Genetic diversity in populations of Isatis glauca Aucher ex Boiss. ssp. from Central Anatolia in ...

13 Pages·2013·2.22 MB·English

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RESEARCH Open Access
Genetic diversity in populations of Isatis glauca
Aucher ex Boiss. ssp. from Central Anatolia in
Turkey, as revealed by AFLP analysis
Özlem Özbek1*, Elçin Görgülü1 and Şinasi Yıldırımlı2
Abstract
Background: Isatidae L.is a complexand systematically difficult genus inBrassicaceae.The genus displays great
morphological polymorphism, which makes theclassification ofspeciesand subspecies difficult as it is observed in
Isatis glaucaAucherex Boiss.The aim ofthis study is characterization of thegenetic diversity in subspecies of Isatis
glauca Aucher exBoiss.distributed widely inCentral Anatolia,in Turkey byusing Amplified Fragment Length
Polymorphism (AFLP) technique.
Results: Eight different EcoRI-MseIprimer combinations produced 805 AFLP loci, 793 (98.5%) of which were
polymorphic in 67 accessions representing nine different populations. The data obtained byAFLP was computed
withusing GDA (Genetic Data Analysis) and STRUCTURE(version2.3.3) software programs for population genetics.
The mean proportion of the polymorphic locus (P),the mean number ofalleles (A),the number ofuniquealleles
(U) and themeanvalue ofgene diversity (He)were 0.59, 1.59,20, and 0.23 respectively.The coancestry coefficient
(ϴ)was 0.24. The optimal numberof K was identified as seven. The principal component analysis (PCA) explained
85.61% of thetotal genetic variation.
Conclusion: Isatis glaucassp. populationsshowed a high level ofgenetic diversity,and the AFLP analysis revealed
that high polymorphismand differentiated subspecies could be used conveniently for population genetic studies.
The principal coordinate analysis (PCoA) based on the dissimilarity matrix, the dendrogram drawnwith UPGMA
method and STRUCTURE cluster analysis distinguished the accessions successfully. The accessions formeddistinctive
population structures for populationsAA, AB,E, K, and S. Populations AG1 and AG2 seemed to have similar genetic
content, inaddition, inboth populations severalhybrid individuals were observed. The accessions did not formed
distinctive population structures for both populations AI and ANP.Consequently, Ankara province might be the
area, where species Isatis glaucaAucher ex Boiss. originated.
Keywords: AFLP; Genetic diversity; Isatis glauca subspecies; ssp. galatica;ssp. glauca;ssp. sivasica
Background TaxonomicandcytologicalinvestigationsontheIsatisL.,
Isatis glauca Aucher ex Boiss., is a diploid biennial or species grown in the Eastern and South Eastern Anatolia
perennial herbaceous species with a chromosome num- and across the Eastern Mediterranean region were made
ber set of 14 (2n=28). Isatis glauca has four recognized byMısırdalı(1985)(Figure1,theregionontheleft sideof
subspecies, glauca, galatica Yıldırımlı, sivasica (Davis) red diagonal line on the map of Turkey). Mısırdalı (1985)
Yıldırımlıandiconia(Boiss.)Davis.Thedistributionofthis displayed that there were 31 species, 14 subspecies, and
polymorphic species in Turkey is represented in Figure 1 2 varieties in Isatis collections from East & Southeast
(Yıldırımlı1988). Anatolia, and the Eastern Mediterranean - regions in
Turkey. Yıldırımlı and Doğan (1986) investigated palyno-
logical polymorphism in 790 pollen grains collected from
*Correspondence:[email protected] Isatis glauca ssp. glauca flowers in Sivas province of
1DepartmentofBiology,FacultyofArtandScience,HititUniversity,Ulukavak
Turkey. They observed that 505 (62.92%) of those pollen
Mah.ÇiftlikÇayırıCd.No:61,Çorum,Turkey
Fulllistofauthorinformationisavailableattheendofthearticle grains were tricolpate (normal), 129 (16.33%) were syn-
©2013Özbeketal.;licenseeSpringer.ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproduction
inanymedium,providedtheoriginalworkisproperlycited.
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Figure1DistributionofIsatisglaucaAucherexBoiss.subspeciesinTurkey.Thesubspeciesontherightsideofthediogonallinewere
reportedbyMısırdalı(1985)andthesubspeciesontheleftsideofthediogonallinewerereportedbyYıldırımlı(1988).
colpate, 87 (11.01%) were stefanocolpate and 69 (8.73%) the chromosome elimination, addition, and introgression
weredicolpate.Gilbertetal.(2002)screenedgeneticdiver- in intertribal partial hybrids between Brassica rapa and
sity in Isatis ssp. (Isatis tinctoria L., dyer’s woad) by using Isatisindigotica.Moazzeni et al. (2010) analysedthephyl-
AFLP.Al-Shehbazetal.(2006)discussedtheevaluationof ogenyof28IraniantaxaofIsatidaebyusingITSsequence
characters and their utilisation in infrafamilial classifica- of ribosomal DNA and morphological characters. Rocha
tions, delimitations of genera, the collection of molecular etal.(2011)detectedgeneticdiversityinwoad(Isatistinc-
data and major subdivisions of the family, problematic toria L.) by using ISSR markers. All these studies reveal
taxaandfuturechallengesintheBrassicaceae(Cruciferae). that although there are many reports on morphological
Kızıl (2006) reported morphological and agronomical and genetic variation in the Brassicaceae family, very few
characteristics of some wild and cultivated Isatis species. studiesarefoundonmorphologicalorgeneticvariationin
Moazzeni and Zarre (2006) studied the presence of Isatis IsatisglaucaAucherexBoiss.
tinctoria in Iran by using its morphological characters. The aim of this study was to analyse genetic diversity
Moazzeni et al. (2007) examined the systematic applica- and population structure using molecular marker tech-
tionofseed-coatsurfacesof23species(41populations)in niques among the I. glauca, subspecies populations from
four genera of tribe Isatidae using scanning electron mi- Central Anatolia in Turkey. AFLP method was used to
croscopy (SEM). Spataro et al. (2007) displayed genetic estimate genetic diversity (He), population structure and
variationandpopulationstructureinaEurasiancollection genetic differentiation between populations using coan-
ofIsatistinctoriaL.byusingAFLPandSAMPLmolecular cestry coefficient ϴ (= F ). In addition, the study also
P ST
markers. Spataro and Negri (2008) analysed phenotypic aimed to find the effects of climate (temperature T,
andgeneticdiversityofaEurasiancollectionofIsatistinc- humidity HU, and rainfall R), and geography (altitudes
toria as well as its adaptability according to a wide range AL, latitudes LA and longitudes LN) on the genetic di-
of altitudes. Tu et al. (2008) assessed intertribal somatic versity of nine I. glauca populations.
hybridsbetweenBrassicarapaandRaphanussativuswith
dye and medicinal plant Isatis indigotica. Du et al. (2009) Materials and Methods
presentedtheproductionandcytogeneticcharacterisation Materials
ofintertribalsomatichybridsbetweenBrassicanapusand In this study, genetic diversity of 67 accessions from nine
Isatisindigotica, and backcrosses. Tu et al. (2009) showed I. glauca subspecies populations collected from Ankara/
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Ayas (AA), Ankara/Beytepe (AB) valley, Ankara/Incek 50 μM) and 1 μL EcoR I adaptor (5 pmol/L=5 μM). The
(AI), Ankara/Golbasi1 (AG1), Ankara/Golbasi2 (AG2), RLMMwaspreparedbyusing1.1μL aliquotsT4DNAlig-
Ankara/Polatlı (ANP), Eskisehir (E), Konya (K) and Sivas asebuffer10x,4.267μLa-RLMM,0.05μLMseI(20U/μL)
(S)(Figure2)inMayandJulyof2011.Foramplifiedfrag- enzyme, 0.05 μL EcoR I (100 U/μL) enzyme, and 0.033 μL
ment length polymorphism (AFLP) analysis, one individ- T4 DNA ligase enzyme (30 weissU/μL). 96 well PCR mi-
ual was used from each accession. The Additional file 1: crotiter plates were used for 67 samples. For each sample
Table S1 contains also population codes (PC), accession a 5.5 μL RLMM aliquot was dispensed into the wells and
numbers (AN), and subspecies names. Throughout the the 5.5 μL of the diluted sample genomic DNA (50 ng)
text, the different populations were identified based on was added. PCR microtiter plates wereshort spinned in a
those codes. Plant specimens, are kept in Hacettepe Uni- centrifuge (Sigma 1–14) and left for incubation (Techne
versityHerbarium(HUB)andidentifiedbyProf.Dr.Şinasi TC-412)at37°Cfor6hours.
Yıldırımlı.
Methods PreselectivePCRamplification
FluorescentbasedAFLPanalysis Before the preselective PCR amplification, the 5-μL
Genomic DNA was extracted from 1 to 1.5 month old restriction-ligationproductsweredilutedbyadding95μL
leaves of individual plants from each of 67 accession TE (1X). For the preselective PCR amplification, the
usingCTABmethod,asdescribedbyKidwellandOsborn primers, were complementary to the sequences of the
(1992).AFLPanalysiswascarriedoutasdescribedbyVos EcoR I and Mse 1 adaptors, that contained one selective
etal.(1995),withminormodifications.Theprimers,adap- nucleotide. Preselective PCR, reaction consisted of 2 μL
tors, and altogether, eight primer combinations used in (10X)PCRbuffer,1.6μL(2mM)MgCl 1.2μL(2.5mM)
2,
this study are given in Additional file 2: Table S2 and dNTPs,1μL(5pmol)EcoRIprimer,1μL(5pmol)MseI
Additionalfile3:TableS3respectively. primer, 0.2 μL (1 μ/μL) Taq DNA polymerase, 9 μL
ddH O, and 4 μL diluted restriction-ligation products.
2
Restriction-ligationreaction Thedilutedrestriction-ligationproductswerekeptat−20°C
The aliquots of the restriction-ligation master-mix until used. Preselective amplification for 20 cycles was
(a-RLMM) were prepared in a 2 mL Eppendorf tube with- carried out through PCR. Initial extended denaturation
out enzymes and the T4 DNA ligase buffer 10x. In a- was carried out at 94°C for 2 min, followed by denatur-
restriction-ligationmaster-mix(RLMM)foronesample,the ation at 94°C for 30 sec, annealing at 56°C for 30 sec,
following were added; 0.617 μL H2O, 1.1 μL NaCl (0.5 M), extension at 72°C for 2 min, and the final extension at
0.55μLBSA10×(1g/L),1μLMseIadaptor(50pmol/μL= 60°Cfor30min.
Figure2Presentationofthelocations,wherenineIsatisglaucaAucherexBoiss.subspeciespopulationswerecollectedfromCentral
AnatoliainTurkey.ThecolouredlinesrepresentsthegeneflowbetweenpopulationsaccordingtoSTRUCTUREanalysisresults.
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SelectivePCRamplification or ordination method that starts with a matrix of simi-
Selective PCR amplification was carried out using 10 μL larities or dissimilarities between a set of individuals.
preselectivePCRamplificationproductsdilutedin190μL The method aims to produce a low-dimensional graph-
TE(1X).ThedilutedpreselectivePCRamplificationprod- icalplotofthedata insuch awaythatdistances between
ucts were kept at −20°C until used. The selective PCR the points in the plot are close to the original dissimilar-
amplification was done with EcoR I and Mse I primers, ities. For PCoA, the dissimilarity matrix values were
which contained three selective nucleotides. For the used toordinate 67accessions representing nineTurkish
fluorescent-based fragment analysis, the EcoR I selective Isatis glauca subspecies populations on a scattered plot
primers of 39, 36, 39, and 36 were labelled with FAM-1, using XLSTAT (version 2013, Addinsoft™), a software
FAM-2,FAM-3,andFAM-4respectively,whiletheEcoRI package for Windows (Figure 3). Second, a dendrogram
selective primers 33, 36, 33, and 36 were labelled with (Figure 4) was constructed according to Nei’s (1978)
VIC-1, VIC-2, VIC-3, and VIC-4 fluorescent dye. Mse I geneticdistanceandUPGMAmethodusingthesoftware
primerswereusedwiththeunlabelledones. Theselective program GDA (Genetic Data Analysis; Lewis and Zaykin
PCRamplificationusedinthisstudyisgiveninAdditional 2001) version 1.1., which is a software package for ana-
file4:TableS4. lysis of discrete population genetics data according to
Weir(1996).
Capillaryelectrophoresis
Capillary electrophoresis of selective amplification prod-
ucts were performed by using ABI 3130 × l (Applied Principalcomponentanalysis(PCA)
Biosytems Inc., Foster City, CA). For capillary electro- PCAwascalculatedbyusingthefollowinggeneticindices,
phoresis; 1μL selective PCR amplification productswere and the sample sizes: P, A and He, and N respectively, as
denatured using 0.2 μL LIZ-500 size standard at 95°C wellasgeographical(AL,LA,andLN)andclimatic(RA,T,
for 5 min followed by heat shock applied by chilling on and HU) data (Additionalfile5:TableS5) as variables ac-
iceat0°Cfor5min.Then,permanentlydenaturedsingle cording to the Pearson’s (one-tailed) correlation matrix
strandedDNAwasloadedonABI3130Xl.Aftercapillary usingXLSTAT(version2013,Addinsoft™)asoftwarepack-
electrophoresis,AFLPfragmentswerescoredintoabinary ageforWindows.
data matrix as 1 (present) or 0 (absent) using Gene Map- Since AFLPs are dominant markers, data were scored
per4.0softwarepackage(AppliedBiosystems). as binary data. Polymorphic bands, exhibiting presence
or absence of bands, were scored as alternative alleles.
Statisticalanalysis The data thus obtained were considered as haploid and
For investigation of population structures of nine I. analysedbyGDA.
glauca ssp. populations, we used the software program Gene diversity (He) (Nei 1973), estimating within-
STRUCTURE (Pritchard et al. 2000). STRUCTURE pro- population diversity, was computed as the expected het-
vides a model-based Bayesian approach to explain popu- erozygosity based on allele frequencies for each locus and
lation structure by using our entire AFLP markers data forallloci,polymorphicandnon-polymorphicones.
set to identify K clusters to which the program then as- Genetic differentiation between populations is often
signs each individual. We used 67 individuals to infer estimated with the Nei’s coefficient G (Nei 1973) for
ST
the optimal value of K (ie., the number of clusters) by dominantly inherited DNA markers. This coefficient may
evaluating K=1–9. For parameter set, in our model, we tell us how genetic variation is partitioned within- and
selected with admixture as ancestry model and the allele between-populations; a high G value indicates that
ST
frequencies were assumed to be correlated, since it is plants within a population are relatively similar, but
more reasonable to assume common ancestry of such populations are considerably different. When there are
closelyrelatedpopulations.Thelengthof burn-inMarkov two alleles (1 and 0), this G is identical to Wright’s
ST
Chain Monte Carlo (MCMC) replications was set to (1951) F (Nei 1973). Thus, F statistics was used to esti-
ST
10000anddatawerecollectedover100000MCMCrep- mate the extent of differentiation, between groups (popu-
lications in run, based on previous literature suggesting lations and sub-populations) (Hartl and Clark 1997). The
that this level is sufficient (Evanno et al. 2005). We de- coancestry coefficient ϴ (= F ) was measured for every
ST
termined the optimal value of K using the second order locusandforalllociineachpopulation.
statistics(ΔK)developedbyEvannoetal.(2005)andthe Multiple regression analysis (MR) (SPSS version 11
adhocproceduredescribedbyPritchardetal.(2000). for Windows) was conducted with AFLP diversity and
To confirm the results obtained from STRUCTURE, allele diversity in nine populations. MR employed the
we conducted two additional analyses enabling us to genetic variables (P, A and He) as dependent and the en-
visualise distribution of individuals. First, we performed vironmental variables (AL, LA, LN, RA, T, and HU) as
Principal Coordinate Analysis (PCoA). PCoA is a scaling independent.
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Figure3ThescatteredplotofthefirstandsecondprincipalcoordinatesobtainedfromeightAFLPprimercombinationsin67
accessionsfromnineIsatisglaucaAucherexBoiss.subspeciespopulations.
ReproducibilityofAFLPprofiles E36xM34 produced the highest number (194) of frag-
The reproducibility of scoring of the AFLP profiles was ments, while the primer combination E36xM33 produced
checked by analysing the genotyping error rate in repeated theleast number(32)offragments(datanot shown).The
runs of the same extracts from eight plants. In the begin- primercombinationE36xM34displayedthehighestnum-
ning of the study, 12 different primer combinations were ber (510) of fragments in population AA in total of eight
scanned for eight samples (accessions) and the eight poly- individuals, while the primer combination E39xM41 dis-
morphic primer sets selected and applied for all of the 67 played the least number (81) of fragments in population
accessions. The data were obtained using selected eight AIintotalofsixindividuals.Averagenumberoffragments
primer combinations in both pre-scanning and applied per population was calculated according to total present
throughallaccessionsincludingthoseeightaccessionswere fragment numbers produced by eight primer combina-
comparedtoevaluatethereproducibilityofAFLPmarkers. tions in all individuals of each population and ranged
The percent of reproducibility was calculated for each pri- 252.17(inpopulationAI)to314.75(inpopulationAA).
mer combination. Average percent of reproducibility was
calculated as 90.62% that ranged 86.38% (E36XM35) to
97.78%(E36XM34)amongtheprimercombinations. Geneticanalysis
InAFLPanalysis,eightEcoRIandMseIprimercombina-
Results tionsproduced805fragments(loci),793(98.5%)ofwhich
AFLPfragmentanalysis werepolymorphicand12(1.5%)weremonomorphic.The
InAFLPanalysis,eightEcoRIandMseIprimercombina- proportion of the mean number of the polymorphic loci
tions generated 805 fragments. The primer combination (P=0.59), the mean number of alleles (A=1.59) and the
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Figure4Adendrogram,whichwasconstructedaccordingNei’s(1978)geneticdistanceandUPGMAmethoddisplayingthe
relatednessbetweennineIsatisglaucaAucherexBoiss.subspeciespopulationscollectedfromCentralAnatoliainTurkey.
mean value of the genetic diversity (He=0.23) for nine Uniquealleles
populations, are represented in Table 1. Population K In AFLP analysis of nine I. glauca ssp. populations, 19
has the highest proportion of polymorphic loci (P= unique alleles were observed (Additional file 6: Table S6).
0.68), the highest number of alleles (A=1.68), and the Abouttheuniquealleles, two existedinpopulationAA,1
highest value (He=0.26) of genetic diversity (Table 1). in each of populations AG1 and E, 3 in population AG2
Population AI has the lowest proportion of poly- and 12 in population S. The frequencies of unique alleles
morphic loci (P=0.50) and the lowest number of alleles range 0.13 (in populations AG1, AG2 and S) to 1.00
(A=1.5), while population S has the lowest genetic di- (populationS).
versity value (He=0.20).
The mean value of the coancestry coefficient ϴ (= F ) Populationstructure
ST
or genetic differentiation between the populations is 0.24 The second order statistics developed by Evanno (2005)
(95% confidence interval) (Table 1). The highest genetic for STRUCTURE in order to determine the number of
distance (GD=0.24) was observed between population subpopulations identified the optimal value for K=7
AA and population S, while the lowest genetic distance (Figure 5). It was confirmed by also the ad hoc proced-
(GD=0.04) was observed between AG1 and AG2 (Data ure (Figure 6) developed by Pritchard et al. (2000). This
notshown). means that the set of accessions was partitioned into
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Table1Thenumberofsamplesize(N),theproportionof there is no such a distinctive population among the pop-
polymorphiclocus(P),numberofalleles(A),thegenetic ulations analysed in this study. This might be because of
diversityvalue(He)anduniqueallele(U)numbersper there is no any sample from this population among the
population,andcoancestrycoefficient(ϴ)forstudied analysedpopulationsorthatmightbeextinctintheevolu-
nineTurkishIsatisglaucasubspeciespopulations
tionaryhistoryofsubspeciespopulations.Therefore,some
POP N P A He U ϴ individuals are still carrying on the genetic material on
AA 8 0.60 1.60 0.22 2 theirgeneticbackground.
AB 8 0.60 1.60 0.22 -
AG1 8 0.58 1.58 0.21 1 Clusteranalysis(UPGMA)
Inthedendrogram,therearetwomaingroups(Figure4).
AG2 8 0.61 1.61 0.23 3
All the populations except, population S clustered
AI 6 0.50 1.50 0.22 -
together in the first main group, while population S
ANP 7 0.60 1.60 0.23 - (I. glauca ssp. sivasica) clustered in the second main
E 5 0.56 1.56 0.25 1 group. In the first main group, population AA (I. glauca
K 9 0.68 1.68 0.26 - ssp. galatica) separated clearly from rest of the popula-
tionsinthe first subgroup. Inthe secondsubgroup popu-
S 8 0.54 1.54 0.20 12
lations clustered into further subgroups. The populations
Mean 0.59 1.59 0.23 0.24
AG1 (I. glauca ssp. glauca) and AG2 (I. glauca ssp. gala-
tica),populationsAB(I.glaucassp.glauca)andANP(Isa-
seven clusters, which corresponded to the ssp. galatica, tis glauca ssp. galatica), and population AI (I. glauca ssp.
ssp. glauca and ssp. sivasica. When the coloured individ- glauca) clustered together in the second subgroup. In
ualbarplot(Figure7)wasexamined,someaccessionshad third subgroup, population K (Isatis glauca ssp. galatica)
varyingproportionsoftheirgenomefromotherclusters. andE(Isatisglaucassp.galatica)clusteredtogether.
Accessions formed distinctive population structures
for populations AA, AB, E, K, and S. Populations AG1 Principalcomponentsanalysis(PCA)
and AG2 seemed to have similar genetic content, in The three extracted principal components according to
addition in both populations several hybrid individuals PCA, explained 85.62% of the genetic variation (Table 2).
were observed. Populations AI and ANP did not display The first component, which accounted for 37.06% of the
distinctive population structures. In these populations, totalvariance,wasformedbyP,A,He,andAL(Additional
all individuals, except one individual in population AI, file 7: Table S7). The second component, representing
were seemed to be hybrids. In general, all populations 29.49% of the total variance, was formed by N, LN, and
havehybridindividuals. RA, whilethethirdcomponent represented 19.06%of the
The hybrid individuals detected in this study is an in- totalvariance,whichcomposedofLA,T,andHU.
dication of gene flow between populations. Particularly,
extensive gene flow was observed between AG1, AG2, Principalcoordinatesanalysis(PCoA)
AI, and ANP. It is noteworthy that there is a population The three coordinates, which obtained according to the
(lightgreencolour)foundinhybridindividuals,although PCoA, explained 29.23% of total variation. The first axis
(Eigen vector) displayed 11.96% of the variance, while
the second axis (Eigen vector) and the third axis (Eigen
vector) showed 9.41% and 7.85% of the variance respect-
ively (data not shown). The codes of 67 accessions are
plotted according to the first and the second axis that
corresponded to different regions, from where nine I.
glauca subspecies populations collected (Figure 3). The
accessions from populations AA, E, K and S seperated
clearly, the accessions from populations AB, AG1, AG2,
AIandANPwere clusteredinthesame region.
Multipleregressionanalysis
We employed the multiple regressions (MR) to deter-
mine AFLP allelic diversity. The results of MR analysis
Figure5Thesecondorderstatistics(ΔK)developedbyEvanno showed that the combination of altitude, latitude, longi-
(2005)forSTRUCTUREinordertodeterminethenumberof
tude, temperature, humidity, and rainfall accounted for a
subpopulationsidentifiedtheoptimalvalueforK.
considerableproportionofthevariationobservedinAFLP
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Figure6TheadhocproceduredescribedbyPritchardetal.(2000)todeterminethenumberofsubpopulationsidentifiedtheoptimal
valueforK.
fragments diversity (Table 3). When combined, these six diversity at a considerable level in Turkish Isatis glauca
variableshadeffectsonthevariancesofthemeanpropor- ssp.populations.
tion of polymorphic locus (92.8%), the mean frequency of
alleles (74.9%), and the mean genetic diversity (74.9%); Discussion
Overall,MRresultsindicatedthatclimaticandgeographic The AFLP technique is a reliable method, which com-
variables in combination with each other affected genetic bines the reliability of RFLPs and the power of PCR in a
Figure7IsatisglaucaAucherexBoiss.subspeciespopulationstructurebasedonBayesianinferenceamong67accessionsanalysed
with8AFLPprimercombinationsassumingK=7.
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Table2Totalvarianceexplainedbyprincipalcomponent genetransfer viapollinationorothertypesoftransfers.In
analysis(PCA)wasperformedbyusingdataofsample addition, ecogeographical conditions in Sivas, where the
sizeN,geneticindices(P,A,andHe),climatic(T,RA,and populationScollectedarehardercomparedtootherloca-
HU),andgeographical(AL,LA,andLN)asvariables tions, from which the other populations collected. In
accordingtoPearson’scorrelation(one-tailed)matrix
addition observation of high number of unique alleles in
withEigenvalues,percentageofvarianceandcumulative
population S displayed that ecogeograhical conditions
percentageofvariance
mighthaveeffecsontheadaptationofdifferentgenotypes
Component Eigenvalue Variance(%) Cumulativevariance(%)
andcausingthegeneticdifferentiationinthepopulationS.
1 3.72 37.15 37.15 AFLP fragment analysis indicated that the primer
2 2.95 29.45 66.59 combinations E39XM40, E36XM34 and E36XM33 amp-
3 1.90 19.03 85.62 lified more present fragments compared to other primer
combinations. However, amplification of fragments by
single method (Krumm et al. 2008) to produce highly primer combinations varied according to populations. In
polymorphic markers, which are used to analyse genetic general the lowest amplified present fragments were ob-
diversity in plants. Garcia et al. (2004) reported that servedinpopulation AI that had thelowestpolymorphic
AFLP seemed to be the most appropriate molecular locus and also lower genetic diversity. According to
essay for fingerprinting and analysing genetic relation- STRUCTURE analysis results, all individuals, except one
ships among tropicalmaize inbred lines with high accur- are hybrid plants in population AI. This might cause the
acy. It generates fingerprints of DNA from any origin or decrease in amplified fragments. During the hybridisa-
complexity (Vos et al. 1995) and produces high number tion process, the members of the population AI might
of polymorphic loci. By this way, it overcomes the loss havelost somefragments.ThepopulationAImostprob-
of information because of its dominance (Gerber et al. ably inhabited in the area in the recent past according to
2000) was the main reason for giving preference to previous field observations (personel communication
AFLP method. Taking into account, the results of this with Şinasi Yıldırımlı 2013). According to fragment ana-
study, the AFLP molecular markerscould beusedeffect- lysis, the highest number of present fragments were ob-
ively to distinguish I. glauca subspecies in agreement servedinpopulationAAfromAnkara/Ayaş.STRUCTURE
with Gilbertet al.(2002)andSpataro et al.(2007). analysis results indicated that accessions formed a distinct-
When we compared the results of this study having iveandstablepopulationstructureforpopulationAA.Al-
793 polymorphic loci, and 98.5% polymorphism, using 8 though, high number of fragments were amplified, low
AFLP primer combinations on 67 accessions, with previ- level of genetic diversity was observed. There might be
ous studies (Gilbert et al. 2002; Spataro et al. 2007), it low or no gene flow with other populations due to geo-
was observed that this study reported a greater poly- graphic distance or some other reasons. Therefore, ob-
morphism among nine Turkish Isatis glauca subspecies served homozygosity might be higher than the other
populations. The genetic data analysis provides import- populations analysed in this study. We also considered
ant information regarding genetic structure of the popu- that anthropogenic stresses might have important role on
lations. This methodology also helps in screening the the level of genetic diversity observed in population AA.
populations for genetic diversity within and among pop- There is a paper factory in the area and also the plants
ulations. The populations E and K displayed the higher generally grow on the highway road sides. These factors
values of genetic diversity. Population S, being a remote might cause to reduce genetic diversity in the population.
populationtotherestofthepopulations,showedtheleast On the other hand amplification of higher number of
genetic diversity. The geographic distance may prevent present fragments might refer that accesions from the
populationAAthatstillcontaingeneticuploadoftheori-
Table3Displayingtheeffectsofeco-geographicalfactors
ginalpopulationofssp.galaticainthearea.
ongeneticindicesbymultipleregressionanalysis(Abbre-
In outcrossing populations, a higher genetic variation
viations:DependentvariableDV,independentvariable
is observed within than among populations or subpopu-
IV,coefficientofmultipleregressionR2,proportionof
lations (Hamrick and Godt 1990). The finding of this
polymorphiclocusP,themeannumberofalleleA,the
meanvalueofgeneticdiversityHe,temperatureT, study showed that the gene diversity within-populations
rainfallRA,humidityHU,altitudeAL,latitudeLAand (0.76) were much larger compared to the gene diversity
longitudeLN) (0.24) between-populations is in agreement with Ham-
DV R2 IV rick and Godt (1990) and Spataro et al. (2007). Although
to date there is no report of detailed research about the
P 0.928 T,RA,HU,AL,LA,andLN
breeding system of I. glauca as in the case of I. tinctoria
A 0.749 T,RA,HU,AL,LA,andLN
(Spataro et al. 2007), these results might indicate that I.
He 0.749 T,RA,HU,AL,LA,andLN
glauca isanoutcrossing species.
Özbeketal.BotanicalStudies2013,54:48 Page10of13
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The presence and frequency of unique alleles reveal IsatisL.speciesfromtheBrassicaceae,displaysmorpho-
useful and important information about the differenti- logicalpolymorphismandthesemorphologicaldifferences
ation in genetic structure of a population. The new al- are often masked, even in fruits, which display the most
leles in a population exist because of mutations and if valuablediagnosticfeatures(Davis1965;Hedge1968;Jafri
they have ability to adapt to their environment, they 1973:citedinMoazzenietal.2007).Thesevariationsindi-
have a chance to be fitted in the population gene pool. cate that hybridization is playing a significant role in the
In this study, the population S had a large number (12) evolution of the genus Isatis L. (Moazzeni et al. 2007).
of unique alleles, which had frequencies higher than Theoutcrossingmatingsystem,associatedwiththeperen-
0.05. The population S is geographically distant from the nial habit and vegetative reproduction is a significant fac-
rest of the populations analysed in this study. Therefore, tor for hybridization, constitution, and emplacement of
existed unique alleles in populations will drive genetic thepolyploids(Ančev2006).Consequently,thegreatmor-
differentiation between populations as it was observed phological (Görgülü et al. 2013) and genetic variation ob-
between population S and the other populations in this served in subspecies of I. glauca in this study might be
study. related to hybridization process. This was confirmed with
Unique alleles have potential as favourable genes for the STRUCTURE analysis results, which showed that in
tolerance to severe conditions, especially at higher alti- all populations hybrid individuals were observed. Particu-
tudes (Mondini et al. 2010). The number and frequency larly, hybridisation was observed extensively in popula-
of unique alleles have important features, which may be tionsAG1,AG2,AI,andANP.
used for improvement of cultivated forms. Isatis L. is According to UPGMA, PCoA and STRUCTURE ana-
economically important plant especially for natural dye- lysis results, the population S was clustered as a distant
stuff beside its medicinal uses. The unique alleles found population to the rest of the populations. Also, the gen-
in I. glauca ssp. populations as molecular markers might etic distances between population S and rest of the pop-
be an indication of desirable traits, which can be used in ulations were quite high (Data not shown). This might
thefuturebreedingprogramsofIsatisL.plant species. show that formation of I. glauca ssp. sivasica is genetic-
In the Brassicaceae, hybridization, introgression and ally differentiated obviously more than the rest of the
hybrid speciation are reported as significant evolutionary subspecies populations analysed in this study. If the
forces (Marhold and Lihová 2006). Interspecific gene unique AFLP fragments detected in only one subspecies,
flow and hybridization promote evolution and species were extracted, cloned, and sequenced to prepare spe-
diversity of some genera, e.g. hybrids in Rorippa, as re- cificprimers,whichmightbeusedforsimplediagnostics
ported by Bleeker and Hurka (2001); Bleeker (2003); PCR assay of large-scale samples to identify species/sub-
Schranz et al. (2005); C. x insueta Urbanska and C. species orlandraces(Gilbertetal. 2002).
schulzii, (Urbanska et al. 1997) (cited in Marhold & Accordingtotheresultsofthisstudyandpreviousstud-
Lihova, 2006) and causes genetic variation (Marhold and ies (Yıldırımlı 1988; Görgülü et al. 2013), we suggested
Lihová 2006). In breeding programs, landraces or wild that Ankara/Ayaş (population AA) was the germplasm
relatives have desirable traits such as resistance to cold, centre of I. glauca ssp. galatica. Some genotypes from
drought etc., have been used for improvement of culti- Ankara/Ayaş (pop AA) migrated through Ankara/Polatlı
vated plants (Du et al. 2009). By this way, transfer of (populationANP)andEskişehir(populationE)(Figure2).
favourablegenes/genecomplexesfromwildalliestoculti- On the other hand, some genotypes from Ankara/Ayaş
vated plant gene pools could be possible. However, in (pop AA) migrated through Ankara/Gölbaşı1 (AG1),
most species, incompatibility barriers between wild forms Ankara/Gölbaşı2(AG2)andAnkara/Incek(AI)andKonya
and cultivated forms usually resulted with observation of (population K). We also considered that Ankara/Beytepe
low fertility in F1 generations that constraint the possible was the germplasm centreof I.glauca ssp.glauca (Yıldır-
transferof desirable traits (Inomata, 1993; Rieseberg et al. ımlı 1988). The genotypes migrated from population AB
1995; Shivanna, 1996; Choudharyand Joshi2001: cited in through the area, where the populations AG1, AG2, AI,
Du et al. 2009). Therefore, intertribal somatic hybrids be- andANPwerecollected.Bythiswayssp.galaticaandssp.
tween Brassica napus and I. indigotica (Du et al. 2009), glaucapopulationsareoverlapedinthatareacausingtoa
between Brassica rapa and Raphanus sativus with dye hybridzoneformationforI.glaucasubspecies.
and medicinal plant I. indigotica (Tu et al. 2008), and The populations from Eskişehir (E) and Konya (K)
between Brassica rapa and I. indigotica (Tu et al. 2008) have distinctive population structures now and they can
assessed to overcome genetic barriers by previous re- be recognised as the varieties of I. glauca ssp. galatica.
searchers. If intertribal hybridization is possible under The gene flow rates between the population ANP and
control of breeders, the possibility to see natural hy- the populations AG1, AG2,and AI seemed to be consid-
brids among Isatis L. species or subspecies in the na- erably high. Therefore, in populations AI and ANP all
ture should be the expected case. individualsexcept,one inpopAI,werehybrids.