Fourteen polymorphic allozyme loci were identified in yellow catfish, Horabagrus

brachysoma. The genetic variation detected at each allozyme locus was assessed

for samples collected from three rivers. The observed heterozygosities per locus

ranged from 0.0286 to 0.4000. Significant genotype heterogeneity indicated that

the samples are not drawn from same gene pool. The results suggest the potential

of the identified loci to analyze stock structure of natural populations of

H. brachysoma

Abdul Muneer, P M

Basheer, V S

Gopalakrishnan, A

Kapoor, D

Lal, Kuldeep Kumar

Mohindra, Vindhya

Ponniah, A G

Thomas, P C

CMFRI Digital Repository

253Identification of polymorphic allozyme markersfor population structure analysis in Horabagrusbrachysoma (Gunther, 1864).*A. GOPALAKRISHNAN, P.M. ABDUL MUNEER, P.C.THOMAS**, KULDEEP KUMAR LAL, VINDHYA MOHINDRA,V.S. BASHEER*, D. KAPOOR AND A.G. PONNIAH***National Bureau of Fish Genetic Resources (NBFGR), P.O. Dilkusha, Lucknow -226002, India. nbfgr@sancharnet.in* NBFGR Cochin Unit, CMFRI Campus, Kochi - 682 018, Kerala.  nbfgrcochin@eth.net** Central Marine Fisheries Research Institute (CMFRI), Kochi - 682 018, Kerala.*** The World Fish Centre, Penang, Malaysia. a.ponniah@cgiar.orgABSTRACTFourteen polymorphic allozyme loci were identified in yellow catfish, Horabagrusbrachysoma. The genetic variation detected at each allozyme locus was assessedfor samples collected from three rivers. The observed heterozygosities per locusranged from 0.0286 to 0.4000. Significant genotype heterogeneity indicated thatthe samples are not drawn from same gene pool. The results suggest the potentialof the identified loci to analyze stock structure of natural populations ofH. brachysoma.Indian J. Fish., 53(3) : 253-261, Jul.-Sep., 2006IntroductionHorabagrus brachysoma (Family:Bagridae) popularly called ‘yellow catfish’or ‘sun-catfish’ is endemic to riversoriginating from southern part of theWestern Ghats. The Western Ghats isrecognized as one of the thirty-fourbiodiversity rich hotspots of the world(Myers et al., 2000). H. brachysoma, withhigh food and ornamental value, hasdeclined in abundance and now restrictedto a few rivers viz. Nethravathi,Chalakkudy and Meenachil Rivers(Gopalakrishnan and Ponniah, 2000).The fish, though a freshwater inhabitant,is also reported from brackishwater,especially in the Vembanad lake, Keraladuring south-west monsoon (Kurup andSamuel, 1982). Unnithan (2001)estimated annual landing of this speciesfrom Vembanad lake as 2150 kg (0.56%of the total fish landing from the lake).H.brachysoma is categorized asendangered, based on the IUCN(International Union for Conservation ofNature and natural resources) criteriadue to restricted distribution, loss ofhabitat, over-exploitation, destructivefishing practices and trade (Anon., 1998).In view of the significance, H.brachysoma is a potential candidate254A. Gopalakrishnan et al.species for research on artificialpropagation, for use in developing itsaquaculture and rehabilitation of naturalstocks. The present work is a part ofintegrated plan covering differentaspects, including captive breeding,sperm cryopreservation protocols,documenting life history traits andinformation on genetic markers as wellas stock structure. Identification ofmarkers with scorable alleles is pre-requisite to generate stock structure datafor any species (Ferguson et al., 1995).There is no information available on anyclass of genetic markers in H.brachysoma.  Allozyme loci are proventools to determine population structureand estimate intra-population gene flowin natural fish populations. These havebeen consistently used to developinformation on genetic structure of manyfish species (Menezes, 1993,Gopalakrishnan et al., 1997; Jerry, 1997;Van der Bank et al., 1997; Cashihlo andMcAndrew, 1998; Hawkins et al., 2002;Lal et al., 2004; Salini et al., 2004). Thepresent study analyses allozyme markersin H. brachysoma, to identifypolymorphic loci and suitability of theidentified polymorphic loci in analyzingpopulation structure of the species.Material and methodsSpecimens of H.brachysoma wereobtained through commercial catchesfrom two rivers viz., Chalakkudy (n=26)at Kanakkankadavu, Kerala (10008’N;76007’E) and Nethravathi (n=25) atKankanadi, Karnataka (12052’N;74054’E). The liver samples werecollected from the specimens  and wereimmediately frozen in liquid nitrogen(-1960C). The riverine locations werechosen to cover geographically isolatedpopulations of H. brachysoma. Thesamples were transported to laboratoryand stored at -800C until analysis. Frozenliver samples (approximately 100 mg)were homogenized in 250 mg/ml ice-coldextraction buffer (0.17 M sucrose, 0.2 MEDTA, 0.2 M Tris-HCl, pH 7.0).Homogenized samples were centrifugedfor an hour at 12,000 rpm at 40C(Heraeus Biofuge-Stratos, Germany) andthe supernatant was recentrifuged for 45minutes. The enzyme systems wereassessed using vertical nativepolyacrylamide gel electrophoresis(PAGE) (Amersham Biosciences Ltd.).Gels of 10 x 8 cm. size were used for allthe enzymes. Investigations were doneusing 7.25% PAGE and electrophoresiswas done using constant voltage (150V)at 40C using TBE buffer (90 mM Tris-borate and 2 m M EDTA, pH 8.0) systemexcept in SOD, that was resolved in TGbuffer (5 mM Tris-Cl and 0.038 MGlycine, pH 8.3) system. To increase theresolution of the bands, NAD or NADPwas added in the gel mixture and in theupper tank of the electrophoresisapparatus based on the nature of theenzyme (Gopalakrishnan et al., 1997). Atotal of 25 enzyme systems wereexamined (Table1). Visualization ofdifferent alleles of enzyme was done byhistochemical staining following theprocedures outlined by Whitmore (1990)and Shaw and Prasad (1970). Agaroverlay method was used to detectzymograms in GPI and PGM. The gelswere documented using Imagemaster 1DElite gel documentation system(Amersham Biosciences, USA).Nomenclature of loci and alleles wasfollowed as recommended by Shaklee etal. (1990). At all the loci, most commonallele was assigned as 100. Alternatealleles were designated as per theirmobility, in relation to the most commonallele. Parameters of genetic variationlike proportion of polymorphic loci (P 0.95and P 0.99) in each population andheterozygosity at individual locus and255TABLE 1: The names of enzyme loci, enzyme commission (E.C.) number and observed alleles forallozyme analysis in Horabagrus brachysoma. The enzymes mark ‘ns’ did not yield any scorableactivity.Enzyme Loci E.C. Locus AllelesnumberAcid phosphatase 3.1.3.2 ACP* nsAdenylate kinase 2.7.4.3 AK* nsAlcohol dehydrogenase 1.1.1.1 ADH* nsAlkaline phosphate 3.1.3.1 ALP* nsAspartate amino transferase 2.6.1.1 AAT-1* 100AAT-2* 100,117,126Creatine kinase 2.7.3.2 CK* nsEsterase EST-1* 083, 100EST-2* 100,1063.1.1.1 EST-3* 095,100EST-4* 100EST-5* 100Fumerase 4.2.1.2 FUM* nsGlutamate dehydrogenase 1.4.1.3 GDH* nsGlucose-6-phosphate dehydrogenase 1.1.1.49 G6PDH* 086,100Glucose phosphate isomerase 5.3.1.9 GPI-1* 100GP1-2* 096,100Glucose dehydrogenase 1.1.1.47 GLDH* 080,089,100,117-Glycerophosphate dehydrogenase 1.1.1.8 G3PDH* 088, 100Glyceraldehyde-3-Phosphate dehydrogenase 1.2.1.12 GAPDH –1* 100GAPDH –2* 100Hexokinase 2.7.1.1 HK* nsIsocitrate dehydrogenase 1.1.1.42 ICDH* nsLactate dehydrogenase 1.1.1.27 LDH-1* 100LDH-2* 100,112,134Malate dehydrogenase 1.1.1.37 MDH* 086,100Malic enzyme 1.1.1.40 ME* 100Octonol dehydrogenase 1.1.1.73 ODH-1* 100ODH-2* 091,100ODH-3* 100Phosphogluconate dehydrogenase 1.1.1.44 6PGDH* nsPhosphogluco mutase 5.4.2.2 PGM* 093,100Pyruvate kinase 2.7.1.40 PK* nsSuperoxide dismutase 1.15.1.1 SOD* 093,100Xanthine dehydrogenase 1.1.1.204 XDH* 093, 100Allozyme markers in Horabagrus brachysomamean over all loci for each populationwere calculated with GENETIX (version4.0, Belkhir et al., 1997). GENEPOPversion 3.4 (Raymond and Rousset,1995a) was used to assess conformity ofallele frequencies to that expected underHardy- Weinberg equilibrium andgenetic differentiation. Genetichomogeneity of the sample sets wasdetermined through an exact test (Gbased test) that assumes randomsamples of genotypes (Genepop version3.4, Genotype differentiation test,Raymond and Rousset, 1995a). This testwas performed on genotype tables andpossible non- independence of alleleswith in genotypes will not affect testvalidity (Raymond and Rousset, 1995b;Goudet et al., 1996).256A. Gopalakrishnan et al.TABLE 2 : Parameters of genetic variation for two riverine populations at each allozyme locus inHorabagrus brachysoma.Locus Population Alleles Hexp. Hobs. HW Genetic(p) heterogeneity(p)AAT-2* CH 3 0.4800 0.0800 <0.0000**NT 3 0.5318 0.6538 0.0552 0.0178EST –1* CH 1 0.0000 0.0000 —NT 2 0.2604 0.0769 0.0035 <0.0001**EST –2* CH 2 0.3432 0.1200 0.0037NT 2 0.1738 0.1154 0.1901 0.2015EST-3* CH 2 0.3200 0.4000 1.000NT 2 0.0000 0.0000 — 0.0003**G3PDH* CH 2 0.5000 0.3600 0.1327NT 2 0.3550 0.1538 0.0087 0.0258*G6PDH* CH 2 0.3432 0.1200 0.0037NT 2 0.4933 0.1154 0.0001** 0.0780GLDH* CH 4 0.4224 0.4000 0.0172NT 2 0.4734 0.3077 0.0686 <0.0001**GPI-2* CH 2 0.3200 0.1600 0.0272NT 2 0.3336 0.0385  <0.0001** 1.0000LDH-2* CH 3 0.4864 0.0800 0.0104 0.0034**NT 2 0.0740 0.0000 0.0196MDH* CH 2 0.4712 0.1200 0.0002**NT 2 0.3550 0.0769 0.0003 0.2678ODH-2* CH 2 0.4872 0.2000 0.0033NT 2 0.3107 0.0769 0.0009** 0.0028**PGM* CH 2 0.3848 0.1200 0.0014**NT 2 0.4260 0.3077 0.1419 0.7269SOD* CH 2 0.4992 0.1600 0.0006**NT 2 0.3750 0.2692 0.1494 0.0712XDH-1* CH 3 0.4968 0.2800 0.0277NT 3 0.5688 0.1923 <0.0001** 0.1543*Mean over CH 1.60 0.2222 0.1040all loci NT 1.60 0.1892 0.0954 — <0.0001*** Significant at (P<0.05); ** Significant after critical significance level is adjusted for sequentialBonferroni correction (Lessios, 1992). CH – Chalakkudy River, Kerala; NT – Nethravathi River,Karnataka.Results and discussionFourteen out of 25 enzymes werefound to give scorable activity thatprovided 25 loci (Table 1). The name ofenzyme loci, enzyme commissionnumbers and alleles observed are givenin Table1. Eleven of twenty five loci weremonomorphic represented by a singleallele in all the studied populations. Alocus was considered polymorphic, if thefrequency of most common allele was 0.99 (Hartl and Clark, 1997).  Fourteenloci (56%), AAT-2*, EST-1*, EST-2*,EST-3*, G3PDH*, G6PDH*, GLDH*,GPI-2*, LDH-2*, MDH*, ODH-2*, PGM*,SOD*, XDH-1* were polymorphic(Table1). The banding pattern ofheterozygous in polymorphic loci,confirmed to that expected as per thestructure of the respective protein. Of the14 polymorphic loci, EST-1*, EST-2*,EST-3*, G6PDH*, GPI-2*, G3PDH*,MDH*, ODH-2*, PGM* and SOD* had257Allozyme markers in Horabagrus brachysomatwo alleles; AAT-2*, LDH-2* and XDH-1* had three alleles and GLDH* had fouralleles.  The zymograms of the studiedproteins are shown in Fig. 1-14. ExceptEST-1* and EST-3*, all otherFig. 1. Aspartate amino transferase (AAT)pattern in H. brachysoma. Lanes 1-5samples from Meenachil River, 6-10Chalakkudy River and 11-15Nethravathi River.Fig. 2. Esterase  (EST) pattern in H.brachysoma. Lanes 1-5 samples fromMeenachil River, 6-10 Chalakkudyriver and 11-15 Nethravathi River.Fig. 3. Glucose dehydrogenase (GLDH)pattern in H. brachysoma. Lanes 1-5samples from Meenachil River, 6-10Chalakkudy river and 11-15Nethravathi River.Fig. 4. Glucose 6-phosphate dehydrogenase(G6PDH)  pattern in H. brachysoma.Lanes 1-5 samples from MeenachilRiver, 6-10 Chalakkudy River and 11-15 Nethravathi River.Fig. 5.  -Glycerol 3-phosphate dehydrogenase( G3PDH)  pattern in H. brachysoma.Lanes 1-5 samples from MeenachilRiver, 6-10 Chalakkudy River and 11-15 Nethravathi River.Fig. 6. Lactate  dehydrogenase (LDH)  patternin H. brachysoma. Lanes 1-5 samplesfrom Meenachil River, 6-10Chalakkudy River and 11-15Nethravathi River.polymorphic loci were polymorphic in allpopulations. EST-1* locus waspolymorphic only in Nethravathipopulation whereas, EST-3* locus waspolymorphic only in Chalakkudy258population. Parameters of geneticvariation are given in Table 2. Theproportion of polymorphic loci at P0.99criteria was 0.52 and the mean numberof alleles per locus is 1.60, for both theFig. 7. Glucose phosphate isomerase (GPI)pattern in H. brachysoma. Lanes 1-5samples from Meenachil River, 6-10Chalakkudy River and 11-15Nethravathi River.Fig. 8. Malate dehydrogenase (MDH)  patternin H. brachysoma.  Lanes 1-5 samplesfrom Meenachil River, 6-10Chalakkudy River and 11-15Nethravathi River.Fig. 9. Phosphoglucomutase (PGM) patternin H. brachysoma. Lanes 1-5 samplesfrom Meenachil River, 6-10Chalakkudy River and 11-15Nethravathi River.Fig. 10. Superoxide dismutase (SOD) patternin H. brachysoma. Lanes 1-5 samplesfrom Meenachil River, 6-10Chalakkudy River and 11-15Nethravathi River.Fig. 11. Octanol dehydrogenase (ODH)pattern in H. brachysoma. Lanes 1-5samples from Meenachil River, 6-10Chalakkudy River and 11-15Nethravathi River.Fig. 12. Xanthine dehydrogenase (XDH)pattern in H. brachysoma.  Lanes 1-5samples from Meenachil River, 6-10Chalakkudy River and 11-15Nethravathi River.sample sets. The observed meanheterozygosity values ranged from 0.09to 0.10 (Table 2). These values were foundto be marginally higher than the valuedescribed for teleost fishes (Nevo, 1978)A. Gopalakrishnan et al.259but are common. The probability testprovided the evidence that the observedallele frequencies significantly (P<0.05)deviated from that expected underHardy-Weinberg equilibrium. Deviationwas observed at least in five to six locifor both the sample sets. Significantdeficiency of heterozygote was observedat these loci. This indicates the possibilityof genetic bottlenecks existing in thenatural populations of Horabagrusbrachysoma.The genetic heterogeneity was testedbased on the genotype rather than allelefrequencies, in view of the observed non-conformity to Hardy-Weinberg expecta-tions (Raymond and Rousset, 1995b;Goudet et al., 1996). Significantheterogeneity (P<0.05) in genotypeproportions was observed (Table 2), afterthe sequential Bonferroni correction wasmade to the probability levels. Highlysignificant probability (P<0.0001) over allthe loci suggests that the three samplesets are not part of the same gene pool.Coefficient of genetic differentiation (Gst)calculated for sample size (Nei andChesser, 1983), was found to be 0.1303confirming the existence of populationsub-structuring in H.brachysoma (Table2).In conclusion, the present studyidentified fourteen polymorphic allozymeloci, useful in determining geneticdivergence in natural populations of H.brachysoma. Nevertheless, analysis oflarger sample size, from other regions ofdistribution will provide better insights,not only for genetic divergence but alsofor pinpointing the genetic bottlenecks inthe natural populations of the species.AcknowledgmentThe authors gratefully acknowledgethe constant help rendered by Mr.Devassy, Fish Hobbyist at Kanakkan-kadavu, Kerala during collection ofspecimens.ReferencesAnonymous, 1998. Report of the workshop“Conservation Assessment andManagement Plan (CAMP) forfreshwater fishes of India 1997”organized by Zoo Outreach Organizationand National Bureau of Fish GeneticResources, Lucknow held at NBFGR inSept. 1997. Zoo Outreach Organization,Coimbatore, India, 156 pp.Belkhir, K., P. Borsa, J. Goudet, J., L. Chikhi,and F. Bonhomme 1997. GENETXlogicielsous windows pour Ia ge’ne’tiquedes populations, http://www.univ-montp2.fr/~gentix/genetix/html.Cashihlo, R. and B. J. McAndrew 1998.Population structure of sea bass inPortugal, Evidence from allozymes. J.Fish Biol., 53: 1038-1049.Fig. 13. Glyceraldehyde 3-phosphatedehydrogenase (GAPDH) pattern in H.brachysoma.  Lanes 1-5 samples fromMeenachil River, 6-10 ChalakkudyRiver and 11-15 Nethravathi River.Fig. 14. Malic enzyme (MEP) pattern in H.brachysoma. Lanes 1-5 samples fromMeenachil River, 6-10 ChalakkudyRiver and 11-15 Nethravathi River.Allozyme markers in Horabagrus brachysoma260Ferguson, A., J.B. Taggart, P. A. Prodohl, O.McMeel, C. Thompson, C. Stone, P.McGinnity, and R.A. Hynes, 1995. Theapplication of molecular markers to thestudy and conservation of fishpopulations, with special reference toSalmo. J. Fish Biol., 47(Suppl. A): 103-126.Gopalakrishnan, A and A.G. Ponniah 2000.Cultivable, food, sport and ornamentalfish species endemic to Peninsular Indiawith special reference to the WesternGhats. P. 13 - 32. In: A.G. Ponniah, andA. Gopalakrishnan (Eds.).  Endemic FishDiversity of the Western Ghats. 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Fishand fishery resources of the VembanadLake. 72 pp., In: K. Ravindran, N.Unnikrishnan Nair, P.A. Perigreen, P.Madhavan, A.G. Gopalakrishna Pillai,P.A. Panicker, and Mary Thomas (Eds.)Proc.  Symp. Harvest and Post-harvestTechnology of Fish. Society of FisheriesTechnologists (India), Cochin – 682029,Kerala, India.Lal, K.K, D. Kumar, S. K. Srivastava, A.Mukherjee, V. Mohindra, Shree Prakash,M. Sinha and A. G. Ponniah 2004.Genetic variation in Tenualosa ilisha(Hamilton-Buchanan) population inriver Ganges. Indian J. Fish. 51(1): 33-42.Lessios, H.A. 1992. Testing electrophoreticdata for agreement with HardyWeinberg expectations. Marine Biol.,112: 517-523.Menezes, M. R. 1993. Inter-specific geneticdivergence in three pomfret species fromthe Goa region. Aquacult.Fish.Management,  24: 341-346.Myers, N., R.A. Mittermiler, C.G. Mittermiler,G.A.B. Da-Fonseca and J.Kent 2000.Biodiversity hotspots for conservationpriorities. Nature, 403: 853-858.Nei, M. and R. K. Chesser 1983   Estimationof fixation indices and gene diversities.Ann. Hum. Genet., 47, 253-259.Nevo, E. 1978. Genetic variation in naturalpopulations: Patterns and theory.Theoretical Pop. Biol., 13: 121- 177.Raymond, M. and F. Rousset 1995a.GENEPOP (ver. 1.2): A  populationgenetics software for exact test andecumenicism. J. Heredity, 86:  248-249.http //www.cefe.cnrs-mop.fr/genepop.htmlRaymond, M. and F. Rousset 1995b. An exacttest for population differentiation.Evolution,  48: 1280-1287.Salini J.P.,  D.A. Milton,  M.J. Rahman andM.G. Hussain 2004. Allozyme andmorphological variation throughout thegeographic range of the tropical shad,Hilsa (Tenualosa) ilisha. Fish. Res.,66(1), : 53-69.Shaklee, J. B., F. W. Allendorf, D. C. Morizonand G. S. Whitt 1990. Genenomenclature for protein - coding loci inA. Gopalakrishnan et al.261fish. Trans.  Amer. fish. Soc., 119: 2-15.Shaw, C. R. and R. Prasad 1970. Starch gelelectrophoresis of enzymes – acompilation of recipes. Biochem. Genet.,4 : 297 – 320.Unnithan, V. K. 2001. Ecology and fisheriesinvestigation in Vembanad Lake.Bulletin No. 107, Central Inland CaptureFisheries Research Institute,Barrackpore, India, 38pp.Van der Bank, F.H., G.D. Engelbrecht, H.Sauer- Guerth, M. Wink, P. F.S. Mulder1997. Allozyme and DNA sequences datasupport speciation of Northern Southernpopulations of silver catfish, Schilbeintermedius (Ruppell, 1832). Comp.Biochem. Physiol., 120A No. 3: 531-543.Whitmore, D. H. 1990. Electrophoretic andIsoelectric focusing techniques infisheries   management. P. 23-80, CRCPress, Inc., Florida, USA.Allozyme markers in Horabagrus brachysomaDate of Receipt : 03-12-04Date of Acceptance : 16-09-05

Identification of polymorphic allozyme markers

for population structure analysis in Horabagrus

brachysoma (Gunther, 1864)

Identification of polymorphic allozyme markers

for population structure analysis in Horabagrus

brachysoma (Gunther, 1864).

Identification of polymorphic allozyme markers for population structure analysis in Horabagrus brachysoma (Gunther, 1864).

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