Cytogenetics and Plant Breeding

Cytogenet Genome Res 109:378–384 (2005)DOI: 10.1159/000082423

Cytogenetics of Hordeum chilense: currentstatus and considerations with reference tobreedingA. Martına and A. Cabrerab

a Departamento de Agronomıa y Mejora Genética Vegetal, Instituto de Agricultura Sostenible,Consejo Superior de Investigaciones Cientıficas, Cordoba, and b Departamento de Genética,Escuela Técnica Superior de Ingenieros Agronomos y de Montes, Universidad de Cordoba, Cordoba (Spain)

We acknowledge the support of the Spanish Ministry of Science and Technology andthe European Regional Development Fund (FEDER) grants Nos. AGL2001-2419-C02-01 and AGL2004-03361-C02-02.

Received 3 November 2003; manuscript accepted 25 February 2004.

Request reprints from: Dr. A. MartınDepartamento de Agronomıa y Mejora Genética VegetalInstituto de Agricultura SostenibleConsejo Superior de Investigaciones Cientıficas, Apdo. 4084ES–14080 Cordoba (Spain); telephone:+ 34-957-499-207fax: +34-957-499-252; e-mail: ge1mamua@uco.es

ABC Fax + 41 61 306 12 34E-mail karger@karger.chwww.karger.com

© 2005 S. Karger AG, Basel0301–0171/05/1093–0378$22.00/0

Accessible online at:www.karger.com/cgr

Abstract. Hordeum chilense Roem. et Schult. has a numberof characteristics interesting for breeding: high crossability withother Triticeae, resistance to biotic and abiotic stresses andhigh variability for quality traits such as endosperm storageproteins or carotenoid content. ×Tritordeum, the amphiploidsbetween H. chilense and different Triticum spp, are bridge spe-cies which facilitate the transfer of traits from H. chilense towheat or triticale. The chromosome pairing between H. chi-lense and wheat chromosomes is very low (if existing) even inthe absence of the action of the Ph1 gene. Nevertheless, translo-cation between H. chilense and wheat chromosomes has beenobserved frequently in genomic combinations where unival-

ents of both species are present and therefore a method is avail-able for using H. chilense in wheat or triticale breeding. Hybridsand amphiploids with other crop species of the Triticeae, suchas rye or barley, have also been obtained, although to date theproduction of stable introgression stocks has not been com-pleted. The technique of chromosome painting, using bothhigh- and low-repeated DNA sequences in combination withgenomic in situ hybridization have been used as effective meth-ods for basic cytogenetic research in H. chilense, allowing anal-ysis of genome evolution, and monitoring H. chilense chromo-somes in interspecific hybridization breeding programs.

Copyright © 2005 S. Karger AG, Basel

Introduction

Hordeum chilense Roem. et Schult. is a diploid wild barleyincluded in the section Anisolepis Nevski, native to Chile andArgentina. Three ecotypes are recognized, based on morpholo-gy and molecular markers (Vaz Patto et al., 2001). Hordeumchilense show a high chromosome pairing with the other ninediploid South American species as well with the North Ameri-can species and with the Asiatic H. roshevitzii but not with H.vul-

gare. The main interest in this species relates to its high crossa-bility with other members of the Triticeae tribe, Aegilops,Agropyron, Dasypirum, Secale, Triticum and × Triticosecale(von Bothmer and Jacobsen, 1986; Martın et al., 1998a). Fertileamphiploids named Tritordeums (× Tritordeum Ascherson etGraebner) were obtained after chromosome doubling of hy-brids between H. chilense and tetraploid and hexaploid wheats.Chromosome addition and substitution lines of H. chilense inbread wheat have been developed (Miller et al., 1981, 1985).Together with these substitution lines, hexaploid tritordeum(2n = 6x = 42, AABBHchHch) can be used as a genetic bridge tointroduce promising characteristics from H. chilense into breadwheat (Martın and Cubero, 1981).

Several studies have demonstrated a high variability forstorage endosperm proteins in both tritordeum (Alvarez et al.,1993) and H. chilense (Atienza et al., 2000, 2002). The carote-noid content in tritordeum seed is much higher than in itswheat parent (Alvarez et al., 1999). Analysis of addition lineshas shown that the · arm of chromosome 7Hch is responsiblefor the elevated carotenoid content (Alvarez et al., 1998).

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A number of other traits of agronomic interest have beenidentified in H. chilense. It is a weak perennial, appreciated bycattle and resistant to several significant pests and diseasesincluding Schizapis graminum (Castro et al., 1995), Diuraphisnoxia (Clement and Lester, 1990; Castro et al., 1998), Fusa-rium culmorum, Septoria nodorum (Rubiales et al., 1996) andSeptoria tritici (Rubiales et al., 1992). In addition, H. chilenseshows elevated tolerance to abiotic stresses such as salt (Forsteret al., 1990) and drought (Gallardo and Fereres, 1989).

In recognition of the number of interesting traits present inH. chilense germplasm, over 250 amphiploids have been gener-ated using different accessions of the species in order toincrease the genetic variability available for breeding (Martınet al., 1998b).

Effective and reliable methods for chromosome identifica-tion are critical for the identification and characterization ofintrogression lines of H. chilense in Triticeae crops. The appli-cation of chromosome painting using both high- and low-repeated DNA sequences in combination with genomic in situhybridization has proven to be effective methods for basic cyto-genetic research, genome evolution studies and monitoringH. chilense chromosomes in breeding programs based on inter-specific hybridization.

The cytology of Hordeum chilense

Physical mapping of repetitive sequences in HordeumchilenseHordeum chilense (2n = 2x = 14) has three metacentric chro-

mosomes (2Hch, 6Hch and 7Hch) and four submetacentric chro-mosomes (1Hch, 3Hch, 4Hch and 5Hch) of which 5Hch and 6Hch

have satellites. The chromosomes are medium size, with lengthsof 5–7 Ìm at the mid-metaphase stage (Fig. 1). Conventional N-and C-banding cytogenetic methods have been applied for iden-tification of H. chilense chromosomes. Differences in the C-banding (Fernandez and Jouve, 1984; Armstrong et al., 1987;Linde-Laursen et al., 1989; Cabrera et al., 1995) and N-banding(Gonzalez and Cabrera, 1999) patterns, with respect to the num-ber of bands per chromosome and band size, have been foundbetween different accessions of H. chilense. As a result of thisstructural variation, all H. chilense chromosome pairs can beidentified by their characteristic N- and C-banding patterns.Some of these differences in banding patterns may reflect thelarge amount of polymorphism in this species.

The presence and distribution of highly repeated DNAsequences families are especially useful as cytological markersfor separate chromosomes and for analyzing genome organiza-tion and evolution. The physical distribution of various repeti-tive DNA sequences has been studied by FISH (fluorescent insitu hybridization) in H. chilense. They include conserved repe-titive DNA sequences, such as rRNA genes or telomeric repeatsand non-functional or coding tandem repeats (ie. pAs1 andGAA-microsatellite).

rDNA Ribosomal RNA genes (rRNA genes, rDNA) in eukaryotes

are tandemly arrayed in hundreds (to thousands) of copies at

chromosomal loci known as nucleolar organizer regions(NORs). Based on the presence of secondary constrictions andthe analysis of nucleolar activity by Ag-NOR banding (Cabreraand Martın, 1991), two pairs of satellite chromosomes wereidentified in H. chilense. In situ hybridization with pTa71probe to H. chilense chromosomes revealed the presence of twomajor 18S–26S rDNA sites in this species (Fig. 1). Analysis ofthe Chinese Spring-H. chilense addition lines using the 18S–26S probe also confirmed the presence and position of only twomajor sites of hybridization on the short arms of chromosomes5Hch and 6Hch in H. chilense (Cabrera et al., 1995).

Nucleolar activity was also studied in interspecific hybridsinvolving H. chilense and wheat genomes using the silver salt-nylon technique (Lima-Brito et al., 1998). The two H. chilenseloci and rRNA genes from the major NORs from wheat (chro-mosome pairs 1B and 6B) were active in the genomic constitu-tions AABBHchHch, AABHch and AABBDHch, indicating thatthese four major rDNA loci are expressed together.

The physical location of 5S sequences in H. chilense hasbeen examined by in situ hybridization using pTa794 clone as aprobe. Two 5S rDNA sites have been observed on the short armof chromosome pair 5Hch, one of them overlapping with strong18–25S rDNA (Taketa et al., 1999, 2001).

Telomeric and subtelomeric sequencesThe telomeric regions of eukaryotic chromosomes, includ-

ing the telomeric repeat and telomere-associated sequenceshave important functions in the protection, replication and sta-bilization of the chromosome ends and they also provideimportant landmarks for the termini of genetic and physicalmaps of chromosomes. The chromosomal distribution of tel-omeric clone pAtT4, containing the telomeric oligonucleotiderepeat (5)-TTTAGGG-3)) of Arabidopsis thaliana and a barleytelomeric-associated HvT01 sequence (a member of the barleyHvRT subtelomeric tandem repeated family) were used ashybridization probes to chromosomes of H1 and H7 accessionsof H. chilense. Hybridization signals with the pAtT4 probewere found at the ends of all seven chromosome pairs in the twoaccessions showing that the telomere sequence of A. thaliana isalso found in the genome of H. chilense. In contrast, the telo-meric associated sequence homologous to HvT01 showed vari-ability for intensity and position of the signals for each line(Fig. 1). The sequence is present in all seven pairs of chromo-somes in accession H1 whereas only four chromosome arms inaccession H7 were labeled with the barley HvT01 sequence(Prieto et al., 2004). In barley, in situ hybridization of satelliteHvT01 to metaphase chromosomes showed the presence ofhybridization signals on all except for one chromosome arm(Röder et al., 1993). Polymorphism for hybridization signalsobserved among the two H. chilense accessions with the HvT01sequence indicated that these subtelomeric repeat sequencesare much more divergent than the evolutionarily conserved tel-omeric sequences.

GAA-microsatellite and pAs1 repetitive sequencesThe pAs1 sequence was isolated from T. tauschii and

described as a D-genome specific repetitive sequence (Rayburnand Gill, 1986). In situ hybridization of the pAs1 sequence to

380 Cytogenet Genome Res 109:378–384 (2005)

Fig. 1. Ideogram of the seven chromosomes ofHordeum chilense showing chromosome length(in Ìm) and FISH signals with different repetitiveDNA probes. Polymorphic chromosome markersin H1 and H7 accessions are arrowed.

5

pAtT4

pAs1

pTa71

GAA

polymorphicchromosomemarker

HvT01

3 4 5 6 7 1 2

0

1

2

3

1

2

3

4

4

5

Fig. 2. (a) FISH with the p1Dx5 probe to apartial mitotic metaphase of tritordeum (2n = 4x= 42, AABBHchHch). Hybridization signals (indi-cated by arrows) were detected with anti-digoxige-nin-FITC. (b) Double FISH with the pAs1 (red)and GAA-satellite (green) sequences hybridizedto the same metaphase as in a. (c) Double FISHsignals with the ribosomal pTa71 (green) andgenomic H. chilense DNA (red) probes hybridizedto the same metaphase as in a and b. (d) GISHwith genomic H. chilense DNA to meiotic meta-phase I of AABBDHch and (e) GISH with ge-nomic H. chilense DNA to meiotic metaphase I ofAmAuBHch hybrid. (f) GISH with double genomicDNA from H. chilense (green) and H. vulgare(red) to meiotic metaphase I of the HvHchD hy-brid. (g) Centromeric translocations involving H.chilense (green), H. vulgare (red) and wheat (blue)chromosomes. (h) Centromeric translocations be-tween H. chilense (green) and wheat (red) chromo-somes. Terminal (i) and (j) and intercalar (k)translocations involving H. chilense (green) andwheat chromosomes. Chromosomes were coun-tersatined with PI in a, d, e, h, i, j and k and withDAPI in b, c and g, respectively. Bar in a, b, c, d,e and f = 10 Ìm. Bar in g, h, i, j and k = 5 Ìm.

H. chilense and to H. chilense chromosome addition lines inwheat cv. Chinese Spring revealed multiple sites of hybridiza-tion on every chromosome allowing the identification of all sev-en pairs (Cabrera et al., 1995). These results indicate that theH. chilense genome contains a significant amount of repetitiveDNA homologous to the pAs1 sequence (Fig. 1). One practicalimplication of this result is that the conservation of the pAs1sequence may be used for chromosome identification and phys-

ical mapping of species belonging to two distant genera. ThusH. chilense primary trisomics (Cabrera et al., 1999), introgres-sions of D-genome chromosomes into tritordeum (Ballesteroset al., 2003), chromosome rearrangements involving both Dand Hch genome chromosomes (Cifuentes et al., 2002), trans-gene integration sites and chromosome alterations in transgen-ic tritordeum lines (Barro et al., 2003) have been successfullyidentified using pAs1 as a probe.

Cytogenet Genome Res 109:378–384 (2005) 381

The physical distribution of the GAA-satellite sequence onboth H1 and H7 metaphase chromosomes also allowed theidentification of the seven chromosome pairs in these twoH. chilense accessions (Prieto et al., 2004). These GAA hybridi-zation sites corresponded with N-banded regions in H. chilense,as was reported previously in wheat and barley (Dennis et al.,1980). Polymorphism for hybridization signals between thetwo accessions for GAA-banding pattern also confirmed thatH1 and H7 accessions belong to distinct ecotype groups as sug-gested previously, based on morphological and AFLP markers(Vaz Patto et al., 2001).

Using the single-copy probe Glu-D1-1d, the gene coding forthe 1Dx5 HMW glutenin subunit, Glu-1 loci were mapped tothe long arm of the 1AL, 1BL and 1HchL genome chromosomesof the amphiploid tritordeum (Cabrera et al., 2002). Chromo-somes with positive signals were successfully identified by re-probing chromosome preparations using both the GAA-satel-lite and pAs1 sequences, simultaneously. After examinationand photography of the metaphases hybridized with theseprobes, the preparations were again re-probed with pTa71 andgenomic H. chilense DNA, simultaneously. Sequential in situhybridization with these DNA sequences allowed identifica-tion of both wheat and H. chilense genomes and chromosomes(Fig. 2a–c).

Hybrids of H. chilense with other Triticeae

Hordeum vulgare and H. bulbosumSome accessions of H. chilense are non hosts of the barley

leaf rust (Puccinia hordei G. Otth) (Rubiales and Niks, 1996).This trait is an example of the variability of interest for barleybreeding present in H. chilense.

Barley–H. chilense hybrids have been described (Thomasand Pickering, 1985) in which some chromosome pairing is vis-ible at metaphase I. From this report it is not clear if theobserved bivalents are true pairing or achiasmatic associations.Unfortunately, no descendent has been obtained from thesehybrids, either by selfing or backcrossing after colchicine treat-ment for chromosome doubling. We have attempted the pro-duction of amphiploids crossing tetraploid H. chilense by tetra-ploid H. vulgare. Despite using four different tetraploid H. chi-lense stocks and a wide collection of tetraploid barley, noamphiploid was obtained. Although many embryos were cul-tured from these crosses, none of them developed into a plant.Adult plants were obtained from pollinating tetraploid H. chi-lense with diploid barley (Sanchez-Monge Laguna and Martın,1982). Nevertheless, as in previous hybrids, no seed setting wasobtained by selfing or backcrossing to either parent.

Crosses between H. vulgare and tetraploid (HchHchDD),hexaploid (HchHchAABB) and octoploid (HchHchAABBDD) tri-tordeums have been obtained. The hybrids have the expectedgenome constitution, HvHchD, HvHchAB and HvHchABD, re-spectively. Some meiotic chromosome pairing between barleyand H. chilense chromosomes has been revealed by FISH in thehybrid HvHchD (Fig. 2e), but again, no plants could be recov-ered after backcrossing. An amphiploid HvHvHchHchAABB wasobtained from tissue culture of immature inflorescences of the

Fig. 3. Spikes of tetraploids Hordeum bulbosum (left), H. chilense (right)and the amphiploid (center).

hybrid HvHchAB. This amphiploid was selfsterile, but a plantwith 38 chromosomes was obtained after backcrossing to du-rum wheat.

H. bulbosum is closely related to barley and has been used asa source of genetic variability. Its possibilities as a bridge spe-cies between H. chilense and H. vulgare have been tested.Hybrids between diploids H. chilense and H. bulbosum havebeen obtained (Padilla and Martın, 1983; Thomas and Picker-ing, 1985) in which at meiosis there was virtually no chromo-some pairing. An H. chilense-H. bulbosum amphiploid wasdirectly synthesized by crossing at the tetraploid level (Cabreraand Martın, unpublished results). This amphiploid was alsosterile, although in some favorable conditions some fertile poll-en was produced (Fig. 3).

Agropyron Amphiploids between H. chilense and Agropyron cristatum

and A. desertorum (unpublished results) have been synthesizeddirectly by crossing tetraploids of the two species. Allosyndeticchromosome meiotic pairing is absent in the amphiploids(Martın et al., 1999).

The difference in chromosome size, with those of Agropyronbeing smaller than those of H. chilense, allows the confirmationof the absence of chromosome pairing between species in meio-sis using classical staining. The lack of chromosome affinitybetween the genomes of these species was confirmed by FISH

382 Cytogenet Genome Res 109:378–384 (2005)

Fig. 4. Spikelets of Hordeum chilense (left), Dasypirum villosum (right)and its hybrid (center).

Fig. 5. Metaphase I of the amphiploid Hordeum chilense–Secale cereale.Bar = 10 Ìm.

in the HchDP hybrids obtained by crossing H. chilense (HchHch)with the fertile amphiploid T. tauschii-A. cristatum (DDPP)(Martın et al. 1998a).

Dasypirum villosum Hybrids with Dasypirum villosum (L.) Borbas are easily

obtained but amphiploids after colchicine treatment have notbeen obtained. The spikelet of the hybrid is similar to Dasypi-rum (Fig. 4).

RyeHordeum chilense has been crossed successfully with rye

(Finch and Bennett, 1980; Thomas and Pickering, 1985; Mar-tın et al., 1988; Pohler and Schrader, 1988; Linde-Laursen etal., 1993). Amphiploids between both species have been direct-ly synthesized by crossing tetraploid H. chilense and S. cereale(Martın et al., 1988) and no significant allosyndetic chromo-some pairing has been observed in hybrids or amphiploids.Cells with full pairing are frequent in the meiosis of amphi-ploids (Fig. 5). Nevertheless, the amphiploid is sterile despiteits regular meiosis. Some seed setting was obtained by back-crossing to rye, giving rise to the monosomic addition for five ofthe seven H. chilense chromosomes to S. cereale (Linde-Laur-sen et al., 1993).

Hybrids between tetra-, hexa- and octoploid tritordeumwith rye have been obtained. In the hybrid HchDR in whichPh1 is absent, the possibilities of homeologous pairing are high-er than in the HchABR or HchABDR combinations. Neverthe-less, no homeologous pairing was observed (Cabrera and Mar-tın, 1992) in agreement with previous analyses in other ge-nomic combinations. All hybrids were sterile and no amphi-ploids were obtained after colchicine treatment.

TriticaleHybrids of H. chilense with hexaploid triticale have been

obtained (Pohler and Schrader, 1988). We also have obtained

such hybrids but the amphiploid has not been produced aftercolchicine treatment.

Hybrids between triticale and tritordeum have been pro-duced at different ploidy levels. No hormonal treatment of fer-tilized flower nor embryo rescue was necessary for developingshrivelled grains which germinate normally.

The hybrid between hexaploid tritordeum and hexaploidtriticale (2n = 6x = 42, AABBHchR) was easily obtained whentriticale was used as female (Fernandez-Escobar and Martın,1985). The hybrid is sterile but set seed when backcrossed toeither parents or bread wheat. FISH with cloned repetitiveDNA probes and total genomic DNA enables the parental ori-gin of all chromosomes to be established in metaphases of triti-cale × tritordeum F1 hybrids (Lima-Brito et al., 1996). Al-though no pairing between rye and H. chilense was observed,some recombinant chromosomes as result of translocationswere identified (Fernandez Escobar, 1988). Therefore thisoffers a route for introgressing rye chromatin into tritordeumand, vice versa, introgressing H. chilense chromatin into triti-cale.

The hybrid between hexaploid triticale and octoploid tritor-deum (2n = 8x = 56 AABBHchRD) was readily obtained (Fer-nandez-Escobar and Martın, 1988). This hybrid is sterile.

The hybrid obtained by crossing octoploid triticale andoctoploid tritordeum (2n = 8x = 56, AABBDDHchR) is self-fertile. In the progeny the chromosome number ranged from 42to 52 with a preferential transmission of rye chromosomes (Fer-nandez-Escobar and Martın, 1989).

WheatH. chilense crosses readily with Triticum spp. at every ploi-

dy level. The hybrid with diploid T. monococcum has beenobtained (Martın, unpublished result) but the amphiploid wasnot produced after colchicine treatment of the hybrid neitherwas an amphiploid obtained from crossing tetraploid H. chi-lense and tetraploid T. monococcum. In contrast, the amphi-

Cytogenet Genome Res 109:378–384 (2005) 383

ploid H. chilense-T. tauschii (HchHchDD) was obtained bycrossing both at the tetraploid level (Cabrera and Martın, 1991)after chromosome doubling of the hybrid H. chilense × T. tau-schii failed (Martın, 1983). The spike morphology of the hybridand the amphiploid resembles the wheat parent with flowers inspikelets.

Hybrids of H. chilense with tetraploid and hexaploid wheatsare readily colchicine doubled and hexaploid (HchHchAABB)and octoploids tritordeums (HchHchAABBDD) are thereforeobtained (Martın and Chapman, 1977; Martın and Sanchez-Monge Laguna, 1982). The morphology of hexaploid and octo-ploid tritordeums resembles hexaploid wheat, with shorterinterspikelet distances in hexaploids. Hexaploid tritordeumhas been the subject of an extensive breeding program. The firststep was the foundation of a pool of wide genetic variability.Over 250 primary tritordeum have been synthesized using 52different H. chilense accessions and a wide collection of durumand bread wheats. At the moment the yield of advanced tritor-deum lines are roughly 90% of current bread wheat varieties.The bread making capacity of tritordeum is similar to breadwheat with a typical yellowish colour due to the high carotenoidcontent of tritordeum flour. Addition and substitution lines ofH. chilense have been produced in bread wheat (Miller, 1981;Miller et al., 1985).

Alloplasmic lines of T. aestivum in the cytoplasm of H. chi-lense line H1 (corresponding to one of the three H. chilense eco-types) are male steriles. The fertility is restored on adding theshort arm of chromosome 6Hch. The cytoplasm of H. chilensehad no significant effect on wheat morphology, although plantheight was reduced and the anthesis time extended.

Wheat-H. chilense meiotic chromosome pairingIn wheat, the Ph1 locus ensures correct homologue pairing

and recombination. The absence of the Ph1 locus in nullisomic5B promotes homeologous pairing among the three genomes ofhexaploid wheat. Nevertheless, in the hybrid H. chilense ×T. aestivum nullisomic for 5B tetrasomic 5D, no increase inchromosome pairing was observed (Martın and Sanchez-Monge Laguna, 1980), indicating this system will not help thetransfer of traits from H. chilense into wheat. The lack of pair-ing between the D and Hch genomes has also been recognized inthe hybrids H. chilense × T. tauschii (Martın, 1983) and T. aes-tivum × hexaploid tritordeum (AABBDHch) (Fernandez andJouve, 1990) by classical analysis and was ratified usinggenomic in situ hybridization (Fig. 2b, c).

Chromosome translocations involving H. chilense, barleyor wheat Due to the lack of pairing between H. chilense and wheat

chromosomes, chromosome rearrangement offers the opportu-nity for interchange of genetic material among these species.Translocations between wheat and H. chilense chromosomesare commonly detected when the genomes from these speciesare together in the same genetic background. Most frequenttranslocations observed are centromeric fusions (Fig. 2g, h).Terminal and intercalary translocations are much more diffi-cult to obtain, i.e. each one requires at least two breaks in thechromosome arms. High frequencies of chromosomal structur-

al changes have been obtained in common wheat by means ofgametocidal factors present in chromosome 2C from Aegilopscylindrica host (2n = 4x = 28, CCDD) (Endo, 1988). Using theAe. cylindrica system for inducing rearrangement between H.chilense and wheat chromosomes, centromeric, terminal andintercalar chromosomes have been obtained in the descendentsof crosses between hexaploid tritordeum with the disomic addi-tion line for chromosome 2C of Ae. cylindrica in “ChineseSpring” (Fig. 2i–k) (Cifuentes et al., 2002). The healing of thebroken chromosome ends achieved by de novo additions oftelomeric repeats led also to the isolation of terminal deletionsin H. chilense chromosomes (unpublished results).

Three integration sites and four translocations were de-tected in two transgenic tritordeum lines transformed indepen-dently with the genes encoding the high molecular weight glu-tenin subunits (HMW-GS), 1Ax1 and/or 1Dx5 by FISH. Allthree integration sites were located on chromosome segmentsof H. chilense translocated (intercalary or terminal) into wheatchromosomes. The remaining translocation with no transgeneintegration was a Robertsonian translocation (Barro et al.,2003).

Interspecific H. vulgare/H. chilense and intergeneric wheat/H. vulgare and wheat/H. chilense translocations occurred spon-taneously in the progeny of crosses between H. vulgare additionlines in T. aestivum and hexaploid tritordeum. Plants with sim-ple, double or triple intergenomic translocations were found.Translocations involving H. vulgare-H. chilense chromosomes(Fig. 2g) were most frequent, followed by wheat-H. chilenseand wheat-H. vulgare intergeneric translocations (Prieto et al.,2001). These lines may be useful for introgressing characters ofinterest from H. vulgare into tritordeum.

Conclusions

The transfer of genetic material from H. chilense to breadwheat and to triticale has been realized using tritordeum as abridge. Introgression into tritordeum of barley, wheat and ryechromatin has also been obtained by crossing tritordeum withaddition lines of barley on wheat, with bread wheat and withtriticale respectively. Genomic DNA in situ hybridization andFISH using the pAs1 and GAA repetitive DNA probes hasmade possible the characterization of these introgressions.

384 Cytogenet Genome Res 109:378–384 (2005)

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