Lactate dehydrogenase gene genotypization for species identification in a fish farm on the river

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 repre- sentatives 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 has also been practised in the river Neretva and stocking activities have never been well documented. There are severale Salmonid species, found in the river Neretva basin, among which S. trutta (brown Lactate dehydrogenase (LDH) C1* gene containing trout), Salmo marmoratus (marble trout), Salmo obtu- parts of exons 3 and 4 with intermediate intron has been sirostris (softmouth trout) Salmo farioides and Salmo found, firstly in brown trout (Mc Meel et al, Ferguson, dentex. 2001), and after that also in S. obtusirostris (Snoj et al., 2002) proven as an informative genetic marker. also known as the Adriatic trout, Adriatic salmon, and softmouth trout, is a spe- A mutation in the intron 3 of the (LDH) C1* gene cies of salmonid fish endemic to the rivers of Western (G/C substitution) was connected with the disruption Balkans. It is found naturally in four drainages of the of the RsaI restriction enzyme recognition site, being , in , and specific for S. obtusirostris (Razpet, 2004; Razpet, 2007). Montenegro: the Neretva- system, the Jadro, The same mutation was found in Salmo obtusiro- the Morača-Zeta system and the river drainages. stris salonitana and in Salmo obtusirostris oxyrhynchus Morphological different characteristics for different soft- (Odak, 2004). mouth trout populations from different river-systems In one fish farm on the river Neretva, S. trutta and resulted in the description of three putative subspecies: S. obtusirostris were decided to breed separately. Salmo obtusirostris oxyrhynchus from the River Neretva, Parental fishes were separated phenotypically on Bosnia and Herzegovina, Salmo obtusirostris salonitana the basis of the morphological signs. The aim of this from the river Jadro, Croatia, and Salmo obtusirostris work was, based on the LDH genotype, to find inter- krkensis from the River Krka, Croatia. crosses or representatives of the other species in two In the river Neretva basin, natural hybrids such groups of juvenille fishes. In the first group, there should as S. obtusirostris x S. trutta have been observed and reported (Vuković, 1982). The hybridization between autochthonous species was confirmed also in an experi- (1) Ph.D. Polonca Margeta ([email protected]), Ph.D. Ivan Bogut, ment performed in the fish farm located in the river Assist. Prof. Margeta Vladimir – Josip Juraj Strossmayer University of Osijek, Faculty of Agriculture in Osijek, Kralja Petra Svačića 1d, 31000 , a tributary of the river Neretva (Kosorić and Osijek, Croatia, (2) Ph.D. Marko Ivanković - Federal Agromediterranean Vuković, 1969). Introduction of non-native brown trout Institute , Biskupa Čule 10, 88000 Mostar, Bosnia and Herzegovina

Poljoprivreda 21:2015(1) Supplement, 56-58 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 be only representatives of S. trutta, while in the second one only representatives of S. obtusirostris.

P.MATERIAL Margeta et al.: LACTATEAND METHODS DEHYDROGENASE GENE GENOTYPIZATION FOR SPECIES ... 57

In the fish farm, juvenille fishes were raised in two groups. In the first group, there were juvenille befishes only representativesof S. trutta, and of S. in trutta the, secondwhile in juvenillethe second fishes DNA, of S. 1,5 obtusirostris mM MgCl2, . 0,2Fin mMclips of were each taken dNTP, from1 µM 21 oneindividuals only representatives from both of groupsS. obtusirostris. (together 42 samples)of and each stored primer in (Ldhxon4R 96% ethanol. and Ldhxon3F), In the lab, and genomic 1U of DNA was isolated from the preserved fin clips usingTaq DNABio Basic polymerase EZ-10 (Fisher Spin scientific). Column AmplificationAnimal DNA MATERIAL AND METHODS was performed in the thermal cycler (Eppendorf) as Mini-Preps Kit (Bio Basic Canada Inc.), following thedescribed supplier’s before instructions. (Mc Meel et al., 2001). In the fish farm, juvenille fishes were raised in two PCR of the lactate dehydrogenase (LDH) C1* genePCR containing products wereparts cleaved of exons by Rsa3 I and restriction 4 with groups. In the first group, there were juvenille fishes of intermediate intron has been performed as describendonuclease,ed by Mc Meelwith a etrecognition al. (2001). site gt/ac.Twenty The ampliµl PCR- S. trutta, and in the second juvenille fishes of S. obtu- contained 100 ng of genomic DNA, 1,5 mM MgClfied2, 0,2 part mM of the of (LDH) each C1* dNTP, gene 1 in µMS. trutta of each contained primer sirostris. Fin clips were taken from 21 individuals from (Ldhxon4R and Ldhxon3F), and 1U of Taq DNA twopolymerase RsaI recognition (Fisher scientific). sites, leading Amplification to three bands was both groups (together 42 samples) and stored in 96% after restriction in the lenght of 74, 135 and 166 bp ethanol.performed In the in lab, the genomic thermal DNA cycler was (isolatedEppendorf from) theas described before (Mc Meel et al., 2001). (genotype NP). On the other hand, PCR product of S. preservedPCR products fin clips wereusing Biocleaved Basic EZ-10by Rsa SpinI restrictionColumn endonuclease, with a recognition site gt/ac. The obtusirostris posessed only one restriction site because Animalamplified DNA partMini-Preps of the Kit (LDH) (Bio Basic C1* Canada gene Inc.), in S.fol -trutta contained two RsaI recognition sites, leading to of the disruption of the second recognition site due to a lowing the supplier’s instructions. three bands after restriction in the lenght of 74, 135G/C and transformation 166 bp (genotype (MP genotype), NP). leadingOn the to other only twohand, PCRPCR product of the lactateof S. dehydrogenaseobtusirostris (LDH)posessed C1* geneonly onebands restriction in the length site becauseof 74 and of300 the bp disruptionafter restriction, of the containingsecond recognition parts of exons site 3due and to 4 a with G/C intermediate transformation respectively (MP genotype), (Figure 1).leading Products to ofonly restriction two bands reactions in the intronlength has of been 74 andperformed 300 bp as after described restriction, by Mc Meel respectivel et werey (Figure checked 1). on Products the 2% agarose of restriction gel. 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 TGTTACCACGACGATACGAGAGTTCGCCGTCACAGAGTAGTCTGACCGTGGGAGAACAAT 60 S.obtusirostris CAATCAATGAGAGTACTGTGTATCATTTGTGTCTAGTATTTCTTCAGTAATTTGTCATAT 120 S.trutta TGTTACCACGACGATACGAGAGTTCGCCGTCACAGAGTAGTCTGACCGTGGGAGAACAAT 60 S.trutta CAATCAATGAGAGTACTGTGTATCATTTGT--CTAGTATTTCTTCAGTAATTTGTCATAT 118 ************************************************************ ****************************** ****************************

S.obtusirostris CAATCAATGAGAGTACTGTGTATCATTTGTGTCTAGTATTTCTTCAGTAATTTGTCATAT 120 S.obtusirostris CATTAATAGATCTAATGGCAGGACTATTACATGTCAAAGTAGGATTTCAGAAATTGCTTT 180 S.trutta CAATCAATGAGAGTACTGTGTATCATTTGT--CTAGTATTTCTTCAGTAATTTGTCATAT 118 S.trutta CATTAATAGATCTAATGGCAGGACTATTACATGTCAAAGTGGGATTTCAGAAATTGCTTT 178 ****************************** **************************** **************************************** *******************

S.obtusirostris CATTAATAGATCTAATGGCAGGACTATTACATGTCAAAGTAGGATTTCAGAAATTGCTTT 180 S.obtusirostris GAGAAACTTCATTCATACATTTCCCTTTCACCCTTTTCCCCCCCATCTCCCTTTCATACA 240 S.trutta CATTAATAGATCTAATGGCAGGACTATTACATGTCAAAGTGGGATTTCAGAAATTGCTTT 178 S.trutta GAGAAACTTCATTCATACATTTCCCTTTCACCC------CCCCCATCTCCCTTTCATACA 232 **************************************** ******************* ********************************* *********************

S.obtusirostris GAGAAACTTCATTCATACATTTCCCTTTCACCCTTTTCCCCCCCATCTCCCTTTCATACA 240 S.obtusirostris CTTCCCCTCTCAGAGAGACTACTTCATTTAACACACAGACATTTGACATGCAGATGGTGT 300 S.trutta GAGAAACTTCATTCATACATTTCCCTTTCACCC------CCCCCATCTCCCTTTCATACA 232 S.trutta CTTCCCCTCTCAGAGAGAGTACTTCATTTAACACACAGACATTTGACATGCAGATGGTGT 292 ********************************* ********************* ****************** *****************************************

S.obtusirostris CTTCCCCTCTCAGAGAGACTACTTCATTTAACACACAGACATTTGACATGCAGATGGTGT 300 S.obtusirostris CCATCTTGGTTTTCTGGTTTCCTGGTGCCCAATTGCTACACACTCACCTTTGCTGGCGAC 360 S.trutta CTTCCCCTCTCAGAGAGAGTACTTCATTTAACACACAGACATTTGACATGCAGATGGTGT 292 S.trutta CCATCTTGGTTTTCTGGTTTCCTGGTGCCCAATTGCTACACACTCACCTTTGCTGGCGAC 352 ****************** ***************************************** ************************************************************

S.obtusirostris CCATCTTGGTTTTCTGGTTTCCTGGTGCCCAATTGCTACACACTCACCTTTGCTGGCGAC 360 S.obtusirostris TATCTTGGGCGTTTTGAGGAA 381 S.trutta CCATCTTGGTTTTCTGGTTTCCTGGTGCCCAATTGCTACACACTCACCTTTGCTGGCGAC 352 S.trutta TATCTTGGGCGTTTTGAGGAA 373 ************************************************************ *********************

S.obtusirostris TATCTTGGGCGTTTTGAGGAA 381 S.trutta 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 underlinedFigure 1 .Rsa ClustalI restriction 2.1 alignment enzyme recognition of the PCR-amplified sites part of the (LDH) C1* gene in S. trutta and S. obtusirostris with underlined RsaI restriction enzyme recognition sites

RESULTSRESULTS AND AND DISCUSSION 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), genotype of the lactate dehydrogenase (LDH) C1* PCR-RFLPIn 21 fishes analysis analyzed of the fromexon 3the to exonfirst group4 part of(S. the trutta ), PCR-RFLP analysis of the exon 3 to exon 4 part gene. However, after PCR amplification and subsecvent lactateof the dehydrogenaselactate dehydrogenase (LDH) C1* (LDH) gene withC1* restricgene -with restriction endonuclease RsaI revealed presence of restriction of the PCR product with RsaI restriction tion endonuclease RsaI revealed presence of only NP endonuclease, 19 samples had MP genotype and two genotype, specific for S. trutta, with two RsaI restriction 2 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 only NP genotype, specific for S. trutta, with two RsaI restriction sites and tree bands on the agarose gel after restriction (Figure 2). In the second group of juvenille fish, there should be softmouth trutt individuals, with characteristic MP genotype of the lactate dehydrogenase (LDH) C1* gene. However, after PCR amplification and subsecvent restriction of the PCR product with RsaI restriction endonuclease, 19 samples had MP genotype and two ones had 58NP genotype (Figure 2), being not speciffic for S. obtusirostris. P. Margeta et al.: LACTATE DEHYDROGENASE GENE GENOTYPIZATION FOR SPECIES ...

(Razpet, 2006). Other genetic methods like microsatellite 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 genotyping, mitochondrial DNA analysis and SNP geno- typing 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 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 16 17 18 19 20 21 22 23 24 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 com- mercially interesting salmonides from the river Neretva. Figure 2. PCR-RFLP results on agarose gel. Samples Figure 2. PCR-RFLP results1-11 on agaroseare from gel.the S.truttaSamples group 1-11 and are they from are the all S.truttaof NP groupREFERENCES and they are all of NP genotype with tree bandsgenotype after res withtriction. tree bandsSamples after 13-24 restriction. are from Samples the S.obtusirostris 1. Kosorić, group. Đ., Vuković,While T. (1969): Ispitivanja moguć- samples 13-22 posess MP genotype,13-24 are fromspecific the S.obtusirostrisfor S. obtusirostris group. ,While samples samples 23 and 24 havenosti NP hibridizacije genotype. salmonidnih vrsta sliva Neretva. Sample 12 is 50bp marker 13-22 posess MP genotype, specific for S. obtusiro- Ichthyologia, 1: 57–67. stris, samples 23 and 24 have NP genotype. Sample 12 2. Mc Meel, O.M., Hoey, E.M., Ferguson, A. (2001): Partial is 50bp marker nucleotide sequences, and routine typing by polymerase In the both analized groups no intercrosses of MP and NP genotypes were found. Thechain assumption reaction-restriction is fragment length polymorphi- that the presence of the NP genotype in the group of juvenille softmouth trutt is eithersm, consequence of the brown trout of (Salmo trutta) lactate dehydro- In the both analized groups no intercrosses of MP genase, LDHC1* 90 and *100 alleles. Molecular Ecology, the presence of pure S. truttaand NP fish genotypes in the wereS. obtusirostris found. The assumption parental is group,that or juvenille fish were accidentally mixed during handling. In the fish fry morphological signs, characteristic10: for 29–34. adult fishes the presence of the NP genotype in the group of juvenille 3. Odak Tomljanović, T. (2004): Molecular-biologic chara- of both species, were not wellsoftmouth defined trutt and is either distinguishing consequence between of the presence representatives of ofcterization both species of endemic is softmouth trout Salmothymus difficult. Based on the results,pure the S. truttasuggestion fish in thewas S. to obtusirostris first devide parental adult group,fishes into twoobtusirostris groups, based salonitana. on Master thesis, Agronomski morphological signs. Further,or juvenilleparental fish lines were of accidentally both species mixed should during be handling. marked and sampledfakultet Sveučilištafor genetic u Zagrebu. analysis for detection of Inintercrosses the fish fry morphologicalor representatives signs, characteristic of other species for 4.in Pustovrh,parental G., groups.Snoj, A., Sušnik Bajec, S. (2014): Molecular Combination of morphologicaladult signsfishes ofand both genetic species, me werethods not was well allready defined and proven to bephylogeny very powerfull of Salmo of the western Balkans, based upon distinguishing between representatives of both species multiple nuclear loci. Genetics Selection Evolution, 46: 7. method in distinguishing betweenis difficult. S. Basedmarmoratus on the results,and hybrids the suggestion with alien was Salmo speciesdoi: http://dx.doi.org/10.1186/1297-9686-46-7 in the Soča river basin (Delling et al., 2000).to first devide adult fishes into two groups, based on 5. Razpet, A., Sušnik, S., Jug, T., Snoj, A (2007): Genetic Disadvantage of the McNeel'smorphological method signs. is that Further, it could parental be lines used of bothonly spe to- elucidatevariation S. obtusirostris among trout in the river Neretva basin, Bosnia representatives from other Salmocies should species, be marked found and in sampled the river for geneticNeretva. analysis While the MPand genotype Herzegovina. of J.the Fish. Biol., 70: 94–110. (LDH) C1* gene is a characteristicfor detection only of forintercrosses S. obtusirostris or representatives, the NP genotypeof other is not doi:present http://dx.doi.org/10.1111/j.1095-8649.2007.01392.x only in S. trutta, but also in S. marmoratusspecies andin parental other groups.Salmo Combinationspecies, found of morphologi in the river- Neretva6. Razpet, basin A. (2004):(Razpet, Genetic diversity of salmonids of the 2006). Other genetic methodscal signslike andmicrosatellite genetic methods genotyping, was allready mitochondrial proven to DNA analysisriver Neretva and drainage. SNP Master thesis, Bitehniška univer- be very powerfull method in distinguishing between S. za v Ljubljani, Oddelek za zootehniko. genotyping were describedmarmoratus in different and phylogenetic hybrids with alien studies Salmo ofspecies Salmo in thespecies 7. in theSnoj, riverA., Melkić, Neretva E., Sušnik, S., Muhamedagić, S., Dovč , (Snoj et al., 2002; Razpet etSoča al., river 2007; basin Pustovrh (Delling et et al., al., 2000). 2014). The described methodsP. (2002): could DNA also phylogeny be supports revised classification used for species identification Disadvantagein fish farms. of theTh McNeel’se problem method is that is thatthese it methodsof areSalmothymus much more obtusirostris. Biological Journal of the Linnaean Society 77: 399–411. could be used only to elucidate S. obtusirostris repre- doi: http://dx.doi.org/10.1046/j.1095-8312.2002.00130.x sentatives from other Salmo species, found in the river 8. Vuković, T. (1982): 3 Sistematika riba. In Slatkovodno Neretva. While the MP genotype of the (LDH) C1* gene is ribarstvo (Habeković, D., ed.), pp. 99–168. Zagreb: a characteristic only for S. obtusirostris, the NP genotype Jugoslavenska medicinska naklada. is not present only in S. trutta, but also in S. marmoratus and other Salmo species, found in the river Neretva basin (Received on 8 June 2015; accepted on 3 August 2015)

Poljoprivreda 21:2015(1) Supplement, 56-58