Extensive Gene Duplications in Diploid Eupatorium (Asteraceae) Title 著者 Yahara, Tetsukazu / Kawahara, Takayuki / Crawford, Daniel J
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Kobe University Repository : Kernel タイトル Extensive Gene Duplications in Diploid Eupatorium (Asteraceae) Title 著者 Yahara, Tetsukazu / Kawahara, Takayuki / Crawford, Daniel J. / Ito, Author(s) Motomi / Watanabe, Kuniaki 掲載誌・巻号・ページ American journal of botany,76(8):1247-1253 Citation 刊行日 1989-08 Issue date 資源タイプ Journal Article / 学術雑誌論文 Resource Type 版区分 publisher Resource Version 権利 Rights DOI JaLCDOI URL http://www.lib.kobe-u.ac.jp/handle_kernel/90001248 PDF issue: 2021-10-04 Amer. J. Bot. 76(8): 1247-1253. 1989. EXTENSIVE GENE DUPLICATIONS IN DIPLOID EUPATORIUM (ASTERACEAE)1 TETSUKAZU YAHARA,2 TAKAYuKI KAWAHARA 2 DANIEL J. CRAWFORD,3 MOTOMI ITO,4 AND KUNIAKI WATANABE5 2BotanicalGardens, Faculty of Science, University of Tokyo, 1842 Hanaishi-cho, Nikko 321-14, Japan;3Department of Botany, The Ohio State University, Columbus,Ohio 43210-1293; 4MakinoHerbarium, Tokyo MetropolitanUniversity, 2-1-1 Fukazawa,Tokyo 158, Japan;and 5Biological Institute,Faculty of GeneralEducation, Kobe University, Kobe 657, Japan ABSTRACT An electrophoreticstudy of isozyme number for seven soluble enzymes revealed extensive gene duplicationsin eight diploid species of American Eupatoriumbelonging to three mor- phologicalgroups. The enzymes isocitrate dehydrogenase,phosphoglucomutase, phosphoglu- cose isomerase, 6-phosphogluconatedehydrogenase, and shikimate dehydrogenaseoccur as three to six isozymes in all species, whereasthe minimal conserved numbertypical of diploid plantsis two isozymes for each. Fructose1, 6-biphosphatealdolase is expressedas multibanded patternsuggesting fixed heterozygosityin all examined species. It was not possible to document gene duplicationfor triosephosphateisomerase from the electrophoreticpatterns. All species examinedhave a chromosomenumber of 2n = 20, which has been regardedas the basic diploid number for Eupatorium.However, the detection of extensive duplicationssuggests that 2n = 10 may be the originaldiploid chromosomenumber in Eupatoriumand that plants with 2n = 20 are of polyploidorigin. This hypothesiswould mean that extensive duplicationsat isozyme geneloci have been maintainedsince the originof the genus,despite chromosomal diploidization having occurred. IT HAS BEEN documented that diploid vascular cannot be employed as the sole criterion. Iso- plants have a minimal highly conserved num- zyme number has been successfullyapplied to ber of isozymes for most of the enzymes rou- systematic questions in several plant groups. tinely examined by electrophoresis(Gottlieb, Recent studies of ferns and fern-allies have 1981a, 1982, 1983, 1984). This means that an shown that plants traditionallyviewed as poly- increasein isozyme numberfor a given enzyme ploids on the basis of high chromosome num- is the result either of gene duplication at the bers are genetically diploids at isozyme loci diploid level or of polyploidy. If a plant is (Haufler and Soltis, 1986; Haufler, 1987; D. polyploid, then it should have duplications at Soltis and P. Soltis, 1987, 1988). Using the many isozyme loci, whereas a diploid plant same criterion, Soltis et al. (1987) and Chase would exhibit much less extensive increase in and Olmstead (1988) rejected the hypothesis isozymenumber(Gottlieb,1981a, 1983,1984). that species with high chromosome numbers While inferences about whether given plants arepolyploids in the Bromeliaceaeand the sub- are diploids or polyploids have often rested tribe Oncidiinae of Orchidaceae,respectively. primarily on their chromosome numbers, On the other hand, Witter (1988) supported Gottlieb (1981b) employed isozyme number the hypothesis that plants of the Hawaiian sil- to infer the ploidy level of plants when chro- verswordalliance, in which 2n = 28, are poly- mosome data per se were inconclusive. Gott- ploid by demonstratingduplicate gene expres- lieb (198 lb) arguedthat an essential featureof sion for four soluble enzymes. polyploid is the number of genomes being ex- In this paper we reportresults of an electro- pressed, and that chromosome number alone phoretic study of isozyme number for seven enzymesfrom eight speciesof Eupatoriumwith a chromosome number of 2n = 20. This has I Received for publication 1 November 1988; revision been regardedwidely as the originalbase num- accepted2 March 1989. ber for the tribe Eupatoriae (King and Rob- We thank V. I. Sullivan for her kind help in the field. This study was partly supportedby grant no. 63041048 inson, 1970a, 1987;King et al., 1976), although from Ministry of Education, Culture and Scientific Re- a number of n = 5 has been reported for the serch, Japan. tribe (Turnerand Irwin, 1960). 1247 1248 AMERICAN JOURNAL OF BOTANY [Vol. 76 MATERIALS AND METHODS -Eight species of well established for each (Randall and Givan, Eupatoriumwere examined;the species, num- 1981 for IDH; Gottlieb, 1982; Mousdale and ber of plants studied from naturalpopulations Coggins, 1985, for SKDH). In all instances, and localities of the populationsare as follows: one isozyme occursin the plastidsand the other E. capillifolium(Lam.) Small (25, Vernon Par- is cytosolic. ish, Louisiana; 24, Guilford Co., North Car- Chloroplasticisozymes were isolated from olina); E. fistulosum J. Barratt (24, Fairfield a single plant of E. perfoliatum collected in Co., Ohio; 24, Hocking Co., Ohio); E. mac- Columbus, Ohio. The method of extracting ulatum L. (12, Franklin Co., Ohio); E. mika- intact chloroplast followed Gastony and Dar- nioides Chapman (12, Wakulla Co., Florida); row (1983) except that the chloroplasts were E. perfoliatumL. (26, FairfieldCo., Ohio; 25, not centrifugedthrough Percoll. The chloro- Hocking Co., Ohio); E. purpureum L. (24, plast-enrichedpellets were broken with the tris- FairfieldCo., Ohio);E. semiserratumDC. (24, HCIgrinding buffer (above). Activities of IDH, Evans Co., Georgia; 18, Allen Parish, Loui- 6-PGD, and SKDH were stronger in flower siana) and E. serotinum Michx. (12, Franklin buds but very weak in leaves and we could not Co., Ohio; 27, Marion Co., Indiana). For E. obtain sufficient activity of these enzymes in fistulosumand E. perfoliatum,progenies grown the chloroplast fraction from leaves. from seeds were also examined (36 from three maternalparents of E.fistulosum; and 36 from RESULTS-PGI- In all eight species, enzyme two plants of E. perfoliatum; seeds were col- activity appearedin two zones in the gels. In lected from Fairfield Co., Ohio). the chloroplast-enrichedfractions of E. per- Flower buds and a small piece of leaf ma- foliatum, isozymesin the fastermigrating (more terial of individual plants were ground in 1.0 anodal) zone only were expressed. This result ml of cold extraction buffer consisting of 0.1 agrees with all the formerly obtained results M tris-HCl,pH 7.5,20 mm 2-mercaptoethanol, on electrophoreticmobilities of PGI isozymes 1.0 mM EDTA, 10 mm KCI, 10 mM MgCl2, from various plants;i.e., the chloroplasticiso- 0.25% (v/v) triton X-100, and 15 mg PVP zyme migrates faster than the cytosolic one. (modified from Odryzykoski and Gottlieb, In the slowermigrating (more cathodal) zone, 1984). For E. capillifolium and E. mikanioi- three or five bands were detected for this di- des, fresh leaves alone were used. meric enzyme in most individuals of the eight Enzymes were resolved in 12% starch gels species examined. This suggests gene dupli- using three buffer systems. System I had a gel cation for cytosolic PGI in diploid Eupato- bufferof 0.033 M tris, 0.005 M citric acid, 0.004 rium. Among progenies grown from a single M lithium hydroxideand 0.3 M boric acid with maternal parent of E. fistulosum, three phe- the pH adjustedto 7.6 and an electrode buffer notypes segregatedin the more cathodal zone of 0.039 M tris and 0.263 M boric acid with the (1-3 in Fig. IA and Fig. 2); one pattern with pH adjusted to 8.0 (P. Soltis and D. Soltis, a seemingly single heavily staining band (1 in 1987). System II had an electrodebuffer of 0.5 Fig. 1 and 2), one phenotypehaving three bands M tris, 0.016 M EDTA and 0.57 M boric acid consisting of a fast migratingvery faint, a slow with the pH adjustedto 8.0. The gel bufferwas migratingvery dark, and a band intermediate a 1:9 dilution of the electrode buffer. System in both mobility and staining intensity (2 in III consisted of an electrode of 0.065 M L- Fig. 1 and 2), and the last pattern having six histidine (free base) and 0.007 M citric acid bands (3 in Fig. 1 and 2). The first phenotype (monohydrate) adjusted to pH 6.5. The gel is interpretedas having three bands that mi- bufferwas a 1:3 dilution of the electrodebuffer grate very closely and the most anodally mi- (Cardy,Sturber, and Goodman, 1981). gratingband is very faint (see dots in Fig. IA System I resolved fructose 1, 6-biphosphate and Fig. 2-1). Also, among progenies grown aldolase (ALD, EC 4.2.1.3), phosphoglucoiso- from a single maternal parent of E. perfolia- merase (PGI, EC 5.3.1.9) and triosephosphate tum, three- and five-banded phenotypes seg- isomerase (TPI, EC 5.3.1.1). System II re- regated in the more cathodal zone (Fig. iB, solved phosphoglucomutase (PGM, EC Fig. 2-4, 2-5). These patternssuggest that fixed 2.7.5.1). System III was employed to resolve heterozygositydue to duplicated gene loci for isocitrate dehydrogenase(IDH, EC 1.1.1.42), cytosolic PGI isozyme occur in E. fistulosum 6-phosphogluconate dehydrogenase (6PGD, and E. perfoliatum. In both species, three- EC 1.1.1.44) and shikimate dehydrogenase banded phenotypes have one faint band, and (SKDH, EC 1.1.1.25). Staining schedules fol- the same pattern was seen in E. mikanioides lowed Soltis et al. (1983). Enzymes for study (Fig. I E), in which one faint band of the three- were selected because the minimal conserved banded phenotype migrated near to a band numberof isozymes in diploid plants has been representinga chloroplasticisozyme. By com- August 1989] YAHARA ET AL. -GENE DUPLICATIONS IN EUPATORIUM 1249 A B~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~A F G I .40 Fig. 1. Electrophoreticpatterns of six solubleisozymes in diploidspecies of Eupatorium.(A-E) PGI, A, E.fistulosum; B, E. perfoliatum;C, E. semniserratum,D, E. capillifolium,E, E. mikanioldes.(F-I) PGM; F, E.