Molecular genetic study on a historical Solanum (Solanaceae) herbarium specimen collected by Paulus Kitaibel in the 18th century

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Molecular genetic study on a historical Solanum (Solanaceae) herbarium specimen collected by Paulus Kitaibel in the 18th century
   Acta Botanica Hungarica 51(3–4), pp. 337–346, 2009DOI: 10.1556/ABot.51.2009.3–4.10 MOLECULAR GENETIC STUDY ON A HISTORICAL SOLANUM   (SOLANACEAE) HERBARIUM SPECIMENCOLLECTED BY PAULUS KITAIBELIN THE 18TH CENTURY P. Poczai, J. T ALLER  and I. S ZABÓ Department of Plant Science and Biotechnology, Georgikon FacultyUniversity of Pannonia, H-8360, Keszthely, HungaryE-mails:,, (Received 25 November, 2008; Accepted 30 January, 2009)The herbarium of Paulus Kitaibel (1757–1817) has been preserved in good condition andcan still be used for the investigation of problematic questions. There happens to be a mis-identifiedspecimenonthebackofoneofthesheets.Althoughithasbeenstudiedbyseveral botanists, it has proved impossible to identify this species because important morphologi-caltraitshavebeenlostduetotheageofthepreservedmaterial.ThecompleteITSregionof the over 200-year-old herbarium specimen has been amplified by PCR. Sequence analysisandthesecondarystructuremodellingoftheITS2RNAtranscriptclearlyprovided,thatthespecimen is Solanumscabrum , thus settling the controversy surrounding this question. Thisresult is particularly interesting, as the presence of  S.scabrum  in the Hungarian flora, hasnever been officially documented.Key words: herbarium, ITS, Paulus Kitaibel, phylogenetics, Solanum INTRODUCTION Herbarium specimens from various collections are valuable sources forplant science. They can be used not only for morphological investigations, butalsoformoleculargeneticstudiesofferingnewopportunitiesforphylogeneticanalysis.Incaseswherenolivingplantmaterialisexists,orisnotavailableforexperiments, DNA can be extracted from preserved herbarium material.These procedures offer great potential to study genetic divergence throughtime, and also to explore problematic taxonomic concepts. 0236–6495/$ 20.00 © 2009 Akadémiai Kiadó, Budapest  However, DNA extracted from herbarium material is frequently highlyfragmented. The extent of DNA degradation seems to be related to the condi-tions under which the specimen was dried and subsequently kept in the her- barium facilities(Lambertini  et al.  2008).Severalprotocols forDNAextractionfrom herbarium samples have been described, e.g. Doyle and Dickson (1987),DoyleandDoyle(1987),Wittzell(1999),Ristaino et al. (2001),Saltonstall(2002,2003), Drabkova  et al.  (2002), De Castro and Menale (2004), Jankowiak  et al. (2005).The section  Solanum  L., commonly known as black nightshades, is one of thelargestandmostvariablespeciesgroupsinthegenus Solanum L.,andcon-sistsofabout40–50species,mostofwhichsrcinatefromtheNewWorldtrop-ics, particularly from South America (Edmonds 1972). The taxonomy of thisgroup is complicated because of polyploidy and phenotypic plasticity (Schil-ling and Andersen 1990). There is also widespread confusion over the preciseidentificationofthetaxa,especiallyinareasinwhichthespeciesaremostcom-monly used as food sources (Edmonds and Chweya 1997), for example in thecase of   Solanum scabrum  Mill., which is native to West and Central Africa. Themorphological distinction now seems to be clear, but it was still not obviousduring the historical examination of the species belonging to sect.  Solanum . Invarious studies many synonyms were used for  S. scabrum , the best known be-ing:  S. guineense  (L.) Mill.,  S. intrusum  J. Soria,  S. melanocerasum  All.,  S. nigrum var.  guineense  L.,  S. nigrum  subsp.  guineense  (L.) Persoon, and  S. tinctorium Welwitsch.In the 18th and 19th century  S. nigrum  and its relatives were extensivelystudied by botanists, including Paulus Kitaibel, whose work around  Solanum wasnoteworthy.ThespeciesnamedbySchultes(1814), S. kitaibelii (nowasyn-onym of   S. villosum  Mill.) preserves his name. Although, his herbarium is stillin excellent condition, one of the  Solanum  pages contains an unresolved para-dox on page 102 of Fasc. IX, two plants were preserved on the same sheet.ThesespecieswerestudiedbySándorJávorka(whowrotenotesontheedgeof the herbarium sheet) and by Polgár (1926), who noted a clear distinction be-tween the two plants preserved on page 102. The first plant on the front of thesheet, is unambiguously  S. nodiflorum  Jacq., having small flowers, yellow an-thers 1.4 mm in length, 1.2 mm styles that do not protrude from the anther-cone. The style hairs are simple, reach only to 1/3 of the organ. The secondplant on the back of page 102, has larger flowers large ovate or occasionallylanceolateleaves,broadlyovoid,purpleberriesandlongpedunclesandstyles(4cm).Afteramicroscopicalexaminationofthespecimens,Polgár(1926)con-cludedthatthetwodifferentplantscouldnotoriginatefromthesamespecies.The plant on the back page might be  S. guineense  Lam., but a positive identifi- 338  POCZAI, P., TALLER, J. and SZABÓ, I.  Acta Bot. Hung. 51, 2009  cation was not possible, for the typical brownish or purplish brown anthers because of the age of the herbarium (Polgár 1926).The biparentally inherited nuclear DNA regions ITS1 and ITS2 (internaltranscribed spacer regions) which are intercalated between ribosomal regions(18S-ITS1-5.8S-ITS2-26S),arethemarkerregionsmostextensivelyusedtoana-lyse phylogenetic relationships at different levels, in different types of organ-isms.AspartofthetranscriptionalunitofnuclearribosomalDNA(nrDNA),itispresentinallorganisms,soitisfrequentlyemployedtoaddressquestionscon-cerningphylogenyaswellasforotherapplicationsinpathologyandepidemi-ology. For further information about applications, concerns and structure seePoczai and Hyvönen (2009). Compared to the 5.8S gene and the ITS1 region,ITS2 has a more variable primary sequence. It is also slightly more conservedthan ITS1, allowing alignments to be made in ranks above the genus level(HershkovitzandLewis1996,Calonje etal. 2008).Thehelicalstructurecreatedduring the maturation of rRNA results in domains with both paired and un-paired bases, implying different evolutionary constrains. Thus, the modellingof the secondary structure of the ITS2 RNA transcript could be useful forphylogenetic analysis, when alignment of the sequences is problematic.The aim of this study was to determine the taxonomic status of the speci-menonthebackofpage102inFasc.IX.oftheKitaibel’sherbariumbyanalys-ing the ITS region. MATERIALS AND METHODS Plant material and DNA extraction The herbarium samples of Paulus Kitaibel were provided by the Depart-ment of Botany of the Hungarian Natural History Museum. Research startedin 2007, the memorial year of the outstanding botanist Paul Kitaibel, the ex-plorer of the Hungaian flora. Total genomic DNA was extracted from smallamounts of the leaves of the herbarium specimens according to Nan etal. (2003) based on the procedure of Doyle and Doyle (1987) with the followingmodifications.Driedleafmaterialwaskeptin800µlof2×CTABextractionso-lution (2% cetyl-trimethyl-ammonium bromide, 0.2% mercaptoethanol, 1.4mMNaCl,pH=8.0)for1week.ThenthesamplesweregroundinaMixerMill(Retsch MM 301, Germany) for 5 min at 30 1/s. The samples were incubatedfor 1 h at 64 °C. Subsequently, 600 µl of chloroform : isoamyl-alcohol (24 : 1v/v)wasadded,andthemixturewasshakengentlyfor2minandcentrifugedat8,000rpmfor15minatroomtemperature.Thesupernatantwasmixedwith2/3 volume ice cold isopropanol. For accurate DNA precipitation the mixturewas stored for 5 days at 4 °C. DNA was then recovered as a pellet by MOLECULAR GENETIC STUDY OF KITAIBEL’S SOLANUM HERBARIUM  339  Acta Bot. Hung. 51, 2009  centrifugation at 15,000 rpm for 10 min and washed with 1 ml washing buffer(99.9% ethanol, 10 mM ammonium acetate). The supernatant was removedand the pellet was washed again with 400 µl 76% ethanol, dried, then resus-pended in 50 µl TE buffer (10 mM Tris (hydroxymethyl)-aminomethane, 1 mMEDTA, pH = 8.0). DNA extraction was done for each sample in three repeti-tions, to allow the detection of sporadic contaminants, and to detect furtherchanges caused by miscoding DNA lesions due to low numbers of templatemolecules.Eachextractionprocessincludedoneextractioncontrol,whichdidnot contain any sample material, but was treated identically. PCR amplification The entire internal transcribed spacer (ITS) region, located between the18S and 26S nrDNA regions and containing ITS1, ITS2 and the 5.8S gene, wasamplified. Each amplification was repeated at least twice. During the PCR re-actiontwonegativecontrolswereincluded,onecontainingnotemplateDNA,andoneextractioncontrol.TheamplificationwasperformedwiththeprimersITSleu1(5’-GTCCACTGAACCTTATCATTTAG–3’;BohsandOlmstead2001)andITS4(5’-TCCTCCGCTTATTGATATGC–3’;White etal. 1990).AllthePCRreactionsweredoneinaMasterCyclerep384(Eppendorf,Germany).The10µlreaction mix contained the followings 5 µl IEW (ion exchanged water, UVtreated and autoclaved), 5 ng DNA template, 1 µM of each primer, 0.2 mMdNTP(Fermentas,Lithuania),2µl10×PCRbuffer(1mMTris-HCl,pH=8.8at25°C,2.0mMMgCl 2 ,50mMKCland0.1%TritonX–100),0.5UofDyNazymeIIpolymerase(Finnzymes,Finland),and1µgBSA.DuringthePCRreactionatouchdown-PCR program was applied: 97 °C for 2 min, followed by 10 cyclesof97°C1min.Theannealingtemperaturestartedfrom60°Cfor1minute,anddecreasedby1°Cpercycleto50°C,andwasfollowedby72°Cfor1min.Thenext25cycleswerepreformedat97°Cfor1min,50°Cfor1minand72°Cfor1min,witha3secextensionpercycle,andafinalextensionat72°Cfor10min. Cloning and sequencing For the cloning procedure, the amplified products of the specimen fromthe back of page 102 was excised from the agarose gel and cleaned using theSpinPrep Gel DNA Kit (Novagen, Germany). The fragment was cloned usingthe pGem-T Easy Vector System (Promega, USA) and a DH5 α  competent E.coli  strain. Plasmids were extracted from the selected white colonies using theEZ-10 Spin Column Plasmid DNA Kit (Bio Basic Inc., Canada). Sequencingwas done using the SP6 and T7 promoter primers, in an ABI3400 by BIOMILtd. (Hungary). 340  POCZAI, P., TALLER, J. and SZABÓ, I.  Acta Bot. Hung. 51, 2009  Phylogenetic analysis Outgroupsfromothersubgenera( S.lycopersicum L., S.aethiopicum L.)andfrom a different, but closely related genus (  Jaltomataprocumbens  (Cav.) J. L.Gentry) were included in the analysis. The ITS1 and ITS2 sequence data wereedited using BioEdit v7.09 (Tom Hall, bioedit.html),thenalignedwithotherGeneBanksequencesfromthedatabaseNCBI (http://www.ncbi., using ClustalW (,becauseof thedifferentevolutionaryratesofthetworegions,andthencombinedtoforma single consensus tree. The aligned sequences were analysed with theMEGA4.1 program (Kumar etal.  2008), using the maximum parsimony (MP)criterion with 10,000 bootstraps. For the construction of ITS2 RNA transcriptsecondarystructure,theITS2DataBasewasused(Schultz etal. 2006).Then,toreconstruct phylogeny based on secondary structures the MARNA software(SiebertandBackofen2005)wasusedtoalignRNAtranscriptsecondarystruc-tures and sequences. Phylogenetic analysis was performed using these datawiththeCBCAnalyser(Wolf  etal. 2005).Thetreewasvisualisedandeditedus-ingTreeView(Page1996).AlldataontheNCBIsequencesandotherplantma- terials used in this study are summarised in Table 1. RESULTS DNAwassuccessfullyextractedfromtheherbariumsamplesandnospo-radic or other contamination was observed during the experiments, so the MOLECULAR GENETIC STUDY OF KITAIBEL’S SOLANUM HERBARIUM  341  Acta Bot. Hung. 51, 2009 Table 1 Accession numbers of the GeneBank sequences of used in the analysisName NCBI accessionnumberMP analysis Structure modeling Solanum aethiopicum  AY996482 + + Solanum americanum  AY863061 + + Solanum lycopersicum  EU760390 + + Solanum nigrum  (1) AY863060 + + Solanum nigrum  (2) AJ300211 + – Solanum nigrum  (3) AY863063 + – Solanum scabrum  Poczai (unpubl.) + + Solanum villosum  (1) AY875752 + + Solanum villosum  (2) AF244736 + –  Jaltomata procumbens  AF244710 + +
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