Lactate dehydrogenase gene genotypization
identification in a fish farm on the river Neretva
for
species
Margeta, P., Bogut, I., Ivanković, M., Margeta, V.
Poljoprivreda/Agriculture
ISSN: 1848-8080 (Online)
ISSN: 1330-7142 (Print)
http://dx.doi.org/10.18047/poljo.21.1.sup.12
Poljoprivredni fakultet u Osijeku, Poljoprivredni institut Osijek
Faculty of Agriculture in Osijek, Agricultural Institute Osijek
ISSN 1330-7142
UDK: 639.3(497.6)
DOI: 10.18047/poljo.21.1.sup.12
LACTATE DEHYDROGENASE GENE GENOTYPIZATION
FOR SPECIES IDENTIFICATION IN A FISH FARM ON THE
RIVER NERETVA
Margeta, P.(1), Bogut, I.(1), Ivanković, M.(2), Margeta, V. (1)
Original scientific paper
SUMMARY
There are severale Salmonid species, found in the river Neretva basin, among
which S. trutta and S. obtusirostris. Also, natural hybrids such as S. obtusirostris
x S. trutta have been observed. In one fish farm on the river Neretva, S. trutta and
S. obtusirostris were decided to breed separately. Parental fishes were separated
phenotypicaly on the basis of the morphological signs. PCR-RFLP analysis of the
exon 3 to exon 4 part of the lactate dehydrogenase (LDH) C1* gene with restriction
endonuclease RsaI was employed to identify the presence of other species representatives or intercrosses in two groups of juvenille fishes. Using this method, we
were able to identify two S. trutta representatives in the S. obtusirostris group.
Key-words: Lactate dehydrogenase gene, PCR-RFLP, S. trutta, S. obtusirostris
INTRODUCTION
There are severale Salmonid species, found in
the river Neretva basin, among which S. trutta (brown
trout), Salmo marmoratus (marble trout), Salmo obtusirostris (softmouth trout) Salmo farioides and Salmo
dentex.
Salmo obtusirostris also known as the Adriatic
trout, Adriatic salmon, and softmouth trout, is a species of salmonid fish endemic to the rivers of Western
Balkans. It is found naturally in four drainages of the
Adriatic Sea, in Croatia, Bosnia and Herzegovina and
Montenegro: the Neretva-Vrljika system, the Jadro,
the Morača-Zeta system and the Krka river drainages.
Morphological different characteristics for different softmouth trout populations from different river-systems
resulted in the description of three putative subspecies:
Salmo obtusirostris oxyrhynchus from the River Neretva,
Bosnia and Herzegovina, Salmo obtusirostris salonitana
from the river Jadro, Croatia, and Salmo obtusirostris
krkensis from the River Krka, Croatia.
In the river Neretva basin, natural hybrids such
as S. obtusirostris x S. trutta have been observed and
reported (Vuković, 1982). The hybridization between
autochthonous species was confirmed also in an experiment performed in the fish farm located in the river
Buna, a tributary of the river Neretva (Kosorić and
Vuković, 1969). Introduction of non-native brown trout
Poljoprivreda 21:2015(1) Supplement, 56-58
has also been practised in the river Neretva and stocking
activities have never been well documented.
Lactate dehydrogenase (LDH) C1* gene containing
parts of exons 3 and 4 with intermediate intron has been
found, firstly in brown trout (Mc Meel et al, Ferguson,
2001), and after that also in S. obtusirostris (Snoj et al.,
2002) proven as an informative genetic marker.
A mutation in the intron 3 of the (LDH) C1* gene
(G/C substitution) was connected with the disruption
of the RsaI restriction enzyme recognition site, being
specific for S. obtusirostris (Razpet, 2004; Razpet, 2007).
The same mutation was found in Salmo obtusirostris salonitana and in Salmo obtusirostris oxyrhynchus
(Odak, 2004).
In one fish farm on the river Neretva, S. trutta and
S. obtusirostris were decided to breed separately.
Parental fishes were separated phenotypically on
the basis of the morphological signs. The aim of this
work was, based on the LDH genotype, to find intercrosses or representatives of the other species in two
groups of juvenille fishes. In the first group, there should
(1) Ph.D. Polonca Margeta ([email protected]), Ph.D. Ivan Bogut,
Assist. Prof. Margeta Vladimir – Josip Juraj Strossmayer University of
Osijek, Faculty of Agriculture in Osijek, Kralja Petra Svačića 1d, 31000
Osijek, Croatia, (2) Ph.D. Marko Ivanković - Federal Agromediterranean
Institute Mostar, Biskupa Čule 10, 88000 Mostar, Bosnia and Herzegovina
two groups of juvenille fishes. In the first group, there should be only representatives of S. trutta,
while in the second one only representatives of S. obtusirostris.
MATERIAL AND METHODS
57
P. Margeta et al.: LACTATE DEHYDROGENASE GENE GENOTYPIZATION FOR SPECIES ...
In the fish farm, juvenille fishes were raised in two groups. In the first group, there were juvenille
mM MgCl2, 0,2
be
only representatives
of S.intrutta,
while in juvenille
the secondfishesDNA,
each taken
dNTP, from
1 µM 21
fishes
of S. trutta, and
the second
of S.1,5obtusirostris.
FinmM
clipsof were
one
only
representatives
of
S.
obtusirostris.
of
each
primer
(Ldhxon4R
and
Ldhxon3F),
and
1U of
individuals from both groups (together 42 samples) and stored in 96% ethanol. In the lab, genomic
Taq
DNA
polymerase
(Fisher
scientific).
Amplification
DNA was isolated from the preserved fin clips using Bio Basic EZ-10 Spin Column Animal DNA
was performed in the thermal cycler (Eppendorf) as
MATERIAL
Mini-PrepsAND
Kit METHODS
(Bio Basic Canada Inc.), following the
supplier’s
described
beforeinstructions.
(Mc Meel et al., 2001).
PCRIn the
of fish
thefarm,
lactate
dehydrogenase
(LDH)
juvenille
fishes were raised
in twoC1* gene containing parts of exons 3 and 4 with
PCR products were cleaved by RsaI restriction
groups.
In the first
group,has
there
wereperformed
juvenille fishes
intermediate
intron
been
as of
described
by Mc Meel
al. (2001).
Twenty
µl PCR
endonuclease,
with a et
recognition
site gt/ac.
The ampliS.contained
trutta, and100
in the
juvenilleDNA,
fishes of
obtu- MgCl2, 0,2 mM of each dNTP, 1 µM of each primer
ngsecond
of genomic
1,5S. mM
fied part of the (LDH) C1* gene in S. trutta contained
sirostris.
Fin clips
taken fromand
21 individuals
from
(Ldhxon4R
andwere
Ldhxon3F),
1U of Taq
DNA two
polymerase
(Fisher scientific).
RsaI recognition
sites, leadingAmplification
to three bandswas
both groups (together 42 samples) and stored in 96%
after restriction
in the
lenght
of 2001).
74, 135 and 166 bp
performed
in
the
thermal
cycler
(Eppendorf)
as
described
before
(Mc
Meel
et
al.,
ethanol. In the lab, genomic DNA was isolated from the
(genotype NP). On
the aother
hand, PCR site
product
of S.The
PCR products
cleaved
by RsaI
endonuclease,
with
recognition
gt/ac.
preserved
fin clips were
using Bio
Basic EZ-10
Spin restriction
Column
obtusirostris
posessed
only
one
restriction
site
because
amplified
of theKit(LDH)
C1*
geneInc.),
in S.
Animal
DNA part
Mini-Preps
(Bio Basic
Canada
fol-trutta contained two RsaI recognition sites, leading to
of the disruption of the second recognition site due to a
lowing
supplier’s
three the
bands
after instructions.
restriction in the lenght of 74, 135
166 bp (genotype
NP). leading
On thetoother
hand,
G/Cand
transformation
(MP genotype),
only two
PCRPCR
product
of S. dehydrogenase
obtusirostris (LDH)
posessed
only onebands
restriction
site
because
of
the
disruption
of
of the lactate
C1* gene
in the length of 74 and 300 bp after restriction,the
containing
parts
of
exons
3
and
4
with
intermediate
second recognition site due to a G/C transformationrespectively
(MP genotype),
leading
to of
only
two bands
in the
(Figure 1).
Products
restriction
reactions
intron
hasofbeen
performed
described
by Mc Meel
et
were
checked
the 2% agarose
gel.
length
74 and
300 bpasafter
restriction,
respectively
(Figure
1).on
Products
of restriction
reactions were
al. (2001). Twenty µl PCR contained 100 ng of genomic
checked on the 2% agarose gel.
S.obtusirostris
TGTTACCACGACGATACGAGAGTTCGCCGTCACAGAGTAGTCTGACCGTGGGAGAACAAT 60
CLUSTAL 2.1 multiple sequence alignment
S.trutta
TGTTACCACGACGATACGAGAGTTCGCCGTCACAGAGTAGTCTGACCGTGGGAGAACAAT 60
************************************************************
S.obtusirostris
S.obtusirostris
S.trutta
S.trutta
S.obtusirostris
S.obtusirostris
S.trutta
S.trutta
S.obtusirostris
S.obtusirostris
S.trutta
S.trutta
S.obtusirostris
S.obtusirostris
S.trutta
S.trutta
S.obtusirostris
S.obtusirostris
S.trutta
S.trutta
S.obtusirostris
S.obtusirostris
S.trutta
S.trutta
S.obtusirostris
S.trutta
TGTTACCACGACGATACGAGAGTTCGCCGTCACAGAGTAGTCTGACCGTGGGAGAACAAT
CAATCAATGAGAGTACTGTGTATCATTTGTGTCTAGTATTTCTTCAGTAATTTGTCATAT
TGTTACCACGACGATACGAGAGTTCGCCGTCACAGAGTAGTCTGACCGTGGGAGAACAAT
CAATCAATGAGAGTACTGTGTATCATTTGT--CTAGTATTTCTTCAGTAATTTGTCATAT
************************************************************
****************************** ****************************
60
120
60
118
CAATCAATGAGAGTACTGTGTATCATTTGTGTCTAGTATTTCTTCAGTAATTTGTCATAT
CATTAATAGATCTAATGGCAGGACTATTACATGTCAAAGTAGGATTTCAGAAATTGCTTT
CAATCAATGAGAGTACTGTGTATCATTTGT--CTAGTATTTCTTCAGTAATTTGTCATAT
CATTAATAGATCTAATGGCAGGACTATTACATGTCAAAGTGGGATTTCAGAAATTGCTTT
****************************** ****************************
**************************************** *******************
120
180
118
178
GAGAAACTTCATTCATACATTTCCCTTTCACCCTTTTCCCCCCCATCTCCCTTTCATACA
CTTCCCCTCTCAGAGAGACTACTTCATTTAACACACAGACATTTGACATGCAGATGGTGT
GAGAAACTTCATTCATACATTTCCCTTTCACCC------CCCCCATCTCCCTTTCATACA
CTTCCCCTCTCAGAGAGAGTACTTCATTTAACACACAGACATTTGACATGCAGATGGTGT
*********************************
*********************
****************** *****************************************
240
300
232
292
CATTAATAGATCTAATGGCAGGACTATTACATGTCAAAGTAGGATTTCAGAAATTGCTTT
GAGAAACTTCATTCATACATTTCCCTTTCACCCTTTTCCCCCCCATCTCCCTTTCATACA
CATTAATAGATCTAATGGCAGGACTATTACATGTCAAAGTGGGATTTCAGAAATTGCTTT
GAGAAACTTCATTCATACATTTCCCTTTCACCC------CCCCCATCTCCCTTTCATACA
**************************************** *******************
*********************************
*********************
180
240
178
232
CTTCCCCTCTCAGAGAGACTACTTCATTTAACACACAGACATTTGACATGCAGATGGTGT
CCATCTTGGTTTTCTGGTTTCCTGGTGCCCAATTGCTACACACTCACCTTTGCTGGCGAC
CTTCCCCTCTCAGAGAGAGTACTTCATTTAACACACAGACATTTGACATGCAGATGGTGT
CCATCTTGGTTTTCTGGTTTCCTGGTGCCCAATTGCTACACACTCACCTTTGCTGGCGAC
****************** *****************************************
************************************************************
300
360
292
352
CCATCTTGGTTTTCTGGTTTCCTGGTGCCCAATTGCTACACACTCACCTTTGCTGGCGAC 360
TATCTTGGGCGTTTTGAGGAA 381
CCATCTTGGTTTTCTGGTTTCCTGGTGCCCAATTGCTACACACTCACCTTTGCTGGCGAC 352
TATCTTGGGCGTTTTGAGGAA 373
************************************************************
*********************
TATCTTGGGCGTTTTGAGGAA 381
TATCTTGGGCGTTTTGAGGAA 373
*********************
Figure 1. Clustal 2.1 alignment of the PCR-amplified part of the (LDH) C1* gene in S. trutta and S. obtusirostris with
Figure 1.RsaI
Clustal
2.1 alignment
of the PCR-amplified
part of the (LDH) C1* gene in S. trutta and S.
underlined
restriction
enzyme recognition
sites
obtusirostris with underlined RsaI restriction enzyme recognition sites
RESULTS
AND
DISCUSSION
RESULTS
AND
DISCUSSION
In the second group of juvenille fish, there should
be softmouth trutt individuals, with characteristic MP
In 21 fishes analyzed from the first group (S. trutta),
of the
lactate ofdehydrogenase
In 21
fishes analyzed from the first group (S. trutta),genotype
PCR-RFLP
analysis
the exon 3 to(LDH)
exon C1*
4 part
PCR-RFLP analysis of the exon 3 to exon 4 part of the
gene. However,
after PCR amplification
and subsecvent
of
the
lactate
dehydrogenase
(LDH)
C1*
gene
with
restriction
endonuclease
RsaI
revealed
presence
of
lactate dehydrogenase (LDH) C1* gene with restricrestriction of the PCR product with RsaI restriction
tion endonuclease RsaI revealed presence of only NP
endonuclease, 19 samples had MP genotype and two 2 genotype, specific for S. trutta, with two RsaI restriction
ones had NP genotype (Figure 2), being not speciffic for
sites
and tree bands on the agarose gel after restriction
S. obtusirostris.
(Figure 2).
Poljoprivreda 21:2015(1) Supplement, 56-58
d group of juvenille fish, there should be softmouth trutt individuals, with characteristic
e of the lactate dehydrogenase (LDH) C1* gene. However, after PCR amplification and
estriction of the PCR product with RsaI restriction endonuclease, 19 samples had MP
d two ones had NP genotype (Figure 2), being not speciffic for S. obtusirostris.
58
P. Margeta et al.: LACTATE DEHYDROGENASE GENE GENOTYPIZATION FOR SPECIES ...
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 (Razpet, 2006). Other genetic methods like microsatellite
genotyping, mitochondrial DNA analysis and SNP genotyping were described in different phylogenetic studies
of Salmo species in the river Neretva (Snoj et al., 2002;
Razpet et al., 2007; Pustovrh et al., 2014). The described
methods could also be used for species identification in
fish farms. The problem is that these methods are much
more expensive than PCR-RFLP genotyping and therefore
they are financially not suitable for the fish farm owners.
CONCLUSIONS
16 17 18 19 20 21 22 23 24 PCR-RFLP analysis of the exon 3 to exon 4 part
of the lactate dehydrogenase (LDH) C1* gene was
employed to identify the presence of other species
representatives or intercrosses in the two groups of
salmonides (S. trutta and S. obtusirostris group), raised
in a fish farm on the river Neretva. Using this method,
we were able to identify two S. trutta representatives in
the S. obtusirostris group. There is also a great interest
of fish farmeres to raise other salmonides from the river
Neretva with a help of genetic methods. Due to financial
limitation, there is a need for developing the low-cost
genetic methods distinguishing between other commercially interesting salmonides from the river Neretva.
Figure 2. PCR-RFLP results on agarose gel. Samples
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