Research Article Application of Microsatellite Loci for

Research ArticleApplication of Microsatellite Loci for MolecularIdentification of Elite Genotypes Analysis of Clonality andGenetic Diversity in Aspen Populus tremula L (Salicaceae)

Dmitry V Politov1 Maryana M Belokon1 Yuri S Belokon1

Tatyana A Polyakova1 Anna V Shatokhina1 Elena A Mudrik1 Anna B Azarova2

Mikhail V Filippov2 and Konstantin A Shestibratov2

1Vavilov Institute of General Genetics Russian Academy of Sciences Gubkin Street 3 Moscow 119991 Russia2Shemyakin amp Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences Pushchino BranchPushchino 142290 Russia

Correspondence should be addressed to Dmitry V Politov dmitrip17gmailcom

Received 31 October 2015 Accepted 2 December 2015

Academic Editor Viktor Korzun

Copyright copy 2015 Dmitry V Politov et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Testing systems for molecular identification of micropropagated elite aspen (Populus tremula L) genotypes were developed onthe base on microsatellite (SSR) loci Out of 33 tested microsatellite loci 14 were selected due to sustainable PCR amplificationand substantial variability in elite clones of aspen aimed for establishment of fast-rotated forest plantations All eight tested cloneshad different multilocus genotypes Among 114 trees from three reference native stands located near the established plantations 80haplotypes were identified while some repeated genotypes were attributed to natural clones which appeared as a result of sproutingThe selected set of SSR markers showed reliable individual identification with low probability of appearance of identical aspengenotypes (a minimum of 48 sdot 10minus10 and 1 times 10minus4 for unrelated and related individuals resp) Case studies demonstrating practicalapplications of the test system are described including analysis of clonal structure and levels of genetic diversity in three naturalaspen stands growing in the regions where plantations made of elite clones were established

1 Introduction

The continued degradation of native forests worldwide dueto the overexploitation requires introduction of intensiveforms of forestry that would favor not only economic effectbut also conservation of woody plant genetic resources Thistask declares obtaining and maintenance of elite genotypesof forest trees aimed for fast-rotated forest plantations Newhighly productive and sustainable to abiotic factors pests andpathogens cultivars and varieties appear as a result of breed-ing selection of mutations andor genetic engineering Theclonal propagation of such outstanding individuals ensuresfixation of their useful traits both for further breeding exper-iments and for establishment of targeted forest plantations Inparticular microclonal propagation via in vitro culture allowsfor rapid large-scale and cost-effective cloning of individuals

with desired traits especially when traditional budding orgrafting is impossible or requires special efforts [1ndash3]

Since morphologically and anatomically different plantclones may look similar it is essential to reliably identifythose including discrimination from each other and fromconspecific individuals or representatives of other closelyrelated species In forestry the problem of individual identi-fication is especially crucial since the external look of treesis highly dependable on environmental parameters [4] Atparticular stages of natural ontogenesis of forest trees forexample seedlings and saplings and especially as calluses orexplants in vitro cell culture individuals may be indiscerniblenot only individually but also as far as species diagnoses areconcerned Long generation time and ontogenetically latematuration makes the task of genetic passportization of eliteclones in woody plants real The complex multistage process

Hindawi Publishing CorporationInternational Journal of Plant GenomicsVolume 2015 Article ID 261518 11 pageshttpdxdoiorg1011552015261518

2 International Journal of Plant Genomics

of obtaining propagation and introduction of elite cultivarsby breeding or genetic engineering increases the probabilityof various errors

Molecular genetic markers (MGM) proved to be veryefficient tools for individual identification Among differentMGM classes microsatellites or simple sequence repeats(SSR) fit best to requirements of testing systems for identifica-tion due to their specificity codominance selective neutralitysufficient allelic richness and heterozygosity caused by highmutation rate Moreover due to relative genome conser-vatismwithin genera and families of plants SSR-markers andPCR primers for their amplification can be transferrable fromone taxon to another

In several poplar species the successful DNA fingerprint-ing and differentiation of clones cultivars and varieties weredemonstrated bymolecularmarkers including SSR loci [5ndash9]

In this paper we report on the development of SSR-based testing system for molecular genetic identification ofelite micropropagated genotypes of aspen Populus tremulaL aimed at the establishment of fast-growing target forestplantations in several regions of Russian Federation andpresent the results of its application for individual genotypesdiscrimination studies of clonal structure in natural aspenstands and estimation of genetic variability in populations

2 Material and Methods

The development of the testing system for the identificationof elite genotypes comprised the selection of a specificmarkerset fitting the requirements of high-resolution discriminationof clones and testing its reliability and identification power ona set of elite genotypes and a sufficient number of representa-tives of a studied species

21 Plant Material Elite aspen and hybrid clones used fordevelopment of testing systems for molecular identificationhave been obtained from micropropagated cell cultures [10]maintained in the Pushchino branch of Shemyakin andOvchinnikov Institute of Bioorganic Chemistry RussianAcademy of Sciences (Pushchino Russia) Origin and shortdescription of eight clones are presented in Table 1

Experimental aspen clonal plantations derived from theseelite genotypes were established in four regions of Europeanpart of Russia (Figure 1) Native aspen stands located closelyto these plots were used for studies of clonal structureevaluation of frequencies of multilocus genotypes and cal-culations of probabilities of appearance of identical alleliccombinations in a single genotype

Prisady Experimental plot located 1 km westward of settle-ment Prisady in Serpukhovsky Raion (district) of Moscowoblastrsquo (Russia) Leaves of 52 young ormedium-aged (approx-imately 10ndash25 yo) aspen trees from a naturalmultiaged standlocated in close proximity (1 km) to this plot have been usedas a reference population

Voronezh Seventeen trees were sampled in a stand adjacentto the experimental plot established in Voronezh Oblast

1

2

3

Figure 1 Map of location of plantations made of elite clones andcorresponding native aspen stands used as reference populations (1)Prisady (2) Voronezh and (3) Yoshkar-Ola

Additional 13 trees were collected along the bank of VoronezhRiver within the city of Voronezh close to the plantation

Yoshkar-Ola Young native stand of aspen was the source oftrees used as a reference population for an experimental plotlocated near the city of Yoshkar-Ola Republic of Mari-ElLeaves from 32 trees were collected

In order to minimize the occasional sampling of indi-viduals having vegetative origin (through sprouting) wecollected leaves from trees at a distance not less than 15ndash20mfrom each other This approach was employed for inclusioninto reference samples of predominantly open-pollinatedseedlings having maximal genetic diversity However basedexclusively on external look of the trees and distance amongthem sampling of the ramets appearing as a result of sprout-ing could not be avoided and subsequent genetic analysisconfirmed this

Shoots with leaves were cut off by means of mechanicalcutter with an aluminium telescopic mast Collected shootswith leaves were placed into plastic bags for no morethan 6 days at +4∘b until processing Sample preparationincludedDNA extraction and placement of reserve leaf tissuefragments into labeled zip-bags with silica gel for long-termstorage Outside the period of active vegetation dormantvegetative buds can be successfully used for DNA extraction

22 DNA Extraction Fragments of leaves approximately350ndash500mg taken from explants growing in vitro on a specialmedia (REF) in Petri dishes were used for DNA extraction

For trees from native populations we extracted DNAfrom 350ndash500mg fragments of fresh or 200ndash300mg of silica-dried leaf tissues by a modified cethyltrimethylammoniumbromide (CTAB) method [13 14]

23 Microsatellite Analysis Microsatellites or SSR representa class of tandemly repeated DNA sequences with short (1ndash6 pairs of nucleotides bp) motifs differing in copy numbersamong individuals due to high mutation rate Multiple allelesusually found in codominant microsatellite loci create a great

International Journal of Plant Genomics 3

Table 1 Elite aspen and hybrid clones and their characteristics

Originalgenotype

Putativespecieshybrid

identityOrigin Description

Clones and clonal lineagesobtained based on original

genotypes

PtV22 PutativelyPopulus tremula

Minsk Oblast Belarus breedingform obtained in Institute ofForest National Academy ofBelarus Gomel Belarusprovided by V E Padutov

Diploid green-bark aspen formCharacterized by fast growth andresistance to heart rot caused bypathogen fungus Phellinustremula

Ptv22-1 Ptv22-2 Ptv22-321mut 2mut 14mut 4mut

12mut

Pt P tremula

Leningrad Oblast Russiabreeding form obtained in StPetersburg Research Institute forForestry provided by D AShabunin

Diploid giant aspen formCharacterized by fast growth andresistance to heart rot caused bypathogen fungus Phellinustremula

Pt2 Pt3

F2 P tremula

Kostroma Oblast Russiabreeding form obtained by S NBagayev provided by D AShabunin

Diploid female clone Highlyproductive (plus 51 by sum ofstem cross section squares andplus 43 by growing stock)Increased wood density of475 kgm3[11]

F2-1 F2-2 F2-3

47 P tremulaLatvian State Forest ResearchInstitute ldquoSilavardquo Latviaprovided by Dr Arnis Gailis

Diploid aspen form Productivityof 180ndash200m3haat age 12 underdensity of 1100 stemshaCharacterized by resistance toheart rot caused by pathogenfungus Phellinus tremula [12]

47-1 47-1-1-31 47-1-1-2247-1-2-27 47-1-1-19

47-1-2-53

b-control P tremuloides timesP tremula -ldquo-

Diploid hybrid formProductivity of 180ndash200m3ha atage 12 under density of1100 stemsha [12]

b-1 b-2 b-3

23 P tremuloides timesP tremula -ldquo-

Diploid hybrid form Maximalproductivity of 200ndash250m3hademonstrated at age 12 underdensity of 1100 stemsha [12]High wood density

L23-1 L23-2 L23-3


Maximal productivity of250ndash300m3ha demonstrated atage 12 under density of2500 stemsha [12]

L4-1 L4-2 L4-3

No 3-understory P tremula

Republic of Tatarstan breedingform by A H Gaziulllinprovided by N R Garipov

Triploid aspen formCharacterized by fast growth andresistance to heart rot caused bypathogen fungus Phellinustremula

No 3-understory-1

variety of unique genotypic combinationswhich ensures theirreliable identification especially when a sufficient numberof loci are employed Technically the analysis of SSR poly-morphism requires only Polymerase Chain Reaction (PCR)and subsequent electrophoresis (gel or capillary) for fragmentanalysis For the development of the testing system we chosethe variant of the method that utilizes only basic equipmentand simple reagents This ensured increased reproducibilityof the procedures in any PCR laboratory and allowed achiev-ing high cost-effectiveness of the analysis which is crucial forlarge-scale practical clone identification

Since the substantial number of nuclear SSR loci forpoplars was found in the literature and primer databases wedecided to select from several publications and test primers

that can be used for routine genotype identification based onvery simple equipment without using of DNA-analyzers Aninitial set of SSR primers for their potential use as elementsof the testing system of molecular identification in aspenwas a result of search in bibliographical databases (Thom-son Reuters Web of Science httpwebofknowledgecom)and in Molecular Ecology Resources Primer Database(httptomatobiotrinityedu)

DNA amplification was performed using PCRCore kits(Isogen Laboratories Ltd Moscow Russia) in BioRad Inc(USA) Dyad Thermo cycler Microsatellite loci (listed inTable 2) from series ORPM [15] andWPMS [16] were ampli-fied with specific primers at concentration of 1mmolmLand 5ndash10 ng of target DNA using the following temperature


Table 2 Microsatellite loci tested for PCR amplification in aspen

Loci Primer sequences (51015840- 31015840) Repeat motif Fragment size (bp)3

ORPM141 F- GGGCTGCAGCAGATATTGAR- CCAAAGGAACCCAAAGAAGA (GCTC)

4

146ndash162

ORPM181 F- AGCAGAGATCGATGCTGAGGR- AATTTCTCGCTTCTCGCATT (TTTA)

4

205

ORPM361 F- AGCCTCCAAACACCATGAACR- ACAGTGGTGTGGATCCTGCT (GAAA)

4

213

ORPM601 F- ATAGCGCCAGAAGCAAAAACR- AAGCAGAAAGTCGTAGGTTCG (AAT)

5

212

ORPM791 F- GAAGCTGAAAACAACAACAAACAR- GGGTTTTTAACATAATAAAAGCTTGG (AAT)

4

160

ORPM811 F- GCTGCAGCCAAACAAAGCR- CAGAAATCCCACCCAAACC (TATT)

4

142ndash158

ORPM841 F- CTGCAGCCTTACCACCATTTR- CGTGTTCGAGATTGGGATTT (AAG)

4

171

ORPM861 F- CCACATCCATAGCTCTGCAACR- GTACTACCTCGCCTGCCAAC (CTT)

5

204

ORPM1071 F- AATCTGGTGGCTTGCCTCTR- TTGAGGAACACGTGCAGACT (TAAA)

4

190

ORPM1171 F- CCCCCTAATTACCTTGGAAACR- TTGTTTGTATCTCCTCCGTTGA (ATTA)

4

210

ORPM1581 F- GCTGAAACATCCTTCATGGTCR- CGAAGCTGCATAAGCATCAA (TTTC)

4

200

ORPM1931 F- CCGCTGGATTTGTTTGTTTTR- TGAGCAGAAAGATGCGAAGA (ATTTT)

4

187ndash207

ORPM2021 F- TCGCAAAAGATTCTCCCAGTR- TTCAAATCCCGGTAATGCTC (TAA)

5

184ndash190

ORPM2061 F- CCGTGGCCATTGACTCTTTAR- GAACCCATTTGGTGCAAGAT (GCT)

7

190ndash208

ORPM2201 F- AGCTAGCCTGTCGTCAAGGAR- CAAGGAAGCATTCTCGCAAT (TTTA)

6

178ndash222

ORPM2961 F- CGAAGCCATTGACCCAGTATR- GGGCATCTCTCTCCTTTCAA (GTTCTG)

4

199

ORPM3121 F- GTGGGGATCAATCCAAAAGAR- CCCATATCAAACCATTTGAAAAA (CCT)

6

194

ORPM3661 F- CCTTGAGGGGACACTTCGATR- AAAGAGTTGAGCCCTTGGTG (TTA)

5

156

ORPM3711 F- CCGGACTCTCACAAATCTCCR- TGCTTTTGCTCCTGGTTCTT (TCTT)

6

192ndash200

ORPM3721 F- AGCTCTTCTGCTGGTGCTGTR- GAGGGAGGGAGGGTAAAAGA (TCTT)

5

190

ORPM4151 F- CTCGGTGCAAATATCGGTTCR- AGATCGATGGTCCTTTCCTG (GGCG)

4

225

ORPM4841 F- CAAAATGGCAATCCAAGGTTR- CCAAGCTTCCAATTGAGTCC (TTAA)

4

190ndash206

ORPM4881 F- CTCCAGCCGCTTCTATCCTTR- TGTCGTGGGAAAGAACCAGT (TTA)

6

200

ORPM4961 F- CAGCAGTGCAAGCTCCTAAAR- GGCCACTGACAGAGACCAAG (GGA)

4

185

WPMS142 F- CAGCCGCAGCCACTGAGAAATCR- GCCTGCTGAGAAGACTGCCTTGAC (CGT)

28

215ndash287

WPMS152 F- CAACAAACCATCAATGAAGAAGACR- AGAGGGTGTTGGGGGTGACTA (CCT)

14

201ndash219


Table 2 Continued


WPMS162 F- CTCGTACTATTTCCGATGATGACCR- AGATTATTAGGTGGGCCAAGGACT (GTC)

8

139ndash184

WPMS172 F- ACATCCGCCAATGCTTCGGTGTTTR- GTGACGGTGGTGGCGGATTTTCTT (CAC)

15

122ndash146

WPMS182 F- CTTCACATAGGACATAGCAGCATCR- CACCAGAGTCATCACCAGTTATTG (GTG)

13

219ndash248

WPMS192 F- AGCCACAGCAAATTCAGATGATGCR- CCTGCTGAGAAGACTGCCTTGACA (CAG)

28

180ndash234

WPMS202 F- GTGCGCACATCTATGACTATCGR- ATCTTGTAATTCTCCGGGCATCT (TTCTGG)

8

210ndash222

WPMS212 F- TGCTGATGCAAAAGATTTAGR- TTGGAACTTCAACATTCAGAT (GCT)

45

287ndash326

WPMS222 F- ACATGCTACGTGTTTGGAATGR- ATCGTATGGATGTAATTGTCTTA (TGA)

23

100ndash135

Comments 1Tuskan et al 2004 [15]2Smulders et al 2001 [16] 3by literature data in different Populus species (P tremula P x canescens and P alba)

Table 3 Results of testing of microsatellite loci in aspen

Locus PCR amplification Fragment size range (bp) Number of alleles Status1 Included in testing systemORPM14 Yes 146ndash162 4 P NoORPM18 No mdash mdash N NoORPM36 Yes 217 1 M NoORPM60 No mdash mdash N NoORPM79 No mdash mdash N NoORPM81 No mdash mdash N NoORPM84 No mdash mdash N NoORPM86 Yes 204ndash216 4 P NoORPM107 No mdash mdash N NoORPM117 Yes 218 1 M NoORPM158 Yes 200 1 M NoORPM193 Yes 182ndash207 6 P YesORPM202 Yes 184ndash193 5 P YesORPM206 Yes 190ndash196 3 P YesORPM220 Yes 178ndash198 5 P YesORPM296 Yes 201ndash183 4 P YesORPM312 Yes 189ndash201 4 P NoORPM366 No mdash mdash N NoORPM371 Yes 192ndash200 3 P NoORPM372 No mdash mdash N NoORPM415 Yes sim280 1 M NoORPM484 Yes 190ndash206 3 P NoORPM488 Yes 197ndash203 2 P NoORPM496 No mdash mdash N NoWPMS14 Yes 224ndash243 3 P YesWPMS15 Yes 189ndash207 5 P YesWPMS16 Yes 139ndash184 9 P YesWPMS17 Yes 122ndash146 7 P YesWPMS18 Yes 219ndash248 7 P YesWPMS19 Yes 210ndash252 9 P YesWPMS20 Yes 210ndash222 4 P YesWPMS21 Yes 196ndash240 5 P YesWPMS22 Yes 115ndash135 3 P Yes1P polymorphic M monomorphic and N no PCR amplification

profile (1) initial denaturation at 94∘b for 3min (2) 30cycles consisting of 30 sec of denaturation at 94∘b primerannealing at variable temperature and time (see Table 3 forfinal annealing temperature recommended after procedureadjustment) and elongation at 72∘b for 15min followed by

(3) final elongation at 72∘b for 10min and (4) chilling the PCRmixture to 4∘b

PCR products were subjected to electrophoresis in 6polyacrylamide gel blocks using Tris-EDTA-borate buffersystem After electrophoresis gels were stained in ethidium


1

190

193 193

193 193

193 196

196

186

186 186

190198

242

309

404

217

201 202

199 202

181

201189

202 202 201207

190

180

160

147

146

186

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

(a)

1

189

204198

201

207

131

140 140

122

147

130

133

127

133

127 127

146

147

160

180

190

201219

233227 227

233242

249

222219

222

222

213210 207 210

198

210210

231237

243

222

309

219

227233 217

131128

131

207207

198

2 3 4 5 6 7 8 9 10 11 12 13 14 1515 16 17 18 19 20 21 22 23 24 25 26 27

(b)

Figure 2 (a) Electrophoretic patterns of PCR-amplified SSR lociORPM202ORPM206ORPM220ORPM296 andORPM312 in aspen Locilanes 1ndash4 ORPM202 lanes 6ndash9 ORPM206 lanes 10ndash13 ORPM220 samples 1 6 10 15 and 19 aspen from native population 2 7 11 16 and20 clone b-control 3 8 12 18 and 21 clone 47-1 4 9 13 18 and 22 clone PtV22 Lanes 5 14 and 23 DNA molecular weight marker (DNAof E coli plasmid pBR322 digested by restriction endonuclease HpaII) (b) Electrophoretic patterns of PCR-amplified SSR loci WPMS15WPMS17WPMS18WPMS19WPMS21 andWPMS22 in aspen Loci lanes 1ndash4WPMS15 lanes 6ndash9WPMS17 lanes 10ndash13WPMS18 lanes15ndash18WPMS19 lanes 19ndash22WPMS21 and lanes 24ndash27WPMS22 Samples 1 6 10 15 19 and 24 aspen from natural population 2 7 11 1620 and 25 clone b-control 3 8 12 16 21 and 26 clone 47-1 4 9 13 18 22 and 27 clone PtV22 Lanes 5 14 and 23 DNA molecular weightmarker (DNA of E coli plasmid pBR322 digested by restriction endonuclease HpaII)

bromide solution and visualized in UV-light graphic imageswere captured and saved using Doc-Print II Vilber Lourmatgel documentation system and processed in graphical editorsFragment size was estimated bymeans of specialized software(Photo-Capt) DNA of E coli plasmid pBR322 restricted byendonuclease HpaII was used as a molecular weight marker

24 Statistical Analysis Clone identity was determined usingmultilocus matches analysis for codominant data Geno-type probability (GP) meaning probability of appearanceof particular multilocus combination in population andprobability of identity estimating probability of randommatching of two unrelated (PI) or related (PIsib) individualsby particular set of loci were calculated based on distributionof allele frequencies in population samples

Correspondence of observed genotype distributions foreach SSR locus to the expected according Hardy-Weinbergequilibriumwas tested by chi-square criterion Allele numberand observed and expected heterozygosities were calculatedfor each native sample We employedWrightrsquos F-statistics forassessment of genetic subdivision among the studied pop-ulation samples All the above-mentioned calculations wereperformed in the add-in for MS Excel GenAlEx 65 [17 18]

3 Results and Discussion

31 Development of SSR-Based Testing Systems for GenotypeIdentification in Aspen For initial testing we selected 33 het-erological tri- tetra- penta- and hexanucleotide microsatel-lite loci from two sets series ORPM was designed first forPopulus trichocarpa [15] and series WPMS was designed forPopulus nigra [16] Characteristics of these loci are given inTable 2 Initial testing was done on DNA samples from threeclones (47-1 PtV22 K b-control) from an in vitro collectionstored and propagated in the Pushchino branch of Shemyakinand Ovchinnikov Institute of Bioorganic Chemistry Russian

Academy of Sciences (PushchinoMoscowOblast Russia) Atthis stage their variability was also tested on 20ndash24 specimensof wild aspen from Novosibirsk Oblast (Western SiberiaRussia) and Krasnoyarsk Krai (Middle Siberia Russia)

As a result of the first phase of testing 24 loci weresuccessfully amplified and nine other loci failed to producePCR products Out of 24 loci that produced PCR fragments20 loci were shown to be variable with a number of allelesfrom two to nine while four loci that have been successfullyamplified were monomorphic (Table 3) After additionaltesting at variable PCR regimes we finally selected 14 loci withreliable amplification and substantial polymorphism level forinclusion into the test system formolecular genetic identifica-tion Examples of the variability of the selected microsatelliteloci in aspen are shown on Figures 2(a) and 2(b)

The obtained multilocus genotypes of eight elite clonesand reference genotypes of wild trees from native standsare listed in Table S1 in Supplementary Material availableonline at httpdxdoiorg1011552015261518 We analyzedup to 8 ramets sampled at different phases of the micro-clonal propagationWithin clones genotypes were stable andunambiguously reproduced among ramets independently ofthe stage of propagation After exclusion of ramets of thesame genet both elite clones and aspen genotypes fromnativepopulations included in the reference samples were 100different Probability of appearance of genotypes (GP geno-type probability) among elite clones varied from 24 sdot 10minus21 to17 sdot 10minus11 among trees in a reference samples from 40 sdot 10minus24to 58 sdot 10minus9 Probability of the occasional coincidence of twounrelated genotypes (PI probability of identity) varied from48 sdot 10minus10 in Yoshkar-Ola to 43 sdot 10minus13 in Prisady Adjusted tothe theoretical probability of descendance of the comparedindividuals from the same ancestors more conservative esti-mate (PIsibs) was within the range of 10 sdot 10minus4 in Yoshkar-Olato 93 sdot 10minus6 in Voronezh All values are quite low so that thetheoretical frequency of appearance of repeatable genotypes


1 2 3 4 5 6 7 8 9 10 11 12 13 14Locus combination

00

01

02

03

04

05

PI

PIsibs PrisadyPIsibs VoronezhPIsibs Yoshkar-Ola

PI PrisadyPI VoronezhPI Yoshkar-Ola

Figure 3 Relationship of PI and PIsibs from the number of usedloci 1 represents locus 1 2 represents loci 1 + 2 and so forth

due to recombination of different gametes in course of seedreproduction was not exceeding about 1 accidentally foundidentical allele combination out of 10000 comparisons Therelationship of PI and PIsibs from the number of loci usedis shown at Figure 3 and demonstrates practically negligibleprobability of appearance of identical genotypes while usingfirst 7 to 8 loci selected for the inclusion into test systemUsing all 14 loci ensures additional reliability and robustnessof the procedure

Development ofmicrosatellite loci for species of the genusPopulus started in late 20th century along with technologiesof molecular genetic markers In 1998 one of the first setsof primers for amplification of dinucleotide SSR loci wasdesigned for American trembling poplar Populus tremuloides[19] In this paper a successful cross-amplification of the samemarkers for several other poplar species P deltoides P nigraP x canadensis and P maximowiczii was demonstrated Theauthors also postulated the broad spectrum of applications ofSSR loci for different purposes including clone identificationanalysis of the controlledmatings genomemappingmarker-assisted selection genetic diversity assays and support of theprograms for conservation and sustainable management offorest genetic resources Subsequent studies provided eightother dinucleotide SSR loci for Populus tremuloides [20]Since that time microsatellite loci were developed for otherspecies including P nigra [16 21] Some of these loci werealso amplified in P deltoides P trichocarpa P tremula Ptremuloides P candicans and P lasiocarpa Specific SSR lociprimers were designed for Populus euphratica [22ndash25] Lateron this set was used for development ofmultiplex panels usedfor genetic diversity estimation in this species [26 27]

Next generation sequencing was the most efficient way ofdetection of tandem repeats in poplar genome and design ontheir base transferrable SSR primers as it was demonstratedin case of balsamic poplar Populus trichocarpa [15 28] Such

universal cross-amplified loci are used for species and hybrididentification and for estimation of genetic differentiationamong congeneric species [5 9 29ndash31]

Microsatellites are also useful for checking of somaclonaldiversity within a pool of ramets obtained by microclonalpropagation from a single donor tree [5] for identificationof individual clones in an aspect of their tolerance to envi-ronmental factors [32] We did not observe any variation inSSR patterns among ramets of the same elite clonal lineageNuclear microsatellites along with other classes of molecularmarkers are employed also for determination of ploidy levelin poplars [33 34] and indications of triploidy were foundby us with respect to several genotypes Triploid aspenhybrids often demonstrate increased vigor and resistance topathogens so this matter should be further studied by meansof karyological and floating cytometry analysis

32 Analysis of Clonal Structure inNative Aspen Stands Sincethe very moment of the field sampling of material in wildstands we tried to avoid inclusion of clonal ramets arised assprouts which is common for aspen In all studied stands thesame sampling scheme was applied keeping at least 15ndash20mbetween the trees Nevertheless ramets of the same cloneindicating common occurrence of vegetative propagationswere found in all of the studied natural populations (Table S1)

Prisady Among the studied 52 trees we found 41 differenthaplotypes one multilocus genotype was found nine timesand one was found three times We concluded that theserepeated multilocus combinations resulted from samplingsprouts being ramets of the same clone

Voronezh Among 17 trees collected at close proximity to thesite of the experimental plantation no clonal individuals werefound Thirteen trees collected at the Voronezh River bankwere represented by eight genotypes four of them were inone replicate three were found to be two ramets of the sameclone and one genotype was found in three copies evidentlyoriginated from sprouting of ever existing progenitors Intotal we identified 25 different haplotypes in this referencepopulation sample

Yoshkar-Ola Thirty-two analyzed trees combined into 13different haplotypes identified by multilocus matches anal-ysis one genotype appeared twice one genotype appearedfive times one genotype appeared seven times and onegenotype appeared nine times Repeated genotypes evidentlycorresponded to ramets resulting from sprouting

We concluded that sprouting and high level of clonalityare a widespread phenomenon in native aspen stands Inaspen as well as in many other poplars vegetative clones areable to occupy large areas Therefore for the collection of asample free of repeated clonal genotypes distances betweentrees should be increased up to at least 40ndash50m Howevermore precise estimation of maximal spread of a single cloneover stand territory also requires special exploration

Among other applications nuclear SSR loci were usefulfor the studies of clonal structure and genetic relationshipsamong clone genotypes in native stands [35ndash38] or between


Table 4 Parameters of genetic variability in aspen populations

Population 119873 119873119886

119873119890

119867119874

119867119864

119880119867119864

119865

Prisady Mean 41 6429 3921 0631 0661 0669 0047se 0850 0602 0059 0044 0045 0065

Voronezh Mean 25 7429 3970 0580 0687 0701 0188se 1274 0584 0068 0035 0036 0076

Yoshkar-Ola Mean 13 4643 2738 0522 0567 0590 0109se 0541 0343 0072 0043 0045 0087

Total mean Mean 26333 6167 3543 0578 0639 0654 0115se 1791 0558 0308 0038 0024 0025 0044

se standard error119873 sample size119873

119886

mean number of alleles119873

119890

effective number of alleles119867

119874

observed heterozygosity119867

119864

expected heterozygosity119880119867

119864

unbiased expected heterozygosity119865 fixation index (intrapopulation coefficient of inbreeding)

native and artificial stands [39 40] Variation in level ofclonality was observed for instance in black poplar P nigra[41] Extensive clonal assemblies were found bymeans of SSRanalysis also in European black poplar [42] in the taxonomiccontinuum P alba ndash P x canescens on the Iberian Peninsula[43] and in other poplar species

33 Levels of Intra- and Interpopulation Genetic VariabilityAll the loci of the selected set were polymorphic in allstudied native population samples Values of average allelenumber effective allele number and observed and expectedheterozygosity were slightly higher in Prisady and Voronezhthan in Yoshkar-Ola (Table 4)

The averaged over loci 119865ST (proportion of inter-population variation in total variation Table 5) was 0058 plusmn0014 with the highest values observed in two loci ORPM202(0164) and WPMS14 (0162) AMOVA test showed that6 of total molecular variation was among populations(significant 119875 = 0001) 10 were among individuals and84 were within individuals

Microsatellites isolated fromnuclear aswell as chloroplastgenomes were employed for the analysis of genetic diversityand genetic structure in various poplar species [42 44ndash60] Levels of intrapopulation genetic diversity observed byus in native aspen stands were within the range of valuescommonly observed in poplars and estimated by SSR locianalysis ([47] and references therein)

A substantial number of studies employing microsatellitetechnique were focused on the detection of hybridization innatural [31 46 61ndash63] or artificial [64] stands of poplars SSRloci help to reveal mechanisms of interspecific crossing andreproductive isolation [65ndash67] In this study we concentratedon among-individual and among-population variation ratherthan on interspecific differences but with respect to some ofthe studied elite clones their hybrid origin should be genet-ically tested using a complex set of markers but of nuclearand cytoplasmic localization Hi-fidelity identification ofinterspecific hybrids and industrial clones was reported in

Table 5 119865-statistics for three natural aspen populations

Locus 119865IS 119865IT 119865ST

ORPM193 0158 0230 0086ORPM202 0514 0593 0164ORPM206 0276 0363 0120ORPM220 0066 0096 0032ORPM296 0046 0067 0023WPMS14 0074 0224 0162WPMS15 minus0187 minus0144 0036WPMS16 minus0105 minus0083 0019WPMS17 minus0066 minus0028 0036WPMS18 0305 0322 0025WPMS19 minus0034 minus0003 0029WPMS20 0111 0131 0023WPMS21 minus0057 minus0027 0028WPMS22 0563 0578 0035Mean 0119 0166 0058se 0060 0062 0014se standard error

a considerable number of publications [5 6 51 68ndash74]A remarkable publication reports on the genetic identityof some industrial clones of poplars previously treated asdifferent [68 69]

The practical task of identity monitoring of commercialclones in in vitro collections [75 76] was similar to thatemployed in the present research In all applications wherereliable individual identification was required SSRs showedhigh effectiveness

4 Conclusion

Application of nuclear microsatellite loci for genetic iden-tification and assessment of genetic variability in aspenelite clones and native stands showed high effectiveness of


the developed low-cost SSR-based testing system Reliableauthentication of clones ensures geneticmonitoring ofmicro-clonal propagation and allows revealing clonality in nativestands We demonstrated that the same set of microsatelliteloci can be successfully employed for estimation of levelsof intra- and interpopulation genetic variability in aspenReconstruction of kinship among individual elite clones orgenetic relationships of naturally mating populations areperspective tasks that can be realized in the future using thesame markers

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research is supported by the Ministry of Education andScience of the Russian Federation (Project no 14607210044from22082014 unique identifier RFMEFI60714X0044)Theauthors also thank E M Romanov A I Shurgin R VSergeev (Volga State University of Technology Yoshkar-OlaRepublic of Mari El Russia) M V Drapaluk A V Tzar-alunga A A Reshetnikov E O Kolesnikova (Voronezh StateForestry Engineering University Voronezh Russia) and GB Kolganikhina (Institute of Forest Science RAS UspenskoeMoscow region Russia) for their help with sample collectionin native stands The authors are also grateful to M ZepsD Auzenbaha and A Gailis (Latvian State Forest ResearchInstitute ldquoSilavardquo Salaspils Latvia) for sharing biologicalmaterial and information on aspen elite clones

References

[1] C C Giri B Shyamkumar and C Anjaneyulu ldquoProgressin tissue culture genetic transformation and applications ofbiotechnology to trees an overviewrdquoTrees vol 18 no 2 pp 115ndash135 2004

[2] K A Shestibratov V G Lebedev and A I Miroshnikov ldquoFor-est biotechnology methods technologies and perspectivesrdquoBiotechnology in Russia vol 5 pp 1ndash34 2008

[3] T A Thorpe I S Harry and P P Kumar ldquoApplication ofmicropropagation to forestryrdquo inMicropropagation TechnologyandApplication pp 311ndash336 Springer AmsterdamTheNether-lands 1991

[4] B Ziegenhagen and M Fladung ldquoDNA markers for identi-fication and evaluation of genetic resources in forest treescase studies in Abies Picea and Populusrdquo in Molecular MarkerSystems in Plant Breeding and Crop Improvement pp 413ndash429Springer Berlin Germany 2005

[5] M H Rahman and O P Rajora ldquoMicrosatellite DNAsomaclonal variation in micropropagated trembling aspen(Populus tremuloides)rdquo Plant Cell Reports vol 20 no 6 pp 531ndash536 2001

[6] M H Rahman and O P Rajora ldquoMicrosatellite DNA finger-printing differentiation and genetic relationships of clonescultivars and varieties of six poplar species from three sectionsof the genus Populusrdquo Genome vol 45 no 6 pp 1083ndash10942002

[7] O P Rajora and M H Rahman ldquoMicrosatellite DNA markersand their usefulness in poplars and conservation of microsatel-lite DNA loci in Salicaceaerdquo in Genetic Response of Forest Sys-tems to Changing Environmental Conditions vol 70 of ForestrySciences pp 105ndash115 Springer Dordrecht The Netherlands2001

[8] O P Rajora and M H Rahman ldquoMicrosatellite DNA andRAPD fingerprinting identification and genetic relationshipsof hybrid poplar (Populus x canadensis) cultivarsrdquo Theoreticaland Applied Genetics vol 106 no 3 pp 470ndash477 2003

[9] H Schroeder and M Fladung ldquoSSR and SNIP markers for theidentification of clones hybrids and species within the genusPopulusrdquo Silvae Genetica vol 59 no 6 pp 257ndash263 2010

[10] K A Shestibratov V G Lebedev and A B Azarova ldquoMethodof in vitro preparation of microshoots of ash aspen willow forsubsequent rooting under ex vitro conditionsrdquo Patent of RussianFederation no 2565806 of 20102015 2012

[11] S N Bagaev and E S Bagaev ldquoA genetic reserve of giant aspenrdquoLesnoe Khozyaistvo no 4 pp 45ndash48 1990

[12] M Zeps D Auzenbaha A Gailis A Treimanis and UGrinfelds ldquoEvaluation and selection of hybrid aspen (Populustremuloides times Populus tremula) clonesrdquoMezzinatne vol 18 no51 pp 19ndash34 2008

[13] M E Devey J C Beil DN Smith D B Neale andG FMoranldquoA genetic linkage map for Pinus radiata based on RFLP RAPDand microsatellite markersrdquo Theoretical and Applied Geneticsvol 92 no 6 pp 673ndash679 1996

[14] J J Doyle and J L Doyle ldquoIsolation of plant DNA from freshtissuerdquo Focus vol 12 no 1 pp 12ndash15 1990

[15] G A Tuskan L E Gunter Z K Yang T Yin MM Sewell andS P DiFazio ldquoCharacterization of microsatellites revealed bygenomic sequencing of Populus trichocarpardquo Canadian Journalof Forest Research vol 34 no 1 pp 85ndash93 2004

[16] M J M Smulders J Van Der Schoot P Arens and B VosmanldquoTrinucleotide repeat microsatellite markers for black poplar(Populus nigra L)rdquo Molecular Ecology Notes vol 1 no 3 pp188ndash190 2001

[17] R Peakall and P E Smouse ldquoGENALEX 6 genetic analysis inExcel Population genetic software for teaching and researchrdquoMolecular Ecology Notes vol 6 no 1 pp 288ndash295 2006

[18] R Peakall and P E Smouse ldquoGenALEx 65 genetic analysis inExcel Population genetic software for teaching and researchmdashan updaterdquo Bioinformatics vol 28 no 19 Article ID bts460 pp2537ndash2539 2012

[19] S Dayanandan O P Rajora and K S Bawa ldquoIsolation andcharacterization of microsatellites in trembling aspen (Populustremuloides)rdquoTheoretical and Applied Genetics vol 96 no 6-7pp 950ndash956 1998

[20] MH Rahman S Dayanandan andO P Rajora ldquoMicrosatelliteDNA markers in Populus tremuloidesrdquo Genome vol 43 no 2pp 293ndash297 2000

[21] J van der Schoot M Pospıskova B Vosman and M J MSmulders ldquoDevelopment and characterization of microsatellitemarkers in black poplar (Populos nigra L)rdquo Theoretical andApplied Genetics vol 101 no 1-2 pp 317ndash322 2000

[22] Q Du C Gong W Pan and D Zhang ldquoDevelopment andapplication ofmicrosatellites in candidate genes related towoodproperties in the Chinese white poplar (Populus tomentosaCarr)rdquo DNA Research vol 20 no 1 pp 31ndash44 2013

[23] F K Du F Xu H Qu S Feng J Tang and R Wu ldquoExploitingthe transcriptome of Euphrates poplar Populus euphratica


(Salicaceae) to develop and characterize new EST-SSR markersand construct an EST-SSR databasrdquo PLoS ONE vol 8 no 4Article ID e61337 2013

[24] Q DuW Pan B Xu B Li and D Zhang ldquoPolymorphic simplesequence repeat (SSR) loci within cellulose synthase (PtoCesA)genes are associated with growth and wood properties inPopulus tomentosardquoNewPhytologist vol 197 no 3 pp 763ndash7762013

[25] Y Wu J Wang and J Liu ldquoDevelopment and characterizationof microsatellite markers in Populus euphratica (Populaceae)rdquoMolecular Ecology Resources vol 8 no 5 pp 1142ndash1144 2008

[26] E Pascal F Steffen and S Martin ldquoDevelopment of twomicrosatellite multiplex PCR systems for high throughputgenotyping in Populus euphraticardquo Journal of Forestry Researchvol 20 no 3 pp 195ndash198 2009

[27] F Xu S Feng R Wu and F K Du ldquoTwo highly validated SSRmultiplexes (8-plex) for Euphratesrsquo poplar Populus euphratica(Salicaceae)rdquoMolecular Ecology Resources vol 13 no 1 pp 144ndash153 2013

[28] TM Yin X Y Zhang L E Gunter et al ldquoMicrosatellite primerresource for Populus developed from the mapped sequencescaffolds of the Nisqually-1 genomerdquo New Phytologist vol 181no 2 pp 498ndash503 2009

[29] T Bruegmann and M Fladung ldquoPotentials and limitations ofthe cross-species transfer of nuclear microsatellite marker in sixspecies belonging to three sections of the genus Populus Lrdquo TreeGenetics amp Genomes vol 9 no 6 pp 1413ndash1421 2013

[30] K Chen Y Peng Y Wang H Korpelainen and C Li ldquoGeneticrelationships among poplar species in section Tacamahaca(Populus L) from western Sichuan Chinardquo Plant Science vol172 no 2 pp 196ndash203 2007

[31] M JM Smulders R Beringen R Volosyanchuk et al ldquoNaturalhybridisation between Populus nigra L and P x canadensisMoench Hybrid offspring competes for niches along the Rhineriver in the Netherlandsrdquo Tree Genetics amp Genomes vol 4 no4 pp 663ndash675 2008

[32] M D Madritch S L Greene and R L Lindroth ldquoGeneticmosaics of ecosystem functioning across aspen-dominatedlandscapesrdquo Oecologia vol 160 no 1 pp 119ndash127 2009

[33] C-B Dong Y-J Suo and X-Y Kang ldquoAssessment of thegenetic composition of triploid hybrid Populus using SSRmark-ers with low recombination frequenciesrdquo Canadian Journal ofForest Research vol 44 no 7 pp 692ndash699 2014

[34] F Kong J Liu Y Chen Z Wan and T Yin ldquoMarker-aidedselection of polyploid poplarsrdquo Bioenergy Research vol 6 no3 pp 984ndash990 2013

[35] N Chenault S Arnaud-Haond M Juteau et al ldquoSSR-basedanalysis of clonality spatial genetic structure and introgressionfrom the Lombardy poplar into a natural population of Populusnigra L along the Loire Riverrdquo Tree Genetics amp Genomes vol 7no 6 pp 1249ndash1262 2011

[36] JDeWoodyCA RoweVDHipkins andK EMock ldquolsquoPandorsquolives molecular genetic evidence of a giant aspen clone inCentral Utahrdquo Western North American Naturalist vol 68 no4 pp 493ndash497 2008

[37] K E Mock C A Rowe M B Hooten J Dewoody and V DHipkins ldquoClonal dynamics in western North American aspen(Populus tremuloides)rdquo Molecular Ecology vol 17 no 22 pp4827ndash4844 2008

[38] G T Slavov S Leonardi W T Adams S H Strauss andS P Difazio ldquoPopulation substructure in continuous and

fragmented stands of Populus trichocarpardquo Heredity vol 105no 4 pp 348ndash357 2010

[39] G Brundu R Lupi I Zapelli et al ldquoThe origin of clonaldiversity and structure of Populus alba in Sardinia evidencefrom nuclear and plastid microsatellite markersrdquo Annals ofBotany vol 102 no 6 pp 997ndash1006 2008

[40] L I Suvanto and T B Latva-Karjanmaa ldquoClone identificationand clonal structure of the European aspen (Populus tremula)rdquoMolecular Ecology vol 14 no 9 pp 2851ndash2860 2005

[41] N Barsoum E Muller and L Skot ldquoVariations in levels ofclonality among Populus nigra L stands of different agesrdquoEvolutionary Ecology vol 18 no 5-6 pp 601ndash624 2004

[42] M JM Smulders J E Cottrell F Lefevre et al ldquoStructure of thegenetic diversity in black poplar (Populus nigra L) populationsacross European river systems consequences for conservationand restorationrdquo Forest Ecology and Management vol 255 no5-6 pp 1388ndash1399 2008

[43] L Santos-del-Blanco A I de-Lucas S C Gonzalez-MartınezR Sierra-de-Grado and E Hidalgo ldquoExtensive Clonal Assem-blies in Populus alba and Populus x canescens from the IberianPeninsulardquo Tree Genetics amp Genomes vol 9 no 2 pp 499ndash5102013

[44] A Alimohamadi F Asadi and R T Aghdaei ldquoGenetic diversityin Populus nigra plantations from west of Iranrdquo Annals of ForestResearch vol 55 no 2 pp 165ndash178 2012

[45] C M Callahan C A Rowe R J Ryel J D Shaw M DMadritch and K E Mock ldquoContinental-scale assessment ofgenetic diversity and population structure in quaking aspen(Populus tremuloides)rdquo Journal of Biogeography vol 40 no 9pp 1780ndash1791 2013

[46] S Castiglione A Cicatelli R Lupi et al ldquoGenetic structure andintrogression in riparian populations of Populus alba Lrdquo PlantBiosystems vol 144 no 3 pp 656ndash668 2010

[47] C T Cole ldquoAllelic and population variation of microsatelliteloci in aspen (Populus tremuloides)rdquo New Phytologist vol 167no 1 pp 155ndash164 2005

[48] D De Carvalho P K Ingvarsson J Joseph et al ldquoAdmixturefacilitates adaptation from standing variation in the Europeanaspen (Populus tremula L) a widespread forest treerdquoMolecularEcology vol 19 no 8 pp 1638ndash1650 2010

[49] Q Du B Wang Z Wei D Zhang and B Li ldquoGenetic diversityand population structure of Chinese white poplar (Populustomentosa) revealed by SSR markersrdquo Journal of Heredity vol103 no 6 pp 853ndash862 2012

[50] P Eusemann A Petzold N Thevs and M Schnittler ldquoGrowthpatterns and genetic structure of Populus euphratica Oliv (Sal-icaceae) forests in NW Chinamdashimplications for conservationandmanagementrdquo Forest Ecology andManagement vol 297 pp27ndash36 2013

[51] T Fossati F Grassi F Sala and S Castiglione ldquoMolecularanalysis of natural populations of Populus nigra L intermingledwith cultivated hybridsrdquo Molecular Ecology vol 12 no 8 pp2033ndash2043 2003

[52] K M Lee Y Y Kim and J O Hyun ldquoGenetic variation inpopulations of Populus davidianaDode based on microsatellitemarker analysisrdquoGenes andGenomics vol 33 no 2 pp 163ndash1712011

[53] D MacAya-Sanz M Heuertz U Lopez-de-Heredia et al ldquoTheAtlanticmdashMediterranean watershed river basins and glacialhistory shape the genetic structure of Iberian poplarsrdquo Molec-ular Ecology vol 21 no 14 pp 3593ndash3609 2012


[54] Y H Peng Z X Lu K Chen O Luukkanen H Korpelainenand C Y Li ldquoPopulation genetic survey of Populus cathayanaoriginating from Southeastern QinghaimdashTibetan plateau ofChina based on SSRmarkersrdquo Silvae Genetica vol 54 no 3 pp116ndash122 2005

[55] D Shen W Bo F Xu and R Wu ldquoGenetic diversity andpopulation structure of the Tibetan poplar (Populus szechuanicavar tibetica) along an altitude gradientrdquo BMC Genetics vol 15supplement 1 p S11 2014

[56] JWang YWu G Ren Q Guo J Liu andM Lascoux ldquoGeneticdifferentiation and delimitation between ecologically divergedPopulus euphratica and P pruinosardquo PLoS ONE vol 6 no 10Article ID e26530 2011

[57] J Wang Z Li Q Guo G Ren and Y Wu ldquoGenetic variationwithin and between populations of a desert poplar (Populuseuphratica) revealed by SSR markersrdquo Annals of Forest Sciencevol 68 no 6 pp 1143ndash1149 2011

[58] Z Wei Q Du J Zhang B Li and D Zhang ldquoGenetic diversityand population structure in Chinese indigenous poplar (Pop-ulus simonii) populations using microsatellite markersrdquo PlantMolecular Biology Reporter vol 31 no 3 pp 620ndash632 2013

[59] J Wyman A Bruneau and M-F Tremblay ldquoMicrosatelliteanalysis of genetic diversity in four populations of Populustremuloides in Quebecrdquo Canadian Journal of Botany vol 81 no4 pp 360ndash367 2003

[60] L Zhang P-P Jiao and Z-J Li ldquoGenetic diversity of Populuspruinosa populations in Xinjiang of China based on SSRanalysisrdquo Shengtaixue Zazhi vol 31 no 11 pp 2755ndash2761 2012

[61] T Fossati G Patrignani I Zapelli M Sabatti F Sala and SCastiglione ldquoDevelopment of molecular markers to assess thelevel of introgression of Populus tremula into P alba naturalpopulationsrdquo Plant Breeding vol 123 no 4 pp 382ndash385 2004

[62] C Lexer M F Fay J A JosephM-S Nica and B Heinze ldquoBar-rier to gene flow between two ecologically divergent Populusspecies P alba (white poplar) and P tremula (European aspen)the role of ecology and life history in gene introgressionrdquoMolecular Ecology vol 14 no 4 pp 1045ndash1057 2005

[63] C Lexer C A Buerkle J A Joseph B Heinze and M F FayldquoAdmixture in European Populus hybrid zones makes feasiblethemapping of loci that contribute to reproductive isolation andtrait differencesrdquo Heredity vol 98 no 2 pp 74ndash84 2007

[64] A Vanden Broeck V Storme J E Cottrell et al ldquoGene flowbetween cultivated poplars and native black poplar (Populusnigra L) a case study along the river Meuse on the Dutch-Belgian borderrdquo Forest Ecology and Management vol 197 no1-3 pp 307ndash310 2004

[65] C Lexer J A Joseph M Van Loo et al ldquoGenomic admixtureanalysis in European Populus spp reveals unexpected patternsof reproductive isolation and matingrdquo Genetics vol 186 no 2pp 699ndash712 2010

[66] D Macaya-Sanz L Suter J Joseph et al ldquoGenetic analysisof post-mating reproductive barriers in hybridizing EuropeanPopulus speciesrdquo Heredity vol 107 no 5 pp 478ndash486 2011

[67] J-F Zhang Z-Z Wei D Li and B Li ldquoUsing SSR markersto study the mechanism of 2n pollen formation in Populus xeuramericana (Dode) Guinier and P x popularisrdquo Annals ofForest Science vol 66 no 5 2009

[68] F Bekkaoui B Mann and B Schroeder ldquoApplication of DNAmarkers for the identification andmanagement of hybrid poplaraccessionsrdquo Agroforestry Systems vol 59 no 1 pp 53ndash59 2003

[69] A I De-Lucas J C Santana P Recio and E Hidalgo ldquoSSR-based tool for identification and certification of commercial

Populus clones in Spainrdquo Annals of Forest Science vol 65 no1 p 107 2008

[70] I Gomez and C-J Tsai ldquoIdentification of microsatellite mark-ers for diagnostic genotyping Populus fremontii x angustifoliabackcross hybridsrdquo Plant Biology pp 236ndash236 2007

[71] H Liesebach V Schneck and E Ewald ldquoClonal fingerprintingin the genus Populus L by nuclear microsatellite loci regardingdifferences between sections species and hybridsrdquoTreeGeneticsamp Genomes vol 6 no 2 pp 259ndash269 2010

[72] G Rathmacher M Niggemann H Wypukol K Gebhardt BZiegenhagen and R Bialozyt ldquoAllelic ladders and referencegenotypes for a rigorous standardization of poplar microsatel-lite datardquo TreesmdashStructure and Function vol 23 no 3 pp 573ndash583 2009

[73] A Vanden Broeck J Cottrell P Quataert et al ldquoPaternityanalysis of Populus nigra L offspring in a Belgian plantation ofnative and exotic poplarsrdquo Annals of Forest Science vol 63 no7 pp 783ndash790 2006

[74] N Wheeler P Payne V Hipkins R Saich S Kenny and GTuskan ldquoPolymix breeding with paternity analysis in Populusa test for differential reproductive success (DRS) among pollendonorsrdquo Tree Genetics amp Genomes vol 2 no 1 pp 53ndash60 2006

[75] T Fossati I Zapelli S Bisoffi et al ldquoGenetic relationships andclonal identity in a collection of commercially relevant poplarcultivars assessed by AFLP and SSRrdquo Tree Genetics amp Genomesvol 1 no 1 pp 11ndash19 2005

[76] A Gomez J A Manzanera E Aguiriano J M Grau andM ABueno ldquoSSR markers for monitoring an in vitro core collectionof Populus tremulardquo Silvae Genetica vol 52 no 5-6 pp 224ndash229 2003

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


of obtaining propagation and introduction of elite cultivarsby breeding or genetic engineering increases the probabilityof various errors

Molecular genetic markers (MGM) proved to be veryefficient tools for individual identification Among differentMGM classes microsatellites or simple sequence repeats(SSR) fit best to requirements of testing systems for identifica-tion due to their specificity codominance selective neutralitysufficient allelic richness and heterozygosity caused by highmutation rate Moreover due to relative genome conser-vatismwithin genera and families of plants SSR-markers andPCR primers for their amplification can be transferrable fromone taxon to another

In several poplar species the successful DNA fingerprint-ing and differentiation of clones cultivars and varieties weredemonstrated bymolecularmarkers including SSR loci [5ndash9]

In this paper we report on the development of SSR-based testing system for molecular genetic identification ofelite micropropagated genotypes of aspen Populus tremulaL aimed at the establishment of fast-growing target forestplantations in several regions of Russian Federation andpresent the results of its application for individual genotypesdiscrimination studies of clonal structure in natural aspenstands and estimation of genetic variability in populations

2 Material and Methods

The development of the testing system for the identificationof elite genotypes comprised the selection of a specificmarkerset fitting the requirements of high-resolution discriminationof clones and testing its reliability and identification power ona set of elite genotypes and a sufficient number of representa-tives of a studied species

21 Plant Material Elite aspen and hybrid clones used fordevelopment of testing systems for molecular identificationhave been obtained from micropropagated cell cultures [10]maintained in the Pushchino branch of Shemyakin andOvchinnikov Institute of Bioorganic Chemistry RussianAcademy of Sciences (Pushchino Russia) Origin and shortdescription of eight clones are presented in Table 1

Experimental aspen clonal plantations derived from theseelite genotypes were established in four regions of Europeanpart of Russia (Figure 1) Native aspen stands located closelyto these plots were used for studies of clonal structureevaluation of frequencies of multilocus genotypes and cal-culations of probabilities of appearance of identical alleliccombinations in a single genotype

Prisady Experimental plot located 1 km westward of settle-ment Prisady in Serpukhovsky Raion (district) of Moscowoblastrsquo (Russia) Leaves of 52 young ormedium-aged (approx-imately 10ndash25 yo) aspen trees from a naturalmultiaged standlocated in close proximity (1 km) to this plot have been usedas a reference population

Voronezh Seventeen trees were sampled in a stand adjacentto the experimental plot established in Voronezh Oblast

1

2

3

Figure 1 Map of location of plantations made of elite clones andcorresponding native aspen stands used as reference populations (1)Prisady (2) Voronezh and (3) Yoshkar-Ola

Additional 13 trees were collected along the bank of VoronezhRiver within the city of Voronezh close to the plantation

Yoshkar-Ola Young native stand of aspen was the source oftrees used as a reference population for an experimental plotlocated near the city of Yoshkar-Ola Republic of Mari-ElLeaves from 32 trees were collected

In order to minimize the occasional sampling of indi-viduals having vegetative origin (through sprouting) wecollected leaves from trees at a distance not less than 15ndash20mfrom each other This approach was employed for inclusioninto reference samples of predominantly open-pollinatedseedlings having maximal genetic diversity However basedexclusively on external look of the trees and distance amongthem sampling of the ramets appearing as a result of sprout-ing could not be avoided and subsequent genetic analysisconfirmed this

Shoots with leaves were cut off by means of mechanicalcutter with an aluminium telescopic mast Collected shootswith leaves were placed into plastic bags for no morethan 6 days at +4∘b until processing Sample preparationincludedDNA extraction and placement of reserve leaf tissuefragments into labeled zip-bags with silica gel for long-termstorage Outside the period of active vegetation dormantvegetative buds can be successfully used for DNA extraction

22 DNA Extraction Fragments of leaves approximately350ndash500mg taken from explants growing in vitro on a specialmedia (REF) in Petri dishes were used for DNA extraction

For trees from native populations we extracted DNAfrom 350ndash500mg fragments of fresh or 200ndash300mg of silica-dried leaf tissues by a modified cethyltrimethylammoniumbromide (CTAB) method [13 14]

23 Microsatellite Analysis Microsatellites or SSR representa class of tandemly repeated DNA sequences with short (1ndash6 pairs of nucleotides bp) motifs differing in copy numbersamong individuals due to high mutation rate Multiple allelesusually found in codominant microsatellite loci create a great



Originalgenotype




genotypes





12mut

Pt P tremula



Pt2 Pt3

F2 P tremula



F2-1 F2-2 F2-3



47-1 47-1-1-31 47-1-1-2247-1-2-27 47-1-1-19

47-1-2-53



b-1 b-2 b-3



L23-1 L23-2 L23-3



L4-1 L4-2 L4-3




No 3-understory-1









4

146ndash162


4

205


4

213


5

212


4

160


4

142ndash158


4

171


5

204


4

190


4

210


4

200


4

187ndash207


5

184ndash190


7

190ndash208


6

178ndash222


4

199


6

194


5

156


6

192ndash200


5

190


4

225


4

190ndash206


6

200


4

185


28

215ndash287


14

201ndash219


Table 2 Continued



8

139ndash184


15

122ndash146


13

219ndash248


28

180ndash234


8

210ndash222


45

287ndash326


23

100ndash135








1

190

193 193

193 193

193 196

196

186

186 186

190198

242

309

404

217

201 202

199 202

181

201189

202 202 201207

190

180

160

147

146

186

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

(a)

1

189

204198

201

207

131

140 140

122

147

130

133

127

133

127 127

146

147

160

180

190

201219

233227 227

233242

249

222219

222

222

213210 207 210

198

210210

231237

243

222

309

219

227233 217

131128

131

207207

198

2 3 4 5 6 7 8 9 10 11 12 13 14 1515 16 17 18 19 20 21 22 23 24 25 26 27

(b)












00

01

02

03

04

05

PI

















Population 119873 119873119886

119873119890

119867119874

119867119864

119880119867119864

119865

Prisady Mean 41 6429 3921 0631 0661 0669 0047se 0850 0602 0059 0044 0045 0065

Voronezh Mean 25 7429 3970 0580 0687 0701 0188se 1274 0584 0068 0035 0036 0076




119886


119890


119874


119864


119864








Locus 119865IS 119865IT 119865ST




4 Conclusion






Acknowledgments


References

























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Originalgenotype




genotypes





12mut

Pt P tremula



Pt2 Pt3

F2 P tremula



F2-1 F2-2 F2-3



47-1 47-1-1-31 47-1-1-2247-1-2-27 47-1-1-19

47-1-2-53



b-1 b-2 b-3



L23-1 L23-2 L23-3



L4-1 L4-2 L4-3




No 3-understory-1









4

146ndash162


4

205


4

213


5

212


4

160


4

142ndash158


4

171


5

204


4

190


4

210


4

200


4

187ndash207


5

184ndash190


7

190ndash208


6

178ndash222


4

199


6

194


5

156


6

192ndash200


5

190


4

225


4

190ndash206


6

200


4

185


28

215ndash287


14

201ndash219


Table 2 Continued



8

139ndash184


15

122ndash146


13

219ndash248


28

180ndash234


8

210ndash222


45

287ndash326


23

100ndash135








1

190

193 193

193 193

193 196

196

186

186 186

190198

242

309

404

217

201 202

199 202

181

201189

202 202 201207

190

180

160

147

146

186

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

(a)

1

189

204198

201

207

131

140 140

122

147

130

133

127

133

127 127

146

147

160

180

190

201219

233227 227

233242

249

222219

222

222

213210 207 210

198

210210

231237

243

222

309

219

227233 217

131128

131

207207

198

2 3 4 5 6 7 8 9 10 11 12 13 14 1515 16 17 18 19 20 21 22 23 24 25 26 27

(b)












00

01

02

03

04

05

PI

















Population 119873 119873119886

119873119890

119867119874

119867119864

119880119867119864

119865

Prisady Mean 41 6429 3921 0631 0661 0669 0047se 0850 0602 0059 0044 0045 0065

Voronezh Mean 25 7429 3970 0580 0687 0701 0188se 1274 0584 0068 0035 0036 0076




119886


119890


119874


119864


119864








Locus 119865IS 119865IT 119865ST




4 Conclusion






Acknowledgments


References

























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





4

146ndash162


4

205


4

213


5

212


4

160


4

142ndash158


4

171


5

204


4

190


4

210


4

200


4

187ndash207


5

184ndash190


7

190ndash208


6

178ndash222


4

199


6

194


5

156


6

192ndash200


5

190


4

225


4

190ndash206


6

200


4

185


28

215ndash287


14

201ndash219


Table 2 Continued



8

139ndash184


15

122ndash146


13

219ndash248


28

180ndash234


8

210ndash222


45

287ndash326


23

100ndash135








1

190

193 193

193 193

193 196

196

186

186 186

190198

242

309

404

217

201 202

199 202

181

201189

202 202 201207

190

180

160

147

146

186

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

(a)

1

189

204198

201

207

131

140 140

122

147

130

133

127

133

127 127

146

147

160

180

190

201219

233227 227

233242

249

222219

222

222

213210 207 210

198

210210

231237

243

222

309

219

227233 217

131128

131

207207

198

2 3 4 5 6 7 8 9 10 11 12 13 14 1515 16 17 18 19 20 21 22 23 24 25 26 27

(b)












00

01

02

03

04

05

PI

















Population 119873 119873119886

119873119890

119867119874

119867119864

119880119867119864

119865

Prisady Mean 41 6429 3921 0631 0661 0669 0047se 0850 0602 0059 0044 0045 0065

Voronezh Mean 25 7429 3970 0580 0687 0701 0188se 1274 0584 0068 0035 0036 0076




119886


119890


119874


119864


119864








Locus 119865IS 119865IT 119865ST




4 Conclusion






Acknowledgments


References

























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 2 Continued



8

139ndash184


15

122ndash146


13

219ndash248


28

180ndash234


8

210ndash222


45

287ndash326


23

100ndash135








1

190

193 193

193 193

193 196

196

186

186 186

190198

242

309

404

217

201 202

199 202

181

201189

202 202 201207

190

180

160

147

146

186

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

(a)

1

189

204198

201

207

131

140 140

122

147

130

133

127

133

127 127

146

147

160

180

190

201219

233227 227

233242

249

222219

222

222

213210 207 210

198

210210

231237

243

222

309

219

227233 217

131128

131

207207

198

2 3 4 5 6 7 8 9 10 11 12 13 14 1515 16 17 18 19 20 21 22 23 24 25 26 27

(b)












00

01

02

03

04

05

PI

















Population 119873 119873119886

119873119890

119867119874

119867119864

119880119867119864

119865

Prisady Mean 41 6429 3921 0631 0661 0669 0047se 0850 0602 0059 0044 0045 0065

Voronezh Mean 25 7429 3970 0580 0687 0701 0188se 1274 0584 0068 0035 0036 0076




119886


119890


119874


119864


119864








Locus 119865IS 119865IT 119865ST




4 Conclusion






Acknowledgments


References

























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1

190

193 193

193 193

193 196

196

186

186 186

190198

242

309

404

217

201 202

199 202

181

201189

202 202 201207

190

180

160

147

146

186

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

(a)

1

189

204198

201

207

131

140 140

122

147

130

133

127

133

127 127

146

147

160

180

190

201219

233227 227

233242

249

222219

222

222

213210 207 210

198

210210

231237

243

222

309

219

227233 217

131128

131

207207

198

2 3 4 5 6 7 8 9 10 11 12 13 14 1515 16 17 18 19 20 21 22 23 24 25 26 27

(b)












00

01

02

03

04

05

PI

















Population 119873 119873119886

119873119890

119867119874

119867119864

119880119867119864

119865

Prisady Mean 41 6429 3921 0631 0661 0669 0047se 0850 0602 0059 0044 0045 0065

Voronezh Mean 25 7429 3970 0580 0687 0701 0188se 1274 0584 0068 0035 0036 0076




119886


119890


119874


119864


119864








Locus 119865IS 119865IT 119865ST




4 Conclusion






Acknowledgments


References

























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



00

01

02

03

04

05

PI

















Population 119873 119873119886

119873119890

119867119874

119867119864

119880119867119864

119865

Prisady Mean 41 6429 3921 0631 0661 0669 0047se 0850 0602 0059 0044 0045 0065

Voronezh Mean 25 7429 3970 0580 0687 0701 0188se 1274 0584 0068 0035 0036 0076




119886


119890


119874


119864


119864








Locus 119865IS 119865IT 119865ST




4 Conclusion






Acknowledgments


References

























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Population 119873 119873119886

119873119890

119867119874

119867119864

119880119867119864

119865

Prisady Mean 41 6429 3921 0631 0661 0669 0047se 0850 0602 0059 0044 0045 0065

Voronezh Mean 25 7429 3970 0580 0687 0701 0188se 1274 0584 0068 0035 0036 0076




119886


119890


119874


119864


119864








Locus 119865IS 119865IT 119865ST




4 Conclusion






Acknowledgments


References

























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Acknowledgments


References

























































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Application of Microsatellite Loci for