analysis of the polymorphism of organelle dna to elucidate...

ISSN 2079�0597, Russian Journal of Genetics: Applied Research, 2015, Vol. 5, No. 4, pp. 430–439. © Pleiades Publishing, Ltd., 2015.Original Russian Text © E.K. Potokina, A.A. Kiseleva, M.A. Nikolaeva, S.A. Ivanov, P.S. Ulianich, A.F. Potokin, 2014, published in Vavilovskii Zhurnal Genetiki i Selektsii, 2014,Vol. 18, No. 4/1, pp. 818–830.

430

INTRODUCTION

Introgressive hybridization processes occurring inthe territory of the Russian Plain in the populations ofNorway spruce (P. abies (L.) H. Karst.) and Siberianspruce (P. obovata Ledeb.) are intensively studiedusing morphometric and molecular�biological meth�ods. Natural hybrids between P. abies and P. obovata,which are often segregated in a separate taxon ofP. fennica (Regel) Kom. (Finnish spruce), are of inter�est not only for taxonomists but also experts in forestry.In particular, the relationship between the productiv�ity and taxonomy of specimens of Siberian spruce,Norway spruce, and Finnish spruce is reported(Egorov et al., 2011). The problem of developingmolecular markers that could identify the species ofspruce and their possible hybrids is of current interest.

In relatively long�lived organisms, such as woodyconifers, the outline of distribution and the populationgenetic structure of the species reflect the history ofpostglacial migrations (Newton et al., 1999). P. abies isone of the first tree species to colonize Central Europeafter the last glacial period about 12 thousand yearsago (according to Gugerli et al., 2001). It is believedthat Norway spruce managed to survive thanks to sev�

eral refugia. One hypothetical refugium was located inthe European part of Russia, where following theretreating Scandinavian ice sheet the northern lineageof Norway spruce (according to Tollefsrud et al.,2008a) migrated to the west and northwest. Currently,this northern group of spruce populations is well rep�resented in Scandinavia, Western Europe, and theBaltic States (Giesecke and Bennett, 2004). Thesouthern lineage of spruce has spread to centralEurope from the refugia in the mountains of Europe,including the Alps, the Carpathians, and the moun�tains of the Balkan Peninsula. Thus, in WesternEurope, the natural habitat P. abies is currently repre�sented by two distinct areas: the northeastern forestpart and the mountains of Central Europe (Schmidt�Vogt, 1977) (Fig. 1). The Middle Polish Upland has aspruceless zone which may have originated as a resultof the postglacial migration of spruce populations in acounter direction (Dering et al., 2009). The recentlypublished phylogenetic studies of genus Picea, whichwere based on the analysis of organelle and nuclearDNA, suggest that the separation of the northern andsouthern lineages of P. abies had happened much ear�

Analysis of the Polymorphism of Organelle DNA to Elucidate the Phylogeography of Norway Spruce in the East European Plain

E. K. Potokinaa, b, A. A. Kiselevaa, d, M. A. Nikolaevab, S. A. Ivanovb,P. S. Ulianichb, and A. F. Potokinb, c

aVavilov Institute of Plant Industry, St. Petersburg, Russiae�mail: [email protected]

bSt. Petersburg State Forest Technical University, St. Petersburg, RussiacSt. Petersburg State University, St. Petersburg, Russia;

dInstitute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, Novosibirsk, RussiaReceived September 9, 2014; in final form, October 3, 2014

Abstract—The history of Norway spruce distribution in the East European Plain is discussed with regard tothe results of the allele diversity survey of the mitochondrial Nad1 gene, which is maternally inherited, andthe chloroplast trnT�trnF region, which is paternally inherited in spruce. The polymorphism of organelleDNAs was examined in 221 genotypes from 28 regions of the former Soviet Union in geographical prove�nances. Alleles common for the northern Picea abies lineage were detected in spruce trees from all the regionsof the European part of Russia. The Nad1 allele typical of the southern lineage of P. abies was discovered justin spruces originating from Zakarpattia. The Nad1 allele species�specific of P. obovata was found only inspruces from the Sverdlovsk (Ural) and Krasnoyarsk (Siberia) oblasts. Among the trees analyzed, some hadchloroplast DNA sequences (trnT�trnF) characteristic of P. abies, others carried cpDNA haplotypes specificof P. obovata. Analysis of polymorphism of organelle DNA allows revealing the hybrid nature of sprucesresulting from the cross�pollination of different species.

Keywords: phylogeography, organelle DNA, molecular markers, Nad1, trnT�trnF, Picea, geographical prov�enances

DOI: 10.1134/S2079059715040176

RUSSIAN JOURNAL OF GENETICS: APPLIED RESEARCH Vol. 5 No. 4 2015

ANALYSIS OF THE POLYMORPHISM OF ORGANELLE DNA 431

lier, about six million years ago, long before the begin�ning of the last glaciation (Lockwood et al ., 2013).

The phylogeography of conifer species is clarifiedby effectively using the polymorphism of mitochon�drial (mtDNA) and chloroplast (cpDNA) genomes.MtDNA is inherited exclusively maternally, whichmeans the distribution of various versions of mtDNAthrough seeds but not pollen. Because of the lack ofrecombination of mtDNA, a single haplotype is inher�ited as a set of closely linked loci (Minchenko andDudareva, 1990). A study of the polymorphism ofmitochondrial genes is a good basis for studying theprocesses of migration and geographical dispersion ofpopulations of this species (Sperisen et al., 2001).CpDNA is also of interest for phylogenetic studies dueto the peculiarities of its inheritance. It is proved thatcpDNA in family Pinaceae is paternally inherited(Sears, 1980; Neale and Sederov, 1989; Neale et al.,1991; Sutton et al., 1991). Summarizing the dataobtained using the markers of cpDNA and mtDNAallows one to estimate the relative contribution of pollenand seeds in the total flow of genes, to identify parentforms of hybrids and thus to monitor processes of intro�gressive hybridization between P. abies and P. obovata.

The aim of our study was to use the results of theanalysis of the polymorphism of the mitochondrialgene Nad1 and intergenic spacer trnT�trnF of cpDNAon samples of spruce populations from differentregions of the European part of Russia to determinethe maternal inheritance of introgression formsP. abies–P. obovata inhabiting the territory of theEuropean part of Russia and to assess the incidence ofhybrid spruce resulting from the cross�pollination ofindividuals of P. abies by the pollen of P. obovata.

MATERIALS AND METHODS

As the object of research we used spruce trees fromgeographical provenances established in the 1970s in var�ious regions of the European part of Russia for the pur�pose of developing and improving the forest�seed zoning.The provenances were established in 1977 according to asingle state procedure. The test included 35 cli�matypes—spruce populations from different regionsand republics of the former Soviet Union. The latitu�dinal difference between the most distanced popula�tions was 19.70° (Murmansk Oblast–Zakarpattia) andthe longitudinal distance was 43.00° (Lithuania–Sver�dlovsk Oblast). Seeds of these populations were sownin different climatic zones of Russia as structuredplantations, which are geographical provenancestoday. One of the studied variants of such geographicalprovenances is located in the Luban Forestry area (theTosno Forestry area, Leningrad Oblast) and anothervariant is located in the Karaidel Forestry area (SouthUral forest�steppe region, Republic of Bashkor�tostan). Geographical provenances are a convenientobject of study of intraspecific genetic diversity as theyare a representative sample of spruce populations from

different regions of the European part of Russia grownunder identical conditions. The study included 28 spruceclimatypes (Table 1). In addition, spruce samples col�lected during an expedition to Krasnoyarsk Krai(Stolby Nature Sanctuary) were tested to increase thenumber of samples of pure species of P. obovata.

Polymorphism Analysis of PCR Products

As a template, molecular genetic analysis usedDNA isolated by CTAB according to Bousquet et al.(1990) from fresh pine needles collected in the geo�graphical provenances of P. abies, P. obovata, and theirhybrids growing in the Luban and Karaidel forestries.

Alleles of the mitochondrial Nad1 gene containingtandem minisatellite repeats were analyzed by poly�merase chain reaction (PCR) using a thermal cyclerGeneAmp PCR system 9700 (Applied Biosystems,Unite States). A reaction mixture of 20 μL contained1× buffer for Taq�polymerase (pH 8.6, 2.5 mM Mg2+)(Silex, Russia), 200 μmol dNTPs, 0.5 μmol of eachprimer, 1 unit of Taq�polymerase (Dialat, Russia), and2 μL (30–60 ng) of the target DNA.

PCR was carried out using primers published byGugerli (2001): forward, CTCTCCCTCACCCATAT�GATG; reverse, AGATCCCCATATATTCCCGG,also PCR was carried out in the mode recommendedby the author: predenaturation for 3 min at 94°C, then26 cycles (1 min at 94°C, 1 min at 57°C, and 2 min at72°C), and the final step of 5 min at 72°C. The result�ing PCR products were digested with EcoRV toshorten the domain containing repetitive elements.This allows more accurately estimating the size of theamplified sequence (Tollefsrud et al., 2008a). Restric�tion with EcoRV was carried out in a volume of 15 μLcontaining 3 μL of the PCR product, 10.25 μL of

N

S

Fig. 1. Distribution of P. abies in Europe with a sprucelesszone in the Middle Polish Upland (according to Shmidt�Vogt, 1977).N and S are the areas of distribution of the northern andsouthern lineages of Norway spruce demarcated by aspruceless zone according to the analysis of mtDNA(Tollefsrud et al., 2008a).

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water, a 1.5 μL buffer, and a 0.25 μL (1 unit) restrictionenzyme for 3 hours at 37°C .

The size of restriction fragments was analyzedusing an automatic station of high resolution capillaryelectrophoresis—QIAxcel System Capillary Electro�phoresis (Qiagen). When using QIAxcel, the length ofthe fragments was calculated using internal standardsin the form of alignment markers (QX AlignmentMarker 15 bp/3 kb) setting the upper (3000 bp) andlower (15 bp) thresholds of detection. Another internalstandard, a set of DNA fragments of known size(QX Size Marker 25 bp/1.8 kb), which differ in lengthby 25 nucleotides, was used simultaneously.

PCR fragments that differ in length according tothe results of the restriction analysis were sequenced intwo replications (Evrogen, Moscow) using forwardand reverse primers. The DNA sequences were alignedusing BioEdit 7.1.

In order to analyze the polymorphism of the inter�genic spacer trnT�trnF of cpDNA, we used universalprimers of Taberlet et al. (1991): a, CATTACAAAT�GCGATGCTCT; and d, GGGGATAGAGGGACT�TGAAC. The reaction mixture of PCR of 50 μL con�tained 1× buffer for Taq�polymerase (pH 8.6, 2.5 mMMg2+) (Sileks, Russia), 200 μM dNTPs, 1 μM of eachprimer, 5 units of Taq�polymerase (Dialat, Russia),and 2 μL (60–80 ng) of the target DNA. PCR was car�ried out under the following conditions: predenatura�toin for 5 min at 94°C, then 35 cycles (45 s at 94°C,1 min at 55°C, and 2 min at 72°C), and the final stageof 5 min at 72°C. The PCR product was sequenced intwo replications using primers a and d.

RESULTS

Analysis of Polymorphism of Mitochondrial Gene Nad1

The mtDNA polymorphism in spruce was ana�lyzed using the markers developed for gene Nad1encoding the first subunit of a key protein of the respira�tory�chain NADH�dehydrogenase (NADH dehydroge�nase subunit 1) (Sperisen et al., 2001). Nad1 has a differ�ent polymorphic sequence of the second intron, whichcontains tandem repeats of 34 and 32 nucleotides (mini�satellites). A section of 34 nucleotides may be repeatedfrom 0 to 10 times in different individuals, and its adja�cent element of 32 nucleotides is characterized by asimilar copy number. In addition, there are 11 addi�tional polymorphic sites flanking the tandem repeats,five of which can affect the size of the fragment(Sperisen et al., 2001). An earlier analysis of polymor�phism of the second intron of mitochondrial geneNad1 in P. abies (Tollesfrud et al., 2008a) revealedalleles specific to the northern and southern lineagesof Norway spruce, as well as an allele species�specificfor P. obovata. All the identified alleles differ in the lengthof the amplified fragment, which varies from 712 bp(P. obovata) to 1027 bp (northern lineage of P. abies).

In our study, the structure of the second intron ofgene Nad1 was analyzed in 221 spruce specimensgrowing in geographical provenances of the Luban andKaraidel forestries, as well as three samples of spruceP. obovata from Krasnoyarsk Krai. The primary anal�ysis was carried out using PCR followed by restrictingthe product by endonuclease EcoRV (Fig. 2) for moreaccurate estimation of the size of the amplified frag�ment. After visualization of the restriction products on1.5% agarose gel their exact size was analyzed using ahigh�resolution capillary electrophoresis (QIAxcel).In total, the analysis of 221 trees revealed nine Nad1alleles differing in length that were sequenced. Theresults of sequencing the identified alleles are shown inFig. 3 and summarized in Fig. 4.

Seven of the nine Nad1 alleles differing in thelength of tandem repeats and nucleotide insertions�deletions of different extension (Fig. 4) have beendescribed previously by Tollefsrud et al. (2008a) ascharacteristic of the northern lineage of P. abies.Another allele (815 bp) described previously for thesouthern lineage of Norway spruce (Tollefsrud et al.,2008a) has been detected in trees grown from seedscollected in Zakarpattia, that is, the most southernarea of those represented in the geographical prove�nances and included in the study. Nad1 allele specificfor P. obovata was detected only in samples of sprucecollected in Krasnoyarsk Krai (Stolby Nature Sanctu�ary) and a single tree of the 36 representatives of theSverdlovsk climatype of spruce specimens studied inthe geographical provenances. This species�specifictype for the P. obovata variant of the sequence of thesecond intron of gene Nad1 of 712 bp has beendescribed previously by Tollefsrud et al. (2008a). As aresult of sequencing, it differs from all the known alle�les of the northern lineage of P. abies by the deletion ofnine nucleotides and two nucleotide substitutions. It

Fig. 2. Mitochondrial gene Nad1 alleles identified in P. abiestrees in the geographical provenances of the Luban andKaraidel forestries using gene�specific PCR (a) and the sub�sequent restriction of the PCR product with EcoRV (b).The identified Nad1 alleles: 1, 2, 3, 6, and 10—721 bp;4 and 5—789 bp; 7—815 bp; 8—857 bp; 9—755 bp;11 and 12—823 bp.

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POTOKINA et al.

can be distinguished from the southern lineage ofP. abies also by two deletions (Fig. 4).

Thus, for the polymorphism of the mitochondrialgene Nad1, the entire diversity of the spruce speci�mens presented in the studied sample can be reducedto three clusters corresponding to population groupswhich probably share a common origin. The northernlineage of P. abies widely represented in the countriesof Northern Europe integrates spruce growing inalmost all regions of the European part of Russia. Thesouthern lineage of P. abies comprises spruce samplesfrom Zakarpattia. Species P. obovata, according to theresults of marking the mtDNA, comprises all speci�mens of spruce from the Stolby Nature Sanctuary(Krasnoyarsk Krai) and a single specimen in the geo�graphical provenances which originated from the mosteastern point of Sverdlovsk Oblast that is presented inthe analysis (pos. Tavda, 65°18′ E).

The distribution of Nad1 alleles among differentclimatypes of spruce in the geographical provenancescan be seen from the table. The most common allele is721 bp in length and is typical of spruce populations ofthe NorthWest and Central regions of Russia; also itdominates populations of the northern lineage ofP. abies in the Nordic countries (Tollefsrud, 2008a).

The remaining alleles of gene Nad1 are presented muchless and do not show any geographical confinement.

Analysis of the Polymorphism of the Intergenic Spacer trnT�trnF of cpDNA

As a result of PCR with universal primers describedby Taberlet et al. (1991), the section of the intergenicspacer trnT�trnF of cpDNA of 1031 bp in length isamplified. Four different versions of the structure ofthis section of cpDNA (haplotypes) have been identi�fied earlier in the analysis of 50 populations of sprucefrom the territory of the East European Plain, which isconsidered the area of introgressive hybridization ofspecies P. abies and P. obovata (Tollefsrud and Spi�rensen, 2011). According to the authors, Siberianspruce is characterized by its own set of species�spe�cific haplotypes of the intergenic spacer trnT�trnF.These nucleotide sequences have been publishedrecently (Volkova et al., 2014) and are currently avail�able from the database GenBank (GenBank Acc.KF896139 and KF896142). Haplotypes of spacertrnT�trnF of Norway spruce and Siberian spruce differby four different polymorphic sites, whereas the chlo�roplast genome of P. abies is characterized by haplo�types _CCC, _CAC, _TCC, GCAC, and GCCC, and

P. abies (Northern) 721 bpKF896145


P. abies (Northern) 789 bpKM588355

P. abies (Northern) 823 bpKM588356




P. abies (Southern) 815 bpKM588357

P. obovata 712 bpKM588358

EcoRV

EcoRV

EcoRV

EcoRV

EcoRV

EcoRV

EcoRV

EcoRV

EcoRV

GA A AG

GA A AG

GA A AG

GA A AG

GA A AG

GA A AG

GA A AG

TC C CG

TC A AG

1 bp101 bp (69 + 32)

34 bp (repeat)

GATATC

9 bp

Fig. 4. Variants of the structure of the second intron of mitochondrial gene Nad1 which were identified in the analysis of sprucespecimens in the geographical provenances of the Luban and Karaidel forestries.The triangle denotes an EcoRV restriction site. Sequences KM588355, KM588356, KM588357, and KM588358 are presentedfor depositing in the database of the National Center for Biotechnology Information (NCBI).



that of P. obovata by haplotypes GCCA and _CCA(Tollefsrud et al., 2008b, cited from Volkova et al.,2014)). Similar conclusions were previously made byRan et al. (2006), who compared the sequence of theintergenic spacer trnT�trnF of cpDNA in species P. abiesand P. obovata (DQ358149 and DQ0106), havingdescribed two species�specific polymorphic sites.

In order to determine how common thesedescribed species�specific haplotypes of the intergenicspacer trnT�trnF are among spruce trees growing in theEast European Plain of Russia, in our study wesequenced the intergenic spacer trnT�trnF in 52 sprucesof different climatypes represented in geographicalprovenances, as well as in three spruces from Krasno�yarsk Krai, which represents in this analysis the purespecies P. obovata. Among the 55 analyzed spruces,37 trees revealed cpDNA haplotypes specific ofP. abies and 18 trees revealed cpDNA haplotypes typ�ical of P. obovata.

The analysis identified individuals that combineP. abies alleles in both the mitochondrial and chloro�plast genome, which in theory can be attributed to thepure species P. abies. Also we found spruces whichcarry mtDNA alleles specific of P. abies and cpDNAalleles specific of P. obovata, which may indicate theirhybrid origin. Thus, according to the results of thecombined analysis of mtDNA and cpDNA, sprucetrees from Komi Republic, as well as Kaluga, Kirov,Arkhangelsk, and Mogilev oblasts, were grown fromseeds set as a result of the cross�pollination of Norwayspruce by pollen of Siberian spruce (table). No geo�graphic patterns in the prevalence of pure P. abies orhybrids P. abies × P. obovata were revealed in the ana�lyzed climatypes of the geographical provenances.According to the results of marking the organelleDNA, only spruce samples from Krasnoyarsk Krai(Stolby Nature Sanctuary) can be referred to as thepure species of P. obovata, as the mtDNA and cpDNAof these samples are characterized by haplotypes typi�cal of P. obovata.

DISCUSSION

The history of the distribution of P. abies in WesternEurope during the Holocene is now a fairly clear pic�ture thanks to the works on the population geneticsand molecular phylogeography of spruce based on theanalysis of the polymorphism of organelle DNA genes(Gugerli et al., 2001; Sperisen et al., 2001; Tollesfrudet al., 2008a). Previously identified patterns of forma�tion of the range of Norway spruce in Central andWestern Europe are maintained in the East EuropeanPlain: almost all regions of the European part of Rus�sia are dominated by genotypes of the northern lineagedescribed by Tollesfrud et al. (2008a). Representativesof the southern lineage—descendants of the sprucetrees that survived the last glaciation in the refugia ofthe Balkan Peninsula (Huntley and Birks, 1983)—are

found only in populations of Zakarpattia. It remainsunknown how far this clade has spread to the northand north�east of the Carpathian Mountains andwhere the border of the distribution of the southernand northern lineages of Norway spruce on the terri�tory of Russia now lies. To date, there are also no workson the phylogeography of the P. obovata species withthe use of methods of marking the organelle DNA.Perhaps this species has its own history of postglacialmigrations, which could be reconstructed in the studyof the polymorphism in mitochondrial and chloro�plast genomes. The obtained data may be of interest indeveloping a strategy of conservation of forest geneticresources in Siberia.

Processes of introgressive hybridization of Euro�pean and Siberian spruce occurring in the territory ofthe East European Plain are of interest from an evolu�tionary point of view (Popov, 2010) and are importantfor the development of genetic and breeding pro�grams, as well as activities for the protection and sus�tainable use of biological resources (Politov, 2007).The specificity of the uniparental inheritance of mito�chondrial and chloroplast genomes in conifers can beused to develop DNA markers that could identifyhybrid individuals resulting from the cross�pollinationof P. abies by the pollen of P. obovata, which undoubt�edly would find its application in breeding practice andseed zoning. Inheritance of the mitochondrial genomein P. abies strictly maternally was confirmed by specialstudies in controlled crosses (Bobola et al., 1996; Grivetet al., 1999) and the parental inheritance of cpDNAmarkers in spruce was proved in experiments carried outto obtain interspecific spruce hybrids of P. mariana andP. rubens (Bobola et al., 1996), P. pungens andP. glauca (Stine et al., 1989), and P. engelmannii andP. sitchensis (Sutton et al., 1991). Using a combinationof these markers could allow identifying the hybridnature of plants that have developed as a result of thecross�pollination between individuals of different spe�cies of spruce.

The question of the species�specificity of the hap�lotypes of the intergenic spacer trnT�trnF of cpDNA,which were specified as typical of P. obovata spruces ofSiberian origin (Tollefsrud and Spirensen, 2011) andlater described by P. Volkova (Volkova et al., 2014) onthe material from Karelia remains unclear. The avail�able published data, unfortunately, do not conclusivelyanswer the question on how reliably the describedhaplotypes GCCA and _CCA of the intergenic spacertrnT�trnF are associated with the P. obovata species.The available information is limited to a short message(Tollefsrud and Spirensen, 2011) and links to anunpublished dissertation (Tollefsrud, 2008b, citedfrom Volkova et al., 2014). Nevertheless, in our study,the use of the markers of organelle DNA proposed bythese authors confirms the previously publishedreports of the intensive processes of the introgressivehybridization of Siberian and Norway spruce on the

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territory of the East European Plain (Shcherbakova,1973; Popov, 2010; Il’inov et al., 2011). According tothe analysis of organelle DNA polymorphism in asample of trees presented in the geographical prove�nances, populations of spruce in the European part ofRussia have been formed by genotypes P. abies andhybrid forms of P. abies × P. obovata. Both of them canbe identified for their specific differences in thesequence of mitochondrial and chloroplast DNA. Theresults of our study confirm the previously publishedreports that the native species of P. obovata is probablynot present in the European part of Russia and isfound to the east of the Ural Mountains (Tollefsrudand Spirensen, 2011). The obtained findings are basedon the analysis of population samples presented in thegeographical provenances of spruce. A more accuratepicture of the distribution of hybrid forms of spruce inthe European part of Russia and the information aboutthe geographical gradient of the incidence of interspe�cific hybrids requires a more detailed analysis of thenatural populations.

ACKNOWLEDGMENTS

The study was supported by the Russian Founda�tion for Basic Research under research project no. 14�04�01418a and scientific project no. 37.1521.2014/Kof the Ministry of Education of the Russian Federa�tion as part of a state�assigned project.

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Translated by K. Lazarev

analysis of the polymorphism of organelle dna to elucidate...

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