UC Davis UC Davis Previously Published Works

Title Genome analysis of South American () and (Triticeae) species based on variation in repeated nucleotide sequences.

Permalink https://escholarship.org/uc/item/54w48156

Journal Genome, 40(4)

ISSN 0831-2796

Authors Dubcovsky, J Schlatter, AR Echaide, M

Publication Date 1997-08-01

DOI 10.1139/g97-067

Peer reviewed

eScholarship.org Powered by the Digital Library University of California Genome analysis of South American Elymus (Triticeae) and Leymus (Triticeae) species based on variation in repeated nucleotide sequences

Jorge DU~COVS~~,A.R. Schlatter, and M. Echaide

Abstract: Variation in repeated nucleotide sequences (RNSs) at the level of entire families assayed by Southern blot hybridization is remarkably low within species and is a powerful tool for scrutinizing the origin of allopolyploid taxa. Thirty-one clones from RNSs isolated from different Triticeae genera were used to investigate the genome constitution of South American Elymus. One of these clones, pHch2, preferentially hybridized with the diploid H genome species. Hybridization of this clone with a worldwide collection of Elymus species with known genome formulas showed that pHch2 clearly discriminates Elymus species with the H genome (StH, StHH, StStH, and StHY) from those with other genome combinations (Sty, StStY, StPY, and StP). Hybridization with pHch2 indicates the presence of the H genome in all South American Elymus species except Elymus erianthus and Elymus mendocinus. Hybridization with additional clones that revealed differential restriction fragments (marker bands) for the H genome confirmed the absence of the H genome in these species. Differential restriction fragments for the NS genome of Psathyrostachys were detected in E. erianthus and E. mendocinus and three species of Leymus. Based on genome constitution, morphology, and habitat, E. erianthus and E. mendocinus were transferred to the Leymus. Key words: Triticeae, Elymus, Leymus, repeated sequences.

Resume : La variation des stquences nucltotidiques rtptttes (RNS) prtsente au niveau de familles botaniques enti&res,telle que rtvtlte par hybridation Southern, est ttonnamment faible entre esp2ces et constitue un outil puissant pour analyser l'origine de taxons allopolyploides. Trente et un clones RNS isolts chez difftrents genres appartenant aux Triticeae ont ttt employts pour analyser la composition gtnomique des esp2ces du genre Elymus en Amtrique du Sud. Un de ces clones, pHch2, montre une hybridation prtftrentielle avec les esp2ces du genre Hordeum h gtnome H diploides. L'emploi de ce clone comme sonde lors For personal use only. d'hybridations avec une collection internationale d'esp2ces d'Elymus ayant une composition gtnomique connue a montrt que pHch2 permet de distinguer nettement les tlymes portant le gtnome H (StH, StHH, StStH et StHY) des tlymes ne posstdant pas un tel gtnome (Sty, StStY, StPY et StP). L'hybridation avec la sonde pHch2 a montrt la presence du gtnome H chez toutes les esp2ces sud-amtricaines h l'exception du Elymus erianthus et du Elymus mendocinus. Des hybridations additionnelles avec des clones qui rtv2lent des bandes sptcifiques au gtnome H ont confirm6 l'absence de ce gtnome parmi ces esp2ces. Des fragments de restriction sptcifiques au gtnome NS du Psathyrostachys ont ttt dttectts chez les esp2ces E. erianthus et E. mendocinus de meme que chez trois esp2ces du genre Leymus. En se fondant sur leur composition gtnomique, leur morphologie et leur habitat, les esp2ces E. erianthus et E. mendocinus sont considtrtes comme faisant partie du genre Leymus. Mots clbs : Triticeae, Elymus, Leymus, stquences rtptttes. [Traduit par la Redaction]

times as many as the second largest genus, Hordeum L. Genome Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 03/28/17 Introduction (approximately 40 species). Species of Elymus are allopoly- The genus Elymus L. is the largest among the perennial ploids cytologically characterized by genome combinations of Triticeae. It contains approximately 150 species, almost four five basic genomes: S' from Pseudoroegneria (Nevski) A. Love, H from Hordeum L., Y from an unknown diploid spe- Corresponding Editor: G. Fedak. cies, P fror? Gaertn., and W from Austmlopyrum Received January 3, 1996. Accepted April 14, 1997. (Tzvelev) A. Love (Dewey 1984; Love 1984; Jensen 1989; Jensen 1990a, 1990b, 1990~;Lu 1993). Genome symbols are J. ~ubcovsk~.'Department of Agronomy and Range Science, University of California, Davis, CA 95616, U.S.A. in accordance with the system proposed at the 2nd Interna- tional Triticeae Symposium (Wang et al. 1994). A.R. Schlatter and M. Echaide. Instituto de Recursos Biol6gicos. Elymus species with different genome combinations differ Centro de Investigaciones en Recursos Naturates (CIRN), Instituto in their geographic distribution. The basic genome W has been Nacional de Tecnologia Agropecuaria (INTA), Villa Udaondo, 17 12 Castelar, Buenos Aires, . identified only in one Australasian hexaploid (S'YW; Torabinejad and Mueller 1993) and the basic genome P is ' Author to whom all correspondence should be addressed restricted to some Central Asiatic hexaploids (S'PY; Jensen (e-mail: [email protected]).

Genome, 40: 505-520 (1997) 01997 NRC Genome, Vol. 40, 1997

1990b, 1990~).The Y genome has a wider distribution and is NSgenome specific repeated nucleotide sequences and H and found in many Asiatic tetraploids and hexaploids (s'Y, S'S'Y, NS genome specific restriction fragments of repeated se- S'HY, and S'PY; Lu 1993). Finally, species with St-H quences (marker bands). We also investigate the use of these genome combinations (S'H, S'S'H, S'HH, and S'S'HH) have repetitive sequences as a tool for genome analysis in Elymus the widest geographic distribution, being found in Europe, by using a worldwide set of species with known genome con- , North America, South America, Australia, and New stitutions. Zealand (Dewey 1984; Love 1984). Genome formulas are known only for a small number of Materials and methods South American Elymus species. Genome analysis has resulted in the tetraploid species Elymus agropyroides materials Presl, Elymus tilcarensis (Hunziker) A. Love, and Elymus The species analyzed, together with their genome constitution, acces- magellanicus (Desv.) A. Love being assigned the genome sion number, and origin, are listed in Table 1. Voucher specimens of formula S'H (Dewey 1970b, 1977; Jensen 1993) and the the South American Elymus species are deposited in the herbarium of the Instituto de Recursos Biologicas (IRB), Instituto Nacional de hexaploid species Elymus patagonicus Speg. and Elymus Tecnologia Agropecuaria (INTA) (BAB, Castelar, Buenos Aires, scabriglumis (Hakel) A. Love being assigned the genome for- Argentina). Herbarium acronyms are according to Holmgren et al. mula S'HH (Hunziker 1953, 1966; Dewey 1972c; Seberg (Holmgren et al. 1990). Seeds of the Elymus species from North 1991). Gupta et. a1 (1988) suggested that the Y genome was America, Europe, and Asia were provided by Dr. R.C. Johnson of the present in E. scabriglumis, but later concluded that the genome United States Department of Agriculture, Agricultural Research formula of this species was S'HH (Lee et al. 1994). In spite of Service, Western Regional Plant Introduction Station, Pullman, this apparent genome uniformity, poor chromosome pairing in Wash., U.S.A. Seeds of the Hordeum species were provided by Dr. R. meiosis of interspecific hybrids has revealed the presence of von Bothmer of the Swedish University of Agricultural Sciences, genome differentiation among some tetraploid South Ameri- Svalov, Sweden. can Elymus species (Hunziker et al. 1965; Hunziker and South American Elymus species were identified using the keys of Nicora (1978) and Hunziker (1966). Elymus angulatus Presl did not Ferrari 1986). flower and it was not possible to verify the identification provided by Karyotype analysis of the South American Elymus species the collector, Dr. 0. Seberg (University of Copenhagen, Denmark). suggested that the S'H genome was present in all South Delimitation of the genus Elymus followed Love (Love 1984; American tetraploids analyzed, but indicated differential char- Zuloaga et al. 1994), and Hordeum was considered in sensu Bothmer acteristics in the karyotypes of the hexaploid Elymus erianthus (Bothmer et al. 1991). When all the South American species of Philippi and the octoploid Elymus mendocinus (Parodi) Agropyron were transferred to Elymus (Love 1984), Agropyron A. Love (Dubcovsky et al. 1989; Lewis et al. 1996). C-banding scabrifolium (Doll) Parodi wa: included as a subspecies of Elymus and N-banding of E. erianthus chromosomes (Seberg and breviaristatus (A.S. Hitchc.) A. Love. This is probably incorrect, Linde-Laursen 1996) and hybridization with a GAA satellite because A. scabrifolium and Agropyron breviaristatum A.S. Hitchc. (Pedersen et al. 1996) and four repetitive DNA sequences are reproductively isolated species (Hunzik~ret al. 1965). Since E. breviaristatus ssp. scabrifolious (Doll) A. Love (Love 1984; For personal use only. cloned from barley (Svitashev et al. 1996) revealed additional Zuloaga et al. 1994) is the only published name for this taxon under differences and suggested the absence of the St and H genomes Elymus, we temporarily use it for the sake of consistency. A taxo- from this species (Seberg and Linde-Laursen 1996). nomic review of this species is in preparation (0. Seberg, personal Clayton and Renvoize (1986; also, S.A. Renvoize, personal communication). communication in Seberg and Linde-Laursen (1996)) sug- gested the inclusion of E. erianthus in Leymus Hochst., a Clones genus of about 30 polyploid species occurring naturally in The clones used in this study, together with the origin of the genomic and North America. More recently, E. erianthus was libraries, vector, cloning site, size, and references are listed in transferred from Elymus to a new monotypic genus, Eremium Table 2. Seberg (Seberg and Linde-Laursen 1996), on the basis of its differential morphology and cytology. To decide between Methods these alternative taxonomic treatments it is important to deter- Nuclear DNAs were isolated from of single following mine the genome constitution of E. erianthus. As Leymus is the procedure of Dvoiiik et al. (1988) and digested with Tag1 and HaeIII. DNA loading in the gel was adjusted by ploidy level (dip- Genome Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 03/28/17 morphologically, cytologically, and genomically affiliated loids, 2 kg; tetraploids, 4 kg; hexaploids, 6 kg; and octoploids, with the NS genome (Dewey 1970a, 1972a, 1972b, 1976; 8 kg). DNAs were electrophoretically fractionated in 1.7% agarose Wang and Hsiao 1984; Zhang and Dvoiik 199 1; Sun et al. gel and transferred to Hybond N+ nylon membrane (Amersham, lll., 1995; Mason-Gamer and Kellog 1996), it is particularly U.S.A.) by capillary transfer. Fluorescence intensity of ethidium bro- important, to establish the presence or absence of the NS mide under UV light was integrated for each lane after electro- genome of the diploid genus Psathyrostachys Nevski in phoretic fractionation with an IBM PC based image processing E. erianthus. The presence of a differential EcoRI site in system (FotoIEclipse, Fotodyne) with the program NIH Image (ver- the intergenic spacer (IGS) of the rDNA units of diploid sion 1.55). Prehybridization and hybridization were performed in a Psathyrostachys stoloniformis C. Baden (Orgaard and Heslop- rotary hybridization oven at 65OC in a solution containing 1% SDS, Harrison 1994) and E. erianthus (Dubcovsky et al. 1992), 2.5~SSPE buffer (lx SSPE: 0.18 M NaC1, 10 mM NaPO,, plus which is absent in the IGS of other diploid Triticeae and most 1 rnM EDTA, pH 7.7), 0.1% polyethylene sulfonic acid, and 0.01% sodium pyrophosphate. The immobilized DNAs were hybridized South American Elymus species, provides preliminary evi- overnight with [~x-~~~l-labeledprobes by the random primer method dence for the presence of the NSgenome in E. erianthus. (Feinberg and Vogelstein 1983). The membranes were washed in 2x In this paper we investigate the distribution of the H and NS SSC (lx SSC: 1.15 M NaCl plus 0.015 M sodium citrate) plus 0.5% genomes among South American Elymus species, using H and SDS for 30 min, lx SSC plus 0.5% SDS for 30 min, and 0.2~SSC

01997 NRC Canada Genome Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 03/28/17 For personal use only. Table 1. Plant species used in this study. - H-genome bandsa Barley bandsb Ns-genome bandsc 2o

Identification Genome Accession Origin No.

Hordeum vulgare L. I cv. Morex U.S.A. Hordeum bulbosum L. I DV 1322-3 Unknown Hordeum marinum ssp. marinum Huds. X" H 90-1 Greece Hordeum marinum ssp. marinum X" H 515 Spain Hordeum marinum ssp. gussonoeanum (Parl.) Thell. X" Hordeum murinum L. ssp. glaucum (Steud.) Tzvelev Xu Georgia Hordeum murinum ssp. glaucum Xu Hordeum bogdanii Wilensky H People' s Republic of Hordeum roshevitzii Bowden Hordeum brevisubulatum (Trin.) Link ssp. violaceum (Boiss & Hohen.) Tzvelev Hordeum brachyantherum Nevski ssp. californicum (Covas & Stebbins) Bothmer, Jacobsen & Seberg U.S.A. Hordeum intercedens Nevski U.S.A. Hordeum pusillum Nutt. U.S.A. Hordeum chilense Roem. & Schult. Hordeum cordobense Bothmer, Jacobsen & Nicora Argentina Hordeum erectifolium Bothmer, Jacobsen & M. Jflrg. Argentina Hordeum euclaston Steud. Argentina Hordeum jlexuosum Nees ex Steud. Uruguay Hordeum muticum J. Presl Bolivia Hordeum patagonicum (Hauman) Covas ssp. magellanicum (Parodi & Nicora) Bothmer, Giles & Jacobsen Argentina Hordeum pubijlorum Hook. f. Argentina Hordeum stenostachys Godr. Argentina Pseudoroegneria stipifolia -4 (Czern. ex Nevski) A. Love Former n3 U.S.S.R. Genome Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 03/28/17 For personal use only.

UI Table 1 (continued). 0 03 H-genome bandsa Barley bandsb Ns-genome bandsc

Agropyron cristatum (L.) Gaertn. P PI 406450 Former U.S.S.R. Australopyrum retrofractum (Vickery) A. Love W PI 531553 Australia Lophopyrum elongatum (Host) A. Love Ee Dl-1-2 Unknown Thinopyrum bessarabicum (Savul. & Rayss.) A. Love Eb D-3483 Former U.S.S.R. (Fisch.) Nevski N" PI 3 14668 Former U.S.S.R. Psathyrostachys juncea N" H 8721 People' s Republic of China Leymus alaicus ssp. karataviensis (Roshev.) Tzvelev NXm PI 314667 Former U.S.S.R. (Kar. & Kir.) Tzvelev NT" PI 440324 Former U.S.S.R. (Scrib. & Merr.) A. Love NsXm CS-18-61-70 U.S.A. Elymus erianthus Phil. NsXmXm? H 213 Argentina Elymus erianthus NT"Xm? Rene Argentina Elymus mendocinus (Parodi) A. Love N"XmXmXm? J 601 Argentina Elymus andinus Trin. StH J 825 Chile Elymus angulatus J. Presl StH H 6419 Argentina Elymus antarticus Hook. f. StH H 203 Argentina Elymus araucanus (Parodi) A. Love StH J 650 Argentina Elymus breviaristatus (Hitchc.) A. Love ssp. scabrifolius (Doll) A. Love Argentina Elymus canadensis L. U.S.A. 9 Elymus cordilleranus Davidse & Pohl Perii 4 Elymus gayanus Desv. Chile Elymus mutabilis (Drobov) Tzvelev People' s Republic [ of China Elymus rigescens Trin. Argentina Genome Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 03/28/17 For personal use only. Table 1 (continued). C0 0- H-genome bandsa Barley bandsb Ns-genome bandsc 9o

Elymus siribicus L. StH Former U.S.S.R. Elymus sitanion Schult. StH U.S.A. Elymus tilcarensis (J.H. Hunz.) A. Love StH Argentina Elymus trachycaulus (Link) Gould ex Shinners StH U.S.A. Elymus trachycaulus ssp. trachycaulus (Link) Gould ex Shinners StH U.S.A. Elymus trachycaulus ssp. subsecundus (Link) Gould StH Canada (Elymus trachycaulus ssp. violaceus (Hornem.) A. Love & D. Love) Agropyron spp. ? PP? Former U.S.S.R. Elymus patagonicus Speg. StHH Argentina Elymus scabriglumis (Hack.) A. Love StHH Argentina Elymus dahuricus Turcz. ex Griseb. StYH People' s Republic of China Elymus drobovii (Nevski) Tzvelev StYH Former U.S.S.R. Elymus nutants ,Griseb. StYH Former U.S.S.R. Elymus tsukushiensis Honda StYH Elymus tsukushiensis var. transiens (syn. A. kamoji (Ohwi) Ohwi) StYH China Elymus glaucissimus (M. Pop.) Tzvelev Ststy Europa Elymus tschimganicus (Drobov) Tzvelev Ststy People' s Republic of China Elymus alatavicus (Drobov) A. Love STY China 2 Elymus batalinii (Krasn.) Nevski StPY Elymus grandiglumis (Keng) A. Love STY China 4 Elymus kengii Tzvelev STY People' s w C] Republic P of China & Psathyrostachys tauri a (Boiss. & Bal.) A. Love StP Iran Genome Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 03/28/17 For personal use only.

Table 1 (concluded). H-genome bandsa Barley bandsb Ns-genome bandsc

Elymus abolinii (Drobov) Tzvelev StY PI 3 14617 Former U.S.S.R. 67 - ns (+) ns ns + ns ns ns - ns - Elymus barbicallus (Ohwi) S. L. Chen StY PI 499380 People's Republic of China 68 - ns - ns ns + ns ns ns - ns ns Elymus caucasicus (C. Koch) Tzvelev StY PI 531572 Armenia 69 - ns (+) + ns + ns - - - - - Elymus ciliaris (Trin.) Tzvelev StY PI 531575 People's Republic of China 70 - - (+) + (+) + ------Elymus fedtschenkoi Tzvelev StY PI 531607 Pakistan 7 1 - ns (+) ns ns + ns ns ns - ns ns Elymus gmelinii (Ledeb.) Tzvelev StY PI 499607 People's Republic of China 72 - ns (+) ns ns + ns ns ns - ns ns Elymus gmelinii ssp. ugamicus (Drobov) A. Love StY PI 3 146 18 Former U.S.S.R. 73 - - - (+> (+> + ------Elymus longearistatus (Boiss.) Tzvelev StY PI 40 1277 Iran 74 - ns (+) ns ns + ns ns ns - ns ns Elymus nipponicus Jaaska StY PI 275776 Japan 75 - ns - ns ns + ns ns ns - ns ns Elymus pendulinus (Nevski) Tzvelev StY PI547309 Former U.S.S.R. 76 - ns - ns ns + ns ns ns - ns ns Elymus praeruptus Tzvelev StY PI 531651 77 - ns (+) ns ns + ns ns ns - ns ns Elymus semicostatus (Nees ex Steud.) Melderis ssp. foliosus (Keng) A. Love StY PI 531664 People's Republic of China 7 8 - ns - ns ns + ns ns ns - ns ns Elymus semicostatus ssp. striatus (Nees ex Steud.) A. Love StY PI 207453 Afghanistan 79 - ns + ns ns + ns ns ns - ns ns

Note: +, present; ++, present at relatively higher intensity; (+), present as a faint band; -, absent; ns, not scored. ' pHch2 (HaeIII): H genome differential intensity: the intensity of the hybridization signal in species with the H genome is at least one order of magnitude higher than in those without the H genome 3 (Fig. 1); pHch93 (HaeIII), pHchl.3 (HaeIII), and AKM60A9 (TaqI): restriction fragments are present in all H genome diploid species and absent or faint in other diploid Triticeae. 0 0 bp~l~C~2(TaqI) and pHvUCD32 (TaqI): restriction fragments present in all Hordeurn (H, I, X",and Xugenomes) diploid species and absent or faint in other diploid Triticeae; pHvUCD16a (HaeIII) 5, (Fig. 2): differential hybridization intensity in all Hordeurn (H, I, X",and Xugenomes) diploid species, faint hybridization signals in Agropyron and Pseudoroegneria species, and absent in other diploid -2 Triticeae analyzed 'pPjUCD5 (TaqI) (Fig. 3): Ns genome tandem repeat (165-bp ladder); pPjUCD6 (HaeIII) (Fig. 3): Ns genome marker bands: one restriction fragment not included in the table is present only in Lqvrnus 5 species, E. erianthus. and E. rnendocinus; pPjUCDll (HaeIII): four Ns genome marker bands: one restriction fragment not included in the table is present only in both accessions of P. juncea; pLmUCDl g (TaqI) (Fig. 3): Nbenome marker band; pTa71 (EcoRI) (Fig. 4): additional EcoRI restriction site in the intergenic spacer of the rDNA repeat unit. 2 s CD fk .J Dubcovsky et al. For personal use only. Genome Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 03/28/17

01997 NRC Canada Genome, Vol. 40, 1997

plus 0.5% SDS for 30 rnin, at 65OC. Inserted DNA fragments were profiles of all South American tetraploid Elymus species and amplified using PCR with M 131pUC sequencing primer (-20) 17-mer in the hexaploid species E. patagonicus and E. scabriglumis. and M131pUC reverse sequencing primer (-48) 24-mer (New These H genome specific restriction fragments had more England Biolabs, Beverly, Mass., U.S.A.). PCR products were puri- intense hybridization signals in the hexaploid species fied with the Magic PCR purification kit (Promega, Madison, Wis., (Table I). U.S.A.). The amount of probe hybridization in each lane of the auto- radiographs was integrated with a Molecular Imager GS-250 (Bio- Rad) and then adjusted with the estimates of DNA loading that were Genome analysis of E. erianthus and E. mendocinus calculated from the measures of ethidium bromide fluorescence in the The H genome specific clone pHch2 showed no hybridization gel. signal when hybridized with E. erianthus and E. mendocinus (Figs. IA and lB, Nos. 33 and 35). Restriction fragments Results absent in the hybridization profiles of diploids with the NS genome, but present in the hybridization profiles of all diploid Identification of an H genome specific repeated nucleotide H genome species (clones pHch93, pHchl.3, and AKM60A9) sequence or all diploid barley species (genomes I, Xa, XU,and H; clones Thirty-two clones of repeated nucleotide sequences (RNSs) pTlUCD2 and pHvUCD32), were also absent in the hybridiza- from different Triticeae species were screened for differential tion profiles of E. erianthus and E. mendocinus (Table I). hybridization with the H genome (Table 2). Clone pHch2 These restriction fragments were present in all the S'H-, (Fig. 1) produced a hybridization signal with all H-genome S'HH-, and S'YH-genome Elymus species analyzed (Table 1). Hordeum species that was at least one order of magnitude The hybridization profiles of E. erianthus and E. mendocinus greater than those observed for other Hordeum species (I, Xa, differed not only from the Elymus species with the H genome and XU genomes) or for the species of Pseudoroegneria (s'H, S'HH, and S'YH) but also from the Elymus species with (St genome), Agropyron (P genome), Australopy rum (W the S'Y genome (Fig. 2). Clone pHvUCD16 from Hordeum genome), Thinopyrum (E~genome), or Psathyrostachys (NS vulgare revealed a strong hybridization signal in the hybrid- genome) (Table 1). The hybridization signal of pHch2 with ization profiles of all the diploid Hordeum species and also in Lophopyrum elongatum (Ee genome) was only threefold lower the hybridization profiles of the S'H- and S'Y-genome Elymus than that detected in the H-genome species. species analyzed (Fig. 2). This clone showed a relatively The amount of probe hybridization in each lane in Fig. 1A lower hybridization signal with the St- and P-genome diploids was integrated with a Molecular Imager Analyzer (Fig. 1B) but no signal at all with E. erianthus, E. mendocinus (Fig. 2, and adjusted by measures of DNA content that were estimated Nos. 33 and 33, Psathyrostachys juncea, and the three Ley- by ethidium bromide fluorescence in the gel. In Fig. IB, mus species analyzed (Table 1, Nos. 28-32). Clone pLeUCDl corrected values are displayed as a histogram imposed over the from L. elongatum showed a very strong hybridization signal curve. Hybridization signal was at least one order of magni- with DNAs from Lophopyrum (Ee genome) and Thinopyrum tude greater in Elymus species with genome formulas (E~genome) (data not shown) and a lower hybridization

For personal use only. (Lu 1993) that included the H genome (S'H, S'HH, and S'HY) signal with DNAs from Pseudoroegneria (St genome), than in those without it (s'Y, S'S'Y, and S'PY) (Fig. 1A). The Agropyron (P genome), and all the Elymus species analyzed only exception was accession PI 276712 from the former (Fig. 2). The hybridization signal of clone pLeUCDl with U.S.S.R. that was identified as Elymus trachycaulus ssp. DNAs from E. erianthus, E. mendocinus (Fig. 2, Nos. 33 and violaceurn. Though this accession, PI 276712, had the 33, P. juncea, and Leymus (data not shown) was either zero expected chromosome number (2n = 28), the closely imbricate (Fig. 2) or one order of magnitude lower than that observed small spikelets and relatively large anthers (2.5 mm) indicated with DNAs from the Elymus species when autoradiographs that it had been misclassified and it is likely a tetraploid were overexposed (data not shown). Agropyron species. Inclusion of this accession in Agropyron RNSs cloned from Psathyrostachys or Leymus that hybrid- was also supported by the similar hybridization patterns ized with NS genome specific bands (marker bands) (Zhang found for the tetraploid accession PI 276712 and the dip- and Dvofiik 1991) revealed a complementary hybridization loid Agropyron cristatum, when hybridized with clones pattern to that previously described for the Hordeum genome pPjUCD 11, pHch2 (Fig. 1, Nos. 52 and 24), and pLeUCD 1 specific marker bands (Fig. 3). Hybridization with clone Genome Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 03/28/17 (data not shown). The two other E. trachycaulus subspecies pPjUCD5 (Fig. 3A) revealed a ladder pattern typical of a tan- included in this study (E. trachycaulus ssp. trachycaulus and dem repeated sequence family with a monomeric unit of E. trachycaulus ssp. subsecundus) showed the hybridization 165 bp. This characteristic ladder was observed only in the intensity expected for Elymus species with S'H genomes hybridization profiles of P. juncea, Leymus, E. erianthus, and (Fig. 1, Nos. 50 and 5 1). E. mendocinus (Fig. 3A, Nos. 28-35). Almost no hybridiza- A highly intense hybridization signal was detected for all tion signal was detected in Elymus (S'PY-, S'YH-, Sty-, or South American tetraploids analyzed (Table I), suggesting the S'H-genome combinations) or in the other diploid Triticeae presence of an H genome in all these species. The hybridiza- analyzed (Fig. 3A; Table I). Clones pPjUCD6 and pLmUCD1 tion signal found in hexaploid species E. patagonicus and did not show ladder patterns, suggesting interspersion of the E. scabriglumis was more intense than those found in most copies of the repeated nucleotide family. Both clones revealed tetraploid species and probably resulted from the double dose marker bands for the NS genome that were present in of H genome in these species (Fig. lB, Nos. 53 and 54). the hybridization profiles of Leymus, E. erianthus, and Restriction fragments present in the hybridization profiles of E. mendocinus but absent from Elymus or other diploid all diploid H genome species (clones pHch93, pHchl.3, and Triticeae analyzed (Figs. 3B and 3C; Table 1). Clone AKM60A9; Table 1) were also present in the hybridization pPjUCD6 revealed bands that were present in all three

01997 NRC Canada Dubcovsky et al

472 ell > P% -CD Z 3 b"' 5 LC)> 00 h a b * WE% s 2 2 "a KG ,ab Z* -WZCD * a* bZ Y0 0 a Z -a> (D " -b rn (D 'n :3 (DP ~a (Dm -02 -bZ bf" b * 3 5 gE -lnxln t -X% - a .s (D $" -00 -00-5; 5 - b *0 ell lnz a .9 a%cv * - 8 -5 -b = z 2 m In 5; 8 2 c7 -5 c9 h: -a= -cob LC) % 64 m= 3 z -5 -3; -a*CD 2 *- 5 -2 b x In > rn -3 3 wk -3% -cog 2 .s *ou z5 ,CV E -In E a.3 4 In a g .; CV = -co 7 - -x a *'n *w - ->s w * Gk $5 a6 32 Lij -m " -% ma i hi= In ,COX $22 2 ma -* 3 w'n z 2 .- -CDF 232 %% 4% c7 * a a c gz .E 7 - -2% -S& * aZL In ';< -b k -CV - $2" * rnLij 33 .f -ax -mk .3 0 * 225 fi -% x ,CVI7 aa rn g+s dmN-0 mcvo 2 ki,E Y22Z n 2 .s m aa8 aleas AJ!SU~JU! ~~JJ!~JV a 5 - 3gg 3aW acg For personal use only. C 0 A $5 ha- & g 5 a;r ello* corn .s 2 .z oco B2:0 C pj4A c, .s % a %.5 .s 5z 5 .3 3g.2 rncm 2 .s 2 Genome Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 03/28/17 2 ij .g 9 .c 5 m ",% 245ellc Y T3 L.Z g 2 .; 2zm cq -;F, g N Q)a '" C 2%P g. =ehC .s g sg,:h a k ri 5 P Oh-g GSz - 01997 NRC Canada 514 Genome, Vol. 40, 1997

Fig. 2. Hybridization of HaeIII-digested DNAs with RNSs pHvUCD16 (A) and pLeUCD1 (B). DNA loading per lane was adjusted by ploidy level (diploids, 2 p,g; tetraploids, 4 p,g; hexaploids, 6 p,g; octoploid, 8 p,g). Numbers correspond to accession identification as given in Table 1 and capital letters to genome formulas (eri, E. erianthus; men, E. mendocinus). Molecular marker size (at the left) is given in basepairs.

S'H S'H S'H Sh S'H S'H S'HH S'HH en' men I X' Xu H S' P Sv S'Y S'H S'H For personal use only.

Leymus species, E. erianthus, and E. mendocinus (Fig. 3B, detected by hybridization with clones pHvUCD32 and Nos. 30-35) but absent in P. juncea (Fig. 3B, Nos. 28 and 29) pHch93 (data not shown). and all other species analyzed. These bands were also present Genome Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 03/28/17 in (= giganteus) (Lam.) Tzvelev (D2949) Restriction site variation in the rDNA repeat unit and absent in Psathyrostachys fragilis (Boiss.) Nevski Restriction maps of the rDNA repeat unit were constructed (PI 401 397) (H.-B. Zhang, personal communication). using restriction enzymes EcoRI, BamHI, and SstI (single- In spite of the similar hybridization patterns in E. erianthus enzyme digests), and EcoRI-SstI and EcoRI-BamHI (double and E. mendocinus revealed by many NS genome specific digests) (Fig. 4). All accessions of Psathyrostachys, Leymus, clones (Fig. 3, Nos. 33 and 33, numerous differences were and E. erianthus analyzed exhibit a second EcoRI site located observed when DNAs from these two species were hybridized in the IGS at 2.7, 2.55, and 2.4 kb from the 18s SstI site, with clones pTa250.15, pTlUCD2, pHch 1.3, pHch950, respectively (Fig. 4). No additional EcoRI site was found in pHVUCD4 1, AKM60A4, pPjUCD4, and pPjUCD 13, E. mendocinus, other diploid Triticeae species, or any of indicating a large differentiation between some of the the Elymus species analyzed, with the exception of Elymus genomes of these two polyploid species. Differences among cordilleranus. However, in the last species the additional the Leymus accessions analyzed were detected with clones EcoRI restriction site is approximately 600 bp closer to the pLmUCD1, pPjUCD4, pPjUCD8, and pTa71. Restriction 18s SstI site (Fig. 4). fragments present in all three Leymus species analyzed but Additional SstI restriction sites were found in Australo- absent in E. erianthus, E. mendocinus, and P. juncea were pyrum retrofractum and Leymus alaicus ssp. karataviensis,

01997 NRC Canada Dubcovsky et al. 515

Fig. 3. (A) TaqI-digested DNAs hybridized with clone pPjUCD5. (B) HaeIII-digested DNAs hybridized with clone pPjUCD6.(C) TaqI- digested DNAs hybridized with clone pLmUCD1. DNA loading per lane was adjusted by ploidy level (diploids, 2 pg; tetraploids, 4 pg; hexaploids, 6 pg; octoploid, 8 pg), but accessions 30 and 63 were overloaded in A and C. Numbers correspond to accession identification as given in Table 1 and capital letters to genome formulas (eri, E. erianthus; men, E. mendocinus). Molecular marker size (at the left) is given in basepairs.

1 3 22 26 27 23 24 25 29 28 30 31 32 33 35 63 57 69 38 I X H E' Eb St P W N' N' N'X" N'Xm N'Xn eri men SPY SYH SY SOI For personal use only. Genome Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 03/28/17

but in different regions of the repetitive unit (Fig. 4). An addi- At the interspecific level, divergence and amplification or tional IGS BamHI site was found in P. juncea (Fig. 4). deletions of sequences eventually result in an emergence of RNS families that can be detected by hybridization in only a Discussion single species or a group of closely related species (Prosnyak et al. 1985; Rayburn and Gill 1986; DvoEAk et al. 1988; Zhao Genome specific RNSs et al. 1989; Hueros et al. 1990; Zhang and DvoEAk 1990; At the level of entire families, variation in the RNSs assayed Crowhurst and Gardner 1991; Iwabuchi et al. 1991; Raz et al. by Southern blot hybridization is remarkably low within spe- 199 1; Talbert et al. 199 1; Baldauf et al. 1992; Zentgraf et al. cies and is a powerful tool for scrutinizing the origin of 1992; Talbert et al. 1993). Since numerous species can be ana- allopolyploid taxa (DvofAk and Dubcovsky 1996). Homoge- lyzed simultaneously in the same Southern blot (Fig. l), these neity within species is maintained through homogenization genome specific RNS families can be very useful for screen- and recombination of RNS families (Dover 1982; Strachan et ing large germplasm collections. Elymus species with genome al. 1985). formulas not including the H genome are not always easy to

01997 NRC Canada 516 Genome, Vol. 40, 1997

Fig. 4. Restriction map of the repeated unit of the rDNA. Restriction sites indicated with black symbols are constant and those indicated with white symbols are variable within the set of species analyzed. Numbers after the species correspond to accession identification as given in Table 1.

A Sstl 1 Psathyrostachys juncea (28,29) 2 Leymus (30,3 1, 32) 0 5 kb 7 ? BamHl 43 Elymus conlilleranuserianthus (33, (42)34) 5 Australopyrum retrofractum (25) f EcoR l 6 Leymus alaicus ssp. karataviensis (30) 7 Psathyrostachys juncea (29)

differentiate morphologically from Elymus species with the H pHch2 (Fig. 1, Nos. 53 and 54). Stronger hybridization inten- genome (Salomon and Lu 1992). The H genome specific RNS sity was also found in the H genome specific marker bands. clone pHch2 is a new tool for differentiating these groups. These results confirm the S'HH-genome formula previously Hybridization with clone pHch2 detected the Agropyron assigned to these species by studies of chromosome pairing in accession PI 276712 that had been erroneously labeled at the interspecific hybrids (Hunziker 1953, 1966; Dewey 1972c; germplasm collection as E. trachycaulus ssp. violaceus. Seberg 1991) and variation in the BamHI sites of the rDNA (Dubcovsky et al. 1992). South American Elymus Restriction fragments characteristic of the H-genome All South American tetraploid Elymus species analyzed using Hordeum species from the New World but absent in the three pHch2 showed a strong hybridization signal indicating the diploid species from the Old World (Hordeum bogdanii, presence of an H genome. This result is in accordance Hordeum roshevitzii, and Hordeum brevisubulatum) were with those previously obtained by chromosome pairing in detected by hybridization with clones pHch3 (HaeIII),

For personal use only. interspecific hybrids, including Elymus agropyroides, Elymus pHch950 (HaeIII), and pHchl.3 (AluI, but different from tilcarensis, and E. magellanicus (Dewey 1970b, 1977; Jensen pHchl.3 HaeIII; Table 1). Differentiation of the New World 1993). Particularly interesting is the demonstration that H genome Hordeum species from the Old World diploid spe- E. breviaristatus ssp. scabrifolius has an H genome, as this cies was also indicated by meiotic analysis of interspecific species shows very low chromosome pairing in interspecific hybrids within and between these regions (Bothmer et al. hybrids with other South American tetraploid species 1991). These restriction fragments specific to the American H (Hunziker and Ferrari 1986). genome species were also found in all the South American RNSs that hybridize differentially to single genomes, such Elymus species with the H genome, with the exception of as pHch2, are not easy to isolate. Fortunately, phylogenetic E. tilcarensis. These restriction fragments were not detected in information can also be obtained from the interspecific varia- the few Elymus species with S'H or s'HY genomes from tion in the hybridization patterns of RNSs that readily cross- Europe, Asia, or North America that were analyzed. It is hybridize among different species. Restriction fragments in tempting to speculate that the presence of American H the Southern hybridization profiles that are unique for a dip- genome specific bands in a large group of South American tet-

Genome Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 03/28/17 loid species or group of species, designated marker bands, can raploid Elymus species indicates an independent origin for this also be used to investigate the origin of polyploid taxa (Talbert group. However, at present there is insufficient data to con- et al. 199 1; Zhang and Dvo2ik 199 1,1992; Zhang et al. 1992; firm the polyphyletic origin of Elymus. Dvofhk et al. 1993; Dubcovsky and DvoZik 1994, 1995; DvoFhk and Dubcovsky 1996). South American Leymus The H genome in Hordeum represents a phylogenetic lin- Hexaploid E. erianthus and octoploid E. mendocinus differ eage differentiated from the I, Xa, and XUgenomes and, con- from all other Elymus species in the hybridization patterns sequently, shared marker bands among H-genome Hordeum detected with clone pHch2. The RNS family detected with species were readily found in this study and by other authors pHch2 is strongly amplified in the H genome but seems to be (Bothmer et al. 1986; Svitashev et al. 1994; Vershinin et al. absent, or present in a very low copy number, in E. erianthus 1994). The presence of these marker bands in South American and E. mendocinus. Restriction fragments present in all barley tetraploid Elymus species further confirmed the presence of species or in all H-genome Hordeum species are also present the H genome in these species. Hexaploids E. patagonicus and in Elymus species with S'H, S'HH, or s'HY genomes, but are E. scabriglumis generally showed stronger hybridization absent in E. erianthus and E. mendocinus. These results, intensity than S'H tetraploids when hybridized with clone together with failure of the chromosome N-banding technique

01997 NRC Canada Dubcovsky et al.

to produce bands in E. erianthus (Seberg and Linde-Laursen (1984) suggested, on morphological grounds, the presence of 1996) and the lack of hybridization with a GAA satellite abun- an Eb genome in tetraploid Leymus species. However, recent dant in the H genome (Pedersen et al. 1996), confirm the studies based on RNSs have shown the absence of the absence of the H genome in E. erianthus. Eb genome from this genus (Zhang and Dvoiiik 199 1; Wang Differences between E. erianthus and the other South and Jensen 1994). Based on the hybridization intensity of American Elymus species were also suggested by their partic- Ns-genome marker bands, Zhang and Dvoiik (1991) sug- ular morphological characteristics (Nicora 1978; Seberg and gested that the second pair of Leymus genomes was also from Linde-Laursen 1996), unique restriction sites in the rDNA Psathyrostachys, and designated their genomes NSlNSlNS2NS2. repeat unit (Dubcovsky et al. 1992), and sequence variation in Other authors preferred to designate Leymus genomes NSXm, the chloroplast gene rbcL (Seberg and Linde-Laursen 1996). based on meiotic analysis of triploid hybrids (Wang and Both E. erianthus and E. mendocinus also differ from the Jensen 1994; Sun et al. 1995; Wang et al. 1994). South American Elymus species in their karyotype parameters A close relationship between E. erianthus and E. mendocinus (Lewis et al. 1996). Values of interchromosomal asymmetry with Leymus was further indicated by the presence of marker in the karyotype of E. erianthus and E. mendocinus were sig- bands in all three Leymus species, E. erianthus, and E. mendo- nificantly higher than in the karyotypes of other South Amer- cinus that were absent in P. juncea and all other species ican Elymus species. This suggests relatively large differences analyzed. Heterogeneity within Leymus species in the ob- in chromosome size between the genomes of the progenitors served restriction patterns was evident even in the few species of these two species. Although all these differences clearly analyzed here. Numerous differences were also observed segregate E. erianthus and E. mendocinus from the S'H- between E. erianthus and E. mendocinus, suggesting a large and S'~~-~enomeSouth American Elymus species, they genome differentiation within Leymus species. do not rule out a relationship between E. erianthus and Seberg and Linde-Laursen (1996) pointed out that the satel- E. mendocinus and the Asiatic Elymus species that do not have lite (SAT) chromosome types of E. erianthus are similar to an H genome (S'PY, S'S'Y, or s'Y). Hybridization with clone those present in species of the genus Psathyrostachys pLeUCD 1 differentiates E. erianthus and E. mendocinus from and Leymus. However, they found differences between the S'PY-, S'S'Y-, or Sty-genome Asiatic Elymus species and E. erianthus and Psathyrostachys in the distribution of the suggests the absence of the St genome from these two South C-bands. Results from the same authors on sequence variation American polyploids. in the chloroplast gene rbcL did not exclude the possibility Hybridization with RNSs specific for the NS genome that E. erianthus could be related to Leymus (Seberg and of Psathyrostachys also differentiates E. erianthus and Linde-Laursen 1996). E. mendocinus from Asiatic Elymus species with the S'PY, S'S'Y, or S'Y genomes. All the RNSs that hybridized with NS Taxonomic treatment genome specific restriction fragments (Zhang and Dvoiiik Leymus is a segregate genus of the Triticeae that can be recog- 1991) also hybridized with E. erianthus and E. mendocinus, nized morphologically and genomically and is now accepted

For personal use only. but showed no hybridization signal with any of the other by most European, Russian, Chinese, and American taxono- Elymus species analyzed. An NS genome in E. erianthus was mists (Pilger 1954; Keng 1965; Tzvelev 1976; Melderis 1980 ; also suggested by the presence of a second EcoRI restriction Barkworth and Atkins 1984; Dewey 1984; Love 1984). site in the IGS of all repeating units of the rDNA genes from Elymus erianthus and E. mendocinus share with Leymus not E. erianthus, P. juncea, and the three Leymus species ana- only similar genomes, as revealed by hybridization patterns lyzed. Orgaard and Heslop-Harrison (1 994) have also reported with RNSs, but also ecological and morphological character- the presence of a second EcoRI site in the IGS of all the istics. Both South American species are found in dry habitats repeated units in P. stoloniformis. This additional site is absent (stony slopes and dry steppes) similar to those inhabited by in E. mendocinus and in some of the repeated units of the many Northern Hemisphere Leymus species (Barkworth and dodecaploid (Trin.) Pilg. (Orgaard and Atkins 1984; Dewey 1984). Elymus erianthus and E. mendoci- Heslop-Harrison 1994). Almost all other Triticeae species nus share with Leymus species their stiff and glaucous analyzed in this study and by Orgaard and Heslop Harrison blades, narrow 1-3 nerved glumes, shortly awned lemmas, (1994) have a single EcoRI restriction site in the 26s rDNA. and long anthers (E. erianthus: 3-4.5 mm; E. mendocinus:

Genome Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 03/28/17 The only exception is E. cordilleranus (= Elymus attenuatus), 5-6 mm) (Parodi 1940; Barkworth and Atkins 1984; which has an IGS EcoRI site but at a shorter distance from the Barkworth and Dewey 1985; Clayton and Renvoize 1986). 18s SstI restriction site (Fig. 4). Both South American species differ from most species of The absence of the H and St genomes and the presence of Leymus in that they lack long rhizomes. However, long the NS genome in E. erianthus and E. mendocinus clearly indi- rhizomes are also absent in North American Leymus species cate that these two species do not belong to the genus Elymus that grow in rocky habitats, for example, Leymus am- and suggest their inclusion in the genus Leymus. The presence biguus (Scrib. & Merr.) D.R. Dewey and Leymus salinus of an NS genome in polyploid species of Leymus has been (M.E. Jones) A. Love (Hitchcock 195 1; Barkworth and Atkins repeatedly demonstrated by meiotic analysis in interspecific 1984). hybrids (Dewey 1970a, 1972a, 1972b, 1976; Wang and Hsiao Similar ecological and morphological characteristics com- 1984; Sun et al. 1995) and by variation in RNSs (Zhang and bined with similar hybridization patterns with RNSs in Dvoiiik 1991; Wang and Jensen 1994; Mason-Gamer and Psathyrostachys, Leymus, and E. erianthus suggest that the Kellog 1996). Though the presence of an NS genome in creation of the new monotypic genus Eremium (Seberg and Leymus is well established, the identification of the other Linde-Laursen 1996) is not justified. Therefore, we transfer genome in tetraploid Leymus is a matter of controversy. Love E. erianthus and E. mendocinus to the genus Leymus.

01997 NRC Canada Genome, Vol. 40, 1997

Leymus erianthus (Phil.) Dubcovsky comb.nov. Crowhurst, R.N., and Gardner, R.C. 1991. A genome-specific repeat Basionym: Elymus erianthus Phil., An. Mus. Nac. Santiago sequence from kiwifruit (Actinida deliciosa var. deliciosa). Chile. 2 Secc. Bot. 13. 1892. TYPE: Argentina, Mendoza, Las Theor. Appl. Genet. 81: 71-78. Heras, in andibus uspallatensis ad thermas Baiios del Inca, Dewey, D.R. 1970a. Genome relations among diploid Elymus invenit 1866, A. Borchers (clastotypus, BAA!). junceus and certain tetraploid and octoploid Elymus species. Am. J. Bot. 57: 633-639. Elymus barbatus Kurtz, Bol. Acad. Nac. Cienc. (Argent.), 15: Dewey, D.R. 1970b. Hybrids of South American Elymus 506. 1894. Nomen nudum. agropyroides with Agropyron aespitosum, Agropyron Cryptochloris spathacea auct. non Benth. 1882; Speg., Rev. subsecundum, and Sitanion Hystrix. Bot. Gaz. 131: 210- Fac. Agron. Vet. La Plata, 3: 584. 1897. 216. Elymus spegazzinii Kurtz, Bol. Acad. Nac. Cienc. (Argent.), Dewey, D.R. 1972a. Cytogenetic and genomic relationships of 16: 259.1899. Nomen nudum. Elymus giganteus with E. dasystachys and E. junceus. Bull. Elymus erianthus Phil. var. aristatus Hicken, Physis (Buenos Torrey Bot. Club, 99: 77-83. Aires), 2: 41. 1915. Dewey, D.R. 1972b. Genome analysis of hybrids between diploid Elymus erianthus Phil. var. spegazzinii Kurtz ex Hauman, An. Elymus junceus and five tetraploid Elymus species. Bot. Gaz. Mus. Nac. Hist. Nat. Buenos Aires, 29: 410. 1917. 133: 4 15420. Dewey, D.R. 1972c. Genome analysis of South American Elymus Eremium erianthus (Phil.) Seberg and Linde-Laursen, Syst. patagonicus and its hybrids with two North American and two Bot. 21: 1996 Asian Agropyron species. Bot. Gaz. 133: 436-443. Dewey, D.R. 1976. The genome constitution and phylogeny of Leymus mendocinus (Parodi) Dubcovsky comb.nov. Elymus ambiguus. Am. J. Bot. 63: 626-634. Basionym: Agropyron mendocinus Parodi, Rev. Mus. La Plata Dewey, D.R. 1977. Genome analysis of South American Agropyron Secc. Bot. 3:14. 1940. TYPE: Argentina, Mendoza, San tilcarense with American and Asian Agropyron species. Bot. Rafael, Pampa del Plateado, Nevado hill, altitude 2500 m., Gaz. 138: 369-375. T. et B. Macola, no P.8, 17/1/1926 (isotype SI!) Dewey, D.R. 1984. The genomic system of classification as a guide Elymus mendocinus (Parodi) A. Love, Feddes Repert. to intergeneric hybridization with the perennial Triticeae. Stadler 95(7-8): 47 1.1984. Genet. Symp. 16: 209-279. Elytrigia mendocina (Parodi) Covas ex J. H. Hunz. & Xifreda, Dover, G. 1982. Molecular drive: a cohesive mode of species evolu- Darwiniana, 27(14): 562. 1986 tion. Nature (London), 299: 11 1-1 17. Dubcovsky, J., and Dvoi&, J. 1994. Genome origin of Triticum Acknowledgements cylindricum, Triticum triunciale, and Triticum ventricosum () inferred from variation in repeated nucleotide The authors are grateful to Dr. Richard C. Johnson, Harold E. sequences: a methodological study. Am. J. Bot. 81: 1327- Bockelman, and Dr. Roland von Bothmer for the seeds; to Dr. 1335. Andrzej Kilian, Dr. Jan Dvoiak, Dr. Hong-Bin Zhang, and Dr. Dubcovsky, J., and Dvoiiik, J. 1995. Genome identification of the Triticum crassum complex (Poaceae) with the restriction patterns Esther Ferrer for the RNS clones; to Renee Fortunato, Zulma of repeated nucleotide sequences. Am. J. Bot. 82: 131-140. For personal use only. Rdgolo, and Elisa Nicora for their advice on the taxonomic Dubcovsky, J., Soria, M.A., and Martinez, A.J. 1989. Karyotype treatment; to Dr. Ole Seberg for sharing his unpublished analysis of the Patagonian Elymus. Bot. Gaz. 150: 462468. results; and to Dr. Esteban Hopp and Ing. E.Y. Suarez for their Dubcovsky, J., Lewis, S.M., and Hopp, E.H. 1992. Variation in the support. restriction fragments of 18s-26s rRNA loci in South American Elymus (Triticeae). Genome, 35: 88 1-885. References Dvoi&, J., and Dubcovsky, J. 1996. Genome analysis of polyploid species employing variation in repeated nucleotide sequences. Appels, R., and Dvoiik, J. 1982. The wheat ribosomal DNA spacer: Methods of genome analysis in plants. CRC Press, New York. its structure and variation in populations and among species. pp. 133-145. Theor. Appl. Genet. 63: 337-348. Dvoiik, J., McGuire, P.E., and Cassidy, B. 1988. Apparent sources Baldauf, F., Schubert, V., and Metzlaff, M. 1992. Repeated DNA of the A genomes of wheats inferred from polymorphism in sequences of Aegilops markgrafii (Gruter) Hammer var. abundance and restriction fragment length of repeated nucleotide markgrafii - cloning, sequencing and analysis of distribution in sequences. Genome, 30: 680-689. Poaceae species. Hereditas, 116: 71-78. Dvoiik, J., di Terlizzi, P., Zhang, H.B., and Resta, P. 1993. The evo- Genome Downloaded from www.nrcresearchpress.com by Calif Dig Lib - Davis on 03/28/17 Barkworth, M.E., and Atkins, R.J. 1984. Leymus Hochst. lution of polyploid wheats: identification of the A genome donor (Gramineae: Triticeae) in North America: and distribu- species. Genome, 36: 2 1-3 1. tion. Am. J. Bot. 71: 609-625. Feinberg, A.P., and Vogelstein, P. 1983. A technique for radiolabel- Barkworth, M.E., and Dewey, D.R. 1985. Genomically based genera ing DNA restriction endonuclease fragments to high specific in the perennial Triticeae of North America: identification and activity. Anal. Biochem. 132: 6-13. membership. Am. J. Bot. 72: 767-776. Gerlach, W.L., and Bedbrook, J.R. 1979. Cloning and characteriza- Bothmer, R., von, Flink, J., and Landstrom, T. 1986. Meiosis in tion of ribosomal RNA genes from wheat and barley. Nucleic interspecific Hordeum hybrids. I. Diploid combinations. Can. J. Acids Res. 7: 1869-1885. Genet. Cytol. 28: 525-535. Gupta, P.K., Balyan, H.S., and Fedak, G. 1988. A study on genomic Bothmer, R., von, N, J., Baden, C., Jorgensen, R., and Linde- relationships of Agropyron trachycaulum with Elymus Laursen, I. 1991. An ecogeographical study of the genus scabriglumis, E. innovatus, and Hordeum procerum. Genome, Hordeum. International Board for Plant Genetic Resources (Food 30: 525-528. and Agriculture Organization), Rome, Italy. Hitchcock, A.S. 1951. Tribe 3. Hordeae. Manual of the grasses of Clayton, W.D., and Renvoize, S.A. 1986. Genera graminum. the United States. U.S. Dep. Agric. Misc. Publ. No. 200. 2nd ed. Grasses of the world. Royal Botanic Gardens, Kew, United States Government Printing Office, , D.C. London. pp. 230-280.

01997 NRC Canada Dubcovsky et al.

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