Chromosome Research 1996, 4, 491-499

Physical mapping of repetitive DNA sequences and 5S and 18S-26S rDNA in five wild species of the genus Hordeum

A. de Bustos, A. Cuadrado, C. Soler & N. Jouve

Received 7 March 1996; received in revised form 29 May 1996; accepted by J.S. (Pat) Heslop-Harrison 31 May 1996

The genetic relationships between several wild spe- cies and subspecies of the genus Hordeum were as- sessed using fluorescence in situ hybridization (FISH). Plant material included natural populations of wild barley growing in Spain of the annual species, H. marinum ssp. maHnum (2n=14) and gussoneanum (2n = 14), and H. murinum ssp. murinum (2n = 28), and leporinum (2n = 28) and the" perennial species H. bul- bosum (2n=14) and H. secalinum (2n=28), plus the South American perennial species H. chilense (2n-14) . FISH was used to locate the chromosomal

sites of two rDNA multigene families 5S and 18S-26S (pTa71 and pTa794) and three repetitive DNA se- quences (pSc119.2, pAsl and pHch950) isolated from different species and genera. The seven chromo- somes of the diploid species were readily distin- guished by their external morphology and hybridization patterns to pTa71, pTa794, pSc119.2 and pAsl. These DNA probes were also useful for the identification of homologous chromosomes and in differentiating these from unidentified chromosomes in the tetraploid taxa~ The use of the probe pHch950 permitted intergenomic differentiation in tetraploids and supports the diphyletic origin of H. murinum and H. secelinum. The in situ experiments yielded the fol- lowing conclusions: (1) differences between the sub- species marinum and gussoneanum; (2) close relationships between the subspecies murinum and leporinum; and (3) major differences in physical map- ping between H. bulbosum and the remaining taxa. The genomic and phylogenetic relationships between taxa, as inferred from the results, are discussed.

Key words: barley, fluorescence in situ hybridization, Hordeum bulbosum, Hordeum chilense, Hordeum marinum, Hordeum murinum, Hordeum secalinum, repetitive DNA

Int roduct ion

The classification of species in the genus Hordeum is still a matter of debate (Bothmer et aI. 1991). However, the classification of Bothmer et al. (1986, 1987, 1991), which divides the genus into four major groups re- presenting the basic genomes H, I, 'X' and 'Y', is generally accepted. The system is based on the meiotic

behaviour of interspecific hybrids and Giemsa C-band- ing (Linde-Laursen et al. 1986, 1989, 1990, Bothmer et al. 1987). This technique enables the unequivocal identi- fication of the seven chromosome pairs in cultivated barley (H. vulgare) and other diploid species. However, its use in the identification of chromosomes, and their allocation to specific genomes in certain species of wild barley, is rather limited. Phylogenetic studies of this type on different barley species have used molecular hybridization techniques employing repetitive DNA sequences (Molnar et al. 1989, 1992, Gupta et al. 1989, Monte et al. 1993, Vershinin et al. 1994). These analyses take advantage of the existence of conserved sequences of DNA repeats present in the genomes of eukaryotes. Repetitive DNA sequences from H. vulgare and H. chilense were previously used to analyse a series of Hordeum species (Vershinin et al. 1990, Shcherban & Vershinin 1992, Svitashev et al. 1994, Anamthawat- J6nsson & Heslop-Harrison 1993, Ferrer et al. 1995). Genomic and phylogenetic studies of barley species have also been performed, employing repetitive DNA sequences from other Triticeae species, taking advan- tage of cross-hybridization phenomena. The probe pSc119 from rye (Gupta et al. 1989), the ribosomal gene DNA sequence pTa71 (Molnar et al. 1992), pTal (Vershinin et al. 1994) from wheat, and the probe pAsl from Triticum tauschii (Cabrera et al. 1995) have all been used.

Fluorescence in situ hybridization (FISH) has been an important tool in physical mapping of chromosomes in cereals (Leitch et al. 1991, Mukai et al. 1993, Cuadrado & Jouve 1994, 1995, Cuadrado et al. 1995, Castilho & Heslop-Harrison 1995). FISH offers the possibility of combining two or more DNA probes labelled with different fluorochromes in simultaneous and /o r suc- cessive treatments on the same cells. This technique combines the advantages of direct chromosome iden- tification using different markers (which can be map- ped relative to one another) and direct genome analysis by determining the degree of conservation of repetitive DNA sequences. The aim of this work was to analyse phylogenetic affinities in a group of species and subspecies of the genus Hordeum using FISH.

A de Bustos and C. Soler are at the Department of Plant Breeding, CIT, INIA, La Canaleja, PO Box 1100, 28800-Alcald de Henares (Madrid), Spain. A. Cuadrado and N. Jouve (corresponding author) are at the Department of Cell Biology and Genetics, University of Alcald de Henares, 28871-AlcaM de Henares (Madrid) Spain. Tel: ( + 34) 1 8854750; Fax: ( + 34) 1 8854799; Emaih bcjouve@alcala.es.

1996 Rapid Science Publishers Chromosome Research Vol 4 1996 491

A . de Bus to s et al.

Materials and methods

Plant material The plant material used in this investigation consisted of 11 populations belonging to four species of the genus Hordeum growing in Spain and an accession of the South American wild species H. chilense (2n = 14). This species was included because of its interest as donor of the DNA probe pHch950 used in the in situ hybridization experiments. The Spanish material is part of the living collection of the authors that consists of wild relatives of cultivated Triticeae, collected from the wild during recent years. It is being maintained at the Plant Breeding Unit of the INIA (La Canaleja, Madrid, Spain). The code of accessions per species, chromosome number, localities and altitude of the habitats where the samples were collected are shown in Table 1. The material consisted of three perennial species and two annual species, each represented by two subspecies. The perennials used included two accessions of H. bulbosum (2n = 14), collected from the south-west of continental Spain growing on acid and neutral soils, and one population of H. secalinum (2n = 4x = 28) collected in humid soil from central Spain. The populations of H. murinum ssp. murinum (2n =4x=28) were found in habi- tats at altitudes above 1000 m with high rainfall. The acces- sion of H. murinum ssp. leporinum (2n = 4x = 28) was collected in the south-west of Spain. Finally the populations of H. marinum ssp. marinum (2n=14) and ssp. gussoneanum (2n =14), were found in different climates in continental Spain and on the Balearic Islands.

DNA probes The probes employed in FISH analyses included pSc119.2, pAsl and pHch950 containing, respectively, highly repeated DNA sequences derived from Secale cereale (Bedbrook et al. 1980, McIntyre et al. 1990), Triticum tauschii (Rayburn & Gill 1985) and Hordeum chilense (Hueros et al. 1993). Two different rDNA probes were also used: pTa71 containing the 18S-5.8S- 26S and pTa794 containing the 5S ribosomal sequences (Ger- lach & Bedbrook 1979, Gerlach & Dyer 1980).

Chromosome preparation and FISH Root tips from numerous plants of each taxonomic unit were excised and pretreated in distilled water for 24 h at 0~

Before squashing, pretreated roots were fixed in a 3:1 mixture of ethanol (99%) and glacial acetic acid, washed and trans- ferred to an enzyme solution containing 2% cellulase and 20% liquid pectinase. Only well-spread somatic metaphases from not fewer than five plants per population were processed to follow FISH experiments. After removal of the coverslip and subsequent RNAase treatment, slides were post-fixed in 4% (w/v) paraformaldehyde, dehydrated in a graded ethanol series and air dried. Chromosome and probe denaturation and in situ hybridization steps were carried out as described by Heslop-Harrison et al. (1991) and by Cuadrado & Jouve (1994). Chromosome preparations were denatured before in situ hybridization using a programmable thermal cycler (PT- 100, M.-J. Research Inc.). Simultaneous hybridization (with probes added just before use) and reprobing was then carried out. Chromosome lengths were measured from DAPI images using a MICROM IP chromosome image-analysing system (MIP) following the technique previously described by Ber- nardo et al. (1992). A group of unidentified chromosomes from the two subspecies of H. murinum were excluded from the measurements.

Results

FISH mapping The physical ch romosome maps showing the hybridi- zat ion sites of the repet i t ive DNA sequences pSc119.2, p A s l and pHch950, and 5S and 18S-5.8S-26S rDNA are g iven in Figures 1 and 2. The external morpho logy was v isual ized and the chromosomes measured in the same metaphase cells s tained with DAPI and hybr idized s imul taneous ly wi th the probes. Chromosomes are ordered relat ively in the maps fol lowing previous descr ipt ions for the same species by different authors.

The samples of Hordeum marinum (2n= 14) were d ip lo id and be longed to either subspecies ssp. marinum or ssp. gussoneanum. The karyotypes of both subspecies were in agreement wi th Bothmer & Jacobsen (1985) and Linde-Laursen et al. (1989). The external morpho logy of the chromosomes of the haplo id genome of both subspecies, wi th five metacentr ic chromosomes, one submetacentr ic and one SAT chromosome are similar (Figure l a and b). However , FISH with different probes

Table 1. Wild barley accessions analysed for the physical mapping of repetitive DNA sequences, and 5S and 18S-26S rDNA by FISH PBU, Plant Breeding Unit, INIA; UAH, Department of Cell Biology and Genetics, University of Alcala.

Species Code 2n Geographical Source Altitude (m)

H. marinum ssp. marinum PBU-327 14 Las Cabezas de San Ju~.n (Sevilla) 50 PBU-329 14 Puerto de Santa Maria (C~.diz) 40 PBU-345 14 Tarifa (C&diz) 250 PBU-176 14 Lluc (Palma de Mallorca) 300 PBU-303 14 Obejo-Villaviciosa (C6rdoba) 550 PBU-307 14 Et Alamo (C6rdoba) 150 PBU-356 14 Grazalema (C&diz) 780 PBU-380 28 Burgo Ranero (Le6n) 870 PBU-362 28 Santiago de la Espada (Jaen) 1300 PBU-390 28 La Poveda (Soria) 1300 PBU-337 28 Jer6z de la Frontera (C~.diz) 30 UAH-22 14

H. marinum ssp. gussoneanum

H. bulbosum

H. secalinum H. murinum ssp. murinum

H. murinum ssp. leporinum H. chilense

492 Chromosome Research Vol 4 1996

a) H. m a r i n u m ssp. m a r i n u m

b) H. m a r i n u m ssp. g u s s o n e a n u m

C) H. bulbosum

d ) H. ch i lense

~1~ v a n m t ~ n

i O0 pro~ ~ pTa71

pAml I ~ p s e ~ 1 9 . 2

Figure 1. The physical map of the chromosomes of diploid species or subspecies of the genus Hordeum showing the hybridization sites and interstitial regions with the repeti- tive sequences inserted in the DNA probes pTa71 and pAsl (left), pTa794 and pSc119.2 (right): a H. marinum ssp. marinum, b H. marinum ssp. gussoneanum, c H. bulbosum, d H. chilense.

reveals differences in the pa t t e rn of d i s t r ibu t ion of sequences be tween co r respond ing chromosomes of the two subspecies. The probe p A s l hyb r id i zed to more segments in H. marinum ssp. marinum than in ssp. gussoneanum (Figure 3h). pSc119.2 hyb r id i zed prefer- ential ly to the te lomeric region, showing coincidences in five chromosomes of both subspecies . However , H. marinum ssp. gussoneanum presen ted some extratelo- meric signals and an interst i t ia l hybr id i za t ion site to pSc119.2 in the long arm of the submetacent r ic chro- mosome. Other differences were encounte red for the 5S rDNA hybr id iza t ion sites. Two signals were located in identical posi t ions: one on the long a rm of the smallest metacentr ic ch romosome and one at a d is ta l pos i t ion near the nucleolus organizer region (NOR) of the SAT chromosome. The third locus was located at different posit ions: an interst i t ia l site on the long arm

Physical mapping in wild species of H o r d e u m by FISH

of the longest metacentr ic ch romosome in ssp. marinum and the te tomere of the long arm of the SAT chromo- some in the ssp. gussoneanum (Figure 3g). The loci for the 18S-5.8S-26S rDNA sequences showed no differ- ences. Both subspecies p resen ted one major site on the secondary constr ic t ion of the SAT chromosomes and the absence of minor NORs (Figure 3f).

The hap lo id ka ryo type of H. bulbosum (2n = 14) has five metacentr ic , one submetacent r ic and one SAT chromosome (Vosa 1976, L inde-Laursen et al. 1990) (Figure lc). The probe p A s l showed a te lomeric dis- t r ibution. The s ignals were, in general , less intense than in the other species (Figure 3o). The hybr id i za t ion of pSc119.2 was rest r ic ted to the te lomeres of four chromosomes , inc luding the satel l i te of the nucleolar ch romosome (Figure 3o). This marke r and the s ignal at the te lomere of a metacentr ic ch romosome were poly- morphic , poss ib ly as a resul t of quant i ta t ive differen- ces in the number of repeats at homologous sites. As expected, pTa71 h y b r i d i z e d to the nucleolar con- s tr ict ion of the SAT chromosome. No minor hybr id i - za t ion sites were found for this probe (Figure 3n). This resul t agrees wi th the observa t ion of L inde-Laursen et al. (1990), who scored a m a x i m u m of two s t anda rd nucleol i in somatic cells of this species. The 5S ribo- somal rRNA site was revea led by pTa794 at a d is ta l pos i t ion on the shor t a rm of a d is t inct ive submeta - centric ch romosome (Figure 3n) that could be consid- ered to be long to homoeo logous group 5 of o ther Triticeae.

The ident i f icat ion of each H. chilense (2n = 14) chro- mosome was based on the pa t t e rn of ISH us ing p A s l and enzyme detec t ion as r epor ted by Cabrera et al. (1995) (Figure ld) . The probe pSc119.2 marked a series of ve ry useful d iagnost ic s ignals at the te lomeres of a rms 1HchS, 4HchS, 4HchL, 5HchS and at a subtelo- meric pos i t ion of the arm 6HchL. Two major 18S-5.8S- 26S rDNA sites at the secondary constr ict ions of the SAT chromosomes 5Hch and 6Hch were seen (Figure 3p). The relat ive size of the s ignal was larger in 5Hch than in 6Hch.

The ka ryo type of H. secalinum (2n = 4x = 28) showed 10 pa i rs of metacentr ic chromosomes , three submeta - centric pa i rs and one SAT ch romosome pair (Linde- Laursen et al. 1986) (Figure 3a). At least six of the 14 pai rs exhibi ted d iagnost ic hyb r id i za t ion sites to pTa71, pSc794 and pSc119.2 (Figure 3j-m). P o l y m o r p h i s m be tween the homologues of the same or different p lants was observed for the p resence-absence of the 120-bp repea ted sequence at the te lomere of the long arm of the mos t submetacent r ic ch romosome pai r (ar- rowed in Figure 2a). As in H. chilense, all the chromo- somes of H. secalinum hybr id i zed to p A s l , showing a d is t inc t ive pa t t e rn that easi ly ident i f ied homologous chromosomes . In add i t i on to the major NOR site at the nucleolar region of the SAT ch romosome pairs , two minor NORs were found in each of two non-sa te l l ized ch romosome pairs. The first appea r s near the te lomere of the shor t a rm on a submetacent r ic ch romosome and

C h r o m o s o m e Research Vol 4 1996 493

A. de Bustos et al .

Figure 2. The physical map of the chromosomes of tetra- ploid species of the genus Hordeum showing the hybri- dization sites and interstitial regions with the repetitive se- quences inserted in the DNA probes pTa71 and pAsl (left), pTa794 and pSc119.2 (right), and pHch950 (dark grey): a H. secalinum, b H. murinum ssp. murinum, c H. murinum ssp. leporinum.

a)

b)

H. s e c a l i n u m

H. m u r i n u m ssp . m u r i n u m

C) H. r n u r i n u m ssp . l e p o r i n u m

l pSc110.2 CND pTa 794

~1~ pHchg~O ( ~ pTa71

pA=l labelling chromc6on'~= recognized by other probes

pA=l I~olling Unidontifled ChromosornN

variation

Figure 3. Multicolour results of FISH on metaphase plates of various species and subspecies of the genus Hordeum. a-d A metaphase of Hordeum murinum ssp. leporinum counterstained with DAPI (a), digoxigenin-labelled pAsl (b), digoxigenin-labelled pTa71 (r and simultaneous visualization of rhodamine-labelled pTa794 (5S loci are arrowed) and pSc119.2 (arrowheads) (d). e-h H. marinum ssp. gussoneanum counterstained with DAPI (e), rhodamine-labelled pTa794 (5S loci are arrowed) (f) and pTa71 (NOR loci are arrowed) (g) and digoxigenin-labelled pAsl (h). i A metaphase of Hordeum murinum ssp. murinum after hybridization with rhodamine-labelled pHch950 (chromosomes labelled are arrowed). ~ A metaphase of Hordeum secalinum counterstained with DAPI (chromosomes labelled by pHch950 are arrowed) (j), rhodamine-labelled pSc-119.2 and digoxigenin-labelled pTa794 (arrows) (k), rhodamine-labelled pHch950 (I) and simultaneous visualization of hybridization sites to pSc119.2 and pTa71, showing two major sites (long arrows) and four minor sites (small arrows) (m). n-o A metaphase plate of Hordeum bulbosum simultaneously visualized for the hybridization sites to pTa794 (green) and rhodamine-labelled pTa71 (red) (n), and simultaneous visualization of pAsl (green) and pSc119.2 (red) (o). p & q A metaphase plate of Hordeum chilense: simultaneous visualization of rhodamine-labelled pTa794 and digoxigenin-labelled pTa71 (figures indicate the SAT chromosomes, 5Hch and 6Hch) (p) and counterstaining with DAPI (q).

4 9 4 C h r o m o s o m e Resea rch Vol 4 1996

Physical mapping in wild species of Hordeum by FISH

C h r o m o s o m e Research Vol 4 1996 495

A. de Bus tos et al.

the second at an interstitial position on a metacentric chromosome. Probe pTa794 detected three sites for the 5S rDNA sequences. One appeared at a distal position at the telomeres of a submetacentric pair.

The karyotypes observed in the 4x populations of H. murinum ssp. murinum (2n =4x=28) and ssp. lepori- num (2n =4x =28) were generally in agreement with Morrison (1958), Rajhathy & Morrison (1962) and Linde-Laursen et al. (1989) with 11 metacentric chro- mosome pairs and three SAT chromosome pairs (Figure 2b and c). The karyotypes of these subspecies are outstanding among the Hordeum karyotypes for their lack of submetacentric chromosomes and small range of variation in size and morphology. Only the homol- ogy of five chromosomes of the haploid set was easily identifiable between subspecies. The probe pSc119.2 hybridized in a more dispersed fashion in ssp. tour- inure than in ssp. leporinum. Consequently, the repe- titive sequence of 120-bp sequence hybridized to more segments in ssp. murinum than in ssp. leporinum. In this subspecies, pSc119.2 showed a large hybridization signal at the telomere of one long chromosome and small signals at the telomeres of other chromosomes. The same probe strongly labelled the telomeres of four chromosomes in ssp. murinum. This result supports the idea of a greater proportion of heterochromatin in ssp. murinum than in ssp. leporinum, as also demonstrated by C-banding (Linde-Laursen et al. 1989). Both sub- species showed three major NOR loci at the secondary constriction of the SAT chromosomes and a minor site at an interstitial position on a small metacentric chro- mosome. Moreover, the probe pTa71 revealed different sizes of the three major NOR loci in both subspecies.

Of special significance and use in FISH analysis were the results obtained using the probe pHch950. Hueros et al. (1993) showed it to have dispersed hy- bridization signals on all the chromosomes of the diploid species H. chilense. The probe contains an insert of 950 bp and is considered a potentially useful marker in differentiating the H genome in the genus Hordeum (Ferrer et al. 1995). The present results show that this sequence hybridizes evenly to only seven chromosome pairs in the three tetraploid taxa assayed, clearly re- cognizing two sets of chromosomes. It hybridized intensely, in a dispersed manner, to the entire length of 14 chromosomes in both H. murinum subspecies. The remaining pairs remained unhybridized (Figure 3i). H. secalinum showed two groups of chromosomes. One set of seven pairs partially hybridized to pHch950, whereas a second set was almost totally unhybridized except for very short segments of three chromosomes (Figure 31).

Discussion

Repetitive DNA to map chromosomes by FISH and to analyse genomic relationships in Hordeum The rye DNA insert of pSc119.2 showed clear signals at the telomeres of several chromosomes of all taxa.

Further, interstitial signals were detected on several chromosomes of H. marinum ssp. gussoneanum, H. chi- lense and H. secalinum. The repetitive sequence of 120 bp obtained from rye (Bedbrook et al. 1980) has also been found in the wild species of the genus Secale as well as in many species belonging to different genera of the Triticeae tribe (Jones & Flavell 1982, Lapitan et al. 1987, McIntyre et al. 1988, Orgaard & Heslop-Harrison 1994, Castilho & Heslop-Harrison 1995, Cuadrado & Jouve 1995, Cuadrado et al. 1995). Moreover, Gupta et al. (1989), using dot-blot and Southern blot procedures, reported homology of DNA obtained from 22 Hordeum species with the repetitive insert of pSc119, of which pSc119.2 is a subclone. The only exception was culti- vated barley, Hordeum vulgare, with the I genome. Because of its outstanding signal, constant position and uneven distribution, pSc119.2 is extremely useful in chromosome identification and mapping. It has been used in mapping chromosomes of other related species (Mukai et al. 1993, Cuadrado & Jouve 1994, Castilho & Heslop-Harrison 1995). Evidence of the in situ hybri- dization of pSc119.2 to the telomeres of some chromo- somes of the genus Hordeum was previously reported by Xu et al. (1990) in H. bulbosum, using biotin labelling techniques.

The results of the present investigation show the pattern of hybridization of pSc119.2 to the chromo- somes of Hordeum to be similar to that found in cul- tivated rye, Secale cereale (Jones & Flavell 1982, Heslop- Harrison et al. 1991, Cuadrado & Jouve 1994, Cuadrado et al. 1995), Secale montanum (Cuadrado & Jouve 1995), Triticum aestivum (Mukai et al. 1993) and Aegilops um- bellulata (Castilho & Heslop-Harrison 1995). All these species show strong telomeric hybridization sites for the 120-bp sequence. In H. bulbosum and H. secalinum, the hybridization signals for pSc119.2 observed at the same locus in the homologues of the same or different plants presented occasional differences in size and intensity. This result reveals the existence of a degree of polymorphism for the number of copies of the repetitive sequence and could be explained by the open pollination system of these species. H. bulbosum is an almost obligate outbreeding species with a self- incompatibility genetic system. H. secalinum is a fa- cultative outbreeding species (Bothmer et al. 1986). Similar results have been reported by Cuadrado et al. (1995) for the in situ hybridization response to pSc119.2 at corresponding sites between homologous chromo- somes of open-pollinated ryes. In contrast, homolo- gous inbred lines of rye showed constant in situ hybridization patterns.

The hybridization of the probe pAsl to specific areas at telomeres and interstitial sites along the D-genome chromosomes in T. aestivum was first reported by Rayburn & Gill (1985). More recently, Cabrera et aI. (1995) found a somewhat similar labelling pattern of hybridization of pAsl to all seven pairs of the H. chilense chromosomes. This probe hybridizes to multi- ple sites in both arms of all chromosomes in H. mur-

496 Chromosome Research Vol 4 1996

Physical mapping in wild species of H o r d e u m by FISH

inum. It reveals fewer signals at both telomeric and interstitial sites in the remaining species. As a conse- quence, the pAsl probe is very useful for identifying chromosomes. However, the best results are obtained when the patterns of pAsl and other DNA probes are combined. The combination of pAsl and pSc119.2 offers very useful diagnostic details for each of the seven chromosomes of the four diploid karyotypes studied.

The 5S and 18S-5.8S-26S rRNA multigenes are formed by highly repeated units arranged in tandem at loci in eukaryote genomes. In the present investi- gation, the rDNA probe pTa71 hybridized to the sites of the major nucleolar organizer regions (NORs) in the SAT chromosomes of all species investigated. One or two minor loci were also found in chromosomes with- out visible secondary constrictions in both subspecies of H. murinum and H. secalinum. This results are similar to that found by Linde-Laursen et al. (1992a), who reported the position of the NORs in a satellized chromosome of diploid populations of H. marinum

ssp . gussoneanum using ISH to pTa71. Moreover, one additional weak signal in a chromosome without visi- ble nucleolar constriction was also detected. The 5S rRNA system has been localized in a subtelomeric position on the short arm of the SAT chromosome of H. bulbosum. This chromosome is homoeologous to the chromosome 5H of H. vulgare (Kasha & Sadasivaiah 1971). Its homoeologue in Hordeum chilense, according to gene contents and C-banding, is assigned as 5Hch (Fernandez & Jouve 1984, Tercero et al. 1991, Cabrera et al. 1995), and also shows the 5S rRNA site at the NOR. It is interesting to note the common occurrence of a 5S rRNA site in the homoeologous group 5 in many species of Triticeae (Mukai et al. 1990, 1991, Leitch et al. 1992, Castilho & Heslop-Harrison 1995, Cuadrado & Jouve 1995, Cuadrado et al. 1995) with the exception of H. vulgare (Leitch & Helsop-Harrison 1993). The 5S and 18S-5.8S-26S ribosomal multigene families appeared close to one another on the single SAT chromosome in both subspecies of H. marinum, in one SAT chromo- some of H. chilense (5Hch) and in two of the three SAT chromosomes of both H. murinum subspecies. H. se- calinum also showed both ribosomal multigene loci located distally on the short arm of a very submeta- centric chromosome. When present on the same chro- mosome, the relative position of both loci with respect to each other was variable. H. marinum and H. chilense presented the 5S rDNA in a distal position relative to that of the 18S-5.8S-26S whereas the remaining ma- terials showed the reverse order.

Studies on the location and number of NORs in the genus Hordeum have mainly been performed using C- banding and silver staining (Linde-Laursen et al. 1986, 1989, 1990). More recently, in situ hybridization has been used to investigate the physical location of both rRNA multigene systems in cultivated barley, H. vul- gare (Leitch & Heslop-Harrison 1992, Pedersen & Linde-Laursen 1994). In this species the 18S-5.8S-

26S rRNA sites were located on six pairs of chromo- somes, whereas pTa794 was mapped on four pairs (Leitch & Heslop-Harrison 1993). In wild barleys there is generally good agreement between the number of major NORs on the SAT chromosomes as revealed by conventional techniques (Linde-Laursen et al. 1989) and the results of FISH using the probe pTa71.

Genome relationships and possible diphyletic origin of H. murinum and H. secalinum The use of FISH on the chromosomes of both subspe- cies of H. marinum seems to demonstrate substantial differences in the distribution patterns of hybridiza- tion sites. This seems to contradict two earlier obser- vations reported by Linde-Laursen et al. (1989) and Bothmer et al. (1989), who used meiotic analysis and C- banding. In situ hybridization permits the distinction between different repetitive DNA sequences involved in the heterochromatic regions. The divergence ob- served between marinum and gussoneanum in the dis- tribution of hybridization sites for the probes pAsl, pSc119.2, pTa71 and pTa794 suggests a clear separation that questions their taxonomic relationship as a mono- typic group of specific rank. On the contrary, the results of the present study agree with previous in- vestigations that consider both taxonomic units as, at least, subspecies of H. marinum (Nevski 1941, Bothmer & Jacobsen 1985, Symeonidis & Moustakas 1986, Both- mer et al. 1989), or even as separate species (Jaaska & Jaaska 1986, Jorgensen 1986, Doebley et al. 1992).

Results from the use of the probe pHch950 in the tetraploid cytotypes of H. secalinum and H. murinum generally supported the case for their alloploid origin. pHch950 clearly shows two sets of seven chromosomes in the cytotypes of Hordeum secalinum and H. murinum. This result provides new evidence for the alloploid origin of these species, as was previously assumed by other authors using meiotic analysis in interspecific hybrids (Bothmer et al. 1988, Jacobsen & Bothmer 1992), repetitive DNA sequences (Svitashev et al. 1994) and chromosome banding (Linde-Laursen et al. 1992b). The H. secalinum cytotype shows two chromo- some sets that display small regions with interchange responses to hybridization with pHch950. This result shows the occurrence of short interchanges or chro- mosomal rearrangements between chromosomes of different genomes and suggests that Hordeum secalinum is an old segmental allopolyploid. H. secalinum should have the basic H genome in combination with a hi- therto unidentified genome (Bothmer et al. 1991). As the diploid species H. chilense bears the diploid basic H genome and also displays hybridization to pHch950, it could be inferred that the seven chromosomes of H. secalinum that hybridize to this probe belong to this genome.

Similar results were found in H. murinum ssp. mur- inum and ssp. leporinum. The karyotypes show a clear differentiation into two sets of seven chromosomes,

C h r o m o s o m e Research Vol 4 1996 497

A. de Bus tos et al.

based on the p resence -absence of d i spe r se D N A se- quences hyb r id i zed to pHch950. Earlier inves t iga t ions sugges ted an a l lop lo id na ture for the t e t rap lo id and hexap lo id subspecies of the H. mur inum aggregate . Accordingly , Covas (1949) descr ibed three taxa in H. murinum, s u p p l y i n g d iagnos t ic characters and chro- mosome numbers . This au thor sugges ted that ssp. leporinum was an a l lop lo id ar is ing from hybr id i za t ion of ssp. murinum (2n = 28) and ssp. glaucum (2n = 14). Rajhathy & Morr i son (1962) also p r o p o s e d that mur- inum (4x) and leporinum (4x) were a l lop lo ids bu t con- specific, and also that H. glaucum was one of the d ip lo id progeni tors . The genome affinity be tween the ssp. glaucum and leporinum has been also demons t r a t ed by Ferrer et al. (1995), who, us ing Southern blots, obse rved the hybr id i za t ion of pHch950 to genomic D N A of both subspecies . Moreover, Bothmer et al. (1988) only obse rved b iva len ts in the meios is of poly- p lo id cy to types of H. mur inum and conc luded that these forms are pure a t lop lo ids wi th different gen- omes. Bothmer et al. (1987, 1988) p r o p o s e d that the d ip lo id genome donors of the H. mur inum aggregate mus t be closely re la ted, based on the h igh au tosynde t ic pa i r ing obse rved in cer tain hybr ids . In agreement wi th this, Rajhathy & Morr i son (1962) and Bothmer et al. (1987) sugges ted that the p o l y p l o i d taxa of H. mur inum m a y possess a d ip lo id i z ing mechan i sm of great s trength, a l though this has not been verif ied in fur ther s tudies. The resul ts wi th FISH indicate clear interge- nomic di f ferent ia t ion and a d d new evidence in favour of an a l l opo lyp lo id or ig in for the murinum aggregate. Fur thermore , FISH reveals a genera l s imi la r i ty in the phys ica l m a p p i n g of ch romosomes of both subspecies . The genera l s imi l i tude of the maps ob ta ined in both subspecies of H. mur inum, us ing a series of probes that came from different species, conf i rms the re la t ionships of both te t rap lo id cy to types and their close re la t ion- ships. The resul ts of the p resen t s tudy confi rm that H. murinum and H. leporinum, which were t rea ted earl ier as separa te species, mus t be cons ide red as subspecies of the complex species H. mur inum (Jorgensen 1986, L inde-Laursen et al. 1989, Jacobsen & Bothmer 1994, Svi tashev et al. 1994).

Acknowledgements

The authors w o u l d like to thank the INIA (Inst i tuto Nacional de Inves t igaci6n y Tecnologia Agrar ia y Ali- mentar ia) of Spain for its f inancial suppo r t of this work (Grant N u m b e r s 7689 and SC93-176-C2) and Comis i6n Asesora de Ciencia y Tecnologia (CICYT) of Spain for the a w a r d of a grant (No. AGF940638), and Adr i an Burton for he lpfu l l inguist ic assistance.

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