Genetic variability in Scotch pine populations of the Bryansk Region radioactively contaminated in the Chernobyl accident

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<ul><li><p>ISSN 00063509, Biophysics, 2010, Vol. 55, No. 2, pp. 324331. Pleiades Publishing, Inc., 2010.Original Russian Text S.A. Geraskin, J.C. Vanina, V.G. Dikarev, T.A. Novikova, A.A. Oudalova, S.I. Spiridonov, 2009, published in Radiatsionnaya Biologiya. Radioekologiya,2009, Vol. 49, No. 2, pp. 136146.</p><p>324</p><p>INTRODUCTION</p><p>The gene pool of natural populations is incessantlychanging toward states that would be most fit to thecurrent environmental conditions. The adaptability ofa population depends, in particular, on the geneticpolymorphism of the traits involved in selection [1, 2].A chronic stress exposure can alter the magnitude orthe structure of intrapopulation variability [39].Therefore, analysis of these interconnections is promising as regards new tools of environmental monitoring. Besides, it would contribute substantially to elucidating the genetic bases of divergence and adaptationof contemporary natural populations.</p><p>With the advance of electrophoretic analysis,revealing the number and frequency of allelic enzymevariants providing for genetic variability in each locusas well as the homo/heterozygote relation, it is nowpossible to quantitatively assess the genetic variabilityand differentiation of populations in ecologically contrasted areas. Mutations of isozyme loci have codominant inheritance and manifest themselves in firstgeneration seed. This allows the genetic structure of populations to be studied without crossings or genealogies,which greatly shortens the time of analysis without lossof information content. Currently the analysis ofenzyme polymorphism has become a most importanttool for studying genetic processes in natural populations [1013].</p><p>The expanding use of ionizing radiation and radionuclides in various spheres of human activity makestopical the assessment of the ecological consequencesof radioactive pollution. These consequences are real</p><p>ized at the population level, therefore of special interest is the analysis of biological and genetic effects inplant and animal populations on territories contaminated with radionuclides. Longterm studies ofKalchenko and colleagues in the East Urals Radioactive Trail (EURT) [14] and the Chernobyl APP 30kmzone [12] have shown that in plant populations onhighly contaminated sites (dose of several Grayabsorbed in the vegetative period), chronic radiationexposure is an ecological factor affecting the geneticstructure of the populations. However, it is still anopen question what genetic processes take place inpopulations on areas with relatively low contamination. The particular microevolution mechanismsinvolved in populational adaptation also remain notquite clear. There is no full understanding of how theelevated frequency of genetic and cytogenetic aberrations in somatic and germ cells [15] tells on the reproductive capacity, adaptive differentiation, and the general fate of such populations. Likewise, there is noanswer to the question: Why in some radioecologicalconditions a natural population adapts to high radioactive pollution quite rapidly, within a few generations, while in other cases no signs of adaptation areseen even decades later?</p><p>Crosspollinating tree species making large population with high genotypic and phenotypic variabilityand growing in various ecological conditions are aconvenient object for studying the moleculargeneticmechanisms of adaptation. In recent years, conifershave become especially popular as test objects in populationgenetic research [14, 16].</p><p>RADIOBIOLOGYAND RADIOECOLOGY</p><p>Genetic Variability in Scotch Pine Populations of the Bryansk Region Radioactively Contaminated in the Chernobyl Accident</p><p>S. A. Geraskin, J. C. Vanina, V. G. Dikarev, T. A. Novikova, A. A. Oudalova, and S. I. SpiridonovRussian Institute of Agricultural Radiology and Agroecology, Obninsk, 249020 Russia</p><p>Email: stgeraskin@gmail.comReceived September 12, 2008</p><p>AbstractThe method of isozyme analysis of megagametophytes is used to estimate the genetic variabilityin Scotch pine populations (Pinus sylvestris L.) of the Bryansk Region sites with contrasting levels of radioactive contamination (soil 137Cs, 60 to 17 800 Bq/kg) resulting from the Chernobyl accident. All indices ofgenetic variability (heterozygosity, frequency of polymorphic loci, Zhivotovskii index) and frequencies oflossoffunction enzyme mutations increase with the dose absorbed by plant generative organs. The datashow that high mutability is intrinsic for seeds of these pine trees, and genetic diversity in the populations isessentially conditioned by radiation exposure.</p><p>Key words: ChAPP accident, Scotch fir, allozymes, polymorphism, heterozygosity, segregation, phenotypicdiversity, null mutations</p><p>DOI: 10.1134/S0006350910020260</p></li><li><p>BIOPHYSICS Vol. 55 No. 2 2010</p><p>GENETIC VARIABILITY IN RADIOCONTAMINATED PINE POPULATIONS 325</p><p>The aim of the present work was to analyze theisozyme polymorphism in Scotch pine populations inareas of the Bryansk Region radioactively contaminated upon the ChAPP accident.</p><p>EXPERIMENTAL</p><p>Object. Scotch pine (Pinus sylvestris L.) is a majorforestforming species of the Northern Eurasia. Innatural communities, pine is an edificator, definingthe appearance of the phytocenosis and essentiallyinfluencing the life of other plants. Data on the highradiosensitivity of conifers were obtained in early1960s at the Brookhaven lab [17] and confirmed in alargescale Ekos experiment in Southern Urals [18].The most susceptible are the reproductive organs withtheir complex organization and long generative cycle[19]. A distinguishing feature of the conifer reproductive system is that the seed has a haploid endosperm(megagametophyte) genetically identical to thematernal gamete, which allows direct determinationof the haplotype and recessive mutations.</p><p>Region. Test sites were in the Novozybkovskii,Klintsovskii and Krasnogorskii districts of the BryanskRegion (Table 1), chosen on account of forest standhomogeneity and high enough representation of pines(at least 70%) in the phytocenosis, uniformity of soiland climatic conditions, agrochemical properties ofsoils, and the level of soil contamination (2950 to17 800 Bq/kg in 137Cs). The control site was in theVygonichskii district, with soil 137Cs not exceeding60 Bq/kg; exposure rate (ER) varied as 0.430.94 pA/kg. A detailed description has been given previously [20].</p><p>Sampling. Samples of soil and biological material(cones) were taken to determine the contaminationlevels and isozyme profiles. On each site, cones werecollected from 1520 trees of 2050 years within ahomogeneous stand, 3050 cones from each tree at a</p><p>height of 1.52.0 m from the ground. Sampling wasconducted in late November and early December of2005. For maturation and stratification, cones werekept outdoors till the end of February. Then they werekept at reduced humidity and room temperature untilopening and release of seeds, which were dewingedmanually. Only freely released, wellformed seedswere used for electrophoretic analysis.</p><p>Isozyme analysis. Five enzymes were assayed(Table 2). Endosperms were isolated from seeds, 1132 (mean 15) per tree. Each endosperm was homogenized in 75 L of extraction buffer (1% Triton X100,0.2% mercaptoethanol). Extracts were resolved invertical slabs of 7.5% PAG in TrisHCl pH 8.9 on aP9DS2 unit (Owl, USA), with Trisglycine pH 8.3 aselectrode buffer, for 1.53 h at 40 mA. Enzyme activity was visualized as in [21] with modifications. Allozymes were identified by gel mobility. The most frequent allozyme and the corresponding allele of thelocus were designated 1.00, and the others were designated in accordance with their relative mobility. Variants without enzyme activity were denoted n. In segregation analysis the alleles were coded fast (F) and slow(S); inactive variants were disregarded. In total, 4945locus tests were performed.</p><p>Table 1. Test sites in the Bryansk Region</p><p>Site Description ER, pA/kg137Cs in soil, </p><p>Bq/kgD2003, mGy</p><p>D19862008, mGy</p><p>C Control site 4 km away from vlg Alekseevskii (Vygonichskii district), at the left of M3 (Kievskoe highway) leading from Bryansk; edge of mixed forest with prevalent pine aged 5060 and logging plot overgrown with pines aged 1520</p><p>0.430.94 59.8 7.6 0.13 3.4</p><p>VIUA Site just off a local road between vlgs VIUA and Perevoz; edge of mixed forest with prevalent pine aged 5080</p><p>3.65.0 2950 322 7.4 190</p><p>SB Site along a forest road 7 km from vlg Staryie Bobovichi; mixed forest with prevalent pine aged 2060</p><p>8.610.1 4030 439 15.3 390</p><p>ZP Site along the edge of a pine forest 24 km from vlg Zaborie; trees aged 2030 growing on an old logging unit</p><p>12.915.8 17100 1864 28.3 720</p><p>ZK Site along the edge of a pine forest 12 km from vlg Zaborie, 50 m from a graveyard; trees aged 2030</p><p>18.025.2 17800 1940 37.8 970</p><p>Note: ER, exposure rate; D2003, absorbed dose in pine generative organs in 2003; D19862008, absorbed dose accumulated over 19862008.</p><p>Table 2. Enzymes, codes, and number of loci</p><p>Enzyme EC Loci</p><p>Glutamate dehydrogenase (LDH) 1.4.1.2 1</p><p>Leucine aminopeptidase (LAP) 3.4.11.1 2</p><p>Malate dehydrogenase (MDH) 1.1.1.37 4</p><p>Diaphorase (DIA) 1.6.99 2</p><p>6Phosphogluconate dehydrogenase (6PGD)</p><p>1.1.1.44 1</p></li><li><p>326</p><p>BIOPHYSICS Vol. 55 No. 2 2010</p><p>GERASKIN et al.</p><p>Data processing. For every population we calculated the allele frequencies and indices of genetic variability. Expected heterozygosity (Hc) in each locus was</p><p>estimated as Hc = [22], where Pi is</p><p>frequency of the ith allele, N is number of alleles.Observed heterozygosity (H0) was calculated by dividing the number of heterozygous trees by the total number of trees analyzed of this locus. The polymorphismindex (P95) was calculated by dividing the number ofpolymorphic loci by the total number of loci examined; a locus was considered polymorphic if the frequency of the most common allele did not exceed95%. The Zhivotovskii index of intrapopulation diver</p><p>sity () was estimated as = ( + + + )2</p><p>[22]. The significance of differences in allele frequencies between control and test populations was evaluated with a modified 2 test recommended [22] forsmall sample volumes and the presence of rare phenotypes. The test statistic and the number of degrees of</p><p>freedom are calculated as = c2; vG = cv, where c isa correction for the smallness of expected occurrenceof some alleles [22]. Deviation from expected segregation was estimated with the 2 test. Significance of difference between the means was determined by the Students test.</p><p>RESULTS</p><p>Mancaused site contamination. The soils of all testsites are close in agrochemical properties, and thecontent of available mobile forms of heavy metals isbelow admissible limits [20]. The situation is qualitatively different in respect of radioactive contamination, which is mainly represented by 137Cs, the doseforming radionuclide on the territories affected by theChAPP accident. The specific activity of this radionuclide at the most contaminated site ZK is 300 timeshigher than at the control one (Table 1). The soil 137Csat ZP and ZK exceeds the threshold of radiation safetynorias [23].</p><p>The exposures of the pine generative organs wereestimated with a recent dosimetric model [24]. Theabsorbed doses formed by and radiation of 137Cs aregiven in Table 1. By the start of this study, the pine populations have grown for almost 20 years under chroniclowrate irradiation. Note that the annual exposure atthe dirtiest ZK site reached almost 40 mGy. In ourestimates, the dose accumulated from 1986 to 2008 inthe crowns of test trees amounted to 0.21.0 Gy,which agrees nicely with the data of an independentwork [25] at a site very close to one of ours. At all testsites the contribution of radiation into the overalldose rate was about 10%. For pine generative organs,the absorbed dose mostly comes from the 137Cs contained in the upper 10 cm of the soil; the crown radionuclides are less important. Therewith the dose</p><p>2N2N 1 1 pi</p><p>2</p><p>i </p><p>p1 p2 pm</p><p>G2</p><p>absorbed by pine generative organs from radionuclidesdistributed in the forest canopy is formed mainly byradiation.</p><p>Allozyme variability. Electrophoretic analysis offive enzymes has revealed 33 variants, the distributionof which on gels is schematized in Fig. 1.</p><p>Staining for GDH yields a single zone of enzymeactivity, testifying to the monogene control; there arefour enzyme variants, including the null allele. ForLAP there are two activity zones, more mobile Lap1and less mobile Lap2 each with four allozyme variants including null. For MDH, each of the four activity zones has two singleband variants and null. ForDIA, the more mobile zone Dia1 is represented bytwo singleband variants and null, and the less mobileDia2 by one singleband and null. For 6PGD, onepolymorphic zone of activity was represented by foursingleband variants.</p><p>In total, we identified 10 enzyme loci responsiblefor the allozyme diversity in the Pinus sylvestris populations: 6Pgd, Lap1, Lap2, Dia1, Dia2, Mdh1,Mdh2, Mdh3, Mdh4, Gdh. Allele frequencies arelisted in Table 3. Five loci exhibit variability in all populations examined (6Pgd, Lap2, Dia1, Mdh3,Gdh), Lap1 is monomorphic in SB, Dia2 in C andZK, Mdh2 and Mdh4 in C, while Mdh1 shows variability only in ZK.</p><p>The data in Table 3 demonstrate the broad range ofvariability in pine populations. With increasing radioactive contamination, the extent of polymorphismrises from P95 = 0.5 in the control to 0.8 in ZK; thecorrelation with the dose absorbed by critical organs is90.8% (p &lt; 0.05). In all exposures there are significantdifferences from control in allele frequency relationsfor one or several loci (Table 4).</p><p>Segregation of allozymes in pine endosperms. Thegenetic control of enzyme variants was assessed byanalyzing their segregation in endosperms from heterozygous trees. By Mendelian laws, the allozymesencoded by alleles of a monogenic locus should be distributed among such seeds at a 1:1 ratio. Table 5 presents the relationships of the numbers of fast (F) andslow (S) alleles in the progeny of heterozygous pinesand the statistics for deviation from uniform distribution.</p><p>In natural environs, aberrant segregation in pine isquite rare [2628]. Our study has also revealed no reliable deviation in the control population (Table 5),whereas in contaminated sites the segregation wasimpaired for some loci. Thus for 6Pgd a significantdeviation from Mendelian segregation was observed inthree out of four test populations. Significant deviations from the 1 : 1 ratio were noted for Mdh3 in theVIUA and ZK populations, as well as for GDH in ZP.None have been found for Dia1 or Lap2.</p><p>Heterozygosity. The dynamics of the genetic structure of a population is to a large extent determined bythe level of heterozygosity. Both the observed and the</p></li><li><p>BIOPHYSICS Vol. 55 No. 2 2010</p><p>GENETIC VARIABILITY IN RADIOCONTAMINATED PINE POPULATIONS 327</p><p>expected values of pine heterozygosity estimated forten loci increase (Fig. 2) with the degree of contamination (correlation with the absorbed dose was 92.6and 92.0% (p &lt; 0.05) for H0 and Hc respectively), andin all test population both values significan...</p></li></ul>

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