research article retrotransposon-based molecular...

Research ArticleRetrotransposon-Based Molecular Markers for Analysis ofGenetic Diversity within the Genus Linum

Nataliya V Melnikova1 Anna V Kudryavtseva1 Alexander V Zelenin1

Valentina A Lakunina1 Olga Yu Yurkevich1 Anna S Speranskaya12 Alexey A Dmitriev1

Anastasia A Krinitsina2 Maxim S Belenikin13 Leonid A Uroshlev1

Anastasiya V Snezhkina1 Asiya F Sadritdinova1 Nadezda V Koroban1

Alexandra V Amosova1 Tatiana E Samatadze1 Elena V Guzenko4 Valentina A Lemesh4

Anastasya M Savilova5 Olga A Rachinskaia1 Natalya V Kishlyan6 Tatiana A Rozhmina6

Nadezhda L Bolsheva1 and Olga V Muravenko1

1 Engelhardt Institute of Molecular Biology Russian Academy of Sciences Moscow 119991 Russia2 Department of Higher Plants Lomonosov Moscow State University Moscow 119991 Russia3 Research Institute of Physico-Chemical Medicine Moscow 119435 Russia4 Institute of Genetics and Cytology National Academy of Science of Belarus 220072 Minsk Belarus5 Research Center for Obstetrics Gynecology and Perinatology Moscow 117997 Russia6All-Russian Research Institute for Flax of the Russian Academy of Agricultural Sciences Torzhok 172002 Russia

Correspondence should be addressed to Nadezhda L Bolsheva nlbolshevamailru

Received 25 April 2014 Revised 18 July 2014 Accepted 1 August 2014 Published 27 August 2014

Academic Editor Peter F Stadler

Copyright copy 2014 Nataliya V Melnikova 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

SSAP method was used to study the genetic diversity of 22 Linum species from sections Linum Adenolinum DasylinumStellerolinum and 46 flax cultivars All the studied flax varieties were distinguished using SSAP for retrotransposons FL9 and FL11Thus the validity of SSAP method was demonstrated for flax marking identification of accessions in genebank collections andcontrol during propagation of flax varieties Polymorphism of Fl1a Fl1b and Cassandra insertions were very low in flax varietiesbut these retrotransposons were successfully used for the investigation of Linum species Species clusterization based on SSAPmarkers was in concordance with their taxonomic division into sections Dasylinum Stellerolinum Adenolinum and Linum Allspecies of sect Adenolinum clustered apart from species of sect Linum The data confirmed the accuracy of the separation in thesesections Members of section Linum are not as closely related as members of other sections so taxonomic revision of this sectionis desirable L usitatissimum accessions genetically distant frommodern flax cultivars were revealed in our work These accessionsare of utmost interest for flax breeding and introduction of new useful traits into flax cultivars The chromosome localization ofCassandra retrotransposon in Linum species was determined

1 Introduction

The genus Linum comprises about 200 species which are dis-tributed throughout the temperate and subtropical regions ofthe worldThe genus is subdivided by Ockendon andWaltersinto five sectionsLinumDasylinum (Planch) JuzLinastrum(Planchon) Bentham Syllinum Griseb and Cathartolinum

(Reichenb) Griseb [1] Some taxonomists classified themembers of the L perenne group from section Linum toan independent section Adenolinum (Reichenb) Juz [2 3]The species L stelleroides (Planch) distributed in Far Eastand China was classified by Yuzepchuk [2] to a monotypesection Stellerolinum Juz ex Prob The phylogenetic analysesbased on chloroplast (ndhF trnL-F and trnK 31015840 intron) and

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 231589 14 pageshttpdxdoiorg1011552014231589

2 BioMed Research International

nuclear ITS (internal transcribed spacer) DNA sequencesrevealed that genus Linum was not monophyletic It con-tains two major lineages a yellow-flowered clade (sectionsLinopsis Syllinum and Cathartolinum) and a blue-floweredclade (sections LinumDasylinum and Stellerolinum) [4]Thecultivated flax (L usitatissimum L) belongs to sec Linumfrom a blue-flowered clade L usitatissimum is believed tohave originated as a result of domestication of wild speciesL angustifolium Huds approximately 8000 years ago [5ndash8]For a long time flax has been cultivating as a dual-purposecrop grown for its fiber and linseed oil

According to morphological and qualitative traits cul-tivated flax was divided into five main types (1) fiber flax(L usitatissimum subsp usitatissimum) (2) oil flax (L usi-tatissimum L subsp humileCzernom) (3) dual-purpose flax(L usitatissimum L subsp intermedium Czemom) that wasan intermediate form between the first two ones cultivatedfor fiber and oil (4) large seeded flax (L usitatissimum Lsubsp latifolium Snankev) which is characterized by a set ofspecific morphological features and cultivated for oil in theMediterranean region and North Africa (5) winter flax (Lusitatissimum L subsp bienne Mill Snankev) cultivated forfiber and oil in theCaucasus Turkey Balkans and some othersouth regions of Europe [9 10] In addition collections offlax germplasm maintain accessions of primitive flax formswith dehiscent capsules (L usitatissimum convar crepitans[Boenningh] Kulpa et Danert) [11]

The taxonomyof the genus cannot be considered as finallyestablished one because the phylogenetic linkages betweenthe individual taxa have not been sufficiently investigatedThe phylogeny of species of the genus Linum was previouslystudied by the use of molecular and cytogenetic approaches[4 12ndash17] but there are problems that still remain to besolved

Transposon-based molecular markers are successfullyused in phylogenic studies Transposable elements wereshown to influence changing in genomic structure as well astranscriptional regulation occurring during the evolution [1819] The presence of transposons in various species of plantstheir high integration activity conservative sequences and alarge number of copies encouraged the use of transposonsin the studies of genetic diversity and profiling of plantvarieties [20ndash22] Several molecular marker systems basedon the information available for the transposable elementssequences were developed for plants [20 22ndash27] SSAP(sequence-specific amplified polymorphism) method wasshown to have a number of advantages as compared to othermarker systems SSAP method produces many polymorphicfragments and allows differentiation of most samples usingonly a single combination of specific primers [23 28ndash30]Different plants were successfully studied by SSAP analysisbut the method has not been applied for the investigation ofspecies of the genus Linum yet Only recently flax sequenceshave appeared in databases [31ndash34] and development of amarker system based on flax transposable elements for theinvestigation of cultivated and wild species of the genusLinum has become possible

In this study the SSAP method was used for assessmentof genetic diversity Besides the possibilities of application

of marker-based profiling for identification of L usitatissi-mum varieties were analyzed We studied 46 varieties of Lusitatissimummainly bred inRussia and a number of varietieswhich were grown in geographically close or distant regionsWe also analyzed different types of cultivated flax (fiberoilseed large seeded winter and dehiscent flax) togetherwith 21 wild species and subspecies from sections LinumAdenolinum Dasylinum and Stellerolinum to estimate thepossibility of using the SSAP method for the investigation offlax domestication history and phylogenic linkages betweendifferent taxa of the genus Linum

2 Materials and Methods

21 Plant Materials For the investigation of genetic diversityof cultivated flax 46 L usitatissimum varieties mainly ofRussian origin were obtained from the All-Russian ResearchInstitute for Flax (VNIIL) (Table 1) For analyzing the repro-ducibility of the SSAP analysis flax variety ldquoStormont cirrusrdquo(IPK genbank Gatersleben accession number LIN 261) wasused

For the investigation of genetic diversity 47 flax acces-sions belonging to 22 species from sections Linum Ade-nolinum Dasylinum and Stellerolinum were used (Table 2)Most of these accessions were obtained from genebank ofLeibniz Institute of Plant Genetics and Crop Plant Research(IPK) (Gatersleben Germany) seed collections of All-Russian Flax Institute (VNIIL) (Torzok Russian Federation)NI Vavilov Research Institute of Plant Industry (VIR) (StPetersburg Russian Federation) and Institute of Geneticsand Cytology of NAS Belarus (IGC) (Minsk Belarus) Theaccessions of L amurense and L stelleroides were kindlyprovided by Dr L N Mironova Botanic Garden Institute ofthe Far-Eastern Branch of the Russian Academy of Sciences(BGI) (Vladivostok Russian Federation) Some accessionswere collected in the wild by Dr A A Svetlova KomarovBotanical Institute RAS (St Petersburg Russian Federation)by Dr N L Bolsheva Engelhardt Institute of MolecularBiology RAS (Moscow Russian Federation) and by Dr MPavelka Euroseeds (Novy Jicin Chech Rep)

22 Confirmation of Species Determination Species determi-nation of some accessions of wild Linum species was doneduring the course of our earlier cytogenetic investigations[14 17 37] To confirm the species determination the restof the accessions were planted in the ground and addition-ally chromosome analysis (determination of chromosomenumber) using acetocarmine staining according to previouslydeveloped approach was performed [14] Chromosome num-bers of all the studied accessions of wild flax are representedin Table 2

23 SSAP Analysis Genomic polymorphism of differentLinum species was studied using SSAP method [23] withmodifications described earlier through genomic studies ofwheat [38] and strawberry [39] Total genomic DNA wasextracted from young flax leaves according to Edwards etal [40] with minor modifications 30 ng of genomic DNA

BioMed Research International 3

Table 1 Studied flax varieties

Number Variety Type Originator Variety pedigree1 Alfa Fiber VNIIL L-1120 Tomskij 4 T-10 Torzhokskij-4 G-4887

2 Gorizont Fiber VNIIL L-1120 T-5 T-10 I-2 I-17 Tvertsa Zarya Marit Smolenskij Severyanin SilvaNatasha Viera

3 Lavina Fiber Smolenskaja GOSHOS Sadko times S-108 [X-5 (selected from L-1120) times 1-7]4 Vasilyok Fiber Belarus I-7 L-1120 X-5 (selected from L-1120) 221-84-45 Soyuz Fiber Smolenskaja GOSHOS6 Lada Fiber VNIIL Viking Fibra7 Slavnyj 82 Fiber VNIIL from Shokinskij variety by single plant selection8 Krom Fiber Pskovskij NIISH9 Tverskoj Fiber VNIIL L-1120 T-4 T-6 T-10 Torzhokskij-4 I-2 G-278210 Rosinka Fiber VNIIL L-1120 T-5 I-17 Fibra 12881211 Lenok Fiber VNIIL L-1120 M-34 (L-1120 times T-4) T-6 Lazurnyj12 A-93 Fiber VNIIL L-1120 T-4 T-6 T-10 Torzhokskij-413 Tost 3 Fiber Sibirskij NIISH14 Mogilevskij-2 Fiber Belarus Stahanovets L-1120 T-5 T-915 Belochka Fiber Vjatskaja GSHA L-1120 Tvertsa16 Torzhokskij-4 Fiber VNIIL M-34 (L-1120 times T-4) times T-10 (G-360 times G-354)17 Tvertsa Fiber VNIIL T-5 times L-112018 Smolich Fiber Smolenskaja GOSHOS Zarya (X-5 times T-5) mutant A-710 8063 severyanin19 A-29 Fiber VNIIL L-1120 T-4 T-5 T-1020 Antey Fiber Pskovskij NIISH

21 Rusich Fiber Pskovskij NIISH Pskovskij 83 (Pryadilshhik L-1120 T-5 Pobeditel) times Rodnik(T-9 Progress I-9 L-1120 VNIIL11 Spartak)

22 Veralin Fiber Netherlands Torzhokskij-4 times Lidiya23 Merilin Fiber Netherlands24 Smolenskij Fiber Smolenskaja GOSHOS Tvertsa (T-5 times L-1120) times Zarya (X-5mdashselected from L-1120 times T-5)25 Tost-5 Fiber Sibirskij NIISH26 S-108 Fiber Smolenskaja GOSHOS X-5 (selected from L-1120) times I-727 Impuls Fiber Smolenskaja GOSHOS S-108 X-5 I-7 Zarya28 Praleska Fiber Belarus29 Lider Fiber Smolenskaja GOSHOS Tayga (France selection) mutagen treatment selection

30 Voronezhskij1308138 Oil VNIIMK

31 Svetoch Fiber VNIIL Selected from Cherskij kryazh32 Ki-5 Oil Ukraine33 LM-98 Oil VNIIL34 Novotorzhskij Fiber VNIIL L-1120 I-2 I-17 selected from Bogotolskij kryazh G-2307 G-4523-6-1335 TMP 1919 china 1 Fiber china36 k-1147 local form Oil Ethiopia37 v-29 Oil China38 Aleksim Fiber VNIIL L-1120 T-4 T-5 T-10 Tekstilshhik M-34 Pryadilshhik Tvertsa39 Ocean k-4497 Oil France40 Orshanskij-2 Fiber Belarus I-16 times L-112041 Κ-6 Fiber Pskovskij NIISH L 1120 times T-542 Vih oil v-2 Oil France43 Donskoj-95 Oil Donskaja opst44 Diplomat Fiber VNIIL Viking times Fibra (with subsequent selection)45 Ford Fiber Belarus46 Tomskij-16 Fiber Sibirskij NIISH and T T-9 times G-1077

4 BioMed Research InternationalTa

ble2Th

eaccessio

nsof

studied

specieso

fgenus

Linu

m

Num

berAc

cessionname

Source

Genebanknu

mber

Orig

inCh

romosom

enum

ber(2n

)SectA

denolin

um1

Lperenn

eL

IPK

LIN1807

RussianFederatio

n2n

=18

[14]

2Lperenn

eLsub

spextraaxillare(Kit)

Nym

anIPK

LIN1651

Poland

2n=36

[17]

3Laltaicu

mLedebex

Juz

IPK

LIN1632

Unk

nown

2n=18

[17]

4LkomaroviiJuz

IPK

LIN1716

Unk

nown

2n=18

[17]

5Lperenn

eLsub

spperenne

IPK

LIN1521

Slovakia

2n=18

[17]

6Lperenn

eLsub

spalpinum

(Jacq)

Stojand

Stef

IPK

LIN1905

Austr

ia2n

=18

[17]

7Lperenn

eLsub

spanglicum

(Mill)

Ockendo

nIPK

LIN1524

GB

2n=36

[17]

8Lperenn

eL

VNIIL

K5500

Unk

nown

2n=36

[17]

9Lleo

niiFW

Schultz

IPK

LIN1672

Germany

2n=18

[14]

10LpallescensB

unge

IPK

LIN1645

Tajik

istan

2n=18

[17]

11LpallescensB

unge

Wild

popu

latio

ncollected

byNLB

olsheva

RussianFederatio

nAltai

2n=18

(present

study)

12Lmesostylum

Juz

IPK

LIN1774

Tajik

istan

2n=18

[17]

13Lmesostylum

Juz

IPK

LIN1662

Tajik

istan

2n=18

[17]

14Llew

isiiP

ursh

IPK

LIN1648

USA

2n=18

[17]

15Llew

isiiP

ursh

IPK

LIN1550

USA

2n=18

[17]

16Lau

striacum

LIPK

LIN1608

Germany

2n=18

[17]

17Lau

striacum

LWild

popu

latio

ncollected

byAA

Svetlo

vaRu

ssianFederatio

nRo

stov

2n=18

(present

study)

18Lau

striacum

Lsubspeuxinu

m(Ju

z)Ockendo

nIPK

LIN1546

Ukraine

2n=18

[17]

19Lau

striacum

LWild

popu

latio

ncollected

byAA

Svetlo

vaCr

imea

2n=18

(present

study)

20Lau

striacum

LWild

popu

latio

ncollected

byAA

Svetlo

vaUkraine

2n=18

(present

study)

21Lam

urenseAlef

BGI

outdoo

rscultivatedcollection

RussianFederatio

nFar

East

near

Pokrovka

settlem

ent

2n=18

[17]

SectD

asylinu

m22

Lhirsutum

Lsubsphirsutum

LIPK

LIN1676

Hun

gary

2n=16

[35]

23Lhirsutum

subsphirsutum

LIPK

LIN1649

Romania

2n=16

[36]

24Lhirsutum

Lsubsppseudoanatolicu

mPH

Davis

Wild

popu

latio

ncollected

byMPavelka

TurkeyK

aram

an2n

=32

(present

study)

25Lhirsutum

Lsubspan

atolicu

m(Boiss)Hayek

Wild

popu

latio

ncollected

byMPavelka

TurkeyA

ksaray

2n=32

(present

study)

SectLinum

26Lmargina

leACun

nex

Planch

IPK

LIN1920

Austr

alia

2n=84

(present

study)

27LnarbonenseL

IPK

LIN2002

Unk

nown

2n=28

[14]

28LnarbonenseL

IPK

LIN1653

France

2n=28

[14]

29Ldecumbens

Desf

IPK

LIN1754

Italy

2n=16

[14]

30Ldecumbens

Desf

IPK

LIN1913

Italy

2n=16

[14]


Table2Con

tinued

Num

berAc

cessionname

Source

Genebanknu

mber

Orig

inCh

romosom

enum

ber(2n

)31

Lgrandiflorum

Desf

IPK

LIN2000

Unk

nown

2n=16

[14]

32Lgrandiflorum

Desf

IPK

LIN974

Unk

nown

2n=16

[14]

33Langustifoliu

mHud

sIPK

LIN1692

France

2n=30

(present

study)

34Langustifoliu

mHud

sVNIIL

K5695

Unk

nown

2n=30

(present

study)

35Langustifoliu

mHud

sVNIIL

K3108

Belgium

2n=30

(present

study)

36Langustifoliu

mHud

sIG

C15

Belarus

2n=30

[14]

37Langustifoliu

mHud

sVNIIL

K4731

EastGermany

2n=30

(present

study)

38Lbienne

Mill

(syn

Langustifolium

Hud

s)IG

C14

Hun

gary

2n=30

[14]

39Lusita

tissim

umLsubspbienne

Mill

Snankev

VIR

u-303794

Unk

nown

winterfl

ax2n

=30

(present

study)

40Lusita

tissim

umconvarcrepita

ns[Boenn

ingh

]Ku

lpae

tDanert

IGC

260

Belarus

dehiscentfl

ax2n

=30

(present

study)

41Lusita

tissim

umconvarcrepita

ns[Boenn

ingh

]Ku

lpae

tDanert

IPK

LIN119

Portugal

dehiscentfl

ax2n

=30

(present

study)

42Lusita

tissim

umLvarie

tyGiza

IPK

LIN277

Egypt

larges

eededflax

2n=30

(present

study)

43Lusita

tissim

umLG12Ru

zokvety

IPK

LIN633

Czecho

slovakia

dual-purpo

seflax

2n=30

(present

study)

44Lusita

tissim

umLvarietyColchidsky1

VIR

u099845

Abkh

azia

winterfl

ax2n

=30

(present

study)

45Lusita

tissim

umL

VNIIL

k7131

Morocco

larges

eededflax

2n=30

(present

study)

SectStellerolinum

46Lste

lleroidesPlanchon

BGI

Outdo

orsc

ultiv

ated

collection

RussianFederatio

nFar

East

TelyakovskyInlet

2n=20

(present

study)

47Lste

lleroidesPlanchon

BGI

Outdo

orsc

ultiv

ated

collection

RussianFederatio

nFar

East

near

Kraskino

settlem

ent

2n=20

(present

study)


was treated with 10U of TaqI restriction enzyme (ThermoScientific USA) for 3 h at 65∘b Ligation was performedby adding 25U of T4 DNA-ligase (Thermo ScientificUSA) 5mM ATP and 50 pmol of double-strand adapter[51015840-ACTCGATTCTCAACCCGAAAGTATAGATCCCA 51015840-PO4-CGTGGGATCTATACTT-(C6linker)-NH

2] and incu-

bation for 7 h at 37∘b The product was diluted twice withdeionized water The DNA sequence between the LTR (longterminal repeat) region of retrotransposons and the 119879119886119902120572119868restriction site was amplified using the adapter primer (51015840-GTTTACTCGATTCTCAACCCGAAAG 31015840) and primers tothe LTR regions of FL1a FL1b FL4 Cassandra FL10 FL8FL7 FL12 and FL9 retrotransposons [32]

1826 51015840-ACCCCTTGAGCTAACTTTTGGGGTAAG-31015840 (FL1a FL1b)1833 51015840-CTTGCTGGAAAGTGTGTGAGAGG-31015840(FL4)1838 51015840-TGTTAATCGCGCTCGGGTGGGAGCA-31015840(FL1a FL1b Cassandra)1845 51015840-AGCCTGAAAGTGTTGGGTTGTCG-31015840(FL11)1846 51015840-CTGGCATTTCCATTGTCGTCGATGC-31015840(FL10)1854 51015840-GCATCAGCCTGGACCAGTCCTCGTCC-31015840 (FL8)1868 51015840-CACTTCAAATTTTGGCAGCAGCGGATC-31015840 (FL1a FL1b)1881 51015840-TCGAGGTACACCTCGACTCAGG-31015840 (FL7)1886 51015840-ATTCTCGTCCGCTGCGCCCCTACA-31015840(FL12)1899 51015840-TGAGTTGCAGGTCCAGGCATCA-31015840(FL9)

Amplification was performed in two stages At the firststage only the primer to LTRof the retrotransposonwas usedAmplificationwas carried out in 25120583L of PCRmix containing5 120583L of ligation mix 1 U of TrueStart Hot Start Taq DNApolymerase (Thermo Scientific USA) TrueStart Taq DNApolymerase buffer 05mM MgCl

2 20120583M dNTP (Thermo

Scientific) and 5 pmol of the LTR primer The program foramplification for the first stage was 95∘C for 15min 30 cycles(95∘C for 30 s 62∘C for 1min 72∘C for 2min) and 72∘Cfor 10min At the second stage of amplification the adapterprimer 51015840-GTTTACTCGATTCTCAACCCGA-31015840 and one ofthe primers to the LTR region of retrotransposons wereused Amplification was carried out in 25 120583L of PCR mixcontaining 12 120583L of the first-stage PCR product 1 U ofTaq DNA polymerase (Thermo Scientific USA) Taq DNApolymerase buffer 15mM MgCl


Scientific USA) 25 pmol of LTR primer and 25 pmol of theadapter primerThe program for amplification for the secondstage was 95∘C for 15min 35 cycles (95∘C for 30 s 62∘C for1min 72∘C for 2min) and 72∘C for 10minThePCRproductswere separated in 25 agarose gel using fj buffer andthen stained with ethidium bromide Ten PCR products were

excised from agarose gel and characterized by sequencingon Applied Biosystems 3730 DNA Analyzer to confirm thespecificity of SSAP reaction The Bio-Rad Gel Doc systemwas used for gel documentation and photography as well asfor visual detection of presence or absence of polymorphicfragments in the samples from different accessions Thesedata were recorded in the form of a binary matrix in whichthe presence of a fragment was coded as 1 and its absence as0

The genetic distances between varieties were calculatedbased on the binary matrix of amplified fragments usingDicersquos formula [41]The dendrogramswere constructed usingSplitsTree 410 software [42] Cluster analysis was performedusing neighbor-joining method [43] and bootstrap valueswere determined based on 5000 permutations

24 Preparation of Cassandra Retrotransposon DNA ProbePCR primers were designed for amplification of Cassandraretrotransposon We amplified an internal domain (primersIntDom-F AGTGGTATCCGAGCCTCT and IntDom-R CCCATAGGACTCAACGTC) and the LTR with theexception of the 5S rDNA region of this retrotransposon(primers LTR-1-86-F TGTAATGTAACACGTTAGGCA andLTR-1-86-R TTAGTTAGGGACGGATTGTT LTR-206-279-F AAATAAATCTGTGAGGGATTAGT and LTR-206-279-RACTTGTAACACCCCGTACT) The amplification wascarried out in 20 120583L of PCR mixture that contained 1Uof TaqF DNA polymerase (Amplisens Russia) 1x TaqFbuffer 25 pmol of the forward and reverse primer 200120583MdNTP (Amplisens Russia) and 10 ng of genomic DNA Theprogram for amplification was 95∘C for 15min 40 cycles(95∘C for 10 s 62∘C for 20 s 72∘C for 30 s) and 72∘C for10min The amplicons were analyzed in 2 agarose gel andthen used as a template for biotin PCR labeling to obtainbiotin-labeled probes for FISH PCR labeling was carriedout using Biotin PCR Labeling Core Kit (Jena BioscienceGermany) according to the manufacturersquos protocol LabeledPCR products were precipitated with ethanol

25 FISH with Cassandra Retrotransposon DNA ProbeChromosome preparation was carried out according to thetechnique developed earlier for plants having small-sizedchromosomes [14] The hybridization mixture contained 2xSSC 50 formamide 10 dextran sulphate and 2 ng120583L ofa biotinylated DNA probe of Cassandra retrotransposonTheprobe was hybridized overnight at 31∘C After hybridizationthe slides were washed twice with 01x SSC at 38∘C for10min followed by two washes with 2x SSC at 44∘C for5min and a final 5min wash in 2x SSC at room temperatureThe biotin-labeled DNA probe was detected using a highlysensitive Alexa Fluor 488 Tyramide Signal Amplificationsystem (Invitrogen) according tomanufacturerrsquos instructions

3 Results

31 Analysis of SSAP Fingerprints For analyzing of thereproducibility of the SSAP method DNA of flax variety


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Figure 1 The test of reproducibility of SSAP markers obtained for ldquoStormont cirrusrdquo flax variety Lanes 2 and 3 primer 1899 lanes 4 and 5primer 1826 lanes 6 and 7 primer 1838 lanes 8 and 9 primer 1845 lanes 10 and 11 primer 1846 lanes 13 and 14 primer 1854 lanes 15 and 16primer 1868 lanes 17 and 18 primer 1881 lanes 19 and 20 primer 1886 lanes 1 and 12 100 bp DNA ladder

ldquoStormont cirrusrdquo was restricted twice ligated and ampli-fied with the primers The obtained PCR products werevisualized in acrylamide and agarose gels (Figure 1) Elec-trophoretic spectra of PCR products obtained with primers1826 1838 1845 1868 1886 and 1899 coincided completelydemonstrating high reproducibility of the SSAP results Thefingerprints obtainedwith primers 1846 1854 and 1881 variedin individual amplified fragments so the use of primers1846 1854 and 1881 for SSAP analysis of flax varieties willrequire further optimization of restriction ligation or PCRconditions We selected primers with high reproducibility(1838 1845 1868 and 1899) as they yielded PCR productsthat were easily discernible in an agarose gel These primerswere used for analysis of 46 flax varieties by the SSAPmethod

All the examined varieties produced identical or verysimilar fingerprints with primers 1838 and 1868 (FL1aFL1b and Cassandra) At the same time the PCR productsobtainedwith primers 1845 and 1899were unique for differentflax varieties So primers 1845 and 1899 were chosen foranalyzing of genetic diversity of flax varieties Several ofPCR products have been sequenced (SupplementaryMaterialavailable online at httpdxdoiorg1011552014231589) themajority of obtained sequences are significantly similar to thesequences of corresponding retrotransposons

32 Analysis of Genetic Diversity of Flax Varieties Visualanalysis of SSAP fingerprints based on retrotransposons FL11and FL9 revealed 44 polymorphic retrotransposon insertions(23 fragments for primer 1845 and 21 fragments for primer1899) in 46 flax varieties Each of the 46 varieties had theirown unique spectrum of retrotransposon insertions So wecould differentiate all the 46 varieties using only two SSAPprimers In order to analyze genetic diversity of these vari-eties we compiled a binary matrix of the presenceabsence ofpolymorphic insertions of the above-mentioned retrotrans-posons calculated the genetic distances between the varietiesusing Dicersquos formula [41] and constructed a dendrogram byusing the neighbor-joining method (Figure 2) The obtainedtree branching pattern revealed no distinct clusters amongexamined varieties

33 Genomic Diversity of Species of the Genus Linum Forinvestigation of species of the genus Linum we chose retro-transposons FL1a FL1b and Cassandra which did not showhigh insertion polymorphism within cultivated flax varietiesPrimers 1838 and 1868 were used for SSAP analysis Asthe result 95 bands that originated with primer 1838 and128 bands with primer 1868 (Figure 3) were scored All thebands were polymorphic Based on the SSAP fingerprintsimilarity nine groups of closely related species (A-I) weredistinguished Group A included different species of sectAdenolinum (syn L perenne group) group B consisted ofL hirsutum subsp hirsutum accessions groupC10158401015840 includedL hirsutum subsp pseudoanatolicum and L hirsutum subspanatolicum Groups D E F and G comprised speciesaccessions of sect Linum (L marginale L narbonense Ldecumbens and L grandiflorum resp) Group H includedaccessions of L angustifolium and L usitatissimum (sectLinum) and group I consisted of L stelleroides accessions(sect Stellerolinum)

All the groups contained at least one group-specificmarker The results of phylogenetic analysis of Linum speciesare shown in the dendrogram on Figure 4 As the dendro-gram shows nine clearly distinguished groups of speciessupported by high bootstrap values can be observed

34 FISH with Cassandra Retrotransposon DNA Probe Thehighly sensitive tyramide FISH method was applied for theinvestigation of abundanceof Cassandra retrotransposonsas well as their distribution along chromosomes in threespecies of sect Linum (L usitatissimum L grandiflorumand L narbonense) and L amurense (sect Adenolinum)FISH revealed that Cassandra dispersed along the wholelength of chromosomes in karyotypes of four studied speciesbut its distribution along the chromosomes was nonrandom(Figure 5) In species having small-sized chromosomes (Lusitatissimum L grandiflorum and L amurense) Cassandrawas mainly localized in pericentromeric and subtelomericchromosome regions The patterns of Cassandra distributionwere chromosome specific and were similar in homologouspairs of chromosomes (Figure 5(e)) In L narbonense which


Ant

Tor

Tve

VerTos

Mer

Pra

Dip

A93

Ale

Novk-1147

K6Ors

v29Vik

Imp

Lid

Tom

Vor Kro

GorVasLav

Ros

AlfLadSla

Don

Oce

Sve

LM98 SmolicTvers

Len

Soy

A29

Ki5BelFor

Smo

Tost5

001

MogRus

S108

TMR

Figure 2 A neighbor-joining dendrogram for SSAP markers Flax varieties Alf-Alfa Gor Gorizont Lav Lavina Vas Vasilyok Soy SoyuzLad Lada Sla Slavnyj 82 Kro Krom Tvers Tverskoj Ros Rosinka Len Lenok A93 A-93 Tos Tost 3 Mog Mogilevskij 2 Bel BelochkaTor Torzhokskij 4 Tve Tvertsa Smolic Smolich A29 A-29 Ant Antey Rus Rusich Ver Veralin Mer Merilin Smo Smolenskij Tost5Tost 5 S108 S 108 Imp Impuls Pra Praleska Lid Lider Vor Voronezhskij Sve Svetoch Ki5 Ki-5 Lm98 LM-98 Nov Novotorzhskij TMRTMR-1919 k-1147 k-1147 v29 v-29 Ale Aleksim Oce Ocean k-4497 Ors Orshanskij 2 K6 K-6 Vic Viksoil V-2 Don Donskoj 95 DipDiplomat For Ford Tom Tomskij 16


1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 474543413937353331M M

Figure 3 SSAP markers generated using 1868 primer for Linum species 1 L perenne LIN 1807 2 L perenne subsp extraaxilare LIN 16513 L altaicum LIN 1632 4 L komarovii LIN 1716 5 L perenne LIN 1521 6 L perenne susp alpinum LIN 1905 7 L perenne susp anglicumLIN 1524 8 L perenne K 5500 9 L leonii LIN 1672 10 L pallescens LIN 1645 11 L pallescens Altai 12 L mesostylum LIN 1774 13 Lmesostylum LIN 1662 14 L lewisii LIN 1648 15 L lewisii LIN 1550 16 L austriacum LIN 1608 17 L austriacum Rostov 18 L austriacumsubsp euxinum LIN 1546 19 L austriacum Crimea 20 L austriacum Ukraine 21 L amurense 22 L hirsutum LIN 1676 23 L hirsutumLIN1649 24 L hirsutum subsp pseudoanatolicum 25 L hirsutum subsp anatolicum 26 L marginale 27 L narbonense LIN 2002 28 Lnarbonense LIN1653 29 L decumbens LIN 1754 30 L decumbens LIN1913 31 L grandiflorum LIN 2000 32 L grandiflorum LIN 97433 L angustifolium LIN 1692 34 L angustifolium K 5695 35 L angustifolium K 3108 36 L angustifolium Belarus 37 L angustifolium K4731 38 L biene (syn L angustifolium) 39 winter flax (L usitatissimum subsp biene) 40 dehiscent flax (L usitatissimum convar crepitans)260 41 dehiscent flax (L usitatissimum convar crepitans) LIN 119 42 large seeded flax(L usitatissimum) LIN 277 43 dual-purpose flax(L usitatissimum) LIN 633 44 winter flax (L usitatissimum) u 099845 45 large seeded flax (L usitatissimum) Q 7131 46 L stelleroidesTelyakovsky Inlet 47 L stelleroides Kraskino settlement Mmdash100 bp DNA ladder

L mesostylumL mesostylum

L austriacum

L hirsutumL hirsutum subsp an

L usitatissimum subsp bien

L bienne

L usitatissimum convarcrepL usitatissimum convarcrep

L usitatissimum

L grandiflorum L grandiflorum

L decumbens

L marginale

L decumbens

L usitatissimum L angustifoliumL usitatissimumL angustifolium

L angustifolium

L usitatissimum

L narbonenseL narbonense

L hirsutumL hirsutum subsp ps

L austriacumL altaicum

L perenne subspextrL perenne

L austriacumL austriacum subsp eux

L leonii

L pallescens

L amurenseL lewisii

L komarovii

L pallescens

L stelleroidesL stelleroides

L perenne L pallescensL perenne subsp angl

L perenne susp alp

L austriacum

A

D I

F

G

H

E

С B

32

29

26

30

4647

31

39

45414044

3837433433 35

2728

2524 23 22

20 18

17 161312

193

21

99 9802

99 9

99 9

97

95

100

100 100

100

100

100

99 8871732

81

97 811

6105

21

15

14

4

3642

Sect Stellerolinum

Sect

Lin

um

Sect Dasylinum

Sect

Ade

nolin

um

Figure 4 Neighbor-joining dendrogram for SSAP markers Flax species 1 L perenne LIN 1807 2 L perenne subsp extraaxilare LIN 16513 L altaicum LIN 1632 4 L komarovii LIN 1716 5 L perenne LIN 1521 6 L perenne susp alpinum LIN 1905 7 L perenne susp anglicumLIN 1524 8 L perenne K 5500 9 L leonii LIN 1672 10 L pallescens LIN 1645 11 L pallescens Altai 12 L mesostylum LIN 1774 13 Lmesostylum LIN 1662 14 L lewisii LIN 1648 15 L lewisii LIN 1550 16 L austriacum LIN 1608 17 L austriacum Rostov 18 L austriacumsubsp euxinum LIN 1546 19 L austriacum Crimea 20 L austriacum Ukraine 21 L amurense 22 L hirsutum LIN 1676 23 L hirsutumLIN1649 24 L hirsutum subsp pseudoanatolicum 25 L hirsutum subsp anatolicum 26 L marginale 27 L narbonense LIN 2002 28 Lnarbonense LIN1653 29 L decumbens LIN 1754 30 L decumbens LIN1913 31 L grandiflorum LIN 2000 32 L grandiflorum LIN 97433 L angustifolium LIN 1692 34 L angustifolium K 5695 35 L angustifolium K 3108 36 L angustifolium Belarus 37 L angustifolium K4731 38 L biene (syn L angustifolium) 39 winter flax (L usitatissimum subsp biene) 40 dehiscent flax (L usitatissimum convar crepitans)260 41 dehiscent flax (L usitatissimum convar crepitans) LIN 119 42 large seeded flax (L usitatissimum) LIN 277 43 dual-purpose flax(L usitatissimum) LIN 633 44 winter flax (L usitatissimum) u 099845 45 large seeded flax (L usitatissimum) Q 7131 46 L stelleroidesTelyakovsky Inlet 47 L stelleroides Kraskino settlement Bootstrap values that exceeded 70 are shown in italic


(a) (b)

(c) (d)

1 2 3 4 5 6 7 8

(e)

Figure 5 FISHwith DNA probe of Cassandra retrotransposons (green) (a) L usitatissimum (sect Linum) (b) L grandiflorum (sect Linum)(c) L narbonense (sect Linum) (d) L amurense (sect Adenolinum) (e) a karyotype of L grandiflorum Chromosomes were stained withDAPI (blue) Barmdash5120583m

possessed large chromosomes the patterns of Cassandradistribution resembled the patterns observed in karyotypesof the above-mentioned species (having small-sized chromo-somes) though they were more regular

4 Discussion

41 Use of SSAP Analysis for Identification of Flax Varietiesand Estimation of Genetic Diversity To estimate genetic

polymorphism and to characterize L usitatissimum varietiesthe SSAP method was used and also high reproducibility ofthe method was shown The validity of the SSAP method formolecular genetic studies of flax varieties using FL11 and FL9retrotransposons was demonstrated Obtained with primers1838 and 1868 (retrotransposons FL1a FL1b and Cassandra)PCR products were very similar in all the studied varietiesSuch low diversity of cultivated flax might be a result oflow transposition activity andor creation of bottleneck effectduring flax selection


In analyzed flax varieties 44 polymorphic insertions forFL11 (primer 1845) and FL9 (primer 1899) retrotransposonswere revealed Every studied variety possessed a unique setof SSAPmarkersTherefore the SSAPmethod can be used tomark the genotypes to identify varieties ofL usitatissimum ingenebank collections to exercise control during of flax varietygrowth and to obtain high quality seed material when thevarietal identity is particularly important

The genetic similarity of 46 flax varieties was charac-terized by genetic distances calculated based on the SSAPdata The dendrogram (Figure 3) did not contain clearlyisolated clusters of varieties Thus the studied flax varietiescould not be subdivided into distinct groups Our resultswere in agreement with earlier obtained data shown that flaxaccessions examined by IRAP analysis did not form distinctclusters in studies of their origin or the type of commercialuse (fiber or oil) These data indicated an overlap in geneticdiversity despite of disruptive selection for fiber or seed oiltypes [32] In our study the SSAP method also failed todistinguish fiber or oil seed flax varieties Since varietieswith the best characteristics are commonly used as parentsin breeding practice some valuable flax forms present inthe genealogy of most modern varieties Besides the linesselected for crossing are usually characterized by low geneticdiversity So the commercial flax varieties were shown to beless diverse than wild flax species and landraces [32]

Although the examined flax varieties could not be clus-tered into different groups by SSAP method it might beused for estimation of their genetic similarity based onpolymorphic insertions of retrotransposons The estimationcan be used for choosing the parents in breeding practiceand also for creation of core collections which should includegenetically diverse accessions

42 Diversity and Phylogeny of Linum Species In the presentstudy 20 accessions from sect Linum 21 accessions fromsect Adenolinum 4 accessions from sect Dasylinum and 2accessions from sect Stellerolinum were analyzed by usingSSAP method All the examined species were clustered into9 groups mainly according to common taxonomic divisionof the genus Linum into sections (Figure 4)

43 Section Dasylinum Species from sect Dasylinum clus-tered together and formed two related groups B and CGroup B included L hirsutum subsp pseudoanatolicum andL hirsutum subsp anatolicum and group C included Lhirsutum subsp hirsutum Thus SSAP analysis singled outsect Dasylinum as a well-supported clade Our results werein agreement with the AFLP and ITS data as well as chloro-plast phylogenies chromosome studies and transcriptomeanalysis of Linum species [4 13 37 44] It should be men-tioned that the subdivision of accessions of sect Dasylinuminto two related clusters correlated with their difference inchromosome numbers and the origin of accessions Thusthe accessions of L hirsutum subsp hirsutum (cluster C)from Europe was characterized by chromosome numberof 2n = 16 while accessions from Turkey L hirsutumsubsp pseudoanatolicum and L hirsutum subsp anatolicum

(cluster B) have chromosome number 2n = 32 Chromosomenumbers for L hirsutum subsp pseudoanatolicum and Lhirsutum subsp anatolicum were firstly determined in thepresent study

44 Section Stellerolinum Two accessions of L stelleroideshave rather similar SSAP fingerprints which were differedsignificantly from all the others Linum species and formeda separated clade The similar results were obtained byphylogenetic analyses of chloroplast and ITS DNA sequences[4] Moreover L stelleroideswas shown to have chromosomenumber 2n = 20 which was unique for blue-flowered flaxes[45]

45 Section Adenolinum All the members of sect Adeno-linum formed an independent group clustered separatelyfrom species of sect Linum and other sections Distinctisolation of this species group was also revealed in severalmolecular and karyological investigations [4 12ndash14 17] Thedata were in good agreement with the opinion of Yuzepchuk[2] and Egorova [3] who isolated the group from sect Linuminto an independent section Adenolinum

SSAP fingerprints of the accessions within sect Adeno-linum were highly polymorphic but SSAP markers did notallow us to reveal any species subclusters supported by ahigh bootstrap valueThus SSAP analysis used in the presentstudy as well as AFLP and RAPD analyses [13 17] separatedindividual accessions but did not identify individual speciesinside sect Adenolinum

46 Section Linum Sect Linumwas subdivided into 5 groupsby neighbor-joining clustering Accessions of L marginaleL grandiflorum L decumben and L narbonense formedfour independent single species groups while the fifth groupcombined accessions L angustifolium and L usitatissimumSimilar results had been obtained earlier by AFLP RAPDmolecular phylogeny based on chloroplast RbsL sequenceand molecular cytogenetic methods (CDAPI-banding pat-terns and localization of rRNA genes on chromosomes) [412ndash14]

Within a subgroup consisted of L usitatissimum and Langustifolium the accessions of large seeded flax (breedingcultivar) dual-purpose flax and L angustifolium were rathersimilar Their fingerprints did not differ significantly fromfingerprints of studied 46 flax varieties This data were in agood agreement with the suggestion that L angustifoliumwasthe progenitor of L usitatissimum [5 7]

The accession of large seeded flax landrace the accessionsof winter flax and the accessions of dehiscent flax differedsignificantly from the other members of cluster H Bothaccessions of dehiscent flax grouped together (supported by ahigh bootstrap value) and had species-specific SSAPmarkers

It should be noted that flax accessions which are genet-ically distant from modern flax cultivars are particularimportant for flax breedingThegenetic diversity of cultivatedflax decreased significantly during the last decades It mightlead to the lack of useful alleles in genomes of moderncultivars [13] Therefore introduction of new useful traits


from the ancient primitive forms of cultivated flax and wildspecies could increase the polymorphism of modern flaxvarieties SSAP markers allowed us to identify the uniqueaccessions which are important for the investigation of thehistory of flax domestication

L marginale the last member of sect Linum is a wildflax native to Australia We found that it had the maximalchromosome number (2n = 84) in the genus Linum Thenumber indicated a high level of ploidy of L marginalegenome SSAP patterns of the species were significantlydifferent compared with the other species of sect LinumThereforeLmarginale clustered apart from the other speciesThe obtained results were in contradiction with ITS andchloroplast topologies which clustered the species togetherwith L bienne and L usitatissimum [4] Rogers [46] assumedthat Australian and New Zealand species L marginale Cunnand L monogynum Forst were related to European speciesL hologynum Reichenb (sec Linum)This assumption basedon the fact that diploid chromosome number of L hologynum(2n = 42) corresponded to haploid chromosome numberof L monogynum and L marginale Moreover all the threespecies had fused styles and pantoporate pollen grains thatwere unusual for blue-flowered flaxes Thus all the above-mentioned data indicated that the phylogenic lineages of Lmarginale need further investigation

The data obtained in the present study as well as theresults of other molecular phylogenetic and chromosomalinvestigations indicated thatmembers of section Linumwerenot as closely related asmembers of other sectionsThereforetaxonomic revision of this section is desirable

47 Chromosome Location of Cassandra Retrotransposon Asdifferences in SSAP fingerprints for several flax species werefound we decided to analyze the distribution of Cassandraretrotransposon along the chromosomes of Linum speciesCassandra is a terminal-repeat retrotransposon in miniature(TRIM) that carries conserved 5S rDNA sequences in itsLTRs Cassandra was found in a number of vascular plants[31] In our work we investigated the distribution of thisretrotransposon along the chromosomes of Linum speciesusing tyramide FISHWe revealed thatCassandra localized inpericentromeric and subtelomeric regions of chromosomesthat was typical for transposable elements [47] A more uni-form distribution of Cassandra retrotransposon was foundin L narbonense in comparison with L usitatissimum Lgrandiflorum and L amurense It was probably due to ahigher content of transposable elements correlated with alarger size of its chromosomes (therefore its genome)

5 Conclusions

The availability of LTR sequences of flax retrotransposonsand high polymorphism of SSAP markers offer a promisingpotential for SSAP analysis of genus Linum Applications ofSSAP analysis for example evolutionary and phylogeneticstudies assessment of genetic diversity accession identifica-tion and search for exotic genepools of cultivated flax couldbe applied to L usitatissimum and other Linum species SSAP

analysis was shown to be very useful for characterizationof flax varieties and identification of accession belonging todifferent species or sections and provided new informationabout of phylogenetic relationships within the genus Linum

Conflict of Interests

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

Authorsrsquo Contribution

Nataliya VMelnikova AnnaV Kudryavtseva andAlexanderV Zelenin contributed equally to this work

Acknowledgments

The work was financially supported by the Russian Founda-tion for Basic Research (Grants 12-04-01469-1 13-04-01770-aand 14-08-01167) by Fundamental Research Program of theRussian Academy of Sciences ldquoDynamics of Plant Animaland Human Genofondsrdquo and by the Ministry of Educationand Science of the Russian Federation under state Contract14621210001 Part of this work was performed at the EIMBRAS ldquoGenomerdquo center

References

[1] D J Ockendon and SMWalters Linaceae Cambridge Univer-sity Press Cambridge Mass USA 1968

[2] S A Yuzepchuk ldquoGenus LinummdashLinaceae Dumortrdquo in FloraSSSR (Flora of the Soviet Union) B K Shishkin Ed vol 14 pp84ndash146 Izdatelrsquostvo Akademii Nauk Leningrad Russia 1949

[3] T V EgorovaGenus Linum Linaceae St Petersburg Publishing1996

[4] JMcDillM Repplinger B B Simpson and JW Kadereit ldquoThephylogeny of Linum and Linaceae subfamily Linoideae withimplications for their systematics biogeography and evolutionof heterostylyrdquo Systematic Botany vol 34 no 2 pp 386ndash4052009

[5] H Helbaek ldquoDomestication of food plants in the old worldrdquoScience vol 130 no 3372 pp 365ndash372 1959

[6] W van Zeist and J A H Bakker-Heeres ldquoEvidence for linseedcultivation before 6000 bcrdquo Journal of Archaeological Sciencevol 2 no 3 pp 215ndash219 1975

[7] A Diederichsen and K Hammer ldquoVariation of cultivated flax(Linum usitatissimum L subsp usitatissimum) and its wildprogenitor pale flax (subsp angustifolium (Huds) Thell)rdquoGenetic Resources and Crop Evolution vol 42 no 3 pp 263ndash272 1995

[8] D Zohary and M Hopf Domestication of Plants in the OldWorld The Origin and Spread of Cultivated Plants in West AsiaEurope and the Nile Valley Oxford University Press OxfordUK 2000

[9] I A Sisov Flax Selhosgiz Leningrad Russia 1955[10] N M Cernomorskaja and A K Stankevic ldquoK voprosu o

vnutryvidovoj klassifikacii lna obyknovennogo (Linumusitatis-simum L) To the problem of intraspecific classification ofcommon flax (Linum usitatissimum L)rdquo Selekcija I GenetikaTehnicheskih Kultur vol 113 pp 53ndash63 1987


[11] A Diederichsen ldquoEx situ collections of cultivated flax (Linumusitatissimum L) and other species of the genus Linum LrdquoGenetic Resources andCrop Evolution vol 54 no 3 pp 661ndash6782007

[12] Y Fu G Peterson A Diederichsen andKW Richards ldquoRAPDanalysis of genetic relationships of seven flax species in thegenus Linum Lrdquo Genetic Resources and Crop Evolution vol 49no 3 pp 253ndash259 2002

[13] J Vromans Molecular genetic studies in flax (Linum usitatissi-mum L) [PhD thesis] Wageningen University WageningenThe Netherlands 2006

[14] O V Muravenko O Y Yurkevich N L Bolsheva et alldquoComparison of genomes of eight species of sections Linumand Adenolinum from the genus Linum based on chromosomebanding molecular markers and RAPD analysisrdquoGenetica vol135 no 2 pp 245ndash255 2009

[15] Y Fu and R G Allaby ldquoPhylogenetic network of Linum speciesas revealed by non-coding chloroplast DNA sequencesrdquoGeneticResources and Crop Evolution vol 57 no 5 pp 667ndash677 2010

[16] B J Soto-Cerda H U Saavedra C N Navarro and P MOrtega ldquoCharacterization of novel genic SSR markers in Linumusitatissimum (L) and their transferability across eleven Linumspeciesrdquo Electronic Journal of Biotechnology vol 14 no 2 2011

[17] O Y Yurkevich A A Naumenko-Svetlova N L Bolshevaet al ldquoInvestigation of genome polymorphism and seed coatanatomy of species of section Adenolinum from the genusLinumrdquoGenetic Resources and Crop Evolution vol 60 no 2 pp661ndash676 2013

[18] J L Bennetzen ldquoTransposable element contributions to plantgene and genome evolutionrdquo Plant Molecular Biology vol 42no 1 pp 251ndash269 2000

[19] C Feschotte and E J Pritham ldquoDNA transposons and theevolution of eukaryotic genomesrdquo Annual Review of Geneticsvol 41 pp 331ndash368 2007

[20] C Feschotte N Jiang and S R Wessler ldquoPlant transposableelements where genetics meets genomicsrdquo Nature ReviewsGenetics vol 3 no 5 pp 329ndash341 2002

[21] P S Schnable D Ware R S Fulton et al ldquoThe B73 maizegenome complexity diversity and dynamicsrdquo Science vol 326no 5956 pp 1112ndash1115 2009

[22] R Kalendar A J Flavell T H N Ellis T Sjakste CMoisy and A H Schulman ldquoAnalysis of plant diversity withretrotransposon-based molecular markersrdquo Heredity vol 106no 4 pp 520ndash530 2011

[23] R Waugh K McLean A J Flavell et al ldquoGenetic distributionof Bare-1-like retrotransposable elements in the barley genomerevealed by sequence-specific amplification polymorphisms (S-SAP)rdquoMolecular and General Genetics vol 253 no 6 pp 687ndash694 1997

[24] T H N Ellis S J Poyser M R Knox A V Vershinin and MJ Ambrose ldquoPolymorphism of insertion sites of Ty1-copia classretrotransposons and its use for linkage and diversity analysisin peardquo Molecular and General Genetics vol 260 no 1 pp 9ndash19 1998

[25] R A Queen B M Gribbon C James P Jack and A JFlavell ldquoRetrotransposon-based molecular markers for linkageand genetic diversity analysis in wheatrdquoMolecular Genetics andGenomics vol 271 no 1 pp 91ndash97 2004

[26] NVMelnikova F A Konovalov andAMKudryavtsev ldquoLongterminal repeat retrotransposon Jeli provides multiple geneticmarkers for commonwheat (Triticum aestivum)rdquo Plant Genetic

Resources Characterisation andUtilisation vol 9 no 2 pp 163ndash165 2011

[27] H Kim S Terakami C Nishitani et al ldquoDevelopment ofcultivar-specific DNAmarkers based on retrotransposon-basedinsertional polymorphism in Japanese pearrdquo Breeding Sciencevol 62 no 1 pp 53ndash62 2012

[28] S R Pearce M Knox T H N Ellis A J Flavell and AKumar ldquoPea Ty1-copia group retrotransposons transpositionalactivity and use as markers to study genetic diversity in PisumrdquoMolecular and General Genetics vol 263 no 6 pp 898ndash9072000

[29] A V Vershinin T R Allnutt M R Knox M J Ambroseand T H N Ellis ldquoTransposable elements reveal the impactof introgression rather than transposition in Pisum diversityevolution and domesticationrdquoMolecular Biology and Evolutionvol 20 no 12 pp 2067ndash2075 2003

[30] A M Sanz S G Gonzalez N H Syed M J Suso C CSaldana and A J Flavell ldquoGenetic diversity analysis in Viciaspecies using retrotransposon-based SSAP markersrdquoMolecularGenetics and Genomics vol 278 no 4 pp 433ndash441 2007

[31] R Kalendar J Tanskanen W Chang et al ldquoCassandra retro-transposons carry independently transcribed 5SRNArdquoProceed-ings of the National Academy of Sciences of the United States ofAmerica vol 105 no 15 pp 5833ndash5838 2008

[32] P Smykal N Bacova-Kerteszovac R Kalendar J CoranderA H Schulman and M Pavelek ldquoGenetic diversity of cul-tivated flax (Linum usitatissimum L) germplasm assessedby retrotransposon-based markersrdquo Theoretical and AppliedGenetics vol 122 no 7 pp 1385ndash1397 2011

[33] R Ragupathy R Rathinavelu and S Cloutier ldquoPhysicalmapping and BAC-end sequence analysis provide initialinsights into the flax (Linum usitatissimum L) genomerdquo BMCGenomics vol 12 article 217 2011

[34] L G Gonzalez and M K Deyholos ldquoIdentification charac-terization and distribution of transposable elements in the flax(Linum usitatissimum L) genomerdquo BMC Genomics vol 13 no1 article 644 2012

[35] I V Nosova O Y Semenova T E Samatadze et al ldquoInvesti-gation of karyotype structure and mapping of ribosomal geneson chromosomes of wild linum species by FISHrdquo BiologicheskieMembrany vol 22 no 3 pp 244ndash248 2005

[36] N L Bolsheva O Y Semenova O V Muravenko I V NosovaK V Popov and A V Zelenin ldquoLocalization of telomeresequences in chromosomes of two flax speciesrdquo BiologicheskieMembrany vol 22 no 3 pp 227ndash231 2005

[37] O V Muravenko N L Bolrsquosheva O I Iurkevich et alldquoKaryogenomics of species of the genus Linum LrdquoGenetika vol46 no 10 pp 1339ndash1342 2010

[38] F A Konovalov N P Goncharov S Goryunova A ShaturovaT Proshlyakova andAKudryavtsev ldquoMolecularmarkers basedon LTR retrotransposons BARE-1 and Jeli uncover differentstrata of evolutionary relationships in diploid wheatsrdquo Molec-ular Genetics and Genomics vol 283 no 6 pp 551ndash563 2010

[39] N V Melnikova A V Kudryavtseva A S Speranskaya et alldquoThe FaRE1 LTR-retrotransposon based SSAP markers revealgenetic polymorphism of strawberry (Fragaria x ananassa)cultivarsrdquo Journal of Agricultural Science vol 4 no 11 pp 111ndash118 2012

[40] K Edwards C Johnstone and C Thompson ldquoA simple andrapid method for the preparation of plant genomic DNA forPCR analysisrdquoNucleic Acids Research vol 19 no 6 p 1349 1991


[41] L R Dice ldquoMeasures of the amount of ecologic associationbetween speciesrdquo Ecology vol 26 no 3 pp 297ndash302 1945

[42] D H Huson and D Bryant ldquoApplication of phylogenetic net-works in evolutionary studiesrdquoMolecular Biology and Evolutionvol 23 no 2 pp 254ndash267 2006

[43] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquoMolecular biol-ogy and evolution vol 4 no 4 pp 406ndash425 1987

[44] S Sveinsson J McDill G K Wong et al ldquoPhylogeneticpinpointing of a paleopolyploidy event within the flax genus(Linum) using transcriptomicsrdquo Annals of Botany vol 113 no5 pp 753ndash761 2014

[45] A P Sokolovskaya andN S Probatova ldquoChromosomenumbersin the vascular plants from the Primorye territory Kamchatkaregion Amur valley and Sakhalinrdquo Botanicheskii Zhurnal SSSRvol 70 no 4 pp 997ndash999 1985

[46] C M Rogers ldquoA further note on the relationships of theEuropean Linum hologynum and the Australian species ofLinum (Linaceae)rdquo Plant Systematics and Evolution vol 147 no3-4 pp 327ndash328 1984

[47] J S P Heslop-Harrison and T Schwarzacher ldquoOrganisation ofthe plant genome in chromosomesrdquo Plant Journal vol 66 no1 pp 18ndash33 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

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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


nuclear ITS (internal transcribed spacer) DNA sequencesrevealed that genus Linum was not monophyletic It con-tains two major lineages a yellow-flowered clade (sectionsLinopsis Syllinum and Cathartolinum) and a blue-floweredclade (sections LinumDasylinum and Stellerolinum) [4]Thecultivated flax (L usitatissimum L) belongs to sec Linumfrom a blue-flowered clade L usitatissimum is believed tohave originated as a result of domestication of wild speciesL angustifolium Huds approximately 8000 years ago [5ndash8]For a long time flax has been cultivating as a dual-purposecrop grown for its fiber and linseed oil

According to morphological and qualitative traits cul-tivated flax was divided into five main types (1) fiber flax(L usitatissimum subsp usitatissimum) (2) oil flax (L usi-tatissimum L subsp humileCzernom) (3) dual-purpose flax(L usitatissimum L subsp intermedium Czemom) that wasan intermediate form between the first two ones cultivatedfor fiber and oil (4) large seeded flax (L usitatissimum Lsubsp latifolium Snankev) which is characterized by a set ofspecific morphological features and cultivated for oil in theMediterranean region and North Africa (5) winter flax (Lusitatissimum L subsp bienne Mill Snankev) cultivated forfiber and oil in theCaucasus Turkey Balkans and some othersouth regions of Europe [9 10] In addition collections offlax germplasm maintain accessions of primitive flax formswith dehiscent capsules (L usitatissimum convar crepitans[Boenningh] Kulpa et Danert) [11]

The taxonomyof the genus cannot be considered as finallyestablished one because the phylogenetic linkages betweenthe individual taxa have not been sufficiently investigatedThe phylogeny of species of the genus Linum was previouslystudied by the use of molecular and cytogenetic approaches[4 12ndash17] but there are problems that still remain to besolved

Transposon-based molecular markers are successfullyused in phylogenic studies Transposable elements wereshown to influence changing in genomic structure as well astranscriptional regulation occurring during the evolution [1819] The presence of transposons in various species of plantstheir high integration activity conservative sequences and alarge number of copies encouraged the use of transposonsin the studies of genetic diversity and profiling of plantvarieties [20ndash22] Several molecular marker systems basedon the information available for the transposable elementssequences were developed for plants [20 22ndash27] SSAP(sequence-specific amplified polymorphism) method wasshown to have a number of advantages as compared to othermarker systems SSAP method produces many polymorphicfragments and allows differentiation of most samples usingonly a single combination of specific primers [23 28ndash30]Different plants were successfully studied by SSAP analysisbut the method has not been applied for the investigation ofspecies of the genus Linum yet Only recently flax sequenceshave appeared in databases [31ndash34] and development of amarker system based on flax transposable elements for theinvestigation of cultivated and wild species of the genusLinum has become possible

In this study the SSAP method was used for assessmentof genetic diversity Besides the possibilities of application

of marker-based profiling for identification of L usitatissi-mum varieties were analyzed We studied 46 varieties of Lusitatissimummainly bred inRussia and a number of varietieswhich were grown in geographically close or distant regionsWe also analyzed different types of cultivated flax (fiberoilseed large seeded winter and dehiscent flax) togetherwith 21 wild species and subspecies from sections LinumAdenolinum Dasylinum and Stellerolinum to estimate thepossibility of using the SSAP method for the investigation offlax domestication history and phylogenic linkages betweendifferent taxa of the genus Linum

2 Materials and Methods

21 Plant Materials For the investigation of genetic diversityof cultivated flax 46 L usitatissimum varieties mainly ofRussian origin were obtained from the All-Russian ResearchInstitute for Flax (VNIIL) (Table 1) For analyzing the repro-ducibility of the SSAP analysis flax variety ldquoStormont cirrusrdquo(IPK genbank Gatersleben accession number LIN 261) wasused

For the investigation of genetic diversity 47 flax acces-sions belonging to 22 species from sections Linum Ade-nolinum Dasylinum and Stellerolinum were used (Table 2)Most of these accessions were obtained from genebank ofLeibniz Institute of Plant Genetics and Crop Plant Research(IPK) (Gatersleben Germany) seed collections of All-Russian Flax Institute (VNIIL) (Torzok Russian Federation)NI Vavilov Research Institute of Plant Industry (VIR) (StPetersburg Russian Federation) and Institute of Geneticsand Cytology of NAS Belarus (IGC) (Minsk Belarus) Theaccessions of L amurense and L stelleroides were kindlyprovided by Dr L N Mironova Botanic Garden Institute ofthe Far-Eastern Branch of the Russian Academy of Sciences(BGI) (Vladivostok Russian Federation) Some accessionswere collected in the wild by Dr A A Svetlova KomarovBotanical Institute RAS (St Petersburg Russian Federation)by Dr N L Bolsheva Engelhardt Institute of MolecularBiology RAS (Moscow Russian Federation) and by Dr MPavelka Euroseeds (Novy Jicin Chech Rep)

22 Confirmation of Species Determination Species determi-nation of some accessions of wild Linum species was doneduring the course of our earlier cytogenetic investigations[14 17 37] To confirm the species determination the restof the accessions were planted in the ground and addition-ally chromosome analysis (determination of chromosomenumber) using acetocarmine staining according to previouslydeveloped approach was performed [14] Chromosome num-bers of all the studied accessions of wild flax are representedin Table 2

23 SSAP Analysis Genomic polymorphism of differentLinum species was studied using SSAP method [23] withmodifications described earlier through genomic studies ofwheat [38] and strawberry [39] Total genomic DNA wasextracted from young flax leaves according to Edwards etal [40] with minor modifications 30 ng of genomic DNA











ble2Th

eaccessio

nsof

studied

specieso

fgenus

Linu

m

Num

berAc

cessionname

Source

Genebanknu

mber

Orig

inCh

romosom

enum

ber(2n

)SectA

denolin

um1

Lperenn

eL

IPK

LIN1807

RussianFederatio

n2n

=18

[14]

2Lperenn

eLsub


Nym

anIPK

LIN1651

Poland

2n=36

[17]

3Laltaicu

mLedebex

Juz

IPK

LIN1632

Unk

nown

2n=18

[17]

4LkomaroviiJuz

IPK

LIN1716

Unk

nown

2n=18

[17]

5Lperenn

eLsub

spperenne

IPK

LIN1521

Slovakia

2n=18

[17]

6Lperenn

eLsub

spalpinum

(Jacq)

Stojand

Stef

IPK

LIN1905

Austr

ia2n

=18

[17]

7Lperenn

eLsub

spanglicum

(Mill)

Ockendo

nIPK

LIN1524

GB

2n=36

[17]

8Lperenn

eL

VNIIL

K5500

Unk

nown

2n=36

[17]

9Lleo

niiFW

Schultz

IPK

LIN1672

Germany

2n=18

[14]

10LpallescensB

unge

IPK

LIN1645

Tajik

istan

2n=18

[17]

11LpallescensB

unge

Wild

popu

latio

ncollected

byNLB

olsheva

RussianFederatio

nAltai

2n=18

(present

study)

12Lmesostylum

Juz

IPK

LIN1774

Tajik

istan

2n=18

[17]

13Lmesostylum

Juz

IPK

LIN1662

Tajik

istan

2n=18

[17]

14Llew

isiiP

ursh

IPK

LIN1648

USA

2n=18

[17]

15Llew

isiiP

ursh

IPK

LIN1550

USA

2n=18

[17]

16Lau

striacum

LIPK

LIN1608

Germany

2n=18

[17]

17Lau

striacum

LWild

popu

latio

ncollected

byAA

Svetlo

vaRu

ssianFederatio

nRo

stov

2n=18

(present

study)

18Lau

striacum

Lsubspeuxinu

m(Ju

z)Ockendo

nIPK

LIN1546

Ukraine

2n=18

[17]

19Lau

striacum

LWild

popu

latio

ncollected

byAA

Svetlo

vaCr

imea

2n=18

(present

study)

20Lau

striacum

LWild

popu

latio

ncollected

byAA

Svetlo

vaUkraine

2n=18

(present

study)

21Lam

urenseAlef

BGI

outdoo


RussianFederatio

nFar

East

near

Pokrovka

settlem

ent

2n=18

[17]

SectD

asylinu

m22

Lhirsutum

Lsubsphirsutum

LIPK

LIN1676

Hun

gary

2n=16

[35]

23Lhirsutum

subsphirsutum

LIPK

LIN1649

Romania

2n=16

[36]

24Lhirsutum


mPH

Davis

Wild

popu

latio

ncollected

byMPavelka

TurkeyK

aram

an2n

=32

(present

study)

25Lhirsutum

Lsubspan

atolicu

m(Boiss)Hayek

Wild

popu

latio

ncollected

byMPavelka

TurkeyA

ksaray

2n=32

(present

study)

SectLinum

26Lmargina

leACun

nex

Planch

IPK

LIN1920

Austr

alia

2n=84

(present

study)

27LnarbonenseL

IPK

LIN2002

Unk

nown

2n=28

[14]

28LnarbonenseL

IPK

LIN1653

France

2n=28

[14]

29Ldecumbens

Desf

IPK

LIN1754

Italy

2n=16

[14]

30Ldecumbens

Desf

IPK

LIN1913

Italy

2n=16

[14]


Table2Con

tinued

Num

berAc

cessionname

Source

Genebanknu

mber

Orig

inCh

romosom

enum

ber(2n

)31

Lgrandiflorum

Desf

IPK

LIN2000

Unk

nown

2n=16

[14]

32Lgrandiflorum

Desf

IPK

LIN974

Unk

nown

2n=16

[14]

33Langustifoliu

mHud

sIPK

LIN1692

France

2n=30

(present

study)

34Langustifoliu

mHud

sVNIIL

K5695

Unk

nown

2n=30

(present

study)

35Langustifoliu

mHud

sVNIIL

K3108

Belgium

2n=30

(present

study)

36Langustifoliu

mHud

sIG

C15

Belarus

2n=30

[14]

37Langustifoliu

mHud

sVNIIL

K4731

EastGermany

2n=30

(present

study)

38Lbienne

Mill

(syn

Langustifolium

Hud

s)IG

C14

Hun

gary

2n=30

[14]

39Lusita

tissim

umLsubspbienne

Mill

Snankev

VIR

u-303794

Unk

nown

winterfl

ax2n

=30

(present

study)

40Lusita

tissim

umconvarcrepita

ns[Boenn

ingh

]Ku

lpae

tDanert

IGC

260

Belarus

dehiscentfl

ax2n

=30

(present

study)

41Lusita

tissim

umconvarcrepita

ns[Boenn

ingh

]Ku

lpae

tDanert

IPK

LIN119

Portugal

dehiscentfl

ax2n

=30

(present

study)

42Lusita

tissim

umLvarie

tyGiza

IPK

LIN277

Egypt

larges

eededflax

2n=30

(present

study)

43Lusita

tissim

umLG12Ru

zokvety

IPK

LIN633

Czecho

slovakia

dual-purpo

seflax

2n=30

(present

study)

44Lusita

tissim


VIR

u099845

Abkh

azia

winterfl

ax2n

=30

(present

study)

45Lusita

tissim

umL

VNIIL

k7131

Morocco

larges

eededflax

2n=30

(present

study)

SectStellerolinum

46Lste

lleroidesPlanchon

BGI

Outdo

orsc

ultiv

ated

collection

RussianFederatio

nFar

East

TelyakovskyInlet

2n=20

(present

study)

47Lste

lleroidesPlanchon

BGI

Outdo

orsc

ultiv

ated

collection

RussianFederatio

nFar

East

near

Kraskino

settlem

ent

2n=20

(present

study)



2] and incu-












3 Results



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20









Ant

Tor

Tve

VerTos

Mer

Pra

Dip

A93

Ale

Novk-1147

K6Ors

v29Vik

Imp

Lid

Tom

Vor Kro

GorVasLav

Ros

AlfLadSla

Don

Oce

Sve

LM98 SmolicTvers

Len

Soy

A29

Ki5BelFor

Smo

Tost5

001

MogRus

S108

TMR



1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 474543413937353331M M



L austriacum



L bienne


L usitatissimum


L decumbens

L marginale

L decumbens


L angustifolium

L usitatissimum






L leonii

L pallescens

L amurenseL lewisii

L komarovii

L pallescens



L perenne susp alp

L austriacum

A

D I

F

G

H

E

С B

32

29

26

30

4647

31

39

45414044

3837433433 35

2728

2524 23 22

20 18

17 161312

193

21

99 9802

99 9

99 9

97

95

100

100 100

100

100

100

99 8871732

81

97 811

6105

21

15

14

4

3642

Sect Stellerolinum

Sect

Lin

um

Sect Dasylinum

Sect

Ade

nolin

um



(a) (b)

(c) (d)

1 2 3 4 5 6 7 8

(e)



4 Discussion






















5 Conclusions







Acknowledgments


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











ble2Th

eaccessio

nsof

studied

specieso

fgenus

Linu

m

Num

berAc

cessionname

Source

Genebanknu

mber

Orig

inCh

romosom

enum

ber(2n

)SectA

denolin

um1

Lperenn

eL

IPK

LIN1807

RussianFederatio

n2n

=18

[14]

2Lperenn

eLsub


Nym

anIPK

LIN1651

Poland

2n=36

[17]

3Laltaicu

mLedebex

Juz

IPK

LIN1632

Unk

nown

2n=18

[17]

4LkomaroviiJuz

IPK

LIN1716

Unk

nown

2n=18

[17]

5Lperenn

eLsub

spperenne

IPK

LIN1521

Slovakia

2n=18

[17]

6Lperenn

eLsub

spalpinum

(Jacq)

Stojand

Stef

IPK

LIN1905

Austr

ia2n

=18

[17]

7Lperenn

eLsub

spanglicum

(Mill)

Ockendo

nIPK

LIN1524

GB

2n=36

[17]

8Lperenn

eL

VNIIL

K5500

Unk

nown

2n=36

[17]

9Lleo

niiFW

Schultz

IPK

LIN1672

Germany

2n=18

[14]

10LpallescensB

unge

IPK

LIN1645

Tajik

istan

2n=18

[17]

11LpallescensB

unge

Wild

popu

latio

ncollected

byNLB

olsheva

RussianFederatio

nAltai

2n=18

(present

study)

12Lmesostylum

Juz

IPK

LIN1774

Tajik

istan

2n=18

[17]

13Lmesostylum

Juz

IPK

LIN1662

Tajik

istan

2n=18

[17]

14Llew

isiiP

ursh

IPK

LIN1648

USA

2n=18

[17]

15Llew

isiiP

ursh

IPK

LIN1550

USA

2n=18

[17]

16Lau

striacum

LIPK

LIN1608

Germany

2n=18

[17]

17Lau

striacum

LWild

popu

latio

ncollected

byAA

Svetlo

vaRu

ssianFederatio

nRo

stov

2n=18

(present

study)

18Lau

striacum

Lsubspeuxinu

m(Ju

z)Ockendo

nIPK

LIN1546

Ukraine

2n=18

[17]

19Lau

striacum

LWild

popu

latio

ncollected

byAA

Svetlo

vaCr

imea

2n=18

(present

study)

20Lau

striacum

LWild

popu

latio

ncollected

byAA

Svetlo

vaUkraine

2n=18

(present

study)

21Lam

urenseAlef

BGI

outdoo


RussianFederatio

nFar

East

near

Pokrovka

settlem

ent

2n=18

[17]

SectD

asylinu

m22

Lhirsutum

Lsubsphirsutum

LIPK

LIN1676

Hun

gary

2n=16

[35]

23Lhirsutum

subsphirsutum

LIPK

LIN1649

Romania

2n=16

[36]

24Lhirsutum


mPH

Davis

Wild

popu

latio

ncollected

byMPavelka

TurkeyK

aram

an2n

=32

(present

study)

25Lhirsutum

Lsubspan

atolicu

m(Boiss)Hayek

Wild

popu

latio

ncollected

byMPavelka

TurkeyA

ksaray

2n=32

(present

study)

SectLinum

26Lmargina

leACun

nex

Planch

IPK

LIN1920

Austr

alia

2n=84

(present

study)

27LnarbonenseL

IPK

LIN2002

Unk

nown

2n=28

[14]

28LnarbonenseL

IPK

LIN1653

France

2n=28

[14]

29Ldecumbens

Desf

IPK

LIN1754

Italy

2n=16

[14]

30Ldecumbens

Desf

IPK

LIN1913

Italy

2n=16

[14]


Table2Con

tinued

Num

berAc

cessionname

Source

Genebanknu

mber

Orig

inCh

romosom

enum

ber(2n

)31

Lgrandiflorum

Desf

IPK

LIN2000

Unk

nown

2n=16

[14]

32Lgrandiflorum

Desf

IPK

LIN974

Unk

nown

2n=16

[14]

33Langustifoliu

mHud

sIPK

LIN1692

France

2n=30

(present

study)

34Langustifoliu

mHud

sVNIIL

K5695

Unk

nown

2n=30

(present

study)

35Langustifoliu

mHud

sVNIIL

K3108

Belgium

2n=30

(present

study)

36Langustifoliu

mHud

sIG

C15

Belarus

2n=30

[14]

37Langustifoliu

mHud

sVNIIL

K4731

EastGermany

2n=30

(present

study)

38Lbienne

Mill

(syn

Langustifolium

Hud

s)IG

C14

Hun

gary

2n=30

[14]

39Lusita

tissim

umLsubspbienne

Mill

Snankev

VIR

u-303794

Unk

nown

winterfl

ax2n

=30

(present

study)

40Lusita

tissim

umconvarcrepita

ns[Boenn

ingh

]Ku

lpae

tDanert

IGC

260

Belarus

dehiscentfl

ax2n

=30

(present

study)

41Lusita

tissim

umconvarcrepita

ns[Boenn

ingh

]Ku

lpae

tDanert

IPK

LIN119

Portugal

dehiscentfl

ax2n

=30

(present

study)

42Lusita

tissim

umLvarie

tyGiza

IPK

LIN277

Egypt

larges

eededflax

2n=30

(present

study)

43Lusita

tissim

umLG12Ru

zokvety

IPK

LIN633

Czecho

slovakia

dual-purpo

seflax

2n=30

(present

study)

44Lusita

tissim


VIR

u099845

Abkh

azia

winterfl

ax2n

=30

(present

study)

45Lusita

tissim

umL

VNIIL

k7131

Morocco

larges

eededflax

2n=30

(present

study)

SectStellerolinum

46Lste

lleroidesPlanchon

BGI

Outdo

orsc

ultiv

ated

collection

RussianFederatio

nFar

East

TelyakovskyInlet

2n=20

(present

study)

47Lste

lleroidesPlanchon

BGI

Outdo

orsc

ultiv

ated

collection

RussianFederatio

nFar

East

near

Kraskino

settlem

ent

2n=20

(present

study)



2] and incu-












3 Results



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20









Ant

Tor

Tve

VerTos

Mer

Pra

Dip

A93

Ale

Novk-1147

K6Ors

v29Vik

Imp

Lid

Tom

Vor Kro

GorVasLav

Ros

AlfLadSla

Don

Oce

Sve

LM98 SmolicTvers

Len

Soy

A29

Ki5BelFor

Smo

Tost5

001

MogRus

S108

TMR



1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 474543413937353331M M



L austriacum



L bienne


L usitatissimum


L decumbens

L marginale

L decumbens


L angustifolium

L usitatissimum






L leonii

L pallescens

L amurenseL lewisii

L komarovii

L pallescens



L perenne susp alp

L austriacum

A

D I

F

G

H

E

С B

32

29

26

30

4647

31

39

45414044

3837433433 35

2728

2524 23 22

20 18

17 161312

193

21

99 9802

99 9

99 9

97

95

100

100 100

100

100

100

99 8871732

81

97 811

6105

21

15

14

4

3642

Sect Stellerolinum

Sect

Lin

um

Sect Dasylinum

Sect

Ade

nolin

um



(a) (b)

(c) (d)

1 2 3 4 5 6 7 8

(e)



4 Discussion






















5 Conclusions







Acknowledgments


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table2Con

tinued

Num

berAc

cessionname

Source

Genebanknu

mber

Orig

inCh

romosom

enum

ber(2n

)31

Lgrandiflorum

Desf

IPK

LIN2000

Unk

nown

2n=16

[14]

32Lgrandiflorum

Desf

IPK

LIN974

Unk

nown

2n=16

[14]

33Langustifoliu

mHud

sIPK

LIN1692

France

2n=30

(present

study)

34Langustifoliu

mHud

sVNIIL

K5695

Unk

nown

2n=30

(present

study)

35Langustifoliu

mHud

sVNIIL

K3108

Belgium

2n=30

(present

study)

36Langustifoliu

mHud

sIG

C15

Belarus

2n=30

[14]

37Langustifoliu

mHud

sVNIIL

K4731

EastGermany

2n=30

(present

study)

38Lbienne

Mill

(syn

Langustifolium

Hud

s)IG

C14

Hun

gary

2n=30

[14]

39Lusita

tissim

umLsubspbienne

Mill

Snankev

VIR

u-303794

Unk

nown

winterfl

ax2n

=30

(present

study)

40Lusita

tissim

umconvarcrepita

ns[Boenn

ingh

]Ku

lpae

tDanert

IGC

260

Belarus

dehiscentfl

ax2n

=30

(present

study)

41Lusita

tissim

umconvarcrepita

ns[Boenn

ingh

]Ku

lpae

tDanert

IPK

LIN119

Portugal

dehiscentfl

ax2n

=30

(present

study)

42Lusita

tissim

umLvarie

tyGiza

IPK

LIN277

Egypt

larges

eededflax

2n=30

(present

study)

43Lusita

tissim

umLG12Ru

zokvety

IPK

LIN633

Czecho

slovakia

dual-purpo

seflax

2n=30

(present

study)

44Lusita

tissim


VIR

u099845

Abkh

azia

winterfl

ax2n

=30

(present

study)

45Lusita

tissim

umL

VNIIL

k7131

Morocco

larges

eededflax

2n=30

(present

study)

SectStellerolinum

46Lste

lleroidesPlanchon

BGI

Outdo

orsc

ultiv

ated

collection

RussianFederatio

nFar

East

TelyakovskyInlet

2n=20

(present

study)

47Lste

lleroidesPlanchon

BGI

Outdo

orsc

ultiv

ated

collection

RussianFederatio

nFar

East

near

Kraskino

settlem

ent

2n=20

(present

study)



2] and incu-












3 Results



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20









Ant

Tor

Tve

VerTos

Mer

Pra

Dip

A93

Ale

Novk-1147

K6Ors

v29Vik

Imp

Lid

Tom

Vor Kro

GorVasLav

Ros

AlfLadSla

Don

Oce

Sve

LM98 SmolicTvers

Len

Soy

A29

Ki5BelFor

Smo

Tost5

001

MogRus

S108

TMR



1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 474543413937353331M M



L austriacum



L bienne


L usitatissimum


L decumbens

L marginale

L decumbens


L angustifolium

L usitatissimum






L leonii

L pallescens

L amurenseL lewisii

L komarovii

L pallescens



L perenne susp alp

L austriacum

A

D I

F

G

H

E

С B

32

29

26

30

4647

31

39

45414044

3837433433 35

2728

2524 23 22

20 18

17 161312

193

21

99 9802

99 9

99 9

97

95

100

100 100

100

100

100

99 8871732

81

97 811

6105

21

15

14

4

3642

Sect Stellerolinum

Sect

Lin

um

Sect Dasylinum

Sect

Ade

nolin

um



(a) (b)

(c) (d)

1 2 3 4 5 6 7 8

(e)



4 Discussion






















5 Conclusions







Acknowledgments


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



2] and incu-












3 Results



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20









Ant

Tor

Tve

VerTos

Mer

Pra

Dip

A93

Ale

Novk-1147

K6Ors

v29Vik

Imp

Lid

Tom

Vor Kro

GorVasLav

Ros

AlfLadSla

Don

Oce

Sve

LM98 SmolicTvers

Len

Soy

A29

Ki5BelFor

Smo

Tost5

001

MogRus

S108

TMR



1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 474543413937353331M M



L austriacum



L bienne


L usitatissimum


L decumbens

L marginale

L decumbens


L angustifolium

L usitatissimum






L leonii

L pallescens

L amurenseL lewisii

L komarovii

L pallescens



L perenne susp alp

L austriacum

A

D I

F

G

H

E

С B

32

29

26

30

4647

31

39

45414044

3837433433 35

2728

2524 23 22

20 18

17 161312

193

21

99 9802

99 9

99 9

97

95

100

100 100

100

100

100

99 8871732

81

97 811

6105

21

15

14

4

3642

Sect Stellerolinum

Sect

Lin

um

Sect Dasylinum

Sect

Ade

nolin

um



(a) (b)

(c) (d)

1 2 3 4 5 6 7 8

(e)



4 Discussion






















5 Conclusions







Acknowledgments


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20









Ant

Tor

Tve

VerTos

Mer

Pra

Dip

A93

Ale

Novk-1147

K6Ors

v29Vik

Imp

Lid

Tom

Vor Kro

GorVasLav

Ros

AlfLadSla

Don

Oce

Sve

LM98 SmolicTvers

Len

Soy

A29

Ki5BelFor

Smo

Tost5

001

MogRus

S108

TMR



1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 474543413937353331M M



L austriacum



L bienne


L usitatissimum


L decumbens

L marginale

L decumbens


L angustifolium

L usitatissimum






L leonii

L pallescens

L amurenseL lewisii

L komarovii

L pallescens



L perenne susp alp

L austriacum

A

D I

F

G

H

E

С B

32

29

26

30

4647

31

39

45414044

3837433433 35

2728

2524 23 22

20 18

17 161312

193

21

99 9802

99 9

99 9

97

95

100

100 100

100

100

100

99 8871732

81

97 811

6105

21

15

14

4

3642

Sect Stellerolinum

Sect

Lin

um

Sect Dasylinum

Sect

Ade

nolin

um



(a) (b)

(c) (d)

1 2 3 4 5 6 7 8

(e)



4 Discussion






















5 Conclusions







Acknowledgments


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Ant

Tor

Tve

VerTos

Mer

Pra

Dip

A93

Ale

Novk-1147

K6Ors

v29Vik

Imp

Lid

Tom

Vor Kro

GorVasLav

Ros

AlfLadSla

Don

Oce

Sve

LM98 SmolicTvers

Len

Soy

A29

Ki5BelFor

Smo

Tost5

001

MogRus

S108

TMR



1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 474543413937353331M M



L austriacum



L bienne


L usitatissimum


L decumbens

L marginale

L decumbens


L angustifolium

L usitatissimum






L leonii

L pallescens

L amurenseL lewisii

L komarovii

L pallescens



L perenne susp alp

L austriacum

A

D I

F

G

H

E

С B

32

29

26

30

4647

31

39

45414044

3837433433 35

2728

2524 23 22

20 18

17 161312

193

21

99 9802

99 9

99 9

97

95

100

100 100

100

100

100

99 8871732

81

97 811

6105

21

15

14

4

3642

Sect Stellerolinum

Sect

Lin

um

Sect Dasylinum

Sect

Ade

nolin

um



(a) (b)

(c) (d)

1 2 3 4 5 6 7 8

(e)



4 Discussion






















5 Conclusions







Acknowledgments


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 474543413937353331M M



L austriacum



L bienne


L usitatissimum


L decumbens

L marginale

L decumbens


L angustifolium

L usitatissimum






L leonii

L pallescens

L amurenseL lewisii

L komarovii

L pallescens



L perenne susp alp

L austriacum

A

D I

F

G

H

E

С B

32

29

26

30

4647

31

39

45414044

3837433433 35

2728

2524 23 22

20 18

17 161312

193

21

99 9802

99 9

99 9

97

95

100

100 100

100

100

100

99 8871732

81

97 811

6105

21

15

14

4

3642

Sect Stellerolinum

Sect

Lin

um

Sect Dasylinum

Sect

Ade

nolin

um



(a) (b)

(c) (d)

1 2 3 4 5 6 7 8

(e)



4 Discussion






















5 Conclusions







Acknowledgments


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a) (b)

(c) (d)

1 2 3 4 5 6 7 8

(e)



4 Discussion






















5 Conclusions







Acknowledgments


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















5 Conclusions







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

research article retrotransposon-based molecular...

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