One of the possible reasons for pheasant population number decline in past several years might be loss of adaptability in populations originated from breeding stations caused by inbreeding depression. Due to fact that adaptability is a consequence of genetic structure of the populations, the aim of this paper was the analysis of genetic variability in pheasant population from breeding station Ristovaca using molecular markers. Allozyme variability of 20 putative gene loci was detected by polyacrylamide gel electrophoresis. Polymorphism was revealed in 5 loci: Est-1, Pgd, Sod, Gpi-2 and Odh. The values of genetic variability measures - heterozigosity polymorphism, fixation indices and H/P ratio indicate low level of genetic variability and possible presence of inbreeding depression within pheasant population

Beuković Miloš T.

Obreht Dragana R.

Tošović-Marić Biljana M.

Vapa Ljiljana B.

Vapa Milan M.

Đan Mihajla R.

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Zbornik Matice srpske za prirodne nauke / Proc. Nat. Sci, Matica Srpska Novi Sad,¥ 107, 5—11, 2004UDC 636.594:575.22(497.113 Ristovaåa)Ljiljana B . Vapa 1* , Mihajla R . Djan 1,Dragana R . Obreht 1, Biljana M . Tošoviã-Mariã 1,Milan M . Vapa 2, Miloš T . Beukoviã 21 Faculty of Sciences, Trg Dositeja Obradoviãa 22 Faculty of Agriculture, Trg Dositeja Obradoviãa 8;21000 Novi Sad, Serbia and MontenegroGENETIC VARIABILITY OF PHEASANT (Phasianus spp.)IN BREEDING STATION RISTOVACAABSTRACT: One of the possible reasons for pheasant population number decline inpast several years might be loss of adaptability in populations originated from breeding sta-tions caused by inbreeding depression. Due to fact that adaptability is a consequence of ge-netic structure of the populations, the aim of this paper was the analysis of genetic variabi-lity in pheasant population from breeding station Ristovaca using molecular markers. Al-lozyme variability of 20 putative gene loci was detected by polyacrylamide gel electropho-resis. Polymorphism was revealed in 5 loci: Est-1, Pgd, Sod, Gpi-2 and Odh. The values ofgenetic variability measures — heterozigosity, polymorphism, fixation indices and H/P ratioindicate low level of genetic variability and possible presence of inbreeding depressionwithin pheasant population.KEY WORDS: allozyme, electrophoresis, genetic variability, pheasantINTRODUCTIONIntroduction of pheasant (Phasianus ssp.) into our area has started in midfifties, and today it has become common and well adapted in our country, butalso in large number of European hunting areas. Unfortunately, in the last de-cade, a remarkable decline of pheasant population number has been detected.In Central Serbia, population number in spring season has decrease 44%, andthis trend continues. In Vojvodina, percentage of decline is lower, but popula-tion number in Vojvodina is at constant low level, mostly because of environ-mental conditions (less forest areas, more agricultural areas). Cause for namedphenomenon can be high hunting pressure on the pheasant species in centralpart of Serbia (C e r a n i c, 2001).Investigation of wild animal genomes is significant because of implemen-tation of results in planing, hunting economy, conservation biology and lack ofresults published. Low genetic variability is usually related to inbreeding de-5pression and loss of heterozygosity. This leads to most of characteristics of po-pulation phenotype, such as metabolic efficiency, reproductive efficiency, di-sease resistance etc. (G i l p i n and S o u l e, 1986). In wild animal species,lower genetic variability can have larger consequences, because of small popu-lations and high inbreeding. Loss of certain allele of genotype decrease chan-ces for new better adapted genotypes, in case of environmental condition chan-ges.One of the most commonly used genetic markers in wild animal speciesare isozymes, present in different molecular forms, allozymes (V a p a et al.,1999, 2002).The aim of this paper was estimation of genetic variability of pheasant(Phasianus ssp.) bred in breeding station Ristovaca, based on variability ofisozymes systems.MATERIAL AND METHODSMaterial: Liver samples of forty three individuals were used in this rese-arch. Livers were frozen immediately after death of animal and kept in freezerat –20°C until electrophoresis.Method: Tissue preparation and vertical polyacrylamide gel electrophore-sis (PAGE) were performed according to G r illitsch e t a l . (1992) andM u n s t e r m a n n (1979). After electrophoresis gels were stained accordingto S e l a n d e r et al. (1971).The following isozymes systems were examined (isozyme/-system, abbre-viation, E.C. number and corresponding structural gene loci in parenthesis):Lactate dehydrogenase (LDH, 1.1.1.27, Ldh-1, -2); Malate dehydrogenase(MOR, 1.1.1.37, Mor-1, -2); Malic enzyme (MOD, 1.1.1.40, Mod-1, -2);6-phosphogluconate dehydrogenase (PGD, 1.1.1.44, Pgd); Octanol dehydroge-nase (ODH, 1.1.1.73, Odh); Superoxid dismutase (SOD, 1.15.1.1, Sod); Aspar-tate aminotransferase (AAT, 2.6.1.1, Aat); Hexokinase (HK, 2.7.1.1, Hk-1, -2);Pyruvate kinase, (PK, 2.7.1.40, Pk); Creatine kinase (CK, 2.7.3.2, Ck-1, -2);Adenylate kinase (AK, 2.7.4.3, Ak-1, -2); Esterases (EST, 3.1.1.1, Es-1); Al-dolase (ALDO, 4.2.1.3, Aldo); Glucose-6-phosphate isomerase (GPI, 5.3.1.9,Gpi-2).Statistical analysis: Statistical evaluation of electrophoretic data was sup-ported by the BIOSYS-1 program of Swofford and Selander (release 1.7,1989). We used the BIOSYS-1 p.c. package to calculate allele frequencies,average heterozygosity (H0-observed, He-expected), proportion of polymorphicloci (99% criterion) P, mean number of allele per locus (A), exact test of de-viation of observed genotypes at polymorphic loci from Hardy-Weinberg ex-pectation, as well as basic parameters of F statistics.6RESULTS AND DISCUSSIONAmong 14 isozyme systems, represented by 20 putative loci, five lociwere polymorphic. Polymorphism was revealed within loci: Es-1, Gpi-2, Odh,Pgd and Sod, with two to four alleles per locus (Tab. 1).Table 1. Allele frequencies at polymorphic loci and indices of genetic variability in pheasant po-pulation from breeding station Ristovaåa (for acronyms see Material and Methods). Ho — obser-ved population-specific heterozygosity; He — expected population specific heterozygosity; P99%— rate of polymorphism (99% criterion); A — mean number of alleles per locus; n — number ofindividuals analyzed; FIS,F IT and FST — fixation indicesLocus Allele Ristovaåan=4 3Es-1abc0.1740.6160.209Pgd ab0.833*0.188Gpi-2abcd0.3330.4500.2000.017Odh ab0.804*0.196Sodabc0.2910.5700.140Ho 0.045He 0.097A 1.40P99% 25%H/P 0.18FIS –0.135FIT –0.097FST 0.034* significant deviation of genotype frequencies from Hardy-Weinberg expectation based on fivepolymorphic loci and exact Fisher test, criterion p < PnThe mean He in various non-endangered bird species and subspecies, ascalculated from Table 2. in Evans (1987), amounted to 0.06, with a rangefrom 0.0 to 0.158, with more then 25 loci analyzed. The mean P99% was22.02%, and ranged between 0 and 54.2%. According to He = 0.097 and P99%= 25% in our studied population, it clearly fits the values of genetic variabilityof non-endangered bird species. H a r t and P u c e k (1994) proposed the useof H/P ratio index, in order to overcome the differences in sample size and lo-ci number analyzed in different mammal populations. In non-endangered birdspecies (calculated from Evans, 1987; regarding 53 studies with more than 24loci analyzed) the mean H/P value was 0.303 and ranged between 0.104 and70.5. Although with H/P value of 0.18, the pheasant population from breedingstation in Ristovaca is slightly above bottom level for non-endangered birdspecies, it still have conserved some level of genetic variability. The lack ofvariability at 15 screened protein loci and genotype frequencies deviation intwo, Pgd and Odh loci, indicate the restricted breeding range. The presence ofinbreeding was also proved by negative FIS = 0.135 and FIT = –0.097 values.Considering the FST = 0.034 value, it can be concluded that pheasant popula-tion analyzed belongs to low genetic differentiated populations.The results of allozyme analysis in Golden Eagle population (S u c h e n -t r u n k et al., 1999) data showed He = 0.034, and P99% = 10.8%, that reveallower genetic variability in natural bird population. It can be due to the factthat in their research, variation of 31 isozyme systems represented by 37 puta-tive loci was examined, but only 15 individuals were analyzed and rare allelesare likely to be missed. Number of individuals examined in our research was43, and this high number of individuals gave us reliable results, because chan-ces to miss rare allele are reduced, comparing with small sample size.The analysis of breeding population of Common Snipe (Gallinago galli-nago) revealed the average values of He = 0.461 and P99% = 80% (P a u l a u -s k a s et al., 2002). Considering the smaller number of loci analyzed in thisresearch (n = 6), we have calculated H/P ratio of 0.576. Comparing with ourresults for pheasants in breeding station (H/P = 0.18) it is clear that our phea-sant population has lower level of genetic variability, comparing to other spe-cies breeding population.Due to fact that common pheasant is a game species widely and increa-singly used for restocking of natural populations depleted by hunting, more ef-fort is done in using highly polymorphic molecular markers, e. g. microsatelli-tes. B a r r a t i et al. (2001) reported on genetic variability detected in phea-sant breeds by means of microsatellites obtained by heterologous amplificationusing primers specific to chicken and turkey. This analysis showed that actualheterozygosity of the pheasant populations was lower then expected underHardy-Weinberg equilibrium (Ho = 0.191 and He = 0.271; Ho = 0.165 and He= 0.210). Same phenomenon occurred in our analyzed population (Ho = 0.0458Figure 1. Zymogrames of polymorphic lociand He = 0.097), with generally lower values, even with a less resolutionpower molecular markers. This was probably the effect of poor genetic mana-gement, e.g. the small number of founders, small population size. Inbreedingcould lead to a rapid loss of individual fitness and genetic variability and redu-ced viability of populations utilized for restocking programs.In order to be able to estimate influence of genetic variability in breedingprogram, it is necessary to continue further study of allozymic variability onwide set of isozyme systems, and in several generations of birds. Because ofthe limited part of genome that can be studied, additional RFLP analysis ofnuclear and mitochondrial genome and DNA sequencing, respectively. Theanalysis of tRNAGlu gene at D-loop region of mtDNA in duck and chickenspecies (L i u et al., 1996) shows greatest sequence divergence in birds, and itcould be relevant marker for estimating pheasant genetic variability.CONCLUSIONThe analysis of allozyme variability of 20 putative gene loci in pheasantpopulation bred in breeding station Ristovaca was detected by polyacrylamidegel electrophoresis (PAGE). Polymorphism was revealed in 5 loci: Est-1, Pgd,Sod, Gpi-2 and Odh. The values of genetic variability measures — heterozigo-sity (Ho = 0.045 and He = 0.097), polymorphism (P99% = 25%), fixation indices(FST = 0.034) and H/P = 0.18 ratio indicate low level of genetic variability andpossible presence of inbreeding depression within pheasant population.ACKNOWLEDGEMENTSWe wish to thank colleagues at breeding station Ristovaca for providingsamples used in the analysis and Hunting Society of Vojvodina for financialsupport of this research.REFERENCESBaratti ,M . , Alberti ,A . , Groenen ,M . , Veenendaal ,T . , Fulgheri ,F .D. (2001): Polymorphic microsatellites developed by cross-species amplificationsin common pheasant breeds, Animal Genetics 32: 222—225.Ã e r a n i ã, A. (2001): Fazan (Phasanius spp.). In: V. Šelmiã, Ed., Podaci o divljaåi,Program razvoja lovstva Srbije, Lovaåki savez Srbije, Beograd, pp 32—37.E v a n s, P. G. H. (1987): Electrophoretic variability of gene products. In: F. Cooke, P.A. Buckley, Eds., Avian Genetics. A Population and Ecological Approach, Acade-mic Press, London, UK,pp 105—162.G i l p i n, M. E., S o u l e, M. E. (1986): Minimum viable populations: Processes ofspecies extinction. In: M. E. Soule, Ed., Conservation Biology, The Science ofScarcity and Diversity, Sinayer Assoc. Inc. Sunderland, MA, pp 19—34.Grillitsch , M . , Hartl , G . , Suchentrunk , F . , Willing , R . (1992): Al-lozyme evolution and the molecular clock in the Lagomorpha, Acta theriologica37: 1—13.9H a r t l, G. B., P u c e k, Z. (1994): Genetic depletion in the European bison (Bison bo-nasus) and the significance of electrophoretic heterozygosity for conservation,Conservation Biology 8: 167—174.L i u, H. T., H u, Y. H., W a n g, C. T., L i n, L. Y. (1996): Sequences and Compari-sons of Duck Mitochondrial DNA Control Regions, Comp. Biochem. Physiol.,Vol. 115B, No. 2: 209—241.M u n s t e r m a n n, L. (1979): Isozymes of Aedes aegypti: Phenotypes, linkage, and useof genetic analysis of sympatric populations in East Africa, Ph. D. Disertation,Univ. Notre Dame.Paulauskas , A . , Svazas , S . , Danileviciute , A . (2002): Multilocus al-lozyme analysis of Common Snipe (Gallinago gallinago) in Lithuania, Acta Zoo-logica Lituanica, Vol. 12, No. 3: 318—332.Selander ,R .K . , Smith ,M .H . , Yang ,S .V . , Johnson ,W .E . , Gentry ,J .B. (1971): Biochemical polymorphism and systematics in the genus Peromyscus I.variation in the old field mouse (Peromyscus polionotus), Studies in Genetics, VI.University of Texas Publication 7103: 49—90.S u c h e n t r u n k, F., H a l l e r, H., R a t t i, P. (1999): Gene pool variability of a gol-den eagle (Aquila chrysaetos) population from the Swiss Alps, Biological Conser-vation 90: 151—155.S w o f f o r d, D. L., S e l a n d e r, R. B. (1989): BIOSYS-1. A computer program forthe analysis of allelic variation in population genetics and biochemical systema-tics. Release 1.7. Users Manual, Champaign: Illinois Natural History Survey.V a p a, M., Š e l m i ã, V., O b r e h t, D., V a p a, Lj. (1999): Allozyme variability innatural populations of hares (Lepus europaeus, Pallas), Zbornik radova Maticesrpske za prirodne nauke 97: 85—91.V a p a, Lj., O b r e h t, D., V a p a, M., Š e l m i ã, V. (2002): Genetic variability inbrown hare (Lepus europaeus) populations in Yugoslavia, Z. Jagdwissenschaft 48:261—266.10GENETIÅKA VARIJABILNOST FAZANA (Phasianus spp.)IZ FAZANERIJE RISTOVAÅAQiqana B. Vapa1*, Mihajla R. Ðan1, Dragana R. Obreht1,Biqana M. Tošoviã-Mariã1, Milan M. Vapa2, Miloš Beukoviã21 Prirodno matematiåki fakultet, Trg Dositeja Obradoviãa 22 Poqoprivredni fakultet, Trg Dositeja Obradoviãa 8;21000 Novi Sad, Srbija i Crna GoraRezimeJedan od moguãih uzroka opadawa brojnosti fazana posledwih godina moÿebiti gubitak adaptibilnosti u populacijama poreklom iz fazanerija usled pa-rewa u srodstvu, koje vodi gubitku genetiåke varijabilnosti i smawewu hetero-zigotnosti. Zbog åiwenice da je adaptabilnost posledica genetiåke strukture,ciq ovog rada bio je analiza genetiåke varijabilnosti populacije fazana izfazanerije Ristovaåa primenom molekularnih markera. Alozimska varijabil-nost 20 genskih lokusa detektovana je poliakrilamid gel elektroforezom (PA-GE). Polimorfnost je registrovana u okviru 5 lokusa: Est-1, Pgd, Sod, Gpi-2 iOdh. Vrednosti mera genetiåke varijabilnosti — heterozigotnost, polimorf-nost, indeksi fiksacije i H/P odnos ukazuju na nizak nivo genetiåke varijabil-nosti i moguãeg parewa u srodstvu u okviru populacije fazana.11

Genetic variability of pheasant (Phasianus spp) in breeding station Ristovača

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