wetlands in Northeast Thailand
and reported that only nine fish species were available and caught by
fishing tools of different kinds. It could have been possible that some
other fish species, apart from the nine species, may be found in other
natural inhabitation such as rivers, swamps and other sources of reservoirs,
thus only nine species were collected. Some wetlands in Northeast Thailand
have its natural connection with some rivers e.g., Ubon Ratana Dam at
Khon Kaen Province has its initial connection with Chi River then Mun
River and Khong River eventually (Suksri, 1999) where some other fish
species of the same genus may be found in different years and perhaps
they may be found in other omitted locations since there are approximately
549 locations of its kind in Northeast Thailand such as Kang Lawa at Manjakiri
District, Khon Kaen; Bueng Khong Long at Roi-Et province and Bueng Tam
at Mukdaharn province and many others (Anonymous, 2005). The published
results of Champasri et al. (2007) on morphological identification
revealed that the nine fish species being harvested from fifteen wetlands
in Northeast Thailand did not provide any information where the fish species
could be classified into other different genera as stated by Rainboth
(1996a, b). Thus there is an urgent need to investigate the nine fish
species again with the use of molecular technique hence this work was
carried out. The actual investigation being carried out in laboratory
with the use of molecular identification where DNA bands of the fish samples
were used in the various processes where the results reflected, more or
less, high accuracy and the technique has been recognized and accepted
by a number of authors such as Williams et al. (1993), Isabel et
al. (1999), Almeida et al. (2001), Almeida and Sodre (2002),
Wasco and Galetti (2002), Leuzzi et al. (2004), Matoso et al.
(2004) and Toth et al. (2005).
With the present work, the results on the determinations
of genetic distances revealed that genetic values being established among
the nine fish species and between pair of the fishes ranged from 0.191
to 0.456 for a pair between Puntius gonionotus and Puntius altus
and a pair between Puntius schwanenfeldi and Puntius leiacanthus,
respectively. The differences found were relatively small and not statistically
significant. The results indicated that the nine fish species did not
differ largely among themselves hence the fish could not be categorized
into other genera as reported by Rainboth (1996a, b). Similarly, the results
derived from similarity coefficient values of the fish where the work
was calculated through NTSYS-pc 2.10 Computer Programme (Rohlf, 2000),
it was found that the attained values ranged from 0.109 to 0.231 for the
nine fish species. This narrow range of similarity coefficient values
indicated that the nine tested fish species possess a similar DNA band
pattern in spite of some minor differences on physical appearances of
the fish. With the results found with a Dendrogram structure of clusters,
although the nine fish species gave small differences in values of similarity
coefficients, the nine fish species could still be identified into five
minor groups i.e., group 1 consists of Puntius gonionotus alone,
group 2 includes Puntius leiacanthus and Puntius orphoides,
group 3 includes Puntius partipentazona and Puntius schwanenfeldi,
group 4 consists of Puntius altus and Puntius binotatus
and finally group 5 includes Puntius wetmorei and Puntius aurotaeniatus.
The results indicated that even the Dendrogram structure of clusters were
produced and able to separate the fish into five different groups but
the fish of the nine species could not be split or shifted into other
genera due to its small values of similarity coefficients (0.111-0.231).
Salini et al. (2004) stated that correlation coefficient values
being calculated for the search in differentiating fish not exceeded a
value of 0.78 could be considered to be insignificant. CONCLUSIONS
The Random Amplified Polymorphic DNA (RAPD) Fingerprints
Technique was used in identifying the fish of genus Puntius Hamilton
1822 being collected from different wetlands in Northeast Thailand. The
results revealed that 1,500 individual fish samples were harvested and
collected from the fifteen wetlands (provinces), i.e., (1) Kalasin, (2)
Yasothon, (3) Udon Thani, (4) Chaiya Phume, (5) Sisaket, (6), Mahasarakham,
(7) Sakon Nakhon, (8) Roi-Et, (9) Nong Kai, (10) Nakhon Phanom, (11) Mukdaharn,
(12) Nakhon Ratchasima, (13) Khon Kaen, (14) Nong Bua Lampoo and (15)
Loei. All of the individual fish samples were identified into its respective
species and it was found, after the use of the various complicated calculations
on the results derived from morphological identifications, the collected
fish samples were categorized into nine fish species only. The results
with respect to RAPD analysis showed that the nine fish species should
only belong to the same genus of Puntius Hamilton 1822. Thus the
use of the RAPD molecular technique was able to produce the ultimate results
with high effectiveness in identifying the fish species, particularly
the fish of the genus Puntius Hamilton 1822. Therefore, the nine
fish species could not be split or shifted into other genera as reported
by Rainboth (1996a, b). ACKNOWLEDGMENTS
The authors wish to thank the Academic Board of the Faculty
of Agriculture, Khon Kaen University and Academic Board of the Kasetsart
University, Bangkok, Thailand for their financial assistance. Thanks are
also due to Miss Tassanee Sunonchai of the Department of Fisheries, Ministry
of Agriculture and Cooperatives, Bangkok, Thailand for her kind assistance
in searching for fish locations and also postgraduate students of the
Department of Fisheries, Faculty of Agriculture, Khon Kaen University
for their kind assistance on laboratory works carried out and the Postgraduate
Board of the University of Hertfordshire, College Lane, Hatfield, Herts,
UK for permission to carry out research work in Thailand and lastly Ms
Chamaiporn (wife of the first author) for her kind assistance in searching
for more research publications.
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REFERENCES |
1: Almeida, F.S., M.H.P. Fungaro and L.M.K. Sodre, 2001. RAPD and isoenzyme analysis of genetic variability in three allied species of catfish (Siluriformes: Pimelodidae) from the Tibagi river. Brazil J. Zool., 253: 113-120. Direct Link |
2: Almeida, F.S. and L.M.K. Sodre, 2002. Comparative study by RAPD analysis of six species of the Pimelodidae family (Osteichthyes: Siluriformes) from the Tibagi River, state of parana. Brazil. Maringa., 24: 513-517. Direct Link |
3: Anonymous, 2005. Wetlands in Northeast Thailand. Office of Environmental Policy and Planning. Ministry of Agriculture and Cooperatives, Bangkok, Thailand.
4: Bardakci, F. and D.O. Skibinski, 1994. Application of the RAPD technique in Tilapia fish: Species and subspecies identification. Heredity, 73: 117-123. CrossRef | Direct Link |
5: Barman, H.K., A. Barat, B.M. Yadav, S. Banerjee, P.K. Meher, P.V.G. Krishna Reddy and R.K. Jana, 2003. Genetic variation between four species of Indian major carps as revealed by random amplified polymorphic DNA assay. Aquaculture, 217: 115-123. CrossRef | Direct Link |
6: Champasri, T., R. Rapley, M. Duangjinda and A. Suksri, 2007. A morphological identification in fish of the genus Puntius hamilton 1822 (Cypriniformes: Cyprinidae) of some wetlands in Northeast Thailand. Pak. J. Biol. Sci., 10: 4383-4390. CrossRef | PubMed | Direct Link |
7: Hassanien, H.A., M. Elnady, A. Obeida and H. Itriby, 2004. Genetic diversity of Nile tilapia populations revealed by randomly amplified polymorphic DNA (RAPD). Aquacult. Res., 35: 587-593. CrossRef | Direct Link |
8: Isabel, N., J. Beaulieu, P. Theriault and J. Bousquet, 1999. Direct evidence for biased gene diversity estimates from dominant random amplified polymorphic DNA (RAPD) fingerprints. Mol. Ecol., 8: 477-483. CrossRef | Direct Link |
9: Leuzzi, M.S.P., F.S. Almeida, M.L. Orsi and L.M.K. Sodre, 2004. Analysis by RAPD of the genetic structure of Astyanax altiparanae (Pisces, Characiformes) in reservoirs on the paranapanema river, Brazil. Genet. Mol. Biol., 27: 355-362. CrossRef | Direct Link |
10: Littell, R.C., W.W. Stroup and R.J. Freund, 2002. SAS for Linear Models. 4th Edn., SAS Institute Inc., Cary, NC.
11: Liu, Z.J. and F.J. Cordes, 2004. DNA marker technologies and their applications in aquaculture genetics. Aquaculture, 238: 1-37. CrossRef | Direct Link |
12: Matoso, D.A., R.F. Artoni and P.M. Galetti Jr, 2004. Genetic diversity of the small Characid fish Astyanax sp. and its significance for conservation. Hydrobiologia, 527: 223-225. CrossRef | Direct Link |
13: Nei, M. and W.H. Li, 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. USA., 76: 5269-5273. PubMed | Direct Link |
14: Rainboth, W.J., 1996. Fishes of the Cambodian Mekong. Food and Agriculture Organization of the United Nations, Rome.
15: Rainboth, W.J., 1996. The Taxonomy, Systematics and Zoogeography of Hypsibarbus. A New Genus of Large Barbs (Pesces: Cyprinidae) from The Rivers of Southeastern Asia. University of California Press, Berkeley, USA.
16: Rohlf, F.J., 2000. Numerical taxonomy and multivariate analysis system. NTSYS-pc. Department of Ecology and Evolution. State University of New York, New York.
17: Salini, J.P., D.A. Milton, J.J. Rahman and M.G. Hussain, 2004. Allozyme and morphological variation throughout the geographic range of the tropical shad, hilsa Tenualosa ilisha. Fish. Res., 66: 53-69. CrossRef | Direct Link |
18: Smith, H.M., 1945. The Fresh-water fishes of Siam or Thailand United States Government Printing Office, Washington.
19: Suksri, A., 1999. Some Agronomic and Physiological Aspects in Growing Crops in Northeast Thailand. 1st Edn., Khon Kaen University Press, Khon Kaen, Thailand, Pages: 212.
20: Toth, B., E. Varkony, A. Hidas, E.E. Meleg and L. Varadi, 2005. Genetic analysis of offspring from intra- and interspecific crosses of Carassius auratus gibelio by chromosome and RAPD analysis. J. Fish Biol., 66: 784-797. CrossRef | Direct Link |
21: Wasko, A.P. and P.M. Galetti Jr., 2002. RAPD analysis in the Neotropical fish Brycon lundii: Genetic diversity and its implications for the conservation of the species. Hydrobiologia, 474: 131-137. CrossRef | Direct Link |
22: Welsh, J. and M. McClelland, 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res., 18: 7213-7218. CrossRef | PubMed | Direct Link |
23: Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski and S.V. Tingey, 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res., 18: 6531-6535. CrossRef | PubMed | Direct Link |
24: Williams, J.G.K., M.K. Hanafey, J.A. Rafalski and S.V. Tingey, 1993. Genetic analysis using random amplified polymorphic DNA markers. Methods Enzymol., 218: 704-740. Direct Link |
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