Comparative FISH mapping of two highlyrepetitive DNA sequences in Hordeum chilense(Roem. et Schult.)

S. Marın, A. Martın, and F. Barro

Abstract: Hordeum chilense Roem. et Schult. (2n = 14) is an autogamous wild barley from Chile and Argentina includedin the section Anisolepis Nevski. This species shows interesting agronomic traits that can be incorporated into crop plantspecies. Hordeum chilense has been successfully crossed with species of the genus Aegilops. Among the amphiploids ob-tained, the hexaploid tritordeum (2n = 6x = 42, AABBHchHch) is outstanding and shows good agronomic characteristics,suggesting its potential either as a new crop or as a bridge species to introgress interesting traits into cultivated cereals.The aim of the present work was to study the hybridization patterns of the two repetitive DNA probes pAs1 and pSc119.2to evaluate their utility for the identification of H. chilense chromosomes. Fourteen lines of H. chilense were analyzedwith fluorescent in situ hybridization using probes pSc119.2 and pAs1. The probe pAs1 was more widely dispersed thanpSc119.2 over the H. chilense (Hch) genome. We found 89 different signals for pAs1, distributed evenly over the wholegenome, and 10 for pSc119.2, located mainly over the telomeric regions. Five distinct hybridization signals were found forpAs1 and four distinct signals for pSc119.2. These signals allow the identification of different H. chilense lines. For exam-ple, centromeric signals for pAs1 on the short arms of chromosomes 1 and 7 identify line H46, and a telomeric signal forpSc119.2 on the short arm of chromosome 2 identifies line H1. A high degree of polymorphism in the hybridization pat-terns was found, confirming the extensive variability present in H. chilense. This work provides tools for the identificationof H. chilense chromosomes in different genetic backgrounds.

Key words: FISH, Hordeum chilense, pAs1, pSc119.2, polymorphism, tritordeum.

Resume : Le Hordeum chilense Roem. et Schult. (2n = 14) est une orge sauvage autogame du Chili et de l’Argentine etfait partie de la section Anisolepis Nevski. Cette espece possede des caracteres agronomiques interessants qu’il est possiblede transferer chez des especes cultivees. Le H. chilense a ete croise avec succes avec des especes du genre Aegilops. Parmiles amphidiploıdes obtenus, l’hexaploıde tritordeum est exceptionnel (2n = 6x = 42, AABBHchHch) et presente de bonnescaracteristiques agronomiques ce qui suggere un potentiel soit comme nouvelle culture soit comme pont pour introduireles caracteres d’interet chez les cereales cultivees. Le but du present travail etait d’etudier l’hybridation de deux sondesd’ADN repete, pAs1 et pSc119.2, pour evaluer leur utilite en vue de l’identification des chromosomes du H. chilense.Quatorze lignees du H. chilense ont ete examinees par hybridation in situ en fluorescence a l’aide des sondes pSc119.2 etpAs1. La sonde pAs1 etait plus largement dispersee que pSc119.2 sur le genome du H. chilense (Hch). Les auteurs ontobserve 89 signaux differents pour pAs1, lesquels etaient distribues uniformement sur l’ensemble du genome, et dixsignaux pour pSc119.2 situes principalement dans les regions telomeriques. Cinq signaux d’hybridation distinctifs ont etenotes avec pAs1 et quatre signaux distinctifs pour pSc119.2. Ces signaux permettent d’identifier les differentes lignees duH. chilense. Par exemple, des signaux centromeriques pour pAs1 sur les bras courts des chromosomes 1 et 7 identifient lalignee H46 tandis qu’un signal telomerique pour pSc119.2 sur le bras court du chromosome 2 identifie la lignee H1. Unpolymorphisme considerable dans les motifs d’hybridation a ete note, ce qui confirme l’existence d’une variabilite impor-tante chez le H. chilense. Ce travail contribue des outils pour l’identification des chromosomes du H. chilense dans diffe-rents fonds genetiques.

Mots-cles : FISH, Hordeum chilense, pAs1, pSc119.2, polymorphisme, tritordeum.

[Traduit par la Redaction]

Introduction

Hordeum chilense Roem. et Schult. (2n = 14) is a nativewild barley from Chile and Argentina, included in the Hor-

deum taxonomy (von Bothmer et al. 1980). This species isautogamous (Alvarez et al. 2006) and occupies a great vari-ety of ecosystems forming part of the local pastures, showsa high degree of morphologic and biochemical polymor-

Received 18 January 2008. Accepted 13 May 2008. Published on the NRC Research Press Web site at genome.nrc.ca on 11 July 2008.

Corresponding Editor: P. Gustafson.

S. Marın, A. Martın, and F. Barro.1 Departamento de Mejora Genetica Vegetal, Instituto de Agricultura Sostenible (CSIC), Apdo.4084, 14080 Cordoba, Spain.

1Corresponding author (e-mail: ge1balof@uco.es).

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phism, and exhibits interesting agronomic traits that can beincorporated into crops. It is resistant to many diseases andpests such as Schizaphis graminum (Castro et al. 1995),Diuraphis noxia (Clement and Lester 1990), Fusarium cul-morun and Septoria nodorum (Rubiales et al. 1996b), Septo-ria tritici (Rubiales et al. 1992), and Tilletia tritici (Rubialeset al. 1996a) and shows resistance to powdery mildew (Ru-biales et al. 1993). Hordeum chilense also shows a hightolerance to some types of abiotic stress, such as salt (For-ster et al. 1990) or drought (Gallardo and Fereres 1989). Inaddition, it exhibits a high variability in storage proteins(Alvarez et al. 2001; Atienza et al. 2005) and a high contentof carotenoid pigments (Atienza et al. 2004). Hordeum chi-lense has been successfully crossed with other species ofHordeum and with species of the genera Aegilops, Agro-pyron, Dasypyrum, Secale, Triticum, and Triticosecale (vonBothmer and Jacobsen 1986). Among the amphiploidsobtained, the hexaploid tritordeum (2n = 6x = 42,AABBHchHch) is outstanding (Martın and Sanchez-MongeLaguna 1980) and shows good agronomic characteristics;therefore, it has potential either as a new crop (Martın et al.1999) or for the introgression of desired traits into cultivatedspecies (Martın et al. 2000). In both cases, the correct iden-tification of the H. chilense chromosomes in the new geneticbackground could help to speed up breeding programs.

Genomic in situ hybridization (GISH) and fluorescent insitu hybridization (FISH) are efficient techniques for chro-mosomal identification using highly repetitive DNA sequen-ces. The probe pAs1 (Rayburn and Gill 1986a), isolatedfrom Aegilops squarrosa, has been used for the identifica-tion of D genome chromosomes (Rayburn and Gill 1986b)and the Hch genome of H. chilense (Cabrera et al. 1995).On the other hand, the probe pSc119.2 (Bedbrook et al.1980; McIntyre et al. 1990), isolated from Secale cereale,hybridizes mainly to the B genome of wheat (Mukai et al.1993) and the R genome of S. cereale (McIntyre et al.1990; Cuadrado and Jouve 1994), but it also hybridizes tothe Hch genome of H. chilense, although not so widely(de Bustos et al. 1996; Taketa et al. 2000). Nevertheless,the nucleotide sequence and length of the repetitive sequen-ces targeted by these probes are variable among species ofthe tribe Triticeae (Contento et al. 2005).

The aim of this work was to study the hybridization pat-terns of the probes pAs1 and pSc119.2 in 14 lines of H. chi-lense representing different ecotypes. The results willcontribute to the knowledge of the existing polymorphismin this species to make possible the identification of its chro-mosomes.

Materials and methods

Plant materialFourteen diploid accession lines of Hordeum chilense

were used in this work. The source and localization of theselines are shown in Table 1. All lines were self-pollinated formore than 10 generations and maintained in the germplasmbank at the Instituto de Agricultura Sostenible, CSIC, Cor-doba, Spain. Therefore, no heterozygotes are expected.

DNA probesTwo DNA probes were used for FISH: pSc119.2 and

pAs1. The probe pSc119.2 (Bedbrook et al. 1980; McIntyreet al. 1990) contains an insert of 152 bp (118 of which rep-resent the repetitive sequence) in the pGEM-T Easy Vector(Promega, Madison, Wisconsin, USA). The probe pAs1(Rayburn and Gill 1986a) consists of an insert of 185 bp(118 of which constitute the repetitive sequence) also in thepGEM-T Easy Vector. Competent cells of Escherichia coli(DH5a) were transformed with plasmids containing theprobes, and plasmids were isolated using the Plasmid MiniKit (QIAGEN, Valencia, California, USA).

The probes were labelled with digoxigenin-11-dUTP(Roche Diagnostics GmbH, Mannheim, Germany) andbiotin-11-dUTP (Roche Diagnostics GmbH) by PCR(Schwarzacher and Heslop-Harrison 2000) under the follow-ing conditions: 1� Gold Buffer, 2 mmol/L MgCl2,0.2 mmol/L dATP, 0.2 mmol/L dCTP, 0.2 mmol/L dGTP,0.13 mmol/L dTTP, 0.07 mmol/L digoxigenin-11-dUTP orbiotin-11-dUTP, 0.4 mmol/L of primers M13R and M13F,10 ng of DNA, and 1.25 units of AmpliTaq Gold DNA pol-ymerase. The PCR program was as follows: 94 8C for10 min, and then 35 cycles of 94 8C for 30 s, 55 8C for30 s, and 72 8C for 90 s, followed by an extension step of5 min at 72 8C. Unincorporated labelled nucleotides wereremoved using the QIAquick Nucleotide Removal Kit(QIAGEN). The correct labelling of probes was checked byagarose gel electrophoresis and its intensity was verified bydot-blot test (Schwarzacher and Heslop-Harrison 2000).

Cytogenetic analysesSeeds from each line were germinated at 25 8C and 4–

5 days later, roots shorter than 1 cm were collected. To ac-cumulate cells in metaphase the root tips were treated with a0.05% colchicine solution for 3 h and 30 min at 25 8C andthen fixed in 100% ethanol : acetic acid (3:1, v/v) for atleast 1 week. The root tips were stained with acetocarminefor 3 min and meristems were extracted, extended, andsquashed in 45% acetic acid on ethanol-cleaned slides. Thepreparations obtained were frozen in liquid nitrogen andafter removal of the cover slips were dried at room temper-ature and stored at 4 8C until use.

Chromosomes were prepared for FISH as described inCabrera et al. (2002). Chromosome preparations weretreated with DNase-free RNase (100 mg/mL RNase in 2�SSC) for 2 h at 37 8C, washed in 2� SSC for 10 min atroom temperature and 5 min at 37 8C, and subsequentlytreated with pepsin solution (5 mg/mL in 0.01 mol/L HCl)for 10 min at 37 8C. Slides were then washed twice in 1�PBS for 5 min, post-fixed in 1% formaldehyde in 1� PBSand 0.05 mol/L MgCl2 for 10 min at room temperature,washed with 1� PBS for 5 min at room temperature, de-hydrated through 70% and 100% ethanol (3 min each), andair-dried. The hybridization mixture (50% formamide, 2�SSC, and 10% dextran sulphate) containing 2 ng/mL of theprobes was denatured for 8 min at 80 8C in a thermal cyclerand cooled in ice for 5 min. Finally, 15 mL of the mixturewas aliquoted on each slide. The chromosomes and probeswere incubated in an in situ PCR thermal cycler (GeneAmpSystem 1000, Perkin Elmer) to denature the chromosomesand then taken to the annealing temperature (37 8C) as fol-lows: 75 8C for 7 min, 55 8C for 2 min, 50 8C for 30 s,45 8C for 1 min, 42 8C for 2 min, 40 8C for 5 min, 38 8C

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for 5 min, and 37 8C for 5 min. Hybridization was carriedout for 16 h at 37 8C in a humid chamber.

Labelled probes were detected with 10 ng/mL of anti-digoxigenin–fluorescein, Fab fragments (Roche Diagnos-tics GmbH) and 50 ng/mL of streptavidin–Cy3 conjugate(Sigma-Aldrich, St. Louis, Missouri, USA). Chromo-somes were counterstained with 2.5 ng/mL of DAPI(4’,6-diamidino-2-phenylindole) and mounted with Vecta-shield (Vector Laboratories, Inc., Burlingame, California,USA), and signals were visualized using an LSM 5Pascal microscope with appropriate filters. At least 5metaphase cells from each of the different seeds wereanalyzed per H. chilense line.

Results

Fluorescent in situ hybridizationThe H. chilense lines used in this work were studied pre-

viously and classified according to morphological characters(Vaz Patto et al. 2001). These lines are in the genetic back-ground of many lines of tritordeum and constitute the basefor developing new populations of tritordeum.

The probes pAs1 and pSc119.2 have been used in pre-vious works for the identification of H. chilense chromo-somes (Cabrera et al. 1995; de Bustos et al. 1996).Nevertheless, the variability in a population of 14 linesfrom different geographic areas and well-differentiated mor-phologic groups has not been studied previously. The pAs1signals are widely distributed among the chromosomes.Thus, we have found pAs1 hybridization sites on all thechromosomes of the H. chilense lines studied (Figs. 1 and2, Table 2) and on practically all the chromosome arms,with the exception of the long arm of chromosome 4 of linesH10 and H212 and the short arm of chromosome 1 of lineH212 (Table 2). These hybridization points appear in termi-nal, subterminal, interstitial, and occasionally centromericpositions. We have found hybridization patterns rangingfrom 43 signals per genome in line H212 to 71 signals pergenome in line H46. Chromosome 4 has the lowest numberof hybridization sites (71), whereas chromosomes 6 and 7

have the highest numbers (139 and 138, respectively). Theshort arms of chromosomes 1 and 4 have the lowest num-bers of hybridization points (34 and 35, respectively),whereas the long arm of chromosome 7 shows 86 signals ofhybridization (Table 2).

We have found 89 different hybridization signals forpAs1, but only 5 of them are distinctive. One is subtelo-meric and is located on the short arm of chromosome 4 ofline H46. The other 4 points are centromeric, which is lessfrequent, and they are located on the short arms of chromo-somes 1 and 7 of line H46 and on the long arms of chromo-somes 4 and 5 of lines H56 and H11, respectively. Wefound 17 points conserved in all lines: 8 are telomeric, 5are subtelomeric, 3 are associated with the nucleolar organ-izer, and 1 is an interstitial site on the long arm of chromo-some 5. In contrast, the remaining 72 hybridization signals(more than 80%) are polymorphic (present or absent in dif-ferent lines).

In contrast, pSc119.2 has a simpler distribution thanpAs1, with hybridization signals mainly on telomeres(Figs. 1 and 2), with the exception of the hybridization pointon the long arm of chromosome 6, which is subterminal,and a subtelomeric point on the short arm of chromosome 7of line H1 (Figs. 1 and 2). This subtelomeric point is uniqueand allows us to distinguish H1 from the rest of the H. chi-lense lines studied here (Table 3). There are 3 additionalspecific signals that can be used to distinguish other lines ofH. chilense (Table 3): the first is on the short arm of chro-mosome 7 of line H10, the second is on the short arm ofchromosome 2 of line H1, and the third is on the long armof chromosome 5 of line H208. There is also one point ofhybridization on the long arm of chromosome 1 of line H46that is peculiar because it is not accompanied by a hybrid-ization point on the short arm of the same chromosome, asis seen in the rest of the H. chilense lines (Table 3). How-ever, the reverse situation, i.e., signal on the short arm andnull expression on the long arm, occurs on many other chro-mosomes.

Of the 10 different hybridization points found forpSc119.2, 7 are are polymorphic (Table 3). The 3 conserved

Table 1. Source and localization of the Hordeum chilense accessions used in this work.

LineAltitude(m)

Latitude(N)

Longitude(W) Source

H1 ? ? ? Former Plant Breeding Institute (PBI), Cambridge, UKH7 0 31856’ 71831’ United States Department of Agriculture (USDA), USAH8 300 34804’ 70856’H10 ? ? ?H11 ? ? ?H16 1750 32818’ 71831’ Prof. von Bothmer, Svalov, SwedenH46 ? ? ? David Contreras, Santiago de Chile, ChileH47 83 36845’ 72818’H56 1000 34851’ 70834’H57 800 34845’ 70834’H208 351 32858’ 71810’ Germplasm collection of the Instituto de Agricultura Sostenible

(CSIC), Cordoba, Spain (Gimenez et al. 1997; Tobes et al. 1995)H212 1 32815’ 71832’H299 1300 30815’ 70841’H308 2100 31847’ 70835’ .

Note: ?, origin unknown.

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Fig. 1. Karyotypes of the 14 lines of Hordeum chilense used in this work. Two-colour FISH of complete root tip cells in metaphase of different lines of Hordeum chilense. Red signalsindicate pAs1 hybridization sites and green signals correspond to pSc119.2 hybridization sites. Chromosomes were counterstained with DAPI.

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Table 2. Hybridization signals of pAs1 found on chromosomes in 14 lines of H. chilense.

Hordeum chilense lines

Chromosome arm H1 H7 H8 H10 H11 H16 H46 H47 H56 H57 H208 H212 H299 H308 Arm total Chromosome total1HchS 2 1 2 3 3 3 5 2 3 2 2 0 3 3 341HchL 3 2 3 3 3 3 5 2 2 3 4 2 2 5 42 762HchS 5 2 5 4 4 4 5 3 2 5 5 5 4 5 582HchL 4 4 4 4 4 4 4 2 5 4 4 4 3 4 54 1123HchS 5 5 6 5 5 5 6 5 5 5 5 6 3 6 723HchL 5 3 4 2 2 3 6 3 3 5 3 1 4 4 48 1204HchS 3 1 3 2 3 1 5 2 3 3 3 1 2 3 354HchL 3 1 4 0 2 3 4 2 3 4 4 0 2 4 36 715HchS 3 3 4 4 4 4 4 4 3 3 4 3 4 4 515HchL 6 5 5 5 8 5 5 5 5 6 6 6 4 5 76 1276HchS 5 4 5 6 5 6 5 5 6 5 5 3 5 6 716HchL 5 4 5 3 7 4 5 5 5 5 6 4 4 6 68 1397HchS 3 3 4 5 5 3 6 3 3 3 4 3 3 4 527HchL 6 6 7 6 6 6 6 6 6 5 8 5 6 7 86 138Line total 58 44 61 52 61 54 71 49 54 58 63 43 49 66 .

Table 3. Hybridization signals of pSc119.2 found on chromosomes in 14 lines of H. chilense.

Hordeum chilense lines

Chromosome arm H1 H7 H8 H10 H11 H16 H46 H47 H56 H57 H208 H212 H299 H308 Arm total Chromosome total1HchS 1 1 1 1 1 1 0 1 0 1 1 1 1 1 121HchL 0 1 0 1 1 0 1 1 0 0 0 0 1 1 7 192HchS 1 0 0 0 0 0 0 0 0 0 0 0 0 0 12HchL 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 13HchS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 03HchL 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 04HchS 1 1 1 1 1 1 1 1 1 1 1 1 1 1 144HchL 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14 285HchS 1 1 1 1 1 1 1 1 1 1 1 1 1 1 145HchL 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 156HchS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 06HchL 1 0 1 0 1 1 0 0 0 1 1 1 0 1 8 87HchS 1 0 0 1 0 0 0 0 0 0 0 0 0 0 27HchL 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2Line total 7 5 5 6 6 5 4 5 3 5 6 5 5 6 .

Note: Values in bold are distinctive hybridization points.

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hybridization sites, present in all the lines of H. chilensestudied here, are located on telomeric regions of both armsof chromosome 4 and on the telomere of the short arm ofchromosome 5. Consequently, chromosome 4 has the great-est number of hybridization signals, followed by chromo-some 1. In contrast, chromosome 3 does not show anyhybridization point for pSc119.2 in any line of H. chilense.We also have not found any pSc119.2 hybridization signalon the long arm of chromosome 2, the short arm of chromo-some 6, or the long arm of chromosome 7. Line H1 shows 7hybridization points for pSc119.2, representing the mostcomplex pattern from all the lines studied. In contrast, thesimplest pattern is shown by line H56, with 3 pSc119.2hybridization sites.

A dendrogram was constructed using 10 different hybrid-ization points for the probe pSc119.2 and 89 for the probepAs1, but no clear relationship among H. chilense lines wasfound (data not shown).

Discussion

FISH and GISH are useful tools for chromosome identifi-cation and can be applied for the characterization of hybridssuch as triticale and tritordeum, or addition and substitutionlines. In all cases the correct identification of chromosomesis required. However, examination by optical microscopeand understanding of the results obtained is a complex proc-ess that requires experience and previous knowledge of thechromosomal morphology of the studied species. Thus, armratios (Table 4) and relative size of the chromosomes havespecial importance in this procedure, since in Hordeum chi-lense only chromosomes 5Hch and 6Hch (formerly chromo-some 7) (Armstrong et al. 1987), carrying the nucleolarorganizing regions, can be distinctly identified. The probespAs1 and pSc119.2 have been used to study hybridizationpatterns in different genera from the tribe Triticeae. For ex-ample, Schneider et al. (2003) used 23 cultivars of Triticumaestivum L. and found a low degree of polymorphism bythese probes. In the genus Secale, Cuadrado and Jouve(1997) found variability in 11 taxa, as determined by thedistribution of pSc119.2 and other repetitive sequences. Inthis work we have characterized the patterns of fluorescentin situ hybridization of the probes pAs1 and pSc119.2 in apopulation of 14 lines of H. chilense.

The results have shown great polymorphism in FISH forboth probes. In fact, we found 8 different hybridization pat-terns for the probe pSc119.2 and 14 different hybridizationpatterns for the probe pAs1, of 14 possible ones. This poly-morphism can be observed even considering the chromo-somes individually (Table 4), especially for pAs1. For pAs1there are 7 different patterns of hybridization for chromo-

some 5 and 13 for chromosomes 3 and 4. In contrast, thesetwo chromosomes show perfect conservation of thepSc119.2 pattern (Table 3). Chromosome 1 shows the great-est polymorphism for pSc119.2, with 4 different patterns ofhybridization. The polymorphism found in H. chilense canbe explained by variation in the sequence and the numberof copies of these repetitive sequences. Minor differenceswere also found within lines, but after the study of a largenumber of cells from different seeds of each line we con-clude that these minor differences are due to the FISH pro-cedure. As expected, there is no polymorphism within lines,since H. chilense is considered autogamous and all lineswere propagated by controlled self-pollination for morethan 10 generations.

The hybridization patterns of pSc119.2 and pAs1 werestudied in the genus Hordeum by de Bustos et al. (1996),who found variability within this genus using 12 lines from5 species, including one accession of H. chilense. Thehybridization pattern of pSc119.2 reported by these authorsin this accession of H. chilense is the most frequent in ourwork and similar to that of lines H8, H16, H57, and H212.Taketa et al. (2000) reported the hybridization patterns ofpSc119.2 and dpTa1 in 5 species of Hordeum, includingline H1 of H. chilense. The hybridization pattern ofpSc119.2 reported for line H1 is the same as that seen inour work. The probe dpTa1, isolated from T. aestivum (Ver-shinin et al. 1994), has a repetitive sequence that is homo-logous to that in pAs1; consequently, the distributions ofsignals obtained with these probes using line H1 of H. chi-lense are comparable. The karyotypes for pSc119.2 given byTaketa et al. (2000) for H. chilense, Hordeum marinum, andHordeum murinum (4x) were different from those reportedby de Bustos et al. (1996), showing the intraspecific varia-bility. Other sequences from Hordeum vulgare, such as thetelomeric-associated HvT01 sequence and the satellite se-quence GAA, also showed polymorphism for hybridizationpatterns in lines H1 and H7 of H. chilense (Prieto et al.2004).

The use of molecular markers to identify chromosomes ofH. chilense (Hagras et al. 2005; Atienza et al. 2007) hasbeen complicated by the polymorphism found in the presentwork and for this reason knowledge of the hybridization pat-tern in specific genetic backgrounds is important. In thisconnection, the hybridization sites of lines H1, H10, H11,H46, H56, and H208 of H. chilense are unique and makepossible their identification in different genetic backgrounds.

The variability of H. chilense found in our work agreeswith the variability described by other authors using mor-phologic and AFLP markers (Vaz Patto et al. 2001).

In summary, in this work we report the polymorphism inthe hybridization patterns of 14 lines of H. chilense using

Table 4. Different patterns of hybridization for the probes pAs1 and pSc119.2 on thechromosomes of the 14 lines of H. chilense analyzed in this work.

1Hch 2Hch 3Hch 4Hch 5Hch 6Hch 7Hch

ProbepAs1 10 12 13 13 7 12 12pSc119.2 4 2 1 1 2 2 3pAs1 + pSc119.2 11 12 13 13 7 12 13

Arm ratio (HchL/HchS) 1.51 1.11 1.72 1.33 1.97 1.09 1.15

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two different probe sequences. These results can be veryuseful in the identification of individual chromosomes ofH. chilense in different genetic backgrounds. In addition,they contribute to the knowledge and understanding of thevariability contained in wild species, providing new criteriafor selection of lines for breeding programs.

AcknowledgmentsThis work was supported by the Ministry of Education

and Science of Spain, grant AGL2007-65685-CO-01. Theprobe pAs1 (clone T. tauschii 25-208!1416) and probepSc119.2 (Petkus clone 25-42!3324) were kindly suppliedby T. Schwarzacher of the Department of Biology, Univer-sity of Leicester (UK). The authors thank Dr. Paul Lazzerifor critical review of the manuscript and C. Ramırez fortechnical assistance.

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