Chloroplast genes as genetic markers for inferring patterns of change, maternal ancestry and ...

17 Pages·2014·1.11 MB·English

Research Article
Chloroplast genes as genetic markers for inferring patterns
of change, maternal ancestry and phylogenetic
relationships among Eleusine species
Renuka Agrawal1, Nitin Agrawal2, Rajesh Tandon1 and Soom Nath Raina3*
1 LaboratoryofCellularandMolecularCytogenetics,DepartmentofBotany,UniversityofDelhi,Delhi110007,India
2 ClusterInnovationCentre,UniversityofDelhi,Delhi110007,India
3 Presentaddress:AmityInstituteofBiotechnology,AmityUniversity,Sector125,Noida201303,UttarPradesh,India
Received:28October2012;Accepted:10December2013;Published:19December2013
Citation:AgrawalR,AgrawalN,TandonR,RainaSN.2014.Chloroplastgenesasgeneticmarkersforinferringpatternsofchange,
maternalancestryandphylogeneticrelationshipsamongEleusinespecies.AoBPLANTS6:plt056;doi:10.1093/aobpla/plt056
Abstract. Assessmentofphylogeneticrelationshipsisanimportantcomponentofanysuccessfulcropimprove-
mentprogramme,aswildrelativesofthecropspeciesoftencarryagronomicallybeneficialtraits.Sinceitsdomesti-
cationinEastAfrica,Eleusinecoracana(2n¼4x¼36),aspeciesbelongingtothegenusEleusine(x¼8,9,10),has
heldaprominentplaceinthesemi-aridregionsofIndia,NepalandAfrica.Thepatternsofvariationbetweenthecul-
tivatedandwildspeciesreportedsofarandtheinterpretationsbaseduponthemhavebeenconsideredprimarilyin
termsofnuclearevents.Weanalysed,forthefirsttime,thephylogeneticrelationshipbetweenfingermillet(E.cora-
cana)anditswildrelativesbyspecies-specificchloroplastdeoxyribonucleicacid(cpDNA)polymerasechainreaction–
restrictionfragmentlengthpolymorphism(PCR–RFLP)andchloroplastsimplesequencerepeat (cpSSR)markers/se-
quences. Restriction fragment length polymorphism of the seven amplified chloroplast genes/intergenic spacers
(trnK, psbD, psaA, trnH–trnK, trnL–trnF, 16S and trnS–psbC), nucleotide sequencing of the chloroplast trnK gene
and chloroplast microsatellite polymorphism were analysed in all nine known species of Eleusine. The RFLPof all
sevenamplifiedchloroplastgenes/intergenicspacersandtrnKgenesequencesinthediploid(2n¼16,18,20)and
allotetraploid(2n¼36,38)speciesresultedinwell-resolvedphylogenetictreeswithhighbootstrapvalues.Eleusine
coracana,E.africana,E.tristachya,E.indicaandE.kigeziensisdidnotshowevenasingle changeinrestrictionsite.
EleusineintermediaandE.floccifoliawerealsoshowntohaveidenticalcpDNAfragmentpatterns.ThecpDNAdiversity
inEleusinemultiflorawasfoundtobemoreextensivethanthatoftheothereightspecies.ThetrnKgenesequence
datacomplementedtheresultsobtainedbyPCR–RFLP.Thematernallineageofallthreeallotetraploidspecies(AABB,
AADD)wasthesame,withE.indicabeingthematernaldiploidprogenitorspecies.Themarkersspecifictocertainspe-
cieswerealsoidentified.
Keywords: cpSSR;Eleusine;PCR–RFLP;phylogeny;Poaceae;trnKgenesequence.
Introduction
programmesthroughintrogressionofdesiredgenesfrom
Thewild relatives of crop species often carry beneficial wildtocultivatedcropspecies(DidaandDevos2006).In
allelesthatareeffectiveagainstvariousbioticandabiotic thiscontext,theassessmentofphylogeneticrelationships
stresses.Theyholdthekeytosuccessfulcropimprovement at the inter-specific level and the identification of gene
*Correspondingauthor’se-mailaddress:[email protected]
PublishedbyOxfordUniversityPressonbehalfoftheAnnalsofBotanyCompany.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.org/
licenses/by/3.0/),whichpermitsunrestrictedreuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2013 1
Agrawaletal.—PhylogeneticrelationshipsamongEleusinespecies
pools are considered important. Based on chloroplast HiluandJohnson1997;Lye1999;BishtandMukai2000,
deoxyribonucleic acid (cpDNA) diversity, we obtained 2001a,2002;Devarumathetal.2005;Nevesetal.2005;
informationonthemolecularphylogenyoffinger millet Liu et al. 2011). Eleusine coracana (2n¼4x¼36)
(Eleusinecoracana)vis-a`-visitswildrelatives. (AABB) is considered to be an allotetraploid and has
ThegenusEleusineis amemberofthetribeEragros- been domesticated from its wild progenitor, Eleusine
teae, subfamily Chloridoideae and the family Poaceae. africana (2n¼4x¼36) (AABB) (Chennaveeraiah and
Itisasmallgenusofninespecies,whichincludessixdip- Hiremath 1974; Hilu and deWet 1976; Hilu et al. 1978;
loid(2n¼2x¼16,18,20)andthreepolyploid(2n¼4x¼ Hilu 1988, 1995; Hiremath and Salimath 1992; Werth
36, 38) species (Bisht and Mukai 2002; Liu et al. 2011). et al. 1994; Hilu and Johnson 1997; Bisht and Mukai
Thesespeciesarewidelydistributed inthetropicaland 2000, 2001a, b; Devarumath et al. 2005; Neves et al.
subtropical regions of Africa, Asia and South America 2005;Dida et al. 2007,2008).Thegeneticmaps gener-
(Phillips1972).EastAfricaisconsideredtobethecentre ated byDidaet al.(2007)showedthat E.coracana and
ofdiversityforthegenus(BishtandMukai2002). E. africana are allotetraploids. There is also strong evi-
Eleusinecoracana(2n¼4x¼36),commonlyknownas dence to suggest that Eleusine indica (2n¼2x¼18)
fingermilletorragi,istheonlyeconomicallyimportant (AA) isthe maternal genome donorof E. coracana and
speciesofthegenus.Aftersorghumandpearlmillet,fin- E.africana(Hilu1988;HiremathandSalimath1992;Hilu
germilletranksthirdincerealproductioninthesemi-arid andJohnson1997;BishtandMukai2000,2001a;Neves
regionsoftheworld(BishtandMukai2002).Itiswidely et al. 2005). Bisht and Mukai (2000, 2001a, 2002) indi-
cultivatedinthearidandsemi-aridregionsofEastAfrica, cated that Eleusine floccifolia (2n¼2x¼18) could be
India,NepalandmanyotherAsiancountriesforitsgrain theBBdonorspeciesofE.coracana.Thishas,however,
andfoddervalue(Verma2009).Thegrainiswidelyused been refuted by others (Hiremath and Salimath 1992;
forpreparingbread,cakes,soup,puddings,porridgeand Neves et al. 2005; Devarumath et al. 2010; Liu et al.
fermentedbeverages(HiluanddeWet1976;Chandrashe- 2011). According to these authors, the BB genome
kar2010;Neves2011).Fingermilletisarichsourceofes- donorspeciesremainsunidentifiedandmaypossiblybe
sential amino acids and polyphenols, and it has extinct.Eleusinekigeziensis(2n¼4x¼36or38)(AADD)
comparatively higher levels of calcium and iron than isthethirdtetraploidspeciesofthegenus.Eleusineindica
otherknown cereals (Barbeau and Hilu 1993;Chandra- (2n¼2x¼18)(AA)andE.jaegeri(2n¼2x¼20)(DD)are
shekar 2010). It has a number of medicinal properties proposedtobethewildprogenitorsofE.kigeziensis(Bisht
as well, particularly in controlling blood sugar levels in and Mukai 2002; Devarumath et al. 2010). On the con-
diabeticpatients(Duke and Wain 1981;Chandrashekar trary, Neves et al. (2005) proposed E. kigeziensis to be
2010;Pradhanetal.2010). autotetraploid, with E. indica being closely related to
The assessment of genomic relationships between E. kigeziensis but not the direct genome donor to
E.coracanaanditsallied specieshasbeenasubjectof E. kigeziensis. Liu et al. (2011) have concluded that
comprehensiveinvestigationsatthemorphological,cyto- all three tetraploids (E. coracana, E. africana and
genetic, biochemical and DNA level. Chromosome re- E.kigeziensis)areofallotetraploidorigin.Theysuggested
search has demonstrated the significant role of independent origins of E. kigeziensis and E. africana—
polyploidyandaneuploidyintheevolutionofthegenus E.coracana.Theyareoftheview thatbotheventsmay
(Chennaveeraiah and Hiremath 1974; Hiremath and haveinvolved the diploids E. indica and E. tristachyaas
Chennaveeraiah 1982; Hiremath and Salimath 1991; maternal parents,but thepaternal parents remain un-
BishtandMukai2000,2001a,b,2002).Biochemical,nu- identified.EleusineindicaandE.tristachyaareconsidered
clearandcpDNAmarkershaveprovidedvaluableinsight tobeverysimilarandthedegreeofrelationshipbetween
intorelationships,andon theoriginofthecropspecies thetworemainsunresolved(HiremathandChennaveer-
(Hilu et al. 1978; Hilu 1988, 1995; Hilu and Johnson aiah1982;HiremathandSalimath1991;HiluandJohn-
1992, 1997; Werth et al. 1993, 1994; Salimath et al. son 1992; Werth et al. 1994; Bisht and Mukai 2001a, b;
1995a; Neves et al. 2005; Dida et al. 2007, 2008; Nevesetal.2005;Liuetal.2011).
Liuetal.2011).The2nnumberandthegenomicformula Inthepresentstudy,thecpDNArestrictionsitepattern
proposed on the basis of earlier studies are given in variationofsevenamplifiedchloroplastgenes/intergenic
Table1. spacers, the chloroplast trnK gene sequence and cp
Inspiteoftheenormousamount ofinformation,there microsatellite polymorphism were investigated for the
stillexistsconsiderabledisagreementovertheidentifica- firsttimeinallnineEleusinespecies,withtheobjective
tionofthediploidancestorsofthethreepolyploidspecies ofconstructingthephylogenyofthegenusEleusineand
andthelevelofspeciationandevolutionaryrelationships identifyingthematernalgenomedonorsofthepolyploid
amongtheninespeciesofEleusine(Phillips1972,1995; speciesincludingE.coracana.Bothdirectsequencingof
2 AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2013
Agrawaletal.—PhylogeneticrelationshipsamongEleusinespecies
Table1. Plantmaterialsusedinthepresentstudy.aDr.MathewsM.Dida,Kenya;USDA,UnitedStatesDepartmentofAgriculture,USA;ILRI,
InternationalLivestockResearchInstitute,Ethopia;NBPGR,NationalBureauofPlantGeneticResources,India.bBishtandMukai(2002).
Species Accessionnumber Sourcea 2n Genomebformula Growthhabit
............................................................................................................................................................................
E.coracana PI482594 USDA 36 AABB Annual
PI462778 USDA
PI462779 USDA
E.africana EC541535 Dida,Kenya 36 AABB Annual
EC541536 Dida,Kenya
PI226270 USDA
PI315700 USDA
E.tristachya PI477078 USDA 18 AA Annual
PI331791 USDA
E.indica PI442480 USDA 18 AA Annual
E.floccifolia PI196853 USDA 18 BB Perennial
E.multiflora PI226067 USDA 16 CC Annual
E.jaegeri PI273888 USDA 20 DD Perennial
E.kigeziensis 1112 ILRI 38 AADD Perennial
1079 ILRI
E.intermedia S.No116 ILRI 18 AB Perennial
D.aegyptium IC-285214 NBPGR
thetrnKgeneandpolymerasechainreaction–restriction et al.2007;Angioi et al.2008;Sehgal et al. 2008;Jena
fragment length polymorphism (PCR–RFLP) of cpDNA etal.2009;Liuetal.2011;Poczaietal.2011).
regionsandchloroplastsimplesequencerepeats(cpSSRs)
are considered to be very good markers for detecting Methods
cpDNAvariation.The chloroplast trnK gene (whichalso
containsthematKgenewithinthetrnKgeneintron)se- Plantmaterials
quencehasbeeneffectivelyusedfortheconstructionof Seeds of the Eleusine species and one outgroup species
grassphylogenies(HiluandAlice1999;Hiluetal.1999). (Dactylocteniumaegyptium)utilizedinthepresentstudy
ThematKgenesequenceisoneofthesevenlociwidely wereobtainedfromtheUnitedStatesDepartmentofAgri-
utilized for the DNA barcoding of plants (Babbar et al. culture(USDA)(Beltsville,MD,USA),theInternationalLive-
2012). Specific chloroplast genes and/or intergenic stockResearchInstitute(ILRI)(AddisAbaba,Ethiopia),Dr
spacerscanbeamplified(Taberletetal.1991;Demesure MathewsM.Dida(MasenoUniversity,Maseno,Kenya)and
etal.1995;Tsumuraetal.1995,1996;DhingraandFolta the National Bureau of Plant Genetic Resources (NBPGR)
2005; Heinze 2005). The amplicons can be directly (NewDelhi,India).Theaccessionnumbersandsourceof
sequenced or restriction endonuclease digested (PCR– the seed material are given in Table 1. The seeds were
RFLPor cleaved amplified polymorphic sequence), and grown under controlled conditions in the experimental
theoccurrenceofmicrosatellites(cpSSRs)inthechloro- fieldoftheDepartmentofBotany,UniversityofDelhi.
plastgenomehasbeenwidelyutilizedforspeciesidenti-
DNAextraction
fication, reconstruction of phylogenetic relationships,
taxonomic studies and the identification of maternal ThetotalgenomicDNAoftheEleusinespeciesandD.ae-
parentsinpolyploids(Tsumuraetal.1995,1996;Weising gyptiumwasisolatedfromfreshyoungleavesusingthe
and Gardner 1999; Ishii and McCouch 2000; Lakshmi cetyltrimethylammoniumbromidemethodasdescribed
et al. 2000; Parani et al. 2000, 2001; Komatsu et al. by Murray and Thompson (1980) with some modifica-
2001; Provan et al. 2001; Kishimoto et al. 2003; Nwa- tions.InsteadofaCsCl–ethidiumbromideultracentrifu-
kanmaetal.2003;Zhuetal.2003;AsadiAbkenaretal. gationstep,theDNAwaspurifiedbyphenol–chloroform
2004, 2008; Van Droogenbroeck et al. 2004; Ibrahim extraction.
AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2013 3
Agrawaletal.—PhylogeneticrelationshipsamongEleusinespecies
PCRamplificationofchloroplastgenes/intergenic (exceptforTaqI,whichwasincubatedat608C).Thedi-
spacers,theirdigestionanddataanalysis gestionwasterminatedbyadding1.5mLof10×loading
Seven chloroplast genes and intergenic spacers (trnS– buffer. The digested products were fractionated on
psbC, psaA, 16S, trnK, psbD, trnL–trnF and trnH–trnK) 1.5% agarose gels containing ethidium bromide
wereamplifiedusingpreviouslypublisheduniversalfor- (0.05mgmL21) in 0.5× TBE buffer. A DNA ladder mix
wardandreverseprimers(Table2).Amplificationwascar- wasloadedalongsidethedigestedproductstoserveas
riedoutin100mLreactionmixturescontaining80ngof size markers.Afteragarose gel electrophoresis,the gel
template DNA, 0.1mM dNTPs (Amersham Biosciences, wasphotographedinultravioletlight.Reproducibilityof
UK), 2mM MgCl (Bangalore Genei, India), 1.3mM thepatternswastestedbyrepeatingallthereactionsat
2
each forward and reverse primer (Bangalore Genei), leasttwice.
2.5U of Taq DNA polymerase (Bangalore Genei) and ForPCR–RFLPanalysis,thepresence(1)orabsence(0)
10mLof10× assaybuffer[100mMTrispH9.0,500mM ofarestrictionfragmentwasrecorded.Totalandmean
KCl,0.1%gelatin(BangaloreGenei)].DNAamplification character differences between pairs of species were
wasperformedinaMycycler(Bio-Rad,USA)programmed calculatedusingPAUP*4.0(Swofford2002).NeiandLi’s
to 36 cycles each of 1min (5min for the first cycle) coefficientofgeneticdistance(NeiandLi1979)wascal-
at 948C for template DNA denaturation, 1min at the culatedbetweeneachpairofspeciesaftertheoptimality
annealing temperature (638C for psaA, 558C for trnS– criterionwassettoDISTANCE.Clusteranalysiswascar-
psbC, trnK and psbD, 608C for 16S, and 508C for trnL– riedoutusingtheunweightedpair-groupmethodusing
trnF and trnH–trnK), and 2min at 728C for primer arithmetic averages (UPGMA) (Sneath and Sokal 1973)
extension,followedbyafinalextensioncycleof15min andneighbour-joining(NJ)(SaitouandNei1987)meth-
at728C. ods.Bootstrapvalueswerecalculatedfrom100replicates
Sevengenesandintergenicspacersamplifiedfromthe usingtheBOOTSTRAPcommandinPAUP.
chloroplast genomewere separately restricted with 31
four-,five-andsix-basecutterrestrictionendonucleases PCRamplification,cloningandsequencingofthe
(AluI,AvaI,AccI,AfaI,BamHI,BalI,BglI,BglII,ClaI,DraI, trnKgene,anddataanalysis
EcoRV, EcoRI, HaeIII, HinfI, HindIII, HincII, MspI, KpnI, ThetrnKgenewasamplifiedfromthenineEleusinespe-
MboI,MluI,PstI,PvuII,SacI,SalI,SpeI,SphI,SmaI,SspI, ciesandoneoutgroupasdescribedabove.Theamplifica-
TaqI,XhoIandXbaI).The12.20mLreactionmixcontained tion products were separated on a 1% agarose gel,
10mLof the amplified gene product, 1.2mLof enzyme excisedfromthegelandpurifiedusingaQIAquickgelex-
bufferand 10U of the restriction enzyme. Aftergentle tractionkit(Qiagen,Germany).Thepurifiedamplification
mixing the mixture was incubated overnight at 378C productswereclonedandsequencedwithanABIPRISM
Table2. Listofchloroplastgenes/intergenicspacersamplifiedinthepresentstudy.aSizesinsource.
Genes/intergenicspacers Primerpair Sizeinbpa Source
...................................................................................................................................................................................
trnS–psbC 5′-GGTTCGAATCCCTCTCTCTC-3′ 1600 Oryzasativa(Demesureetal.1995)
5′-GGTCGTGACCAAGAAACCAC-3′
psaA 5′-AAGAATGCCCATGTTGTGGC-3′ 2218 Nicotianatabacum(Shinozakietal.1986;
5′-TTCGTTCGCCGGAACCAGAA-3′ Tsumuraetal.1996)
16S 5′-ACGGGTGAGTAACGCGTAAG-3′ 1375 Nicotianatabacum(Shinozakietal.1986;
5′-CTTCCAGTACGGCTACCTTG-3′ Tsumuraetal.1995,1996)
trnK 5′-AACCCGGAACTAGTCGGATG-3′ 2569 Oryzasativa(Hiratsukaetal.1989;
5′-TCAATGGTAGAGTACTCGGC-3′ Tsumuraetal.1995,1996)
psbD 5′-TATGACTATAGCCCTTGGTA-3′ 1042 Nicotianatabacum(Shinozakietal.1986;
5′-TAGAACCTCCTCAGGGAATA-3′ Tsumuraetal.1995,1996)
trnL–trnF 5′-CGAAATCGGTAGACGCTACG-3′ 995 Nicotianatabacum(Taberletetal.1991)
5′-ATTTGAACTGGTGACACGAG-3′
trnH–trnK 5′-ACGGGAATTGAACCCGCGCA-3′ 1831 Nicotianatabacum(Demesureetal.1995)
5′-CCGACTAGTTCCGGGTTCGA-3′
4 AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2013
Agrawaletal.—PhylogeneticrelationshipsamongEleusinespecies
377 automated DNA sequencer (Applied Biosystems, TBEbuffer),andelectrophoresiswasconductedat55W
FosterCity,CA,USA).BLASTsimilaritysearcheswereper- and558Cfor (cid:3)2h.
formedusingtheNationalCentreforBiotechnologyInfor- Forsilverstaining,thegelwasfixedin10%(v:v)acetic
mation(NCBI)BLASTNalgorithmtoconfirmtheidentity acidfor30min.Itwassubsequentlyrinsedthreetimesin
of the trnK sequences. The nucleotide sequences have de-ionizedwater(2minperrinse).Thegelwasthenkept
been submitted to GenBank with accession numbers forstainingfor30minina2Lsolutioncontaining2gof
KF357736–KF357745. The nucleotide sequence of the silvernitrateand3mLof37%formaldehyde(Promega,
partial trnK gene from Acrachne racemosa available in USA). The stained plate was rinsed with de-ionized
GenBank (accession number JN681616.1) was alsouti- waterfor20sanddevelopedinaprechilled(108C)devel-
lizedforphylogeneticanalysis.Theconsensussequence oper(2L)solutioncontaining60gofsodiumcarbonate,
forthetrnKgenefromthenineEleusinetaxaandoneout- 3mLof37%formaldehydeand400mLofsodiumthio-
group(D.aegyptium)wasgeneratedusingthesoftware sulfate (10mgmL21).When bandsbecame visible, the
CLCSequenceviewer6.1. gelwasimmediatelytransferredto10%aceticacidsolu-
ThesequenceswerealignedusingClustal-X,version1.8 tion to stop further reaction. The gel was finally rinsed
(Thompsonetal.1997).Phylogeneticanalyseswerecar- withdistilledwaterandairdried.
riedoutwiththesoftwareMEGA5(Tamuraetal.2011). ThePCRproductsofeightprimer–templatecombina-
Pairwise sequence divergence rates between species tions were characterized by direct sequencing. A PCR
were calculated using the maximum composite likeli- was performed in 100mLvolumes as described above.
hood method. Phylogeny construction was carried out An aliquot of the PCR product was checked byagarose
usingNJ,minimumevolution(ME),maximumlikelihood gel electrophoresis, and the remainder was purified
(ML) and maximum parsimony (MP) methods. Neigh- throughaQIAquickPCRcleanupkit(Qiagen).Nucleotide
bour-joiningandMEtreeswereobtainedusingthemax- sequencingwasperformedusinganABIPRISM377auto-
imum composite likelihood criterion while ML and MP matedDNAsequencer(AppliedBiosystems).Thenucleo-
trees were constructed using the nearest-neighbour- tide sequences have been submitted to GenBank with
interchange(NNI)andtree-bisection-reconnectionalgo- accessionnumbersKF357730–KF357735.
rithms, respectively. In all the analyses, all positions
containinggapsandmissingdatawereeliminatedfrom
the dataset (complete deletion option).Supportvalues Results
of the internal branches of NJ, ME, ML and MP trees
wereevaluatedbybootstrap(500replicates). ChloroplastPCR–RFLP
Robustamplificationproductswereobtainedforallseven
PCRamplificationofchloroplast genes/intergenic spacers (trnS–psbC, psaA, 16S, trnK,
microsatellites,polyacrylamidegel psbD, trnL–trnFand trnH–trnK) from the nine Eleusine
electrophoresisandsequencing
speciesandanoutgroupspecies,D.aegyptium.Foreach
A total of eight primer pairs (Table 3) were used for gene/intergenicspacer,theamplifiedproductsappeared
amplificationofthechloroplastmicrosatellites.Amplifi- to be monomorphic on a 1.5% agarose gel across the
cation wascarried outin 25mL reactionmixtures con- species. The approximate size of the PCR product for
taining 2.5mL of 10× reaction buffer, 1.5mM MgCl , trnS–psbC, psaA, 16S, trnK, psbD, trnL–trnFand trnH–
2
200mM dNTPs, 200nM each primer, 0.5U of Taq DNA trnK was 1620, 2380, 1480, 2510, 1090, 1090 and
polymerase (Bangalore Genei) and 25ng of template 2270bp,respectively.AliquotsofPCRproductsweredi-
DNA.DNA amplificationwasperformedin aMyCyclerTM gestedseparately with31four-,five-andsix-cutter re-
(Bio-Rad) programmed to initial denaturation at 948C striction endonucleases. Of the 217 amplification
for 5min followed by 35 cycles each of 1min at 948C, product–enzymecombinations,137didnotshowintern-
1min at 558C (658C for ccmp2 and 538C for ccmp9) alrestrictionsitesinanyofthe10speciesinvestigated.In
and 1min at 728C, followed by a final extension cycle theremaining80amplificationproduct–enzymecombi-
of5minat728C. nations, 1–5 restriction sites within the amplification
Anequalvolume(10mL)offormamidedye(98%for- product were obtained. Of these, 57 combinations re-
mamide, 10mM EDTA, 0.026g of bromophenol blue, vealednopolymorphismamongthe10species.There-
0.026gof xylene cyanol) was added toeach amplified maining 23 (trnK/AfaI, trnK/MspI, trnK/DraI, trnK/SpeI,
product. The samples were heated for 5min at 948C trnK/SspI, trnK/KpnI, trnK/TaqI, trnK/BamHI, trnL–trnF/
and immediately placed on ice. A total of 2.5mL of AluI, trnL–trnF/BglII, trnL–trnF/DraI, trnL–trnF/MboI,
each sample was loaded on a 6% polyacrylamide gel psaA/AluI, psaA/TaqI, psaA/MspI, psbD/HaeIII, psbD/
(19:1 acrylamide:bisacrylamide, 7.5M urea and 1× MboI,psbD/TaqI,trnS–psbC/AfaI,trnS–psbC/AvaI,trnS–
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Agrawaletal.—PhylogeneticrelationshipsamongEleusinespecies
Table3. Listofchloroplastmicrosatellitesamplifiedinthepresentstudy.
Locus Location Repeat Primerpair Sizein Sizein Source
Eleusine(bp) source(bp)
............................................................................................................................................................................
ccmp2 5′totrnS (A) 5′-GATCCCGGACGTAATCCTG-3′ 197,200 189 Nicotianatabacum
11
5′-ATCGTACCGAGGGTTCGAAT-3′ (Weisingand
Gardner1999)
ccmp5 3′torps2 (C) (T) 5′-TGTTCCAATATCTTCTTGTCATTT-3′ 145,146 103 Nicotianatabacum
7 10
(T) C(A) 5′-AGGTTCCATCGGAACAATTAT-3′ (Weisingand
5 11
Gardner1999)
ccmp6 ORF77–ORF82 (T) C(T) 5′-CGATGCATATGTAGAAAGCC-3′ 96 98 Nicotianatabacum
5 17
intergenic 5′-CATTACGTGCGACTATCTCC-3′ (Weisingand
Gardner1999)
RCt3 Intergenicregion (A) 5′-TAGGCATAATTCCCAACCCA-3′ 113 129 Oryzasativacv
10
5′-CTTATCCATTTGGAGCATAGGG-3′ Nipponbare(Ishii
andMcCouch2000)
RCt4 Codingregion (T) 5′-ACGGAATTGGAACTTCTTTGG-3′ 131 128 Oryzasativacv
12
(psbG) 5′-AAAAGGAGCCTTGGAATGGT-3′ Nipponbare(Ishii
andMcCouch2000)
RCt5 Intergenicregion (T) 5′-ATTTGGAATTTGGACATTTTCG-3′ 151 143 Oryzasativacv
10
5′-ACTGATTCGTAGGCGTGGAC-3′ Nipponbare(Ishii
andMcCouch2000)
RCt7 Codingregion (T) 5′-GTGTCATTCTCTAGGCGAAC-3′ 126 126 Oryzasativacv
10
(infA) 5′-AAATATGACAGAAAAGAAAAATAGG-3′ Nipponbare(Ishii
andMcCouch2000)
RCt8 Intron(rpl16) (T) 5′-ATAGTCAAGAAAGAGGATCTAGAAT-3′ 125 131 Oryzasativacv
17
5′-ACCGCGATTCAATAAGAGTA-3′ Nipponbare(Ishii
andMcCouch2000)
trnKsequencedata
psbC/HaeIII, 16S/MboI, trnH–trnK/AluI) combinations
were phylogenetically very informative(Table4; Fig.1). ThetrnKgenecodesfortRNA-LysUUU.Inrice,thechloro-
A total of 282 bands were scored for cluster analyses. plasttrnKgeneis2576bplong,ofwhichtheexonisonly
Novariationwasobservedacrossdifferentaccessionsof 72bplong.Theexonisdividedintotwopartsbyalong
thesametaxon.Thepolymorphisminalltheprofileswas intron of (cid:3)2500bp. The 5′ exon consists of 37bp and
theresultofsitemutations. the 3′ exon consists of 35bp. The matK gene, which
Geneticdistance(NeiandLi’s)amongthenineEleusine codesformaturaseK,is1536bpinlengthandislocated
speciesandoneoutgrouprangedfrom0.0000to0.01857 withinthechloroplasttrnKintron.Inthepresentstudy,
(Table5).Basedontherestrictionsitedataoftheampli- thetrnKgenewassequencedfromthenineEleusinespe-
fied gene/intergenic products, UPGMA and NJ dendro- ciesandD.aegyptium.ThealignedtrnKgenesequenceof
gramsweregenerated.Bothshowedsimilartopologies. Eleusinecorrespondedtoposition45atthe5′endtopos-
Theyresolvedintotwomajorclades.CladeI,supported ition2531atthe3′endofrice.ThelengthofthetrnKgene
by 100% bootstrap support, contained E. kigeziensis, in the nine Eleusine species ranged between 2463
E.indica,E.tristachya,E.coracanaandE.africana.Clade (E.jaegeri)and2467 bp(E.tristachyaandE.floccifolia).
IIwascomprisedofE.jaegeri,E.floccifolia,E.intermedia ThelengthoftrnKinA.racemosaandD.aegyptiumwas
and E. multiflora. Eleusine floccifolia and E. intermedia 2466and2472bp,respectively[seeSupportingInforma-
weremorecloselyrelatedtoeachother(supportedbya tion].TheGCcontentofthetrnKgenesequencevaried
bootstrapvalueof100%)thaneitherofthespecieswas from32.3to33%withanaverageof32.6%.Thenucleo-
to E. jaegeri. Dactyloctenium aegyptium was the most tidefrequencieswere0.162forG,0.164forC,0.324forA
divergedspeciesamongtheanalysedspecies(Fig.2). and 0.350 for T. The dataset including alignment gaps
6 AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2013
A
o
B
PL Table4. GroupingofEleusineandoutgroupspeciesbasedonrestrictionfragmentpatternsofsevencpDNAamplifiedgenes/intergenicspacers.
A
N
T
S Species trnS–psbC 16S psbD psaA trnK trnH–trnK trnL–trnF
w ....................... ...... ....................... .................... ............................................................. ............ ..............................
w AfaI AvaI HaeIII MboI HaeIII MboI TaqI AluI MspI TaqI AfaI DraI MspI SpeI SspI KpnI TaqI BamHI AluI AluI BglII DraI MboI
w .................................................................................................................................................................................................................................
.ao E.coracana A1 B1 C1 D1 E1 F1 G1 H1 I1 J1 K1 L1 M1 N1 O1 P1 Q1 R1 S1 T1 U1 V1 X1
b
p
la E.africana A1 B1 C1 D1 E1 F1 G1 H1 I1 J1 K1 L1 M1 N1 O1 P1 Q1 R1 S1 T1 U1 V1 X1
n
ts E.tristachya A1 B1 C1 D1 E1 F1 G1 H1 I1 J1 K1 L1 M1 N1 O1 P1 Q1 R1 S1 T1 U1 V1 X1
.o
x
fo E.indica A1 B1 C1 D1 E1 F1 G1 H1 I1 J1 K1 L1 M1 N1 O1 P1 Q1 R1 S1 T1 U1 V1 X1
rd
jo E.floccifolia A2 B1 C1 D2 E1 F2 G1 H2 I1 J1 K2 L1 M1 N1 O2 P1 Q1 R1 S1 T1 U1 V1 X1
u
rn E.intermedia A2 B1 C1 D2 E1 F2 G1 H2 I1 J1 K2 L1 M1 N1 O2 P1 Q1 R1 S1 T1 U1 V1 X1
a
ls.o E.kigeziensis A1 B1 C1 D1 E1 F1 G1 H1 I1 J1 K1 L1 M1 N1 O1 P1 Q1 R1 S1 T1 U1 V1 X1
rg
E.jaegeri A2 B1 C1 D2 E1 F2 G1 H2 I2 J1 K2 L1 M1 N1 O2 P1 Q1 R1 S1 T1 U1 V1 X1
E.multiflora A2 B1 C1 D1 E2 F2 G1 H1 I1 J2 K1 L2 M2 N2 O2 P1 Q1 R1 S1 T1 U1 V1 X1
D.aegyptium A2 B2 C2 D1 E3 F2 G2 H1 I1 J1 K2 L1 M3 N1 O1 P2 Q2 R2 S2 T2 U2 V2 X2
Combinations I VIII VIII III IV I VIII III V VI VII VI IV VI II VIII VIII VIII VIII VIII VIII VIII VIII
A
g
ra
TheAuthors2013& con–enzymecombinationsfortheKgeneshowedtrnmorphicPCR–RFLPprofilesoftheKgene.Eightampli-trnTheKsequencedatawerecorrelatedwiththepoly-trnand57wereparsimonyinformativesites. outofwhich2254wereconserved,214werevariable andmissingdatacomprised2486nucleotidepositions, catedontheleft.(10).MarkerDNA(M).Thesizeofthefragmentsinbasepairsisindi-(7),(8),(9)andE.kigeziensisE.multifloraE.jaegeriD.aegyptium(3),(4),(5),(6),E.tristachyaE.indicaE.floccifoliaE.intermediafiedSgenewithMboI(D)in(lane1),(2),16E.coracanaE.africanaAfaI(B),theamplifiedLFgenewithMboI(C)andtheampli-trn–trngenewithAfaI(A),theamplifiedSCintergenicspacerwithtrn–psbRestrictionfragmentsizepatternsoftheamplifiedKFigure1.trn wal—Phylogeneticrelationshipsamongspeciesetal.Eleusine
7
Agrawaletal.—PhylogeneticrelationshipsamongEleusinespecies
Table5. Geneticdistance(NeiandLi’scoefficient)matrixgeneratedfromcpDNAPCR–RFLPdataofnineEleusinespeciesandanoutgroup,
D.aegyptium.
1 2 3 4 5 6 7 8 9 10
............................................................................................................................................................................
1 E.coracana
2 E.africana 0.00000
3 E.tristachya 0.00000 0.00000
4 E.indica 0.00000 0.00000 0.00000
5 E.floccifolia 0.00479 0.00479 0.00479 0.00479
6 E.intermedia 0.00479 0.00479 0.00479 0.00479 0.00000
7 E.kigeziensis 0.00000 0.00000 0.00000 0.00000 0.00479 0.00479
8 E.multiflora 0.00650 0.00650 0.00650 0.00650 0.00790 0.00790 0.00650
9 E.jaegeri 0.00655 0.00655 0.00655 0.00655 0.00170 0.00170 0.00655 0.00827
10 D.aegyptium 0.01443 0.01443 0.01443 0.01443 0.01815 0.01815 0.01443 0.01692 0.01857
polymorphicprofileswhichrevealedthatpolymorphism showed almost the same topology as that of the NJ
inalltheprofileswastheresultofsitemutations.Theen- tree (Fig. 4). Maximum parsimony analysis resulted in
tiretrnKgenesequencefromthetaxaanalysedshowed most parsimonioustrees (Fig. 4C) with atree length of
214single-nucleotidepolymorphisms(SNPs).Atotal of 232. The bootstrap consensus tree had a consistency
10SNPsintherestrictionsiteswereresponsibleforthe indexof0.943,aretentionindexof0.859andarescaled
eightpolymorphicprofiles.Thepolymorphicprofilespro- consistencyindexof0.811.
duced were due to gain or loss of the restriction sites
causedbySNPs.TheconsensustrnKgenesequencewas Chloroplastmicrosatellitepolymorphism
generatedfrom the taxa analysed and the locations of andsequencedata
thepolymorphicsitesweremarkedonit(Fig.3). EightconsensusprimerpairswereusedtoamplifycpDNA
Pairwise sequence divergence ranged from 0.003 to microsatellitesfromtheninespecies(Table3).Theprimer
0.058(Table6).Dendrogramsweregeneratedusingfour pairs were able to amplify a product in all 10 species
differentmethods(NJ,ML,MEandMP).Thephylogenyre- (Fig. 5). A major band was produced for each primer–
constructionthroughtheNJmethodresultedinanopti- template combination. The typical stuttering phenom-
maltreewithasumofbranchlengthof0.095.IntheNJ enon which usually occurs upon amplification of
bootstrapconsensustree,allthe Eleusinespecies were mononucleotide- and dinucleotide-type microsatellites
grouped into two distinct clades with 100% bootstrap was observed. Size variation for the amplified products
support. Clade I consisted of E. coracana, E. africana, wasonlyobserved fortwocpSSRloci. Twosizevariants
E.indica,E.kigeziensisandE.tristachya.Eleusinecoracana weredetectedforccmp2andccmp5.Forccmp2,allbut
andE.africanagroupedtogetherinonesubcladewitha one (E. tristachya) species showed a 197-bp allele. In
bootstrapsupportof95%,andE.indicaandE.kigeziensis E. tristachya, it was 200bp. In the case of ccmp5,
groupedtogetherinanothersubcladewithabootstrap E.jaegerishowedabandof146bp,whileeachofthere-
support of 54% within Clade I. Eleusine tristachya did mainingninespecieshada145-bpallele(Fig.5).
notgroupwithinanyofthetwosubcladesandthuswas TotestthepresenceofarepeatmotifinEleusine,nu-
themostdivergedamongthefivespeciesofCladeIwith cleotide sequencing was carried out for amplified pro-
a bootstrap support of 98%. Clade II consisted of ductsfromE.coracanawithalleightcpSSRprimersets.
E. intermedia, E. floccifolia, E. jaegeri and E. multiflora. RCt5 and RCt8 could not be successfully sequenced.
EleusineintermediaandE.floccifoliaweremoreclosely ccmp2, ccmp5, ccmp6, RCt3, RCt4 and RCt7 showed
relatedtoeachotherthantoE.jaegeriwithabootstrap sequencesizesof197,145,96,113,131and126bp,re-
support of 54%. Eleusine multiflora represented the spectively(Tables3and7).Repeatmotifswereidentified
most diverged species within Clade II. Acrachne race- inallsixamplificationproductsattheexpectedpositions
mosaandD.aegyptiumwerethemostdivergedspecies but the degree of conservation was variable (Table 7).
amongallthespecies(Fig.4A). Amongtherepeatmotifs,RCt4andRCt7werehighlycon-
Therobustnessofthepresentdataliesinthefactthat served,ccmp5and ccmp6weremoderatelyconserved,
the dendrograms based on ME, ML and MP analysis and ccmp2and RCt3were least conserved.Inthe case
8 AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2013
Agrawaletal.—PhylogeneticrelationshipsamongEleusinespecies
Figure 2. Unweighted pair-group method using arithmetic averages dendrogram based on the restriction fragment data of amplified
chloroplastgene/intergenicspacers.Numbersatthenodesrepresentbootstrapprobabilityvaluesoutof100replicates.
of ccmp2and RCt3,the numberof mononucleotide re- 18) and E. kigeziensis (AADD,2n¼4x¼38) on the one
peatswasreduced. hand,and betweenE.floccifolia(BB,2n¼2x¼18)and
E.intermedia(AB,2n¼2x¼18) ontheother hand.Al-
thoughthemaximumnumberofpolymorphicandphylo-
Discussion
genetically informative markers was obtained in eight
The restriction site variation identified in the amplified trnK amplicon–enzyme combinations, the amount of
chloroplastgene/intergenicspacershasprovidednewin- variationthatcanbetappedwiththePCR–RFLPmethod
sightsintotheoriginandevolutionofthethreepolyploid waslowerthanthevariationtappedwithsequencingof
species,andthegeneticrelationshipsbetweentheculti- the trnK gene. A total of 214 SNPs were found in the
vatedandwildEleusinespecies.Species-specificmarkers trnKgenesequence,ofwhichonly10werecausativeto
were also identified. None of the amplicon–enzyme 8polymorphicprofiles.Thisclearlydemonstratesthatse-
combinations could discriminate between E. coracana quencingofthetrnKgeneismoreinformativethanthe
(AABB,2n¼4x¼36), E. africana (AABB,2n¼4x¼36), PCR–RFLP method. Moreover, the nucleotide sequence
E.tristachya(AA,2n¼2x¼18),E.indica(AA,2n¼2x¼ of the chloroplast trnK gene fine-tuned the species
AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2013 9
Agrawaletal.—PhylogeneticrelationshipsamongEleusinespecies
Figure3. ConsensussequenceofthetrnKgenefornineEleusinespeciesandoneoutgroup(D.aegyptium)showingmajorrestrictionsites.
10 AoBPLANTS www.aobplants.oxfordjournals.org &TheAuthors2013