Phylogenetic reconstruction of Aegilops sectionSitopsis and the evolution of tandem repeats inthe diploids and derived wheat polyploids

Elena A. Salina, K. Yoong Lim, Ekaterina D. Badaeva, Andrey B. Shcherban,Irina G. Adonina, Alexandra V. Amosova, Tatiana E. Samatadze,Tatyana Yu. Vatolina, Sviatoslav A. Zoshchuk, and Andrew R. Leitch

Abstract: The evolution of 2 tandemly repeated sequences Spelt1 and Spelt52 was studied in Triticum species repre-senting 2 evolutionary lineages of wheat and in Aegilops sect. Sitopsis, putative donors of their B/G genomes. Usingfluorescence in situ hybridization we observed considerable polymorphisms in the hybridization patterns of Spelt1 andSpelt52 repeats between and within Triticum and Aegilops species. Between 2 and 28 subtelomeric sites of Spelt1probe were detected in Ae. speltoidies, depending on accession. From 8 to 12 Spelt1 subtelomeric sites were observedin species of Timopheevi group (GAt genome), whereas the number of signals in emmer/aestivum accessions was sig-nificantly less (from 0 to 6). Hybridization patterns of Spelt52 in Ae. speltoides, Ae. longissima, and Ae. sharonensiswere species specific. Subtelomeric sites of Spelt52 repeat were detected only in T. araraticum (T. timopheevii), andtheir number and chromosomal location varied between accessions. Superimposing copy number data onto our phylo-genetic scheme constructed from RAPD data suggests 2 major independent amplifications of Spelt52 and 1 of Spelt1repeats in Aegilops divergence. It is likely that the Spelt1 amplification took place in the ancient Ae. speltoides beforethe divergence of polyploid wheats. The Spelt52 repeat was probably amplified in the lineage of Ae. speltoides prior todivergence of the allopolyploid T. timopheevii but after the divergence of T. durum. In a separate amplification event,Spelt52 copy number expanded in the common ancestor of Ae. longissima and Ae. sharonensis.

Key words: evolution, RAPD, subtelomeric tandem repeats, Aegilops, wheat, B and G genome.

Résumé : L’évolution de 2 séquences répétées en tandem, Spelt1 et Spelt52, a été étudiée chez des espèces du genreTriticum qui représentent 2 lignages évolutifs du blé et les espèces donatrices des génomes B/G au sein de la sectionSitopsis des Aegilops. Par hybridation in situ en fluorescence (FISH), les auteurs ont observé un polymorphisme consi-dérable quant aux motifs d’hybridation obtenus avec les répétitions Spelt1 et Spelt52 parmi et entre les espèces desgenres Triticum et Aegilops. Entre 2 et 28 sites subtélomériques Spelt1 ont été détectés chez l’Ae. speltoides selonl’accession. Entre 8 et 12 sites subtélomériques Spelt1 ont été observés chez les espèces du groupe Timopheevi (gé-nome GAt), tandis que le nombre de signaux chez les accessions de blés amidonnier/aestivum était significativementinférieur (de 0 à 6). Les motifs d’hybridation de Spelt52 chez les espèces Ae. speltoides, Ae. longissima et Ae. sharo-nensis sont spécifiques à chacune. Des sites subtélomériques de la répétition Spelt52 ont été détectés seulement chez leT. araraticum (T. timopheevii), leur nombre et emplacement variant d’une accession à l’autre. La superposition des don-nées quant au nombre de copies et la phylogénie fondée sur les RAPD suggère deux amplifications majeures indépen-dantes des répétitions Spelt52 et une des répétitions Spelt1 au cours de la divergence des Aegilops. Il est probable quel’amplification de Spelt1 se soit produite au sein de l’ancien Ae. speltoides avant la divergence des blés polyploïdes. Larépétition Spelt52 aurait vraisemblablement été amplifiée chez l’Ae. speltoides avant la divergence de l’allopolyploïdeT. timopheevii, mais après la divergence du T. durum. Lors d’une amplification ultérieure, le nombre de copies deSpelt52 se serait accru chez l’ancêtre commun de l’Ae. longissima et de l’Ae. sharonensis.

Mots clés : évolution, RAPD, répétitions subtélomériques en tandem, Aegilops, blé, génomes B et G.

[Traduit par la Rédaction] Salina et al. 1035

Genome 49: 1023–1035 (2006) doi:10.1139/G06-050 © 2006 NRC Canada

1023

Received 4 August 2005. Accepted 31 March 2006. Published on the NRC Research Press Web site at http://genome.nrc.ca on30 September 2006.

Corresponding Editor: T. Schwarzacher.

E.A. Salina,1 A.B. Shcherban, I.G. Adonina, and T.Y. Vatolina. Institute of Cytology and Genetics, Siberian Branch of RussianAcademy of Sciences. Lavrentiev Ave., 10, 630090 Novosibirsk, Russia.K.Y. Lim and A.R. Leitch. School of Biological and Chemical Sciences, Queen Mary, University of London, E1 4NS, U.K.E.D. Badaeva, A.V. Amosova, T.E. Samatadze and S.A. Zoshchuk. Engelhardt Institute of Molecular Biology, Russian Academyof Sciences. Vavilov Street 32, Moscow 119991, Russia.

1Corresponding author (salina@bionet.nsc.ru).

Introduction

This paper addresses karyotype and repetitive sequencedivergence in Aegilops and Triticum. Five Aegilops speciesbelong to section Sitopsis: Ae. speltoides, Ae. longissima,Ae. sharonensis, Ae. bicornis, and Ae. searsii. Among them,Ae. speltoides (genome SS) is considered to be most closelyrelated to the B and G genomes of polyploid wheats(Feldman et al. 1995; Friebe and Gill 1996; Tsunewaki1996). Plant morphology, chromosome pairing, and geneticmarker analysis indicate that Ae. speltoides differs from theother 4 species, and it has been placed in a separate group(Eig 1929; Kerby and Kuspira 1987). The distinctive statusof Ae. speltoides has since been confirmed by RFLP, C-banding, and fluorescent in situ hybridization (FISH) usingrepetitive DNA, and 45S and 5S ribosomal DNA (rDNA)probes (Dvorak and Appels 1982; Dvorak and Zhang 1992;Friebe and Gill 1996; Badaeva et al. 1996a, 1996b;Sasanuma et al. 1996; Giorgi et al. 2002). Aegilopsspeltoides is also the only outbreeding Aegilops species andshows the broadest genomic polymorphism (Friebe and Gill1996; Sasanuma et al. 1996; Giorgi et al. 2002).

The present day polyploid wheats are represented by 2evolutionary lineages: Emmer wheats (tetraploid Triticumdicoccoides, T. dicoccum, and T. durum, and hexaploidT. aestivum) and Timopheevi wheats (tetraploid T. dicoccumand T. timopheevii, hexaploid T. zhukovskyi, and several arti-ficially synthesized amphiploids). Timopheevi and Emmerwheats differ in a number of morphological, biochemical,molecular, and cytological characteristics (Lilienfeld andKihara 1934; Chen and Gill 1983; Jiang and Gill 1994;Badaeva et al. 1994; Khlestkina and Salina 2001).T. dicoccoides and T. araraticum are thought to have origi-nated from 2 independent crosses involving progenitors ofmodern Ae. speltoides (maternal form) and T. urartu (paternalform) (Dvorak et al. 1988; Jiang and Gill 1994). Subsequentevolution of the 2 species was accompanied by numerousgenomic modifications, including different species-specifictranslocations and elimination or amplification of variousDNA sequences (Naranjo et al. 1987; Liu et al. 1992; Jiangand Gill 1994; Maestra and Naranjo 1999; Salina et al. 2005).

Repetitive sequences can constitute more than 70% of theplant genome (Schmidt and Heslop-Harrison 1998; Gill andFriebe 1998). The emergence of different families of repeti-tive sequences is presumably the result of sequence diver-gence (mutations, deletions, insertions) accompanied bysequence amplifications and reductions (Flavell 1982). Somerepetitive families are useful in defining taxonomic groups(Nagaki et al. 1995; Lim et al. 2000; Taketa et al. 2000;Contento et al. 2005); others are species specific (Heslop-Harrison 2000).

In the Triticeae several families and categories of tandemrepeats are known. Many are subtelomeric in location (theterm “subtelomeric sequences” is used to distinguish themfrom the true telomeric repeat (TTTAGGG)n). However, theirroles are not fully resolved. The tandem repeats known inTriticeae are as follows:(a) the pAs1/Afa family/ pHcKB6/dpTa1 family of repeats

(Rayburn and Gill 1986; Nagaki et al. 1995, 1998;Anamthawat-Jonsson and Heslop-Harrison 1993; Vershininet al. 1994), which localize predominantly to subtelomeric

and intercalary genomic regions of Ae. tauschii,T. aestivum, Hordeum species, Elymus trachycaulus, andseveral other species (Sharma and Raina 2005);

(b) the TaiI family of tandem repeats, which localizes tosubtelomeric regions in the majority of Triticeae speciesexcept in Triticum and Aegilops species, where repeatsare at centromeric regions (Kishii and Tsujimoto 2002);

(c) HvRT repeats, the telomere-associated sequences ofHordeum vulgare (Kilian and Kleinhofs 1992);

(d) subtelomeric repeats in Secale cereale, which are repre-sented by 350 family/pSc200/pSc74 and pSc250(Bedbrook et al. 1980; Appels et al. 1986; Vershinin etal. 1995). In the course of evolution, these sequencesamplified in genomes of individual Secale species aswell as in some species of Agropyron and Dasypyrum ofthe tribe Triticeae (Vershinin et al. 1996);

(e) the 120-bp family of repeats called pSc119.2 (Bedbrooket al. 1980; Appels et al. 1986; McIntyre et al. 1990),which are widely distributed at different chromosomalregions of many species from the tribe Triticeae and thesister tribe Avenae (Jones and Flavell 1982; Mukai et al.1993; Jiang and Gill 1994; Cuadrado et al. 1995;Vershinin et al. 1995; Katsiotis et al. 1997; Badaeva etal. 1996a; Taketa et al. 2000; Contento et al. 2005). Ini-tially, this family was cloned from Secale cereale anddescribed as a family of rye telomeric heterochromatin(Bedbrook et al. 1980). The clone pSc119.2 was usedhere to identify individual chromosomes;

(f) the family of tandemly repeated sequences pAesKB52(including Spelt52 and pGc1R-1), previously located atsubtelomeric regions of some S-genome Triticeae spe-cies (Ae. speltoides, Ae. sharonensis and Ae. longissima)(Anamthawat-Jonsson and Heslop-Harrison 1993;Zhang et al. 2002; Salina et al. 2004b). Spelt52 is ana-lyzed here in Aegilops sect. Sitopsis and derived allo-polyploids; and

(g) the Spelt1 family of repeats, which has been observed inAe. speltoides and is located at subtelomeric chromo-some regions (Salina et al. 1998; Adonina et al. 2004).Spelt1 is analyzed here in Aegilops sect. Sitopsis and de-rived allopolyploids.

Clearly, there has been much divergence of tandem repeatstructure in Triticeae evolution, but whether this has influ-enced any function is unknown. To gain deeper insights intotandem repeat evolution we examine here repetitive se-quence evolution in Aegilops sect. Sitopsis and in derived al-lopolyploid wheats of different evolutionary lineages. Usingphylogenetic reconstructions of species relations enables usto predict patterns of repeat sequence evolution. The under-standing is important because the divergence of repeats, in-cluding copy number expansions or contractions, may beinvolved in reinforcing isolation barriers between species.Furthermore, our analysis gives insight into the genetic con-sequences of allopolyploidy, particularly in wheat (Levy andFeldman 2002). Here we study the chromosomal distributionof both Spelt1 and Spelt52 repeats in different Aegilops spe-cies and accessions of section Sitopsis and in tetraploid andhexaploid wheats representing 2 independent evolutionarylineages. Our data allow predictions of major changes incopy numbers of repetitive DNAs in Triticum–Aegilops di-vergence.

© 2006 NRC Canada

1024 Genome Vol. 49, 2006

Materials and methods

Plant materialThe list of the studied plant materials, their genome con-

stitution, chromosome number, and origins are given inTable 1. Aegilops species were provided by M. Feldman

and maintained at the germplasm collection of the Plant Sci-ences Department, the Weizmann Institute of Science, Israel;Triticum araraticum lines were from the wheat germplasmcollection of the Institute für Pflanzengenetik und Kulturp-flanzenforschung, Gatersleben, Germany; Triticum dicoccoideswas from USDA–ARS (US Department of Agriculture,Agricultural Research Service) Small Grains Collection,Aberdeen, Idaho; and the other materials are maintained inthe Wheat Germ-Plasm Collection of the Vavilov Institute ofPlant Industry, St-Petersburg, Russia.

DNA probesTwo telomere-associated tandem sequences of Ae.

speltoides, Spelt1 and Spelt52, and pSc119.2 sequence iso-lated from S. cereale were used as probes (Salina et al.1998; Salina et al. 2004a; McIntyre et al. 1990).

RAPD analysisEighteen primers were used to develop a phylogenetic

scheme of species relations. These primers were previouslyselected from 115 RAPD primers for their ability to generatepolymorphic amplification patterns in Triticum and Aegilops(Bildanova et al. 2002). The RAPD primer sequences (Insti-tute of Cytology and Genetics, Novosibirsk, Russia) aregiven in Table 2. PCR reaction mixtures (50 µL) containedthe following components: 50 ng of template DNA,67 mmol Tris–HCl/L, pH 8.8, 1.8 mmol MgCl2/L, 0.01%Tween 20, 18 mmol (NH4)2SO4/L, 0.2 mmol of each deoxy-nucleotide triphosphate/L, 0.5 µmol primer/L, and 1 U TaqDNA polymerase (Medigen, Novosibirsk). PCR of totalgenomic DNA of Aegilops species was performed on aPerkin Elmer Cetus Thermocycler using the following mode:2 min at 94 °C, 30 cycles of 1 min at 94 °C, 1 min at 37 °C,and 2 min at 72 °C, followed by a final stage of 5 min at72 °C. PCR products were separated by electrophoresis in2% agarose gels. Each PCR band was treated as a unit char-acter, and all species were scored for the presence (1) or ab-sence (0) of individual bands. Calculation of coefficients ofsimilarity (dis) was based on a data matrix, according to Neiand Li (1979), using the PHYLIP software package (Felsenstein1989). Phylogenetic trees were constructed according to theresulting distance matrices using the neighbor-joiningmethod of the software package MEGA version 2.1 (Kumaret al. 2001). The reliability of the neighbor-joining tree wasassessed by bootstrap analysis (Felsenstein 1989); 100 boot-strap replicates were analyzed.

C-banding analysisAll wheat and most Aegilops accessions were first exam-

ined by C-banding to determine their karyotype structures.The C-banding procedure was as described by Badaeva et al.(1994). Chromosomes were classified according to standardgenetic nomenclature of common wheat (Gill et al. 1991),T. timopheevii (Badaeva et al. 1991) or Aegilops (Badaeva etal. 1996a; Friebe and Gill 1996) chromosomes.

Selection of lines for fluorescent in situ hybridization(FISH) analysis

In total, 44 lines and accessions of Aegilops and Triticumwere mapped for Spelt1 and Spelt52 by FISH. The Aegilopslines were selected for analysis on the basis of differences incopy numbers of the repeats detected in dot-blot hybridiza-tions (Salina et al. 2004b): Ae speltoides TS42 and TS05 con-tained nearly equal amounts of Spelt1 and Spelt52 repeats,TS01 had the lowest Spelt1 content, Ae. sharonensis TH01and Ae. longissima TL03 had high Spelt52 copy numbers, andAe. longissima TL05 had low Spelt52 content. All wheat ac-cessions chosen for analysis were selected at random.

FISHFISH was conducted according to Leitch et al. (1994)

with minor modifications. Vigorously growing young roottips were harvested and pretreated with a saturated aqueoussolution of Lindane® (hexachlorocyclohexane, SigmaAldrich Corp., St. Louis, Mo.) for 4 h or in ice-cold waterfor 20–24 h. Probes were labeled using either PCR or nicktranslation to incorporate biotin-16-dUTP (Life Technol-ogies, Gibco BRL, Gaithersburg, Md.) or digoxigenin-11-dUTP (Roche Diagnostics, Penzberg, Germany). Labeledprobes were used at a concentration of 4 µg/mL in a hybrid-ization mix containing 50% v/v formamide, 10% w/v dextransulphate, and 0.1% w/v SDS in 2× SSC (0.3 mol sodiumchloride/L, 0.03 mol sodium citrate/L). Slides were dena-tured in 70% formamide in 2× SSC at 70 °C for 2 min. Afterovernight hybridization at 37 °C, the slides were washed in20% v/v formamide in 0.1× SSC at 42 °C at an estimatedhybridization stringency of 80%–85%. Sites of probehybridization were detected using 20 µg/mL fluorescein con-jugated anti-digoxigenin IgG (Roche Biochemicals, Sussex,UK) and 5 µg/mL Cy3 conjugated avidin (AmershamPharmacia Biotech, Uppsala, Sweden). Chromosomes werecounterstained with 2 µg/mL DAPI (4′,6-diamidino-2-phenylindole) in 4× SSC, mounted in Vectashield (VectorLaboratories, Peterborough, UK) medium, and examinedusing a Leica DM RA2/. Images were captured usingOpenlab® (Improvision, Coventry, UK) and processed usingAdobe Photoshop® (Adobe Systems, Edinburgh, UK) bytreating all images for colour contrast and brightness uni-formly.

Idiograms of Ae. speltoides, Ae. longissima, andAe. sharonensis chromosomes indicating the position ofSpelt1 and Spelt52 sequences were constructed on the basisof 2 independent FISH experiments carried out on prepara-tions made from the same plants. In the first experiment,probe combinations pSc119.2 + Spelt52 were used toidentify chromosomes carrying Spelt52 signals. The second(probe combination Spelt1 + Spelt52) enabled the relativelocation of Spelt1 and Spelt52 to be determined. Thechromosomes were arranged according to standard geneticnomenclature (Badaeva et al. 1996a; Friebe and Gill 1996).

Results

RAPD analysisIn total, 207 bands were reproducibly generated by

RAPD amplification with 18 primers (Table 2). Of these,

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Salina et al. 1025

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1026 Genome Vol. 49, 2006

Species Accession or line Genome*Karyotypestructure† Origin

Ae. speltoides Tausch TS01

TS05

TS41

TS42

TS47

2n = 2x = 14, SS N

No data

No data

N

No data

Ashqelon, southern Coastal Plain, Israel

Zikhron Yaaqov, Coastal Plain, Israel

Wadi Hilazon, Western Galilee, Israel

Givat Koah, Shefela, Israel

Line from Colin LawAe. longissima Schw.,

and Muschl.TL03

TL04

TL05

TL09

TL15

2n = 2x = 14, SlSl N

No data

N

No data

No data

Rehovot, Coastal Plain, Israel

Gilat, Northern Negev, Israel

Nahariyya, Northern Coastal Plain, Israel

East of the Dead Sea, Jordan Dimona,

Negev, IsraelAe. sharonensis Eig TH01

TH02

2n = 2x = 14, SshSsh N

N

Caesarea, Coastal Plain, Israel

Naaman salt-marsh, near Acre, IsraelAe. bicornis (Forssk.)

Jaub., and Sp.TB05 2n = 2x = 14, +-SbSb N‡ Matruh, Delta area, Egypt

Ae. searsii Feld., and Kis. TE12

TE16

2n = 2x = 14, SsSs No data

No data

Tayasir, Eastern Samaria, Israel

Gabagib, Southwestern SyriaT. araraticum Jakubz. Tri11365

Tri11508

Tri11509

Tri11939

Tri16599

Tri17418

Tri17419

2n = 4x = 28, GGAtAt T3At:7At +T2At:4G:7

N

inv 7At

N

T2At:6At:4G

N

T5At:3G

Azerbaijan, Nakhichevan

Iraq

Iran

Iraq

Armenia

Iraq

Iraq

T. timopheevii Zhuk. Coll.2 N21

Tri26

2n = 4x = 28, GGAtAt N

N

Institute of Agriculture, Georgia

Gatersleben, GermanyT. kiharae Dorof et

Migusch.2n = 6x = GGAtAtDD N Vavilov Institute of Plant Industry,

St-Petersburg, RussiaT. dicoccoides (Koern. ex

Aschers. et Graebn.)Schweinf.

TTD20

PI478703

PI538657

PI467013

PI428126

PI571750

2n = 4x = 28, BBAA N

N

N

N

N

N

Israel

Israel

Turkey

Israel

Lebanon, Raskaya

IsraelT. dicoccum (Schrank)

Schuebl.k-5154

k-1405

2n = 4x = 28, BBAA N

N

Ethiopia unknown

T. durum Desf. ‘Langdon’‘Krasnokutka’

2n = 4x = 28, BBAA N

N

USA

RussiaT. aethiopicum Jakubz. k-19217

k-19289

k-19305

k-19321

k-43767

k-44505

2n = 4x = 28, BBAA T2A:4B:inv 5A

N

N

N

N

N

Ethiopia

Ethiopia

Ethiopia

Ethiopia

Ethiopia

EthiopiaT. aestivum L. Chinese Spring

Courtot

Mironovskaya 808

2n = 6x = 42, BBAADD N

N

N

China

France

Ukraine

*Genomic formulae are given according to Feldman (2001).†N, normal karyotype. Karyotype structure was determined by C-banding.‡Karyotype structure was determined by B. Friebe (Friebe and Gill 1996).

Table 1. Plant material used in this study.

204 (98.5%) were polymorphic. An example of thepolymorphisms obtained using primer RAPD-64 is shown inFig. 1. On the basis of RAPD analysis we constructed adendrogram that shows phylogenetic relations between dif-ferent lines of Aegilops (sect. Sitopsis) and Triticum species(Fig. 2). Eighteen lines fell into 3 clades. The first clade in-cluded 10 lines of 4 Sitopsis species (group 1 on Fig. 2), thesecond clade had five lines of Ae. speltoides (group 2), andthe third clade included the 3 Triticum lines (group 3). Ingroup 1, each species formed a separate clade. The geneticdistance between Ae. sharonensis and Ae. longissima wasshorter than between any other species. The distances be-tween the 5 genotypes of Ae. speltoides were longer than be-tween any of the other 4 Sitopsis species.

C-bandingC-banding analysis of wheat and Aegilops accessions was

carried out to determine their genome and chromosome con-stitution. All diploid Aegilops accessions had normal karyo-types, although C-banding patterns were polymorphic.Wheat species of the Timopheevi group differed in karyo-type structure. Three accessions of T. araraticum (Tri11508,Tri11939, and Tri17418), 2 of T. timopheevii, and T. kiharaehad normal karyotypes. Four different types of chromosomalrearrangements were identified in the other T. araraticumlines. A complex translocation (T3At:7At + T2At:4G:7G)was found in Tri11365 from Nakhichevan, a double cyclictranslocation T2At:6At:4G was detected in Tri 16599 fromArmenia, a single translocation T5At:3G was observed inTri17419 from Iraq, and a paracentric inversion of chromo-some 7At was present in Iranian accession Tri11509(Table 1). In addition to translocation polymorphisms, all ac-cessions differed in the C-banding patterns (data not shown).

In contrast, only 1 accession of Emmer wheat possessedchromosomal rearrangements, i.e., T. aethiopicum (k-19217),had a translocation between 2A and 4B chromosome and a

paracentric inversion of chromosome 5A. All wheats of thisgroup displayed broad polymorphisms in C-banding pat-terns.

Spelt52 and Spelt1 in Ae. speltoidesThe Spelt1 and Spelt52 content differed between

Ae. speltoides accessions. For example, Spelt1 was moreabundant than Spelt52 in accession k-389 (Adonina et al.2004), whereas the reverse was the case in TS01 (Fig. 3). InTS01 Spelt52 occurred at 6 regularly resolved clusters ob-served on 4 chromosomes (no signals were recorded onchromosome 1S). In all Ae. speltoides accessions Spelt52was most prominent at interstitial sites rather than subtelo-meric regions (Figs. 3, 4c, and 4g). Some sites in a numberof lines were heteromorphic between homologous chromo-somes. Spelt1 regularly hybridized to subtelomeric regions,and all chromosomes of TS05 and TS42 possessed Spelt1signals on both arms, although some signals were very faintand not resolved in all cells, i.e., on chromosome 2S in TS42(Fig. 3). In contrast, in line TS01 only 1 major Spelt1 signalwas observed, on 3SL, and an additional minor, inconsis-tently detected interstitial locus on 4SL. The 3 studied linesof Ae. speltoides had distinct Spelt52 hybridization patterns,but several distal Spelt52 sites (on 3SL, 6SS, and 7SS) werecommon to all.

Spelt52 in Ae. longissima and Ae sharonensisThe total number of Spelt52 sites per haploid genome was

8 in Ae. longissima (TL03), 4 in Ae. longissima (TL05), and9 in Ae. sharonensis (TH01). Spelt52 sites were distributedon 1 or both arms of 6 chromosomes (1Sl, 2Sl, 3Sl, 4Sl, 5Sl,and 7Sl) in Ae. longissima (no sites on 2Sl and 5Sl in lineTL05) and all chromosomes except 6Ssh of Ae. sharonensis.In each species 3 to 5 sites were polymorphic. Spelt52 re-peats were subtelomeric, except for minor loci on the distalregions of chromosome arms 1SlS (TL03), 1SlL, and 7SlL(TL05) in Ae. longissima and 4SshL in Ae. sharonensis. Only2 chromosomes of these species showed similar hybridiza-tion patterns: homoeologues of group 3 had 1 or 2 strongsubtelomeric signals, whereas the group 6 chromosomes hadno signal (Figs. 4a, and 4b).

There was no detectable FISH signal using Spelt1 to anyspecies in Aegilops sect. Sitopsis except Ae. speltoides or ofSpelt52 to Ae. bicornis or Ae. searsii (data not shown).

Spelt 52 and Spelt1 in the Timopheevi groupThe distribution of Spelt52 probe was examined in 6 ac-

cessions of 3 species (T. araraticum, T. timopheevii, andT. kiharae). Altogether, 5 hybridization sites were detected:2AtS, 4AtS, 6AtS, 1GS, and 6GL. The total number of sig-nals at metaphase was from 2 to 6 (Table 3). Hybridizationsites were usually small or medium; the only large signalwas on 6GL chromosome (Fig. 4f).

Hybridization patterns of Spelt1 repeat were examined in10 accessions of the Timopheevi group. The signals weredetected on 9 chromosome pairs: 2At, 6At, and 1G–7G (Ta-ble 4). The number of Spelt1 signals varied from 8 to 12among lines. Signal size varied significantly among differentchromosomes and among accessions (Fig. 4d). The lines thatshowed a similar number of hybridization sites (e.g., 10)may nevertheless differ in their chromosome distribution

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Salina et al. 1027

Primer Sequence (5′→3′)No. ofscorable bands

RAPD-31 GTCGACGCATG 8RAPD-33 TGGTGCTGAGA 14RAPD-58 ATCGGTCGGTA 11RAPD-64 ATGCCCTAGAG 18RAPD-68 CAACTAGACGG 8RAPD-74 CCCTGAAACAC 6RAPD-82 GATGGAACGAC 11RAPD-91 CATACGATACG 8RAPD-156 TGCACACTGA 8RAPD-158 TGGTCTCTGA 13RAPD-159 TGGTCAGTGA 6RAPD-177 CCGAACGGGT 11RAPD-180 TCACCGAACG 16RAPD-181 GCCAATCCTG 14RAPD-186 TCGTCACTGA 14RAPD-342 GGCTGCAATG 9RAPD-401 CTGAACCGCT 18RAPD-565 CCAAAATCGTA 14

Table 2. List of primers, their sequence, and the number ofRAPD bands they generate.

(Table 4). Although 2 Transcaucasian accessions ofT. araraticum possessed different translocations (T3At:7At +T2At:4G:7G in Tri11365 and T2At:6At:4G in Tri16599), theSpelt1 repeats were detected in similar positions on the samechromosome arms. The distribution of Spelt1 and Spelt52sites in 2 T. timopheevii accessions and the derived amphi-ploid T. kiharae were identical. At the same time, 4 Iraqi ac-cessions of T. araraticum were highly polymorphic withrespect to number, distribution, and Spelt1 signal intensities.

Spelt1 and Spelt52 in Emmer wheatsThe distribution of Spelt1 repeat was polymorphic. No de-

tectable signals were observed in 8 accessions of 5 species(T. dicoccoides, T. polonicum, T. carthlicum, T. aethiopicum,and T. aestivum), but in the remaining 14 lines hybridizationwas observed on chromosome arms 2BL (6 lines), 6BS (5lines), 3BL (3 lines), 2AL and 2AS (3 and 2 lines, respec-tively), and 6BL (1 line). The total number of Spelt1 siteswas 2 (9 lines), 4 (4 lines), and 6 (1 line) (Fig 4e; Table 4).No hybridization with Spelt52 probe was detected in Emmerwheats.

Discussion

Aegilops species relationsBy RAPD analysis we show that Ae. speltoides is phylo-

genetically distinct from other Aegilops species of sectionSitopsis (Fig. 2). This agrees with (i) the classical taxonomyof Eig (1929), who subdivided the Sitopsis section into 2subsections: Truncata (Ae. speltoides alone) and Emarginata(Ae. longissima, Ae. sharonensis, and Ae. bicornis; Ae. searsiiwas discovered later and also placed in subsectionEmarginata); (ii) microsatellite analysis of the same acces-sions of Ae. speltoides, Ae. searsii, and Ae. longissima(Adonina et al. 2005); (iii) RFLP analysis (Sasanuma et al.1996); (iv) C-banding (Friebe and Gill 1996); and (v) FISHwith highly repetitive DNA sequences and ribosomal RNAgene families (Badaeva et al. 1996a,1996b). Of the 4 speciesof subsection Emarginata, Ae. longissima and Ae. sharonensisare the most closely related, and the genetic distance be-tween them is the shortest (Fig. 2). These 2 species differ

only by a species-specific translocation in Ae. longissimabetween chromosomes 4 and 7 with breakpoints in distalregions of the long arms (Tanaka 1955; Ankory and Zohary1962; Friebe et al. 1993; Friebe and Gill 1996; Zhang et al.2001). Thus, all data support the pattern of branching weobtained in the RAPD phylogenetic analysis (Fig. 2). Thisprovides confidence that the scheme represents a close ap-proximation to the true pattern of divergence in Aegilopssect. Sitopsis (Fig. 5). We use this scheme to interpret themost probable patterns of Spelt1 and Spelt52 divergence.

Spelt1 and Spelt52 in Sitopsis speciesSpelt1 repeats are organized in simple, near identical

tandem arrays (6 PCR products of Spelt1 from various ac-cessions of Ae. speltoides were 98% homologous (Salina etal. 2004a)). In contrast, Spelt52 has a more complex organi-zation involving 2 types of monomers (Spelt52.1 andSpelt52.2) with a conserved region of 280 bp and non-homologous sequences of 96 and 110 bp, respectively(Salina et al. 2004a). But even within a species the con-served region shows less than 84% homology (Salina et al.2004a). The monomer lengths of Spelt1 (178 bp) andSpelt52 (376–390 bp) are thought to be related to DNA fold-ing around 1 or 2 nucleosomes, respectively (Trifonov 1985;Heslop-Harrison 1998).

In Ae. speltodies the Spelt1 sequence is localized exclu-sively to subtelomeric regions, whereas Spelt52 has majorinterstitial and more minor subterminal sites in most lines(Figs. 3 and 4). These sequences can colocalize at subtelo-meric regions as revealed by overlapping FISH signals andby the interspersed sequence within chromosome fiber ar-rays (data not shown). Spelt52 occurs at more distal loca-tions than pSc119.2, and these sequences are probably notinterspersed at either subterminal or interstitial sites (theoverlapping signals in Fig. 4c probably relate to the resolu-tion of the FISH experiment). Thus only Spelt1 and Spelt52are likely to have a subtelomeric structural role, including,perhaps, a role at meiosis, when subtelomeric regions arethought to facilitate pairing (Bass 2003; Cowan et al. 2001;Heslop-Harrison 2000).

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Fig. 1. Amplification products generated with primer RAPD-64. Genotypes used for RAPD analysis: lanes 1–5 Ae. speltoides, (1)TS47, (2) TS42, (3) TS41, (4) TS05, (5) TS01; lanes 6–10 Ae. longissima, (6) TL15, (7) TL09, (8) TL05, (9) TL04, (10) TL03; lanes11–12 Ae. sharonensis, (11) TH02, (12) TH01; lane 13 Ae. bicornis, TB05; lanes 14–15 Ae. searsii, (14) TE12, (15) TE16; lane 16T. durum ‘Langdon’; lane 17, T. timopheevii; lane 18, T. aestivum ‘Chinese Spring’; lane M, molecular weight marker, λ phage di-gested with PstI.

Significant intraspecific polymorphism in the distributionof Spelt1 and Spelt52 signals in Ae. speltoides is evidence ofdynamic amplifications, reductions, or eliminations of entireblocks of repeats. Thus, in line TS42 we found 2 distinctSpelt52 hybridization patterns on 5S chromosomes, one withstrong double signals in the distal quarter of the long arm(variant 1) and a second with an additional, more proximal,fainter signal (variant 2). Both variants occurred in the plantshown in Fig. 4c. Similar chromosomes (variant 1) werefound in lines TS42 and TS01 (Fig. 3) and line No. 829 de-scribed earlier (Friebe et al. 2000), but the double signal wasabsent in TS05.

Comparisons of Spelt1 and Spelt52 content among diploidAegilops species and between polyploid wheats and Aegilopsspecies enable predictions of the patterns of repeat amplifi-cation during Ae. speltoides evolution. The most parsimoni-ous explanation of these data is as follows:(a) In the early Ae. speltoides ancestor Spelt1 was amplified

at subtelomeric loci resulting in higher sequence copynumbers than in other Aegilops species in sect Sitopsis.This is shown by squash-dot (86 accessions checked),Southern hybridization, and FISH, which generates sig-nal only in Ae. speltoides (Pestsova et al. 1998; Fig. 3).Outside sect. Sitopsis, the only other species in which

Spelt1 has been detected (as a minor band by Southernhybridization) is in T. monococcum (Salina et al. 1998).In Ae. speltoides in which Spelt1 is abundant, variationin Spelt1 signal location ranges from 2 to 28 sites, andintensity reflects changing patterns of sequence distribu-tion and copy numbers at individual loci. Thus, ongoingdynamic changes in Spelt1 distribution are associatedwith Ae. speltodies divergence. There is also consider-able intraspecific variation in RAPD patterns inAe. speltoides compared with other Sitopsis species(Fig. 2). This genetic diversity might be related to theoutbreeding nature of this species.

(b) Spelt52 and its analogues pGc1R-1 and pAesKB52 arefound in Ae. speltoides, Ae. longissima, and Ae. sharonensisbut not in Ae. bicornis or Ae. searsii or any other diploidAegilops species (Anamthawat-Jonsson and Heslop-Harrison 1993; Zhang et al. 2002; Salina et al. 2004b).This is best explained by 2 independent Spelt52amplification events, one in the Ae. speltoides lineageand a second in the ancestor to Ae. longissima andAe. sharonensis (Fig. 5). These latter 2 species havesimilar pSc119.2 labeling patterns to Ae. bicornis andAe. searsii but differ from them by having moreheterochromatic chromosomes (Badaeva et al. 1996a;

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Fig. 2. Neighbor-joining dendrogram showing phylogenetic relations among 18 lines and species of Aegilops and Triticum: Asp,Ae. speltoides; Alo, Ae. longissima; Ash, Ae. sharonensis; Ase, Ae. searsii; Abi, Ae. bicornis; Trtimoph, T. timopheevii Coll.2 N21;TraestCS, T. aestivum ‘Chinese Spring’; Trdurum, T. durum ‘Langdon’; the numbers of other accessions are given in Table 1. Thedendrogram was generated from 100 bootstrapped multiple analyses, and the numbers at the forks indicate the number of times thebranch point occurred (out of 100). The scale of genetic distance is given.

Friebe and Gill 1996). The amplification of Spelt52 andperhaps other classes of repetitive sequences in thecommon ancestor of Ae. sharonensis and Ae. longissimamay have contributed to the gain of heterochromatin.

(c) Spelt52 is present in wheat species of Timopheevi groupbut not in Emmer wheats. Thus amplification of Spelt52repeats in Ae. speltoides may have occurred after theemergence of the ancestral form of Emmer wheat butbefore the emergence of the Timopheevi group of wheats(Fig. 5 and see also section on polyploid wheats).

Independent amplification and reductions of repeats indifferent evolutionary lineages may be a feature of tandemrepeat evolution in the Triticeae. For example, the 350family/pSc200/pSc74 repeats are characteristic of S. cereale,S. montanum, Dasypyrum villosum, D. breviaristatum, andAgropyron cristatum. However, these repeats are undetect-able by hybridization methods to other rye species (Vershininet al. 1996). The 2 independent Spelt52 amplification eventsin Sitopsis divergence (Fig. 5) involved various combina-tions of Spelt52.1 and Spelt52.2 repeats. Characteristic ofAe. speltoides (except for the sample TS01) is an alterna-tion of Spelt52.1 and Spelt52.2, whereas in Ae. sharonensisand Ae. longissima there is repetition of only the Spelt52.2monomer (Salina et al. 2004a). What governs the amplifica-tion or reduction of particular subtelomeric tandem repeatsat a locus and more globally is unknown. However, theamount of homology between repeats might influence homo-genization rates within and between individual tandem clus-ters (Polanco et al. 1998; Schlotterer and Tautz 1994). Thechanging patterns of repeats at the telomere of different linesof Ae. speltoides may, over time, help establish isolation bar-riers between Ae. speltoides lines and expedite speciation.

Spelt1 and Spelt52 in polyploid wheatsIn the Timopheevi group Spelt1 generates more signal

than Spelt52 on average (5.3 versus 2 loci per haploid ge-nome), and signal intensities are usually higher (Tables 3and 4). The distribution of the 2 repeats in genomes ofT. timopheevii/T. araraticum was different. The Spelt1 repeatwas detected at subterminal positions on either the short orlong arm of all 7 G-genome chromosomes and on chromo-somes 2At and 6At. Chromosome 6At is known to undergostructural rearrangements as a result of species-specifictranslocation 6AtS/1GS (Jiang and Gill 1994). Therefore, itis likely that the Spelt1 site on 6At was derived from a chro-mosome of the G-genome donor. It is more difficult to ex-plain the occurrence of the repeat on chromosome 2At,which is thought to be nearly unmodified relative to the pa-rental chromosome (Salina et al. 2006). Possibly the Spelt1repeat was already present on chromosome 2A of diploidwheat, which gave rise to T. timopheevii and was amplifiedat this locus after formation of the initial allopolyploid. Indi-rect evidence supporting this assumption is that Spelt1 isdetected in minor quantities (400-fold less than inAe. speltoides) using dot-blot hybridization on T. monococcum(Salina et al. 1998). Intraspecific variation in the distributionof Spelt1 probably results from elimination of the repeat inT. araraticum divergence.

All wheat species of the Emmer group showed no hybrid-ization with the Spelt52 probe, and the number of Spelt1sites was significantly less than wheats of the Timopheevigroup. An examination of 75 accessions of Emmer speciesshowed drastically different Spelt1 content with abundancecomparable to Timopheevi wheat in only 20% accessions(Pestsova et al. 1998). The Spelt1 copy number also differed

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Fig. 3. Generalized idiogram of Ae. speltoides (lines TS42, TS01, TS05), Ae. longissima (line TL03, TL05), and Ae. sharonensis (lineTH01). For Ae. speltoides, schemes show Spelt1 (left) and Spelt52 (right) probe distribution. For the remaining species, schemes showSpelt52 distribution. Asterisks indicate inconsistent FISH sites. Asterisks in black squares indicate heteromorphisms between thehomologue chromosomes.

significantly between and within hexaploid species(Pestsova et al. 1998). In Emmer species the maximum num-ber of chromosomes containing Spelt1 was 6. Four of theSpelt1 sites were on orthologous chromosome arms of the 2wheat lineages (2AL/2AtL, 2BL/2GL, 3BL/3GL, 6BS/6GS).The occurrence of Spelt1 on homoeologous chromosomes

2AL and 2AtL confirm our suggestion that this repeat wasalready present in the ancestral form of diploid wheat.

Polyploid evolutionOne of the most important driving forces in angiosperm

evolution is the formation of allopolyploids. Indeed the evo-

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Fig. 4. FISH to mitotic metaphase chromosomes of (a) Ae. sharonensis, TH01; (b) Ae. longissima, TL05; (c) Ae. speltoides, TS42;(d) T. araraticum, Tri11939; (e) T. dicoccoides, TTD20; (f) T. araraticum, Tri11939; (g) Ae. speltoides, TS05. The probe combinations:(a–c, f) pSc119 and Spelt52; (d, e) pSc119 and Spelt1; (g) Spelt52 and Spelt1. Probes were detected with fluorescein isothiocyanate(green) and cy3 (red): Spelt52 (red) and pSc119 (green) (c); Spelt52 (green) and pSc119 (red) (a, b, f); pSc119 (red) and Spelt1(green) (d, e); Spelt52 (red) and Spelt1 (green) (g). The yellow signal arises from colocalised Spelt1 and Spelt52. Arrows show poly-morphism between homologous chromosomes.

Chromosome arm*

Species AccessionTotal sites perhaploid genome 2AtS 4AtS 6AtS 1GS 6GL

T. araraticum Tri11939 1 – – – – XLTri11508 3 – M S S –Tri17418 2 S – – – XL

T. timopheevii Tri26 2 M – – M –N21 2 M – – M –

T. kiharae 2 M – – M –

*Signal size: S, small; M, medium; L, large; XL, extra large.

Table 3. Distribution of Spelt52 repeats in species of the Timopheevi group.

lution of many, perhaps all angiosperms, has involvedpolyploidy. Evidence for this comes from sequence data in-dicating that apparently “diploid” species are palaeo-polyploids (Lagercrantz and Lydiate 1996). This realizationhas enhanced interest in the genetic consequences ofpolyploidy both in natural and newly synthesized polyploids.In a range of different systems polyploidy has been associ-ated with rDNA gene conversion, the loss, gain, and evolu-tion of markers such as amplified fragment lengthpolymorphisms, and all known forms of chromosomaltranslocation and mutation (Leitch and Bennett 1997; Levyand Feldman 2002; Liu and Wendel 2003; Rieseberg 2001;Soltis and Soltis 1995; Soltis and Soltis 1999; Soltis et al.2004; Wendel 2000). Here we show that there is rather littlechange in the abundance of tandem repeats in wheat that canbe attributed to polyploidy per se.

Wild emmer wheat (T. dicoccoides; genome BBAA), theancestor of macaroni wheat (T. durum; genome BBAA),T. timopheevii, and wild T. araraticum (genome GGAtAt) are

allotetraploid. The most probable parents are thought to beancestral forms of Ae. speltoides (maternal) and T. urartu(paternal) (Dvorak et al. 1988; Tsunewaki 1996; Huang etal. 2002). Although the 2 wheat species have originatedfrom similar parental forms, they are probably from inde-pendent hybridization events separated by several tens orhundreds of thousands of years. Thus, T. dicoccoides proba-bly arose first, and T. araraticum (T. timopheevii) appearedmore recently, about 50 000 to 300 000 years ago (Mori etal. 1995; Huang et al. 2002; Levy and Feldman 2002). Dur-ing this period, some changes must have occurred in the ge-nome of Ae. speltoides. One of these was presumably theloss of the 5S rDNA locus on the chromosome 1S since adistinct 5S rDNA locus is present on chromosomes ofhomoeologous group 1 in all Triticum and Aegilops speciesexcept Ae. speltoides and T. timopheevii (Badaeva et al.1996b; Badaeva 2000). A second change has been in the oc-currence, abundance, and distribution of tandem repeats.

Given that Spelt52 cannot be detected in Emmer wheats,

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Chromosome arm

Species AccessionTotalsites* 2A/At S 2A/At L 6A/At S 1B/G L 2B/G L 3B/G L 4B/G L 5B/G L 6B/G S 6B/G L 7B/G S

T. araraticum Tri11365 5 – – XL† L – – M M S – –Tri11508 5 – M – – – XL M S – – LTri11509 5 – – XL – XL L M – S – –Tri11939 6 – – XL – XL M S S S – –Tri16599 5 – – XL L – – M M S – –Tri17418 4 – – XL S – – M M – – –Tri17419 5 – – XL – M – M M L – –

T. timopheevii N21 6 – S XL M M – M M – – –Tri26 6 – S XL L L – M M – – –

T. kiharae 6 – S XL M L – M M – – –T. dicoccoides TTD20 1 – – – – – – – – L – –

PI478703 1 – – – – – L – – – – –PI538657 1 – – – – – L – – – – –PI467013 1 – – – – – – – – L – –

1 – L – – – – – – – – –PI428126 0 – – – – – – – – – – –PI471750 3 – L – – L – – – L – –

T. dicoccum k-1405 1 – – – – L – – – _ – –k-5154 2 M – – – L – – – _ – –

T. polonicum 0 – – – – – – – – _ – –T. carthlicum k-19726 0 – – – – – – – – _ – –T. durum ‘Langdon’ 1 – – – – – – – – L – –

‘Krasnokutka’ 2 M – – – – L – – _ – –T. aethiopicum k-19217 0 – – – – – – – – _ – –

k-19289 2 – – – – L – – – L – –k-19305 0 – – – – – – – – _ – –k-19321 2 – L – – L – – – _ – –k-43767 1 – – – – L – – – _ – –k -44505 0 – – – – – – – – _ – –

T. aestivum ‘Chinese Spring’ 0 – – – – – – – – _ – –‘Courtot’ 0 – – – – – – – – _ – –‘Mir.808’‡ 1 – – – – – – – – _ L –

*Total number of sites per haploid genome.†Signal size: S, small; M, medium; L, large; XL, extra large.‡Mironovskaya 808.

Table 4. Distribution of Spelt1 repeat on chromosomes of different polyploidy wheat species.

but is found in T. timopheevii and on all 7 chromosomes ofAe. speltoides, indicates that either (i) Spelt52 increased incopy number in Ae. speltoides after the divergence ofEmmer wheats but before the divergence of the Timopheevigroup or (ii) the sequence was eliminated in Emmer wheats.An analysis of 11 newly synthesized amphiploids of Aegilopsand Triticum and their parental forms suggests that the for-mer hypothesis is most likely. In synthetic material Spelt52was found to either amplify or remain stable in copy num-ber, whereas the copy number of Spelt1 stayed the same orwas reduced (Salina et al. 2004b). Thus, if the synthetic ma-terial reflects early evolution of Emmer wheat, then the ab-sence of Spelt52 in Emmer wheat is a consequence of itsinheritance from ancestral Ae. speltoides rather than its sub-sequent loss from Emmer wheat. If so, amplification ofSpelt52 in Ae. speltoides took place after the emergence ofEmmer wheats but before the occurrence of Timopheeviwheats (Fig. 5). There are considerable differences betweenT. timopheevii and modern Ae. speltoides in the number andlocation of Spelt52 repeats indicating subsequent divergencein either or both Timopheevi wheats and Ae. speltodies.

Acknowledgements

We are grateful to Prof. M. Feldman for providing theseeds and for helpful comments about the manuscript. Thiswork was supported by grants from INTAS (the International

Association for the Promotion of Cooperation with Scien-tists from the New Independent States of the Former SovietUnion) (01–0537) and the Natural Environment ResearchCouncil, the Russian State Foundation for Basic Research(projects 02–04–48113 and 05–04–48453), and the program“Dynamics of gene-pools of human, animal and plants”.

References

Ankory, H., and Zohary, D. 1962. Natural hybridization betweenAegilops sharonensis and Ae. longissima: a morphological andcytological study. Cytologia, 27: 314–324.

Adonina, I.G., Salina, E.A., Efremova, T.T., and Pshenichnikova,T.A. 2004. The study of introgressive lines of Triticumaestivum × Aegilops speltoides by in situ and SSR analyses.Plant Breed. 123: 220–224.

Adonina, I.G., Salina, E.A., Pestsova, E.G., and Röder, M.S. 2005.Transferability of wheat microsatellites to diploid Aegilops spe-cies and determination of chromosomal localizations ofmicrosatellites in the S genome. Genome, 48: 959-970.

Anamthawat-Jonsson, K., and Heslop-Harrison, J.S. 1993. Isola-tion and characterization of genome-specific DNA sequences inTriticeae species. Mol. Gen. Genet. 240: 151–158.

Appels, R., Moran, L.B., and Gustafson, J.P. 1986. Ryeheterochromatin I. Studies on clusters of the major repeating se-quences and the identification of a new dispersed repetitive se-quences element. Can. J. Genet. Cytol. 28: 645–657.

© 2006 NRC Canada

Salina et al. 1033

Fig. 5. Divergence of species in Aegilops sect. Sitopsis inferred from RAPD data (as in Fig. 2). The divergence of Emmer wheats (ca.300 000 – 500 000 years ago) occurred before T. timopheevii (50 000 – 300 000 years before the present) (Levy and Feldman 2002)and involved allopolyploidy with AA genome progenitors. The most parsimonious explanation of the tandem repeat data is that therehave been 2 independent amplification events for Spelt52 and 1 for Spelt1 during Aegilops divergence.

Badaeva, E.D., Budashkina, E.B., Badaev, N.S., Kalinina, N.P., andShkutina, F.M. 1991. General features of chromosome substitu-tions in Triticum aestivum x T. timopheevii hybrids. Theor. Appl.Genet. 82: 227–232.

Badaeva, E.D. 2000. Chromosome analysis in investigation of theorigin of the B- and G-genomes of polyploid wheats. Membr.Cell Biol. 18: 216–229. [In Russian.]

Badaeva, E.D., Badaev, N.S., Gill B.S., and Filatenko, A.A. 1994.Intraspecific karyotype divergence in Triticum araraticum. PlantSyst. Evol. 192: 117–145.

Badaeva, E.D., Friebe, B., and Gill, B.S. 1996a. Genome differen-tiation in Aegilops. 1. Distribution of highly repetitive DNA se-quences on chromosomes of diploid species. Genome, 39: 293–306.

Badaeva, E.D., Friebe, B., and Gill B.S. 1996b. Genome differenti-ation in Aegilops. 1. Physical mapping of 5S and 18S-26S ribo-somal RNA gene families in diploid species. Genome, 39:1150–1158.

Bass, H.W. 2003. Telomere dynamics unique to meiotic prophase:formation and significance of the bouquet. Cell. Mol. Life Sci.60: 2319–2324.

Bedbrook, R.J., Jones, J., O’Dell, M., Thompson, R.J., and Flavell,R.B. 1980. A molecular description of telomeric heterochromatinin Secale species. Cell, 19: 545–560.

Bildanova, L.L., Salina E.A., and Pershina, L.A. 2002. Investiga-tion of the backcross progeny of barley-wheat hybrids by theRAPD and RAMPO methods. Rus. J. Gen. 38: 257–263.

Chen, P.D., and Gill, B.S. 1983. The origin of chromosome 4A,and genomes B and G of tetraploid wheats. In Proceedings ofthe 6th International Wheat Genetics Symposium, Kyoto, Japan.Edited by S. Sakamoto. pp. 39–48.

Contento, A., Heslop-Harrison, J.S., and Schwarzacher T. 2005.Diversity of a major repetitive DNA sequence in diploid andpolyploid Triticeae. Cytogenet. Genome Res. 109: 34–42.

Cowan, C.R., Carlton, P.M., and Cande, W.Z. 2001. The polar ar-rangement of telomeres in interphase and meiosis. Rab1 organi-zation and the bouquet. Plant Physiol. 125: 532–538.

Dvorak, J., and Appels, R. 1982. Chromosome and nucleotide se-quence differentiation in genomes of polyploid Triticum species.Theor. Appl. Genet. 63: 349–360.

Dvorak, J., and Zhang, H.-B. 1992. Reconstruction of the phylog-eny of the genus Triticum from variation in repeated nucleotidesequences. Theor. Appl. Genet. 84: 419–429.

Dvorak, J., Mcguire, P.E., and Cassidy, B. 1988. Apparent sourcesof the � genomes of wheats inferred from polymorphism inabundance and restriction fragment length of repeated nucleo-tide sequences. Genome, 30: 680–689.

Eig, A. 1929. Monographisch-kritische Ubersicht der GattungAegilops. Repertorium Specierum Novarum Regni Vegetabilis,55: 1–228.

Feldman, M., Lupton, F.G.H., and Miller, T.E. 1995. Wheats. InEvolution of crop plants. Edited by J. Smartt and N.W.Simmonds. Longman Group, London, U.K. pp. 184–192.

Feldman, M. 2001. Origin of cultivated wheat. In The world wheatbook. Edited by A.P. Bonjean and W.J. Angus. Lavoisier Pub-lishing, Paris, France. pp. 3–56.

Felsenstein, J. 1989. PHYLIP: Phylogeny Inference Package (Ver-sion 3.2). Cladistics, 5: 164–166.

Flavell, R.B. 1982. Amplification, deletion and rearrangement: ma-jor sources of variation during species divergence. In Genomeevolution. Edited by G.A. Dover and R.B. Flavell. AcademicPress, London.

Friebe, B., and Gill, B.S. 1996. Chromosome banding and genomeanalysis in diploid and cultivated polyploid wheats. In Methods

of genome analysis in plants. Edited by P.P. Jauhar. CRC Press,New York. pp. 39–60.

Friebe, B., Tuleen, N., Jiang, J., and Gill, B.S. 1993. Standardkaryotype of Triticum longissimum and its cytogenetic relation-ship with T. aestivum. Genome, 36: 731–742.

Friebe, B., Qi, L.L., Nasuda, S., Zhang, P., Tuleen, N.A., and Gill,B.S. 2000. Development of a complete set of Triticum aestivum-Aegilops speltoides chromosome addition lines. Theor. Appl.Genet. 101: 51–58.

Gill, B.S., and Friebe, B. 1998. Plant cytogenetics at the dawn ofthe 21st century. Curr. Opin. Plant Biol. 1: 109–115.

Gill, B.S., Friebe, B., and Endo, T.R. 1991. Standard karyotypeand nomenclature system for description of chromosome bandsand structural aberrations in wheat (Triticum aestivum). Ge-nome, 34: 830–839.

Giorgi, D., D’ovidio, R., Tanzarella, O.A., and Porceddu, E. 2002.RFLP analysis of Aegilops species belonging to the Sitopsis sec-tion. Genet. Resour. Crop Evol. 49: 145–151.

Heslop-Harrison, J.S. 2000. Comparative genome organization inplants: from sequences and markers to chromatin and chromo-some. Plant Cell, 12: 617–635.

Huang, S., Sirikhachornkit, A., Su, X., Faris, J., Gill, B.,Haselkorn, R., and Gornicki, P. 2002. Genes encoding plastidacetyl-CoA carboxylase and 3-phosphoglycerate kinase of theTriticum/Aegilops complex and the evolutionary history ofpolyploids wheat. Proc. Natl. Acad. Sci. U.S.A. 99: 8133–8138.

Jiang, J., and Gill, B.S. 1994. Different species-specific chromo-some translocation in Triticum timopheevii and T. turgidum sup-port diphyletic origin of polyploid wheats. Chromosome Res. 2:59–64.

Jones, J.D.G., and Flavell, R.B. 1982. The structure, amount andchromosomal localization of defined repeated DNA sequencesin species of the genus Secale. Chromosoma, 86: 613–641.

Katsiotis, A., Hagidimitriou, M., and Heslop-Harrison, J.S. 1997.The close relationship between the A and B genomes inAvena L. (Poaceae) determined by molecular cytogenetic analy-sis of total genomic, tandemly and dispersed repetitive DNA.Ann. Bot. 79: 103–107.

Kerby, K., and Kuspira, J. 1987. The phylogeny of the polyploidwheats Triticum aestivum (bread wheat) and Triticum turgidum(macaroni wheat). Genome, 29: 722–737.

Khlestkina, E.K., and Salina, E.A. 2001. Genome-specific markersof tetraploid wheats and their putative diploid progenitor spe-cies. Plant Breed. 120: 227–232.

Kishii, M., and Tsujimoto, H. 2002. Genus-specific localization ofthe TaiI family of tandem-repetitive sequences in either thecentromeric or subtelomeric regions in Triticeae species(Poaceae) and its evolution in wheat. Genome, 45: 946–955.

Kilian, A., and Kleinhofs, A. 1992. Cloning and mapping oftelomere-associated sequences from Hordeum vulgare L. Mol.Gen. Genet. 235: 153–156.

Kumar, S., Tamura K., Jakobsen, I.B., and Nei, M. 2001. MEGA2:Molecular Evolutionary Genetics Analysis software. ArizonaState University, U.S.A.

Lagercrantz, U., and Lydiate, D.J. 1996. Comparative genomemapping in Brassica. Genetics, 144: 1903–1910.

Leitch, I.J., and Bennett, M.D. 1997. Polyploidy in angiosperms.Trends Plant Sci. 2: 470–476.

Leitch, A.R., Schwarzacher, T., Jackson, D., and Leitch, I.J. 1994.In situ hybridization: a practical guide. BIOS, Oxford.

Levy, A.V., and Feldman, M. 2002. The impact of polyploidy ongrass genome evolution. Plant Physiol. 130: 1587–1593.

Lilienfeld, F., and Kihara, H. 1934. Genomanalyze bei Triticum

© 2006 NRC Canada

1034 Genome Vol. 49, 2006

und Aegilops. V. Triticum timopheevi Zhuk. Cytologia, 6: 87–122.

Lim, K.Y., Matyasek, R., Lichtenstein, C.P., and Leitch, A.R. 2000.Molecular cytogenetic analyses and phylogeny of the Nicotianasection Tomentosae. Chromosoma, 109: 245–258.

Liu, B., and Wendel, J.F. 2003. Epigenetic phenomena and the evo-lution of plant allopolyploids. Mol. Phylogenet. Evol. 29: 365–379.

Liu, C.J., Atkinson, M.D., Chinoy, C.N., Devos, K.M., and GaleM.D. 1992. Nonhomoeologous translocations between group 4,5 and 7 chromosomes within wheat and rye. Theor. Appl. Genet.83: 305–312.

Maestra, B., and Naranjo, T. 1999. Structural chromosome differ-entiation between Triticum timopheevii and T. turgidum andT. aestivum. Theor. Appl. Genet. 98: 744–750.

McIntyre, C.L., Pereira, S., Moran, L.B., and Appels, R. 1990.New secale cereale (rye) DNA derivatives for the detection ofrye chromosome segments in wheat. Genome, 33: 635–640.

Mori, N., Lui, Y.G., and Tsunewaki, K. 1995. Wheat phylogenydetermined by RFLP analysis of nuclear DNA: II. Wildtetraploid wheats. Theor. Appl. Genet. 90: 129–134.

Mukai, Y., Nakahara, Y., and Yamamoto, M. 1993. Simultaneousdiscrimination of the three genomes in hexaploid wheat bymulticolor fluorescence in situ hybridization using total genomicand highly repeated DNA probes. Genome, 36: 489–494.

Nagaki, K., Tsujimoto, H., Isono, K., and Sasakuma, T. 1995. Mo-lecular characterization of a tandem repeat, Afa family, and itsdistribution among Triticeae. Genome, 38: 479–486.

Nagaki, K., Tsujimoto, H., and Sasakuma, T. 1998. H genome spe-cific repetitive sequence, pEt2, of Elymus trachycaulus in part ofAfa family of Triticeae. Genome, 41: 134–136.

Naranjo, T., Roca, A., Goicoecha, P.G., and Giraldez, R. 1987.Arm homoeology of wheat and rye chromosomes. Genome, 29:873–882.

Nei, M., and Li, W.H. 1979. Mathematical model for studing ge-netic variation in terms of restriction endonucleases. Proc. Natl.Acad. Sci. U.S.A. 76: 5269–5273.

Pestsova, E.G., Goncharov, N.P., and Salina, E.A. 1998. Elimina-tion of a tandem repeat of telomeric heterochromatin duringevolution of wheat. Theor. Appl. Genet. 97: 1380–1386.

Polanco, C., Gonzalez, A.I., de la Fuente, A., and Dover, G.A.1998. Multigene family of ribosomal DNA in Drosophilamelanogaster reveals contrasting patterns of homogenization forIGS and ITS spacer regions: a possible mechanism to resolvethis paradox. Genetics, 149: 243–256.

Rayburn, A.L., and Gill, B.S. 1986. Isolation of a D-genome spe-cific repeated DNA sequence from Aegilops squarrosa. Plant.Mol. Biol. Rep. 4: 102–109.

Rieseberg, L.H. 2001. Polyploid evolution: keeping the peace atgenomic reunions. Curr. Biol. 11: R925–R928.

Salina, E.A., Pestsova, E.G., Adonina, I.G., and Vershinin, A.V.1998. Identification of a new family of tandem repeats inTriticeae genomes. Euphytica, 100: 231–237.

Salina, E., Adonina, I., Vatolina, T., and Kurata, N. 2004a. A com-parative analysis of the composition and organization of twosubtelomeric repeat families in Aegilops speltoides Tausch., andrelated species. Genetica, 122: 227–237.

Salina, E.A., Numerova, O.M., Ozkan, H., and Feldman, M. 2004b.Alterations in subtelomeric tandem repeats during early stagesof allopolyploidy in wheat. Genome, 47: 860–867.

Salina, E.A., Leonova, I.N., Efremova, T.T., and Röder, M.S. 2005.Wheat genome structure: translocations during the course of

polyploidization. Funct. Integr. Genomics: doi:10.1007/s10142–005–0001–4.

Salina, E.A., Leonova, I.N., Efremova, T.T., and Röder, M.S. 2006.Wheat genome structure: translocations during the course ofpolyploidization. Funct. Integr. Genomics, 6: 71–80.

Sasanuma, T., Miyashita, N.T., and Tsunewaki, K. 1996. Wheatphylogeny determined by RFLP analysis of nuclear DNA. 3.Intra- and interspecific variations of five Aegilops Sitopsis spe-cies. Theor. Appl. Genet. 92: 928–934.

Schmidt, T., and Heslop-Harrison, J.S. 1998. Genomes, genes andjunk: the large-scale organization of plant chromosomes. TrendsPlant Sci. 3: 195–199.

Sharma, S., and Raina, S.N. 2005. Organization and evolution ofhighly repeated satellite DNA sequences in plant chromosomes.Cytogenet. Genome Res. 109: 15–26.

Schlotterer, C., and Tautz, D. 1994. Chromosomal homogeneity ofDrosophila ribosomal DNA arrays suggests intrachromosomalexchanges drive concerted evolution. Curr. Biol. 4: 777–783.

Soltis, D.E., and Soltis, P.S. 1995. The dynamic nature ofpolyploid genomes. Proc. Nat. Acad. Sci. U.S.A. 92: 8089–8091.

Soltis, D.E., and Soltis, P.S. 1999. Polyploidy: recurrent formationand genome evolution. Trends Ecol. Evol. 14: 348–352.

Soltis, D.E., Soltis, P.S., and Tate, J.A. 2004. Advances in thestudy of polyploidy since Plant speciation. New Phytol. 161:173–191.

Taketa, S., Ando H., Takeda K., Harrison G.E., and Heslop-Harrison J.S. 2000. The distribution, organization and evolutionof two abundant and widespread repetitive DNA sequences inthe genus Hordeum. Theor. Appl. Genet. 100: 169–176.

Tanaka, M. 1955. Chromosome pairing in hybrids between Ae.sharonensis and some species of Aegilops and Triticum. WheatInformation Service, 2: 7–10.

Trifonov, E.N. 1985. Curved DNA. CRC Crit. Rev. Biochem. 19:89–106.

Tsunewaki, K. 1996. Plasmon analysis as the counterpart of ge-nome analysis. In Methods of genome analysis in plants. Editedby P.P. Jauhar. CRC Press, New York. pp. 271–299.

Vershinin, A.V., and Heslop-Harrison, J.S. 1998. Comparativeanalysis of the nucleosomal structure of rye, wheat and their rel-atives. Plant Mol. Biol. 36: 149–161.

Vershinin, A.V., Svitashev, S., Gummesson, P.-O., Salomon, B.,Bothmer, R., and Bryngelsson, T. 1994. Characterization of afamily of tandemly repeated DNA sequences in Triticeae. Theor.Appl. Genet. 89: 217–225.

Vershinin, A.V., Schwarzacher, T., and Heslop-Harrison, J.S. 1995.The large-scale genomic organization of repetitive DNA familiesat the telomeres of rye chromosome. Plant Cell, 7: 1823–1833.

Vershinin, A.V., Alkhimova, E.G., and Heslop–Harrison, J.S. 1996.Molecular diversification of tandemly organized DNA sequencesand heterochromatic chromosome regions in some Triticeae spe-cies. Chromosome Res. 4: 515–525.

Wendel, J.F. 2000. Genome evolution in polyploids. Plant Mol.Biol. 42: 225–249.

Zhang, P., Friebe, B., and Gill, B.S. 2002. Variation in the distribu-tion of a genome-specific DNA sequences on chromosomes re-veals evolutionary relations in the Triticum and Aegilopscomplex. Plant Syst. Evol. 235: 169–179.

Zhang, H., Reader, S.M., Lui, X., Jia, J.Z., Gale, M.D., and Devos,K.M. 2001. Comparative genetic analysis of the Aegilopslongissima and Ae. sharonensis genomes with common wheat.Theor. Appl. Genet. 103: 518–525.

© 2006 NRC Canada

Salina et al. 1035

und Aegilops. V. Triticum timopheevi Zhuk. Cytologia, 6: 87–122.

Lim, K.Y., Matyasek, R., Lichtenstein, C.P., and Leitch, A.R. 2000.Molecular cytogenetic analyses and phylogeny of the Nicotianasection Tomentosae. Chromosoma, 109: 245–258.

Liu, B., and Wendel, J.F. 2003. Epigenetic phenomena and the evo-lution of plant allopolyploids. Mol. Phylogenet. Evol. 29: 365–379.

Liu, C.J., Atkinson, M.D., Chinoy, C.N., Devos, K.M., and GaleM.D. 1992. Nonhomoeologous translocations between group 4,5 and 7 chromosomes within wheat and rye. Theor. Appl. Genet.83: 305–312.

Maestra, B., and Naranjo, T. 1999. Structural chromosome differ-entiation between Triticum timopheevii and T. turgidum andT. aestivum. Theor. Appl. Genet. 98: 744–750.

McIntyre, C.L., Pereira, S., Moran, L.B., and Appels, R. 1990.New secale cereale (rye) DNA derivatives for the detection ofrye chromosome segments in wheat. Genome, 33: 635–640.

Mori, N., Lui, Y.G., and Tsunewaki, K. 1995. Wheat phylogenydetermined by RFLP analysis of nuclear DNA: II. Wildtetraploid wheats. Theor. Appl. Genet. 90: 129–134.

Mukai, Y., Nakahara, Y., and Yamamoto, M. 1993. Simultaneousdiscrimination of the three genomes in hexaploid wheat bymulticolor fluorescence in situ hybridization using total genomicand highly repeated DNA probes. Genome, 36: 489–494.

Nagaki, K., Tsujimoto, H., Isono, K., and Sasakuma, T. 1995. Mo-lecular characterization of a tandem repeat, Afa family, and itsdistribution among Triticeae. Genome, 38: 479–486.

Nagaki, K., Tsujimoto, H., and Sasakuma, T. 1998. H genome spe-cific repetitive sequence, pEt2, of Elymus trachycaulus in part ofAfa family of Triticeae. Genome, 41: 134–136.

Naranjo, T., Roca, A., Goicoecha, P.G., and Giraldez, R. 1987.Arm homoeology of wheat and rye chromosomes. Genome, 29:873–882.

Nei, M., and Li, W.H. 1979. Mathematical model for studing ge-netic variation in terms of restriction endonucleases. Proc. Natl.Acad. Sci. U.S.A. 76: 5269–5273.

Pestsova, E.G., Goncharov, N.P., and Salina, E.A. 1998. Elimina-tion of a tandem repeat of telomeric heterochromatin duringevolution of wheat. Theor. Appl. Genet. 97: 1380–1386.

Polanco, C., Gonzalez, A.I., de la Fuente, A., and Dover, G.A.1998. Multigene family of ribosomal DNA in Drosophilamelanogaster reveals contrasting patterns of homogenization forIGS and ITS spacer regions: a possible mechanism to resolvethis paradox. Genetics, 149: 243–256.

Rayburn, A.L., and Gill, B.S. 1986. Isolation of a D-genome spe-cific repeated DNA sequence from Aegilops squarrosa. Plant.Mol. Biol. Rep. 4: 102–109.

Rieseberg, L.H. 2001. Polyploid evolution: keeping the peace atgenomic reunions. Curr. Biol. 11: R925–R928.

Salina, E.A., Pestsova, E.G., Adonina, I.G., and Vershinin, A.V.1998. Identification of a new family of tandem repeats inTriticeae genomes. Euphytica, 100: 231–237.

Salina, E., Adonina, I., Vatolina, T., and Kurata, N. 2004a. A com-parative analysis of the composition and organization of twosubtelomeric repeat families in Aegilops speltoides Tausch., andrelated species. Genetica, 122: 227–237.

Salina, E.A., Numerova, O.M., Ozkan, H., and Feldman, M. 2004b.Alterations in subtelomeric tandem repeats during early stagesof allopolyploidy in wheat. Genome, 47: 860–867.

Salina, E.A., Leonova, I.N., Efremova, T.T., and Röder, M.S. 2005.Wheat genome structure: translocations during the course of

polyploidization. Funct. Integr. Genomics: doi:10.1007/s10142–005–0001–4.

Salina, E.A., Leonova, I.N., Efremova, T.T., and Röder, M.S. 2006.Wheat genome structure: translocations during the course ofpolyploidization. Funct. Integr. Genomics, 6: 71–80.

Sasanuma, T., Miyashita, N.T., and Tsunewaki, K. 1996. Wheatphylogeny determined by RFLP analysis of nuclear DNA. 3.Intra- and interspecific variations of five Aegilops Sitopsis spe-cies. Theor. Appl. Genet. 92: 928–934.

Schmidt, T., and Heslop-Harrison, J.S. 1998. Genomes, genes andjunk: the large-scale organization of plant chromosomes. TrendsPlant Sci. 3: 195–199.

Sharma, S., and Raina, S.N. 2005. Organization and evolution ofhighly repeated satellite DNA sequences in plant chromosomes.Cytogenet. Genome Res. 109: 15–26.

Schlotterer, C., and Tautz, D. 1994. Chromosomal homogeneity ofDrosophila ribosomal DNA arrays suggests intrachromosomalexchanges drive concerted evolution. Curr. Biol. 4: 777–783.

Soltis, D.E., and Soltis, P.S. 1995. The dynamic nature ofpolyploid genomes. Proc. Nat. Acad. Sci. U.S.A. 92: 8089–8091.

Soltis, D.E., and Soltis, P.S. 1999. Polyploidy: recurrent formationand genome evolution. Trends Ecol. Evol. 14: 348–352.

Soltis, D.E., Soltis, P.S., and Tate, J.A. 2004. Advances in thestudy of polyploidy since Plant speciation. New Phytol. 161:173–191.

Taketa, S., Ando H., Takeda K., Harrison G.E., and Heslop-Harrison J.S. 2000. The distribution, organization and evolutionof two abundant and widespread repetitive DNA sequences inthe genus Hordeum. Theor. Appl. Genet. 100: 169–176.

Tanaka, M. 1955. Chromosome pairing in hybrids between Ae.sharonensis and some species of Aegilops and Triticum. WheatInformation Service, 2: 7–10.

Trifonov, E.N. 1985. Curved DNA. CRC Crit. Rev. Biochem. 19:89–106.

Tsunewaki, K. 1996. Plasmon analysis as the counterpart of ge-nome analysis. In Methods of genome analysis in plants. Editedby P.P. Jauhar. CRC Press, New York. pp. 271–299.

Vershinin, A.V., and Heslop-Harrison, J.S. 1998. Comparativeanalysis of the nucleosomal structure of rye, wheat and their rel-atives. Plant Mol. Biol. 36: 149–161.

Vershinin, A.V., Svitashev, S., Gummesson, P.-O., Salomon, B.,Bothmer, R., and Bryngelsson, T. 1994. Characterization of afamily of tandemly repeated DNA sequences in Triticeae. Theor.Appl. Genet. 89: 217–225.

Vershinin, A.V., Schwarzacher, T., and Heslop-Harrison, J.S. 1995.The large-scale genomic organization of repetitive DNA familiesat the telomeres of rye chromosome. Plant Cell, 7: 1823–1833.

Vershinin, A.V., Alkhimova, E.G., and Heslop–Harrison, J.S. 1996.Molecular diversification of tandemly organized DNA sequencesand heterochromatic chromosome regions in some Triticeae spe-cies. Chromosome Res. 4: 515–525.

Wendel, J.F. 2000. Genome evolution in polyploids. Plant Mol.Biol. 42: 225–249.

Zhang, P., Friebe, B., and Gill, B.S. 2002. Variation in the distribu-tion of a genome-specific DNA sequences on chromosomes re-veals evolutionary relations in the Triticum and Aegilopscomplex. Plant Syst. Evol. 235: 169–179.

Zhang, H., Reader, S.M., Lui, X., Jia, J.Z., Gale, M.D., and Devos,K.M. 2001. Comparative genetic analysis of the Aegilopslongissima and Ae. sharonensis genomes with common wheat.Theor. Appl. Genet. 103: 518–525.

© 2006 NRC Canada

Salina et al. 1035