Molecular diversity of the 5S rDNA units in the Elymus dahuricus complex (Poaceae: Triticeae)...

Molecular diversity of the 5S rDNA units in theElymus dahuricus complex (Poaceae: Triticeae)supports the genomic constitution of St, Y, andH haplomes

Bernard R. Baum, L. Grant Bailey, Douglas A. Johnson, and Alex V. Agafonov

Abstract: The phylogenetic analysis of 118 5S rRNA gene sequences cloned from members of the Elymus dahuricuscomplex containing the St, Y, and H haplomes, and of several related species containing at least one of these threehaplomes, is reported. Differences in sequence pattern, primarily within the nontranscribed spacer, enabled the identifi-cation of six putative orthologous groups that we refer to as unit classes. In previous publications, we have been ableto assign unit classes to haplomes. In addition to four unit classes previously identified in other genera, namely thelong H1, long S1, long P1, and long {Y1, here we document two new unit classes called the long S2 and long W1.Most sequences of the E. dahuricus complex and related tetraploid species are classified as long S1 and assigned tothe St haplome. Both long S1 and long S2 unit classes were identified in the diploid Pseudoroegneria spicata (Pursh)Á. Löve with the St haplome. The long S2 unit class was also identified in the hexaploid Elymus scabrus (R. Br.) Á.Löve with the St, Y,and W haplomes. The long P1 was known from the diploid Agropyron cristatum Gaertn. with theP haplome, and the long W1 was determined in Australopyrum retrofractum (Vickery) Á. Löve, known to contain theW haplome, but was not yet detected in E. scabrus, a hexaploid species with W being one of the three haplomes. Thelong H1 reported earlier from Hordeum was identified in several clones of the E. dahuricus complex. As previously re-ported, the long {Y1 unit class was found to be rare overall, but we identified it in a few clones of Elymus droboviiand in the E. dahuricus complex.

Key words: 5S rDNA, unit classes, haplomes, concerted evolution.

Résumé : Les auteurs présentent l’analyse phylogénétique des séquences de gènes du 118 5S rARN clonés à partir demembres du complexe Elymus dahuricus contenant les haplomes St, Y et H, ainsi que de nombreuses espèces conte-nant au moins un de ces trois haplomes. Les différences entre les patrons de séquence, surtout parmi l’espaceursnon-transcrits, rendent possible l’identification de six présumés groupes orthologues auxquels les auteurs réfèrentcomme classes d’unités. Dans des publications antécédentes, les auteurs ont pu assigner les classes d’unités aux haplo-mes. En plus des quatre classes d’unités précédemment identifiées dans d’autres genres, nommément le H1 long, le S1long, le P1 long et les longs {Y1, les auteurs documentent deux nouvelles classes d’unités appelées le long S2 et lelong W1. On classifie la plupart des séquences du complexe E. dahuricus, et espèces tétraploïdes apparentées, commeS1 long et sont assignées à l’haplome St. On retrouve également la classe d’unités du S2 long chez l’héxaploïdes Ely-mus scabrus (R. Br.) Á. Löve avec les haplomes St, Y et W. On connaît le long P1 chez le diploïde Agropyron crista-tum Gaertn. avec l’haplome P, et on a retrouvé le long W1 chez l’Australopyrum retrofractum (Vickery) Á. Lövereconnu pour contenir l’haplome W, mais pas encore détecté chez l’E. scabrus, une espèce hexaploïde dont le W estun des trois haplomes. On a retrouvé le long H1, rapporté plus tôt chez l’Hordeum, dans plusieurs clones du complexeE. dahuricus. Comme précédemment mentionné, la classe d’unités des longs {Y1 est en général rare, mais a été iden-tifiée dans quelques clones du E. drobovii et du complexe E. dahuricus.

Mots clés : 5S rADN, classes d’unités, haplomes, évolution concertée.

[Traduit par la Rédaction] Baum et al. 1103

Can. J. Bot. 81: 1091–1103 (2003) doi: 10.1139/B03-102 © 2003 NRC Canada

1091

Received 2 January 2003. Published on the NRC Research Press Web site at http://canjbot.nrc.ca on 27 November 2003.

B.R. Baum1 and L.G. Bailey. Eastern Cereal and Oilseed Research Centre, Agriculture & Agri-Food Canada, 960 Carling Ave.,Neatby Building, Ottawa, ON K1A 0C6, Canada.D.A. Johnson. Ottawa-Carleton Institute of Biology, University of Ottawa, 30 Marie Curie, Ottawa, ON K1N 6N5, Canada.A.V. Agafonov. Central Siberian Botanical Garden, 101 Zolotodolinskaya, 630090 Novosibirsk, Russia.

1Corresponding author (e-mail: [email protected]).

J:\cjb\cjb8111\B03-102.vpNovember 24, 2003 12:00:07 PM

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Introduction

The Elymus dahuricus complex contains eight taxonomicentities, which at various times have been recognized as sep-arate species or as infraspecific taxa. Six species are consid-ered to be part of the complex according to Lu (1993):Elymus dahuricus Turcz. ex Griseb., Elymus excelsus Turcz.ex Griseb., Elymus tangutorum (Nevski) Hand.-Mazz.,Elymus cylindricus (Franch.) Honda, Elymus puraristurC.P. Wang et X.L. Yang, and Elymus villifer C.P. Wang etX.L. Yang. Two additional species, Elymus woroschilowiiProbat. and Elymus franchetii Kitag., are also included in thecomplex by some authors (Agafonov et al. 2001). The spe-cies complex possesses three haplomes St, Y, and H with2n = 6x = 42 chromosomes (Löve 1984) and has an Asiaticdistribution, ranging from Iran to Japan and from southernSiberia to central China. The haplome symbols in this paperfollow Wang et al. (1996), where the term haplome is usedto define the monoploid sets in a genome (Heilbronn andKosswig 1966).

Subtle morphological differences have often formed thebasis for taxon recognition within the complex, resulting indifferent taxonomic treatments of the E. dahuricus complex(e.g., Agafonov et al. 2001). Because of the difficulty inproperly assigning taxa to the genus, Elymus as currently de-fined may contain 150 or more species that have differentand unrelated morphologies or genomes. Thus, Elymus canbe considered as a large “dustbin” entity (Davis and Hey-wood 1963), containing mainly tetraploid species withdifferent genome constitutions, some with the St and Yhaplomes and others with the St and H haplomes. Therehave been several attempts to better define the genusElymus. A decade ago, Baum et al. (1991), based upon mor-phological arguments, removed 127 species characterized bythe St and Y, or St and H haplomes, from Elymus andgrouped them into a genus that they called Roegneria. How-ever, Roegneria as a separate genus remains taxonomicallycontroversial and for an alternative viewpoint see Dewey(1984). Since that time, both cytological (Lu and Salomon1992) and molecular approaches, including Southern hybrid-ization patterns of repetitive DNA (Svitashev et al. 1996),have been used to differentiate between the StH and StYHspecies. Elymus also contains hexaploid species with theStYH, StYP, and StYW genomes. Yang et al. (1992) re-moved species containing the St, Y, and P haplomes fromElymus (including Roegneria) and placed them into the ge-nus Kengyilia C. Yen & J.L.Yang.

In previous publications (e.g., Baum and Bailey 1997,2000, 2001; Baum and Johnson 1994, 1996, 1998, 1999,2000, 2002; Baum et al. 2001), we have described the mo-lecular diversity of the 5S rRNA gene sequences in specieswithin Hordeum, Kengyilia, and Triticum. We were able toclassify the sequences into putative orthologous groups,which we call unit classes, based mainly on sequence vari-ability within the nontranscribed spacer (henceforth NTS),and moreover were able to assign the different unit classesto haplomes. The purpose of the present study was to exam-ine the diversity of the 5S rRNA gene sequences, especiallythe NTS region, in the E. dahuricus complex and to comparethis diversity with species related to the E. dahuricus com-

plex and known to possess at least one haplome in commonwith it, i.e., St, Y, or P. For this reason, we have included inour study sequences from Pseudoroegneria, a genus contain-ing diploid and tetraploid species with the St haplome (De-wey 1984; Löve 1984) and sequences from Agropyron, agenus that contains diploid, tetraploid, and hexaploid specieswith the P haplome (Dewey 1984; Löve 1984). As a point ofcomparison with our previous work, we have also includedexamples from the genus Hordeum, which contains severalhaplomes (von Bothmer et al. 1991; Dewey 1984; Löve1984), with most species within the genus containing the Hhaplome. Furthermore, our objectives were to allocate thesequences to putative orthologous groups, i.e., unit classes,to assign them to haplomes (Baum et al. 2001) and to exam-ine whether the resulting unit classes with their assignmentsto haplomes matches with the known cytological haplomeconstitution.

Materials and methods

Plant material and 5S rDNA cloningSeed material from 26 seed accessions (Table 1), most

collected in natural habitats, was used. In addition to mem-bers of the E. dahuricus complex, representatives of specieswith at least one of the three haplomes, namely St, Y, and H(or a different haplome thought to be related in some fashionto them, such as the W haplome) were included for compar-ative purposes (Table 1). Plants were grown and genomicDNA was isolated and PCR-amplified as described in Baumand Johnson (1994, 1996). The primers used targeted thecoding regions in tandem repeats by amplifying a sequencestarting from a region 5′ of the BamH1 site within the tran-scribed region, through the NTS, to a site 3′ of the BamH1site within the adjacent unit in the array. The PCR productswere run on an agarose gel, and fragments were excised andpurified prior to being cut with BamHI to remove the flank-ing primer sequences and to facilitate cloning. This approachrequires that the coding region retains the BamH1 site neces-sary for cloning of the PCR product and that the target ispart of a tandem repeat. The fragments were then ligatedinto a pUC19 vector (Yanisch-Perron et al. 1985) and ligantstransformed into Eschericia coli DH5α. A total of 118clones were isolated and sequenced to achieve ample sam-pling in the absence of any pre-existing knowledge of lo-cus-specific sequences that would have allowed the designof locus-specific primers (Baum et al. 2001).

Sequencing and sequence analysisSequencing reactions were carried out in the forward and

reverse direction using the T7Sequencing™ kit (Amershamplc, Buckinghamshire, U.K.) with 35S-labelled dATP (Dupont,New England Nuclear, Mass., U.S.A.). Sequencing gelswere run manually, and the autoradiographs were read man-ually in both the forward and the reverse lanes. The nucleo-tide sequences of the different 5S DNA units were submittedto GenBank® (Table 1).

The 118 clones were subsequently aligned using CLUSTALW (Thompson et al. 1994). Included in the aligned matrix withthese 118 clones were sequences from the following clones,which were added as representatives of different, previously

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CollectionID No. Origin Clone

Size(bp)a

EMBL–GenBank–DDBJ acc. No. Unit class

Elymus dahuricus Turcz. ex Griseb. St, Y, and H haplomesBAR-8818b Kirghizstan: Tien-Shan, 20 km South Issik-kul Lake Edah94-01 448 AF550766 Long S1

Edah94-02 489 AF550693 Long H1Edah94-03 438 AF550767 Long S1Edah94-05 448 AF550770 Long S1

CUR-8827b Kirghizstan: Tien-Shan, 5 km S Issik-Kul lake Edah95-02 448 AF550741 Long H1Edah95-03 448 AF550738 Long S1Edah95-05 485 AF550712 Long {Y1

BUD-8704b Kirghizstan: Western Tien-Shan, near town of Talas Edah96-01 446 AF550762 Long S1Edah96-02 489 AF550694 Long S1Edah96-03 447 AF550751 Long S1Edah96-04 448 AF550742 Long S1

CHI-8635b Russia: Chitinskaya oblast, near town of Zabaykalsk Edah97-01 448 AF550755 Long S1Edah97-02 448 AF550743 Long S1Edah97-04 448 AF550750 Long S1Edah97-05 448 AF550771 Long S1Edah97-06 448 AF550760 Long S1

ARS-8706b Russia: Primor’ye, near town of Arsen’ev Edah98-01 448 AF550733 Long S1Edah98-02 448 AF550761 Long S1Edah98-03 448 AF550736 Long S1Edah98-04 448 AF550772 Long S1Edah98-05 447 AF550774 Long S1

POP-8403b Russia: Primor’ye, Popov Island Edah101-01 448 AF550768 Long S1Edah101-04 485 AF550711 Long {Y1Edah101-05 489 AF550695 Long H1

MES-8709b Russia: Primor’ye, near town of Pos’yet Edah102-01 489 AF550692 Long H1Edah102-03 448 AF550739 Long S1Edah102-04 448 AF550731 Long S1Edah102-05 447 AF550753 Long S1Edah102-06 448 AF550758 Long S1

Elymus excelsus Turcz. ex Griseb. St, Y, and H haplomesANI-8625b Russia: Primor’ye, near town of Partizansk Eexc99-01 448 AF550763 Long S1

Eexc99-03 449 AF550737 Long S1Eexc99-04u 448 AF550740 Long S1Eexc99-05 448 AF550756 Long S1Eexc99-06 489 AF550797 Long H1

Elymus tangutorum (Nevski) Hand.-Mazz. St, Y, and H haplomesH-8107b China: province of Sichuan Etan103-01 448 AF550759 Long S1

Etan103-02 448 AF550747 Long S1Etan103-03 448 AF550732 Long S1Etan103-04 448 AF550773 Long S1Etan103-05 448 AF550764 Long S1

H-8113b China: province of Tibet Etan104-01 448 AF550748 Long S1Etan104-02 448 AF550754 Long S1Etan104-03 447 AF550745 Long S1

H-8363b China: province of Tibet Etan105-02 448 AF550749 Long S1Etan105-03 485 AF550714 Long {Y1Etan105-04 237 AF550744 Long S1Etan105-05 447 AF550734 Long S1

Table 1. Assignment of 5S rDNA sequences of the Elymus dahuricus complex and related species to unit classes, including passportdata of seed accessions grown for the present study.



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Size(bp)a


Elymus woroschilowii Probat. St, Y, and H haplomesVLA-8642b Russia: Primor’ye, near city of Vladivostok Ewor100-01 448 AF550752 Long S1

Ewor100-02 448 AF550757 Long S1Ewor100-03 448 AF550765 Long S1Ewor100-04 447 AF550735 Long S1Ewor100-06 448 AF550746 Long S1

Elymus drobovii (Nevski) Tzvel. St, Y, and H haplomesPI314196c Uzbekistan: Chatkal Mountains, 70 km east of Tashkent,

nature reserveEdrob56-10 448 AF550776 Long S1

Edrob56-11 443 AF550775 Long S1Edrob56-26 300 AF550715 Long {Y1Edrob56-28 444 AF550728 Long S1

Elymus caucasicus (C. Koch) Tvzel. [Roegneria caucasica Koch] St and Y haplomesPI531572c Armenia: near Dilijan Ecau53-01 452 AF550792 Long S1

Ecau53-02 452 AF550790 Long S1Ecau53-03 448 AF550796 Long S1Ecau53-06 451 AF550794 Long S1Ecau53-08 456 AF550795 Long S1Ecau53-10 443 AF550797 Long S1Ecau53-12 452 AF550798 Long S1Ecau53-13 448 AF550769 Long S1Ecau53-15 453 AF550787 Long S1Ecau53-16 449 AF550789 Long S1Ecau53-19 451 AF550791 Long S1Ecau53-20 450 AF550788 Long S1Ecau53-21 448 AF550793 Long S1

Elymus pendulinus (Nevski) Tzvel. [Roegneria pendulina Nevski] St and Y haplomesY2400d China: Gansu province, Xiahe, Qiaogou to Qingshui, 8 Km Epend31S02 80 AF550799 Long Y2

Epend31S03 447 AF550778 Long S1Epend31S04 447 AF550777 Long S1Epend31L01 445 AF550783 Long S1Epend31L02 448 AF550779 Long S1Epend31L03 445 AF550784 Long S1Epend31L05 445 AF550785 Long S1Epend31M01 80 AF550800 Long Y2Epend31M02 444 AF550786 Long S1Epend31M03 447 AF550781 Long S1Epend31M04 446 AF550782 Long S1Epend31M05 445 AF550780 Long S1

Elymus caninus (L.) L. [Roegneria canina (L.) Nevski] St and H haplomesY1566d China: Xinjiang province, Fuyun, hydropower dam, in desert Ecan52-02 470 AF550718 Long S1

Ecan52-07 447 AF550719 Long S1Ecan52-08 447 AF550720 Long S1Ecan52-11 488 AF550696 Long H1

Elymus confusus (Roshev.) Tzvel. [Roegneria confusa (Roshev.) Nevski] St and H haplomesJAT-8919b Russia: Jacutia-Sakha, 250 km N from town of Nerungry Econ54-09 448 AF550730 Long S1

Econ54-10 447 AF550722 Long S1Econ54-14 445 AF550721 Long S1Econ112-01 450 AF550723 Long S1Econ112-02 450 AF550724 Long S1Econ112-05 450 AF550726 Long S1Econ112-06 450 AF550725 Long S1Econ112-08 441 AF550727 Long S1

Table 1 (continued).



established unit classes. Long H1: HCAL015 from Hordeumcalifornicum and HROS016 from Hordeum roshevitzii;Long H2: HCOR028 from Hordeum cordobense; Long H3:HROS018 from H. roshevitzii; LongY2: HCOR013 fromH. cordobense and HCAL026 from H. californicum; LongP1: 24L01 from Kengyilia batalinii; Long S1: 38S04 from

K. batalinii; and Long {Y1: 38S02 from K. batalinii(Table 1). The alignment was further improved by visualexamination and editing using GeneDoc© Version 2.6.002(Nicholas and Nicholas 1997). GeneDoc© was used to as-sign similar sequences to sequence groups, i.e., unit classes,based on the refined alignments for each putative ortho-

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Size(bp)a


Elymus batalinii (Krassn.) A. Löve [Kengyilia batalinii (Krassn.) J.L. Yang, C. Yen & B.R. Baum] St, Y, and P haplomesPI314623c Kazakhstan: west of Game Preserve headquarters, about

180 km east of Almati24L01 481 AF550705 same as

AF248219eLong P1

PI547361c Kyrgyzstan: Central Tian Shan Mountains 38S02 489 AF550713 same asAF248227

Long {Y1

38S04 448 AF550729 same asAF248228e

Long S1

Agropyron cristatum Gaertn. P haplome‘Fairway’g U.S.A.: Canberra seed collection cultivar ‘Fairway’ Acr_ac10 484 AF550729 Long P1

Acr_ac19 483 AF550703 Long P1Acr_ac20 479 AF550706 Long P1Acr_ac21 480 AF550707 Long P1AcrAC5SDNAB 483 Z11476e Long P1

Elymus scabrus (R. Br.) A. Löve St, Y, and W haplome107972h U.S.A.: Canberra seed collection EscES25S1 483 AF550699 Long S2

EscEST115S 483 AF550702 Long S2EscES35S 485 AF550700 Long S2EscES55S 485 AF550701 Long S2

Pseudoroegneria spicata (Pursh) A. Löve St haplome‘Whitmar’h U.S.A.: Canberra seed collection cultivar ‘Whitmar’ PsZ11438.1 447 Z11438e Long S1

PsZ11439.1 484 Z11439e Long S2Pst11 483 AF550698 Long S2Pst20 446 AF550717 Long S1Pst35 446 AF550716 Long S1

Hordeum cordobense Bothmer, N. Jacobsen & Nicora H “general” haplomeH1702f Argentina: Cordoba province, Cordoba to Jesus Maria HCOR028 488 AY034736e Long H2

HCOR013 455 AY034721e Long Y2

Hordeum roshevitzii Bowden H “general” haplomeY2362d China: Qinghai province., Xiahe to Ganjia 20 km HROS016 480 AY034680e Long H1

HROS018 481 AY034682e Long H3

Hordeum californicum Covas & Stebbins H “general” haplomeCHC3439g U.S.A.: California, Thousand Oaks, 10 km South HCAL015 489 AY034696e Long H1

HCAL026 468 AY034707e Long Y2

Australopyrum retrofractum (Vickery) Á. Löve W haplome86147h Australia: Canberra seed collection AUSretrWT2 487 AF550708 (same as

Z11431e)Long W1

AUSretrWT3 486 AF550710 Long W1AUSretrWT4 485 AF550709 Long W1

aBase pairs.bMaintained at the Central Siberian Botanical Garden, Novosibirsk, Russia.cReceived from the USDA, Logan, Utah, U.S.A.dMaintained at the Triticeae Research Institute, Dujiangyan City, Sichuan, China.eFrom previous submissions to GenBank.fMaintained at the Nordic Gene Bank, Alnarp, Sweden.gCanadian Hordeum collection maintained by the Plant Genetic Resources of Canada, Saskatoon, Canada.hMaintained at the seed collection, Commonwealth Scientific and Industrial Research Organization, Canberra, Australia.

Table 1 (concluded).



logous group. At this stage, the alignment would reveal anysequences that would appear to have been assigned to thewrong unit class. Several sequences representative of eachunit class were then subjected to similarity searches of theGenBank and European Molecular Biology Laboratory data-bases using the National Center for Biotechnology Informa-tion Web-based BLAST service (Altschul et al. 1990) toidentify the unit class with an already established, i.e., pub-lished, unit class. The sequence identified as being the mostsimilar, i.e., having the highest scoring segment pairs andthe lowest P(N) value (as defined in Altschul et al. 1990),was subsequently aligned in toto with the representative unitclass and also with other sequences of interest among previ-ously defined unit class sequences mentioned above. All 118sequences were then reassembled for a final step of align-ment and manual refinement. This process and the methodof unit class determination and recognition has been dis-cussed in more detail in Baum et al. (2001).

Following refinement of the alignments, the guide treeswere used to assess whether the unit classes were distinct.Relationships within unit classes, which are putative ortho-logous sequences, and differences between the unit classes,which are paralogous sequences, were obtained through aphylogenetic analysis of the 118 aligned sequences bymeans of PAUP* Version 4.0b10 (Swofford 2002). The anal-ysis was done using the neighbor-joining algorithm (Saitouand Nei 1987), followed by a bootstrap analysis, with theneighbor-joining search option, of 10 000 replicates to esti-mate bootstrap support (Felsenstein 1985) for the putativeorthologous groups. In this and the following analyses, wetreated alignment gaps as missing data. After estimating allsix substitution-rate classes and the gamma shape parameter,we also performed a neighbor-joining analysis using a maxi-mum likelihood estimator of the distance. The option usedfor the maximum-likelihood analysis was to use the set ofvalues of the six-parameter instantaneous rate matrix alreadyin memory and the shape of the gamma distribution also inmemory. The data were also analyzed using the Bayesianapproach, i.e., based upon the posterior probabilities for theestimation of phylogeny as implemented in MRBAYES(Huelsenbeck and Ronquist 2001) using the following pa-rameters for Markov chain Monte Carlo analysis: number ofcycles (ngen) = 100 000, number of chains for Markov chainMonte Carlo variant (nchain) = 4, temperature parameter forheating the chains (Temp) = 0.6, option to save the branchlengths (savebrlens) = yes. We found that the Temp parame-ter played a major role in obtaining those identical results.By subjecting the data to three methods of analysis, basedupon different assumptions, we obtain a more balancedview. Our approach to phylogenetic analysis is eclectic andstatistical. In this regard we follow Felsenstein (1983, 1984,and his related publications) who advocated analyses withina statistical framework. On the one hand, we have not as-sumed that the conditions for parsimony are met, especiallywhen using phylogenetic methods to unravel putative ortho-logous groups in a multigene family such as the 5S rDNA.On the other hand, we have used parsimony where deemedappropriate.

The neighbor-joining tree initially obtained was then inputinto PAUP* to obtain a (syn)apomorphy list of nucleotidechanges on the branches of the phylogram, particularly to

obtain those changes along the major branches subtendingthe putative orthologous sequences, i.e., the 5S unit classes.The final alignment of all the 118 sequences was submittedto GenBank. They can be retrieved at the National Centerfor Biotechnology Information website http://www.ncbi.nlm.nih.gov (see Brawley 1999 for instructions on alignment re-trieval).

A phylogenetic analysis using the branch-and-bound me-thod (Hendy and Penny 1982), with the optimality criterionset to parsimony, was carried out using PAUP* on a subsetof the sequences to provide further justification for the newunit classes identified by the neighbor-joining analysis.Finally, consensus sequences for each unit class were ob-tained, concatenated, and aligned. This approach provided aclearer summary of the similarities and differences amongthe unit classes by reducing dominance effects due to differ-ences in the number of sequences within each one. To depictthe cladistic relationship among the unit classes in this study,a branch-and-bound analysis and a bootstrap analysis of thealigned consensus sequences were carried out, also withPAUP*, with the following parameters: the bootstrap methodwas carried out with branch-and-bound search, with 1000replicates, and gaps were treated as a fifth base. In the caseof the 5S rDNA in the Triticeae, especially within the vari-able NTS, each gap appeared to be independent from theother. Thus, there was no reason to treat gaps as characterstates. Long continuous gaps in the NTS may appear to indi-cate indels in an alignment. However, the process of align-ment was done first by computer followed by manualadjustments to refine it, aimed at finding patterns within pu-tatively orthologous groups (Baum et al. 2001). Thus, gaps(single or continuous) may be artificial constructs resultingfrom the alignment process.

To obtain a measure of the differences (polymorphisms)between unit classes, three sequence statistics were calcu-lated using the SITES program (http://lifesci.rutgers.edu/~heylab). (i) Group differences, i.e., a matrix with unit classby unit class comparisons yielding the average pair-wise dif-ferences for all the positions in the alignment between theunit classes (the triangle above the diagonal) and the net av-erage pair-wise divergence (the triangle under the diagonal)(Nei 1987, p. 276). The matrix of group differences abovethe diagonal was subject to a cluster analysis by unweightedpair group method using arithmetic averages (Sokal andMichener 1958) to obtain a dendrogram of relationshipsamong unit classes in this study. (ii) Fixed differences calcu-lated for all the polymorphic sites demonstrated how all se-quences in one unit class are different from all sequences inthe other. (iii) Shared polymorphisms was employed to findwhich sequences of two unit classes were found to have atleast two of the same bases for each polymorphic site.

Results

A summary of analyzed sequences along with their lengthin base pairs and GenBank accession Nos. is given in Ta-ble 1. The sequence alignments and subsequent phylogeneticanalyses suggested the presence of the six groups, which areclearly identified in the neighbor-joining tree (Fig. 1)and also obtained in the maximum-likelihood and theMRBAYES analyses (data not shown).

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Most sequences belong to the long S1 unit class or to oneof three previously catalogued unit classes: long H1, longP1, and long {Y1 (Baum and Johnson 1998; Baum andBailey 1997, 2000, respectively). The H “general” haplome(Table 1) refers to an ensemble of different haplomes thatsome authors have loosely designated as the H haplomeoriginating from Hordeum (von Bothmer et al. 1991). Thisensemble contains the following unit classes: long H1, longH2, and long Y2, which reflect the assignment of the respec-tive 5S rDNA units to the H and Y haplomes in Hordeum(von Bothmer et al. 1991). The Y haplome has recently beenrenamed as the Xu haplome (Wang et al. 1996). In additionto these previously described unit classes, this study hasidentified two new unit classes, the long S2 and long W1.

The final sequence alignment indicated clearly that thelong Y2 unit class, represented by HCOR013 andHCAL026, and the long H2 unit class, represented byHCOR028, were not similar to any of the sequences gener-ated in this study. Thus, none of the sequences can be identi-fied as belonging to the long H2 or long Y2 unit classes.In addition, two sequences, Epend31S02 and Epend31M01,which contain very large deletions, can be assigned to a unitclass only with difficulty. They are tentatively assigned tothe long Y2 unit class based on the phylogenetic analysiswith bootstrap support of 62% (Fig. 1). The fourth groupconsists of the long H1 unit class (Fig. 1, Table 1). Close tothe long H1 unit class (Fig. 1) is a representative sequenceof the long H2 unit class and two representative sequencesof the long Y2 unit class.

The two new unit classes are the long W1 assignable tothe W haplome (Dewey 1984; Löve 1984) and present inAustralopyrum and the long S2 identified here in some Pseu-doroegneria spicata and Elymus scabrus clones, assignableto the St haplome (Dewey 1984; Löve 1984). Based on dis-tance, the long W1 unit class is closest to a set containingthree unit classes, long S2, long P1, and long {Y1 (Fig. 1).They differ at several positions that can best be seen in thealignment. Notable examples are positions 42–44 where theAAG found in the long W1 is replaced by TGA/T in thelong P1 and by TAA in the long {Y1; positions 60–63 wherethe long W1 has GTGC instead of ATAT in the other two;positions 184–185 where GG is found in the long W1 in-stead of TG in the long P1; and in other positions. Thesethree unit classes together with the new long S2 unit classhave a gap in common at positions 186–195, which is absentin the other sequences. They also have a stretch of sequencesfilled by GTg/aCAATC at position 344–351. The long S1unit class differs from all other unit classes in this study bythree major gaps at positions 284–296, 301–304, and 324–354 and by other sequence details (refer to alignment).

The six unit classes are well supported in terms of boot-strap analysis as shown on the tree (Fig. 1), except for thelong {Y1, which is supported by 100% after the exclusion ofEdrob56-26 (see Discussion). Within the long S1 unit class,one may discern subgroups of sequences in the tree, butthese are not significantly different based on the sequencepattern.

While the results of the analysis in Fig. 1 provide supportfor the presence of the long W1, long P1, long {Y1, andlong S2 unit classes as indicated in the alignment, the groupsas unit classes may not appear to be significant because of

the dominance of sequences belonging to the long S1 unitclass. To further assess the differences between them, theirsequences were aligned along with only two representativesof the long S1 unit class. The matrix of their aligned se-quences was subjected to a phylogenetic analysis (Fig. 2).The analysis conclusively showed that it is justified to rec-ognize them as distinct groups of putative orthologous se-quences. This analysis was also done to assess whetherEdrob56-26 belongs to the long {Y1 unit class or to the longS1 unit class. Clearly Edrob56-26 belongs to the long {Y1unit class. The reason for its unexpected placement in thephylogenetic analysis carried out with all the sequences wasthe presence of a large deletion at the 5′ end of the NTS. Thejustification for its classification within the long {Y1 unitclass is the presence of GTACAATC that characterizes thefour unit classes, which is deleted in the long S1 units (posi-tion 344–351, see above). Finally, to clearly highlight thedifferences between the unit classes, consensus sequencesfrom each of the unit classes established in this study wereproduced (Fig. 3), and the phylogenetic relationships amongthem were estimated from the resulting single tree (Fig. 4).In this tree, the long H1, long S1, and long {Y1 are groupedtogether and are separated from the other three unit classesby 25 synapomorphic steps. Although these unit classes aredivided into two groups, the number of steps (in terms ofnucleotide changes) that separates the long W1 from thelong {Y1 unit class (113 steps) is not much different fromthe number of steps between it and the long P1 (110 steps)that is in the same group. The relationships among the unitclasses, based on Nei’s average pair-wise divergence,yielded two alternative configurations because of ties in-volving the topologies of the long {Y1 and long S1 and theirrelationship with the long W1 (not shown). A strict consen-sus (Sokal and Rohlf 1981) dendrogram (Fig. 5) was there-fore produced along with the consensus indices CIC(Colless’ index, also known as consensus fork index) andCIM (Mickevich’s consensus index) (Rohlf 1982). The highvalue for each of these two indices (CIC = 0.83 and CIM =0.75) indicates that the two alternative dendrograms (notshown) are very similar. Overall, the shared polymorphismsbetween the unit classes are small, ranging from zero in 13pair-wise comparisons to 22 between the long S1 and longP1 (Table 2, upper triangle). The fixed differences are obvi-ous and in several cases much higher (Table 2, lower trian-gle).

Discussion

Our analysis of the 5S rDNA sequences from species as-signed to the E. dahuricus complex has identified severalunit classes. Four classes were previously identified in otherTriticeae (long S1, long H1, long P1, and long {Y1), where-as two others, long S2 and long W1, are newly identified(Table 1).

Comparisons between the unit classes based on differentdata matrices, i.e., the entire data set or the consensus se-quences of the unit classes or the group differences, pro-vided different views of the relationships between the unitclasses (Figs. 1, 2, 4, and 5). By and large, each view sup-ports the general conclusion that the long P1, long S2, longW1, and long {Y1 form a group of unit classes that are more

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closely associated with each other than with the long S1 andwith the three unit classes long H1, long H2, and long Y2.The identity of the unit classes is supported by the lowshared polymorphisms among them (Table 2, upper triangle)and by their relatively high fixed differences (Table 2, lowertriangle). The highest fixed differences are found where theshared polymorphisms are lowest, providing further justifi-cation for the identity of the unit classes.

The relationships and differences among the six unitclasses can be more clearly seen by reference to the relation-ships among their consensus sequences (Figs. 3 and 4).Aside from the differences in sequence patterns, the long S1unit class is characterized by a long gap at position 253–298(Fig. 3) in the NTS. The S1 unit class is unique in this re-spect. In the analyses reported in Figs. 3 and 4, the long S1unit class is more similar to the long H1 unit class than to

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Fig. 2. Branch-and-bound cladogram (optimality criterion parsimony) of the 5S DNA units classified into the following five unitclasses: long W1, long P1, long S2, long S1, and long {Y1. The two units representing the long S1 unit classes, clones 41L01 and46L01, were taken from GenBank accession Nos. AF248176 and AF122845, respectively. The scale at the bottom left indicates 1 stepchange. The tree length is 252 steps, with a consistency index of 0.8690 and a retention index of 0.9106.

Fig. 1. Neighbor-joining phylogram of the 5S DNA units obtained in the Elymus dahuricus complex study. A total of 10 000 bootstrapsamples were analyzed, and support for the unit classes on the major branches is shown below each branch. The scale at the bottomleft indicates 0.1 distance units. Tree length after being subjected to parsimony analysis is 945 steps, with a consistency index of0.6825 and a retention index of 0.8868.



any other; however, when gaps are treated as missing data,the long S1 sequences becomes closest to the long {Y1 se-quences (Fig. 2). It is noteworthy that the long {Y1 unit

class, assignable to the Y haplome, is closest to the long W1unit class, assignable to the W haplome. The former ha-plome is present in many species in East Asia, whereas the

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Fig. 3. Alignment of the consensus sequences from the six unit classes compared in this study. Shade style: black, 100% identity; darkgrey, 80% identity; light grey, 60% identity; white, less than 50% identity. The 5′ end start of the transcribed region is marked with avertical bar followed by three arrowheads “| > > >”, and the BamH1 restriction site (GGATCC) is indicated between two vertical bars.Full alignment of the 118 sequences can be seen and retrieved from National Center for Biotechnology Information website http://www.ncbi.nlm.nih.gov (click on “PopSet” from the menu).



latter is found in some species in Australia and New Zea-land. Since a diploid carrier of the Y haplome is yet to bediscovered, it may be very rare or extinct. However, it maybe found in the far East or among the species that carry theW haplome. The long P1 and long S2 have the greatest se-quence pattern in common (Figs. 1–4).

When the number of clones in different unit classes is to-taled, it is clear that approximately 75% of the clones are as-signable to the long S1 unit class. Several hypotheses mayexplain the paucity of units in the {Y1 or all H units classes,representing the H or Y haplomes, including the preferentialamplification of long S1 units at one or several loci, loss ofH1 or {Y1 units because of homogenization via gene con-version, or simply the loss of H1 or {Y1 units because of in-breeding of populations. The alternative explanation that H1or {Y1 units were missed because of sampling is consideredto be less likely, as 112 sequences (out of the 118 analyzed)were sampled from 26 collecting sites throughout most ofthe range of the complex. A recent population genetic analy-sis of the E. dahuricus complex using amplified fragmentlength polymorphisms (Agafonov et al. 2001) provided sup-port for the idea of inbreeding within populations. These au-

thors found that the amount of variation within populationswas 3.5 times lower than the amount of variation betweenthe so called species within the complex. {Y1 units werealso found to be rare in Kengyilia (Baum and Bailey 2000).Despite the rarity of the long {Y1 and long H1 units, theirpresence along with the long S1 units mirrors the presenceof the St, Y, and H haplomes in the E. dahuricus complexand support its classification as a member of Elymus, notRoegneria. The scarcity of long H1 units in both tetraploidand hexaploid species may help explain the observation ofSvitashev et al. (1996) that morphological signs of the pres-ence of the H haplome are usually absent in these taxa.

Sequence diversity is observed within all unit classes,including the long S1. Included in this group are long S1units from Elymus caucasicus and Elymus pendulinus, spe-cies with the St and Y haplomes that have been recentlybeen assigned to Roegneria (Baum et al. 1991). Within thelong S1 class, the 5S DNA units from E. caucasicus appearto be a distinct group with 96% bootstrap support (Fig. 1).In spite of its distinct appearance as a cluster, the sequence

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Fig. 4. Branch-and-bound cladogram of the consensus sequencesof the 5S DNA unit classes in the Elymus dahuricus complexstudy. A total of 10 000 bootstrap samples were analyzed, andsupport for the unit classes on the major branches is shown be-low each branch (%). The number of step changes is indicatedabove the branches. The scale at the bottom left indicates 10steps change. Tree length is 324 steps, with a consistency indexof 0.8704 and a retention index of 0.4750.

Fig. 5. Strict consensus dendrogram of two alternative un-weighted pair group method with arithmetic means dendrogramsfrom the group pair-wise differences (see text). CIC = 0.83;CIM = 0.75 (CIC, Colless’ index, also known as consensus forkindex; CIM, Mickevich’s consensus index).

Unitclass 1 2 3 4 5 6 7 8

1 5 9 1 3 15 0 02 78 2 1 1 4 0 03 75 23 0 5 22 0 04 81 46 49 0 1 0 05 75 34 40 54 10 0 16 62 45 50 66 33 0 37 68 107 109 113 101 74 08 130 136 139 148 45 60 141

Note: Insertion and deletions are not included in this table. 1, Long H1;2, Long S2; 3, Long P1; 4, Long W1; 5, Long {Y1; 6, Long S1; 7, LongH2; 8, Long Y2.

Table 2. Shared polymorphisms are represented above the diago-nal, and fixed differences are represented below the diagonal.



pattern is not significantly different from the long S1 as awhole; however, this slight difference may suggest a differ-ent evolutionary history for E. caucasicus that is distinctfrom E. pendulinus.

The phylogenetic implications for the E. dahuricus com-plex from the perspective of the 5S DNA units points to agroup, distinct from other hexaploid Elymus species, thatcontains either the same StYH genome, i.e., E. drobovii, ora different genome, i.e., E. scabrus, that contains the Whaplome instead of the H haplome. We did not detect se-quences that could be assigned to the W haplome (the longW1 class) in E. scabrus, likely because of the few clonesisolated from this species. The available 5S DNA units ofE. scabrus were assigned to a new unit class, i.e., the longS2 (Table 1, Fig. 1) also found in P. spicata, a diploid spe-cies with the St haplome. The long S2 unit class is differentfrom the long S1 in sequence pattern (Figs. 1–4). In P. spi-cata, both long S1 and long S2 unit classes were identified(Table 1).

Among the tetraploid and hexaploid species studied, onlythe long S1 unit class was identified, with the exception ofE. scabrus in which the long S2 appeared to prevail (againthis observation is based upon few clones). It is thus possiblethat the rate of interlocus recombination or gene conversionin the polyploids was high among the already diverged para-logues — long S1 and long S2 in the Pseudoroegneria. Iftrue, this might explain the difference in the 5S unit classesamong E. scabrus, the E. dahuricus complex, and E. dro-bovii among the hexaploid and the tetraploid species exam-ined in this study. Within the long S1 unit class, divergenceprobably continued after speciation but only to a smalldegree as in E. caucasicus (Fig. 1), where the units cannotbe considered different paralogues. Alternatively, differentchromosomal locations may limit concerted evolution(Buckler et al. 1997), as may be the case for several recentexamples taken from the Triticeae (Baum et al. 2001), whereparalogues can be clearly defined and established as unitclasses.

It has been suggested that concerted evolution in the 5SDNA gene has played only a partial role as an evolutionaryforce (Baum and Bailey 1997; Baum et al. 2001). Further-more, we speculated that some orthologous sequencesremained preserved as much as the haplomes themselves,while others remained preserved only in a smaller groupwithin a genome. In this study, we found more support forthis conclusion. The study of 5S rDNA is a useful tool forthe identification of haplomes, at least in the Triticeae. Thisprocess requires that we sample and sequence a substantialnumber of clones followed by assignment of the clones intoputative orthologous groups through careful alignments.Subsequently, orthology is tested by various phylogeneticanalyses. As has been shown previously, such orthologousgroups are defined as unit classes that can be assigned tohaplomes (e.g., Baum et al. 2001). In some cases, two ormore unit classes can be assigned to the same haplome, andin that case, it is their combination that becomes haplome-specific. We continue to believe that the ability to define unitclasses based upon conserved sequences may allow the syn-thesis of molecular probes designed to exploit differencesbetween the unit classes. Furthermore, under the appropriatehybridization conditions, these probes have the potential to

localize unit classes on chromosomes and thus might serveto identify haplomes directly via in situ hybridization.

Acknowledgements

We thank Dr. S. Molnar and Dr. B. Miki for their usefulcomments in an earlier draft. We thank Dr. A. Bruneau, As-sociate Editor of the Journal, for suggestions for improve-ment of the manuscript. This work was supported, in part,by a grant from the Natural Sciences and Engineering Re-search Council of Canada to Douglas A. Johnson.

References

Agafonov, A.V., Baum, B.R., Bailey, L.G., and Agafonova, O.V.2001. Differentiation in the Elymus dahuricus complex(Poaceae): evidence from grain proteins, DNA, and crossability.Hereditas, 135: 277–289.

Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J.1990. Basic local alignment search tool. J. Mol. Biol. 215: 403–410.

Baum, B.R., and Bailey, L.G. 1997. The molecular diversity of the5S rRNA gene in Kengyilia alatavica (Drobov) J.L. Yang, Yen& Baum (Poaceae: Triticeae): potential genomic assignment ofdifferent rDNA units. Genome, 40: 215–228.

Baum, B.R., and Bailey, L.G. 2000. The 5S rDNA units inKengyilia (Poaceae: Triticeae): diversity of the nontranscribedspacer and genomic relationships. Can. J. Bot. 78: 1571–1579.

Baum, B.R., and Bailey, L.G. 2001. The 5S rRNA gene sequencevariation in wheats and some polyploidy wheat progenitors(Poaceae: Triticeae). Genet. Resour. Crop Evol., 48: 35–51.

Baum, B.R., and Johnson, D.A. 1994. The molecular diversity ofthe 5S rRNA gene in barley (Hordeum vulgare). Genome, 37:992–998.

Baum, B.R., and Johnson, D.A. 1996. The 5S rRNA gene units inancestral two-rowed barley (Hordeum spontaneum C. Koch) andbulbous barley (H. bulbosum L.): sequence analysis and phylo-genetic relationships with the 5S rDNA units in cultivated barley(H. vulgare L.). Genome, 39: 140–149.

Baum, B.R., and Johnson, D.A. 1998. The 5S rRNA gene in seabarley (Hordeum marinum Hudson sensu lato): sequence varia-tion among repeat units and relationship to the X haplome inbarley (Hordeum). Genome, 41: 652–661.

Baum, B.R., and Johnson, D.A. 1999. The 5S rRNA gene in wallbarley (Hordeum murinum L. sensu lato): Sequence variationamong repeat units and relationship to the Y haplome in the ge-nus Hordeum (Poaceae: Triticeae). Genome, 42: 854–866.

Baum, B.R., and Johnson, D.A. 2000. The 5S rRNA gene unitsin the native New World annual Hordeum species (Triticeae:Poaceae). Can. J. Bot. 78: 1590–1602.

Baum, B.R., and Johnson, D.A. 2002. A comparison of the 5SrDNA diversity in the Hordeum brachyantherum–californicumcomplex with those of the eastern Asiatic Hordeum roshevitziiand the South American Hordeum cordobense (Triticeae:Poaceae). Can. J. Bot. 80: 752–762.

Baum, B.R., Yen, C., and Yang, J.L. 1991. Roegneria: its genericlimits and justification for its recognition. Can. J. Bot. 69: 282–294.

Baum, B.R., Johnson, D.A., and Bailey, L.G. 2001. Defininggroups among multicopy genes prior to inferring phylogeny,with special emphasis on the Triticeae (Poaceae). Hereditas,135: 123–138.

© 2003 NRC Canada

1102 Can. J. Bot. Vol. 81, 2003



Brawley, S.H. 1999. Submission and retrieval of an aligned set ofnucleic acid sequences. J. Phycol. 35: 433–437.

Buckler, E.S., IV, Ippolito, A., and Holtsford, T.P. 1997. The evolu-tion of ribosomal DNA: divergent paralogues and phylogeneticimplications. Genetics, 145: 821–832.

Davis, P.H., and Heywood, V.H. 1963. Principles of angiospermtaxonomy. D. Van Nostrand Co., Princeton, New Jersey.

Dewey, D.R. 1984. The genomic system of classification as a guideto intergeneric hybridization with the perennial Triticeae. InGene manipulation in plant improvement. Edited by J.P. Gustaf-son. Plenum Publishing Corporation, New York. pp. 209–279.

Felsenstein, J. 1983. Statistical inference of phylogenies (with dis-cussion). J. R. Stat. Soc. Ser. A, 146: 246–272.

Felsentein, J. 1984. The statistical approach to inferring evolution-ary trees and what it tells us about parsimony and compatibility.In Cladistics: perspectives on the reconstruction of evolutionaryhistory. Edited by T. Duncan and T.F. Stuessy. Columbia Univer-sity Press, New York, N.Y.

Felsenstein, J. 1985. Confidence limits on phylogenies: an ap-proach using the bootstrap. Evolution, 39: 783–791.

Heilbronn, A., and Kosswig, C. 1966. Principia genetica. 2nd ed.Grunderkenntnisse und Grundbegriffe der Vererbungswissen-schat, Hamburg.

Hendy, M.D., and Penny, D. 1982. Branch and bound algorithms todetermine minimal evolutionary trees. Math. Biosci. 59: 277–290.

Huelsenbeck, J.P., and Ronquist, F. 2001. MRBAYES: Baysian in-ference of phylogenetic trees. Bioinformatics Applications Note17: 754–755.

Löve, Á. 1984. Conspectus of the Triticeae. Feddes Repert. 95:425–521.

Lu, B.-R. 1993. Biosystematic investigations of Asiatic wheat-grasses — Elymus L. (Triticeae: Poaceae). Ph.D. thesis, SwedishAgricultural University, Alnarp, Sweden. ISBN 91-576-4705-4.

Lu, B.-R., and Salomon, B. 1992. Differentiation of the SYgenomes in Asiatic Elymus. Hereditas, 116: 121–126.

Nei, M. 1987. Molecular evolutionary genetics. Columbia Univer-sity Press, New York.

Nicholas, K.B., and Nicholas, H.B., Jr. 1997. GeneDoc©: a tool forediting and annotating multiple sequence alignments [online].

(Distributed by the authors.) Version 2.6.02. Available fromhttp://www.psc.edu/biomed/genedoc/ [updated July 2001].

Rohlf, F.J. 1982. Consensus indices for comparing classifications.Math. Biosci. 59: 131–144.

Saitou, N., and Nei, M. 1987. The neighbor-joining method: a newmethod for reconstructing phylogenetic trees. Mol. Biol. Evol.4: 406–425.

Sokal, R.R., and Michener, C.D. 1958. A statistical method forevaluating systematic relationships. Univ. Kans. Sci. Bull. 38:1409–1438.

Sokal, R.R., and Rohlf, F.J. 1981. The comparison of dendrogramsby objective methods. Taxon, 11: 33–40.

Svitashev, S., Salomon, B., Bryngelsson, T., and Bothmer, R. von.1996. A study of 28 Elymus species using repetitive DNA se-quences. Genome, 39: 1093–1101.

Swofford, D.L. 2002. PAUP*. Phylogenetic analysis using parsi-mony (*and other methods). Version 4.0b3. Sinauer Associates,Sunderland, Mass.

Thompson, J.D., Higgins, D.G., and Gibson, T.J. 1994. CLUSTALW: improving the sensitivity of progressive multiple sequencealignment through sequence weighting, position-specific gappenalties and weight matrix choice. Nucleic Acids Res. 22:4673–4680.

von Bothmer, R., Jacobsen, N., Baden, C., Jørgensen, R.B., andLinde-Laursen, I. 1991. An ecogeographical study of the genusHordeum. In Systematic and ecogeographic studies on cropgenepools 7. IBPGR, Rome.

Wang, R.R.-C., von Bothmer, R., Dvorak, J., Fedak, G., Linde-Laursen, I., and Muramatsu, M. 1996. Genome symbols in theTriticeae (Poaceae). In Proceedings of the 2nd InternationalTriticeae Symposium, Logan, Utah, 20–24 June 1994. Edited byR.R.-C. Wang, K.B. Jansen, and C. Jaussi. Utah State Univer-sity, Logan, Utah. pp. 29–34.

Yang, J.L., Yen, C., and Baum, B.R. 1992. Kengyilia: synopsis andkey to species. Hereditas, 116: 25–28.

Yanisch-Perron, C., Vieira, J., and Messing, J. 1985. ImprovedM13 phage cloning vectors and host strains; nucleotide se-quence of M13mp18 and pUC19 vectors. Gene, 33: 103–119.

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