Cytologically Integrated Physical Restriction Fragment Length Polymorphism Maps for the Barley Genome Based on Translocation Breakpoints

Corresponding author: Gottfried Künzel, Institute of Plant Genetics and Crop Research, Corrensstr. 3, D-06466 Gatersleben, Germany., kuenzel{at}ipk-gatersleben.de (E-mail)

Communicating editor: B. S. GILL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have developed a new technique for the physical mapping of barley chromosomes using microdissected translocation chromosomes for PCR with sequence-tagged site primers derived from >300 genetically mapped RFLP probes. The positions of 240 translocation breakpoints were integrated as physical landmarks into linkage maps of the seven barley chromosomes. This strategy proved to be highly efficient in relating physical to genetic distances. A very heterogeneous distribution of recombination rates was found along individual chromosomes. Recombination is mainly confined to a few relatively small areas spaced by large segments in which recombination is severely suppressed. The regions of highest recombination frequency (1 Mb/cM) correspond to only 4.9% of the total barley genome and harbor 47.3% of the 429 markers of the studied RFLP map. The results for barley correspond well with those obtained by deletion mapping in wheat. This indicates that chromosomal regions characterized by similar recombination frequencies and marker densities are highly conserved between the genomes of barley and wheat. The findings for barley support the conclusions drawn from deletion mapping in wheat that for all plant genomes, notwithstanding their size, the marker-rich regions are all of similar gene density and recombination activity and, therefore, should be equally accessible to map-based cloning.

MOLECULAR linkage maps of cereals are improved rapidly by adding new types of markers, by merging different species-specific maps and by comparative mapping of markers between related genomes. Efficient use of the resulting dense maps, therefore, requires detailed insights into the relationship between genetic and physical distances. For example, in crop plants with large genomes, the success of positional gene cloning depends on the possibility of defining genomic regions where physical distances between linked loci are short enough to be bridged by molecular techniques (TANKSLEY et al. 1995 ).

Different strategies can be used to relate genetic to physical distances along entire chromosomes of high DNA content. One is physical localization of short, single-copy marker sequences or large insert clones (bacterial or yeast artificial chromosomes) by in situ hybridization on individual chromosomes. In spite of moderate progress in barley (LEHFER et al. 1993 ; LEITCH and HESLOP-HARRISON 1993 ; BUSCH et al. 1994 ; FUKUI et al. 1994 ; PEDERSEN et al. 1995 ; LAPITAN et al. 1997 ), these techniques are laborious and not yet routinely practicable for plants with large genomes. An alternative approach is to integrate physical landmarks into genetic maps. For hexaploid wheat, a system to generate terminal chromosome deletions was developed (ENDO 1988 ; ENDO and GILL 1996 ). Using defined deletions, comprehensive results were obtained as to the distribution of markers and recombination events on chromosomal subregions (WERNER et al. 1992 ; GILL et al. 1993A , GILL et al. 1993B , GILL et al. 1996A , GILL et al. 1996B ; KOTA et al. 1993 ; HOHMANN et al. 1994 , HOHMANN et al. 1995A , HOHMANN et al. 1995B ; DELANEY et al. 1995A , DELANEY et al. 1995B ; MICKELSON-YOUNG et al. 1995 ). Besides suppressed recombination in proximal chromosome regions and frequent recombination in distal chromosome regions, local hot spots for recombination and gene density were identified for small interstitial chromosome regions (GILL and GILL 1994 ; GILL et al. 1996A , GILL et al. 1996B ) On the basis of the high degree of colinearity of genetic markers among Triticeae species (DEVOS et al. 1992 , DEVOS et al. 1993 ; NELSON et al. 1995A , NELSON et al. 1995B ; VAN DEYNZE et al. 1995 ; DUBCOVSKY et al. 1996 ), restriction fragment length polymorphism (RFLP) markers comparatively mapped between wheat and barley also have been used to compare wheat physical maps with barley linkage maps for group-7 chromosomes (HOHMANN et al. 1995B ). Because of the difficulty of developing deletion lines for diploid species, such material is not yet available for all barley linkage groups (SCHUBERT et al. 1998 ).

To achieve direct physical mapping for barley, we devised a polymerase chain reaction (PCR)-mediated technique for integrating translocation breakpoints (TBs) into barley genetic maps (SOROKIN et al. 1994 ; KUNZEL et al. 1995 ; KUNZEL and KORZUN 1996 ). Although >1000 translocation (T) lines are available for barley (KUNZEL 1992 ), this technique was hampered at the beginning since the positions of TBs in most cases could not be determined precisely enough by analysis of Giemsa-banded karyotypes (LINDE-LAURSEN 1988 ; KAKEDA and YAMAGATA 1991 ; MARTHE and KUNZEL 1994 ). After mapping many TBs into different linkage groups, an increasing refinement of the karyologically defined TB positions was possible by the use of a specific software program that was developed to process the corresponding data for that purpose. Evaluation of data obtained from PCR with marker-specific primers and DNA of translocated chromosomes by this software proved to be highly efficient in relating physical to genetic distances within the barley genome. On the basis of 240 TBs of 120 T lines, we provide cytogenetically integrated physical RFLP maps for the seven barley chromosomes with an accuracy comparable to the deletion-based ladder maps of wheat. This article is accompanied by supplementary data (Tables S1–S7) available online at http://www.genetics.org/cgi/content/full/154/1/397/DC1/.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Translocations:
A total of 120 reciprocal translocations (for nomenclature and origin see RAMAGE 1971 , RAMAGE 1975 ; KUNZEL 1992 ) were used (see Tables S1–S7 online at http://www.genetics.org/cgi/content/full/154/1/397/DC1/.). T chromosomes were designated according to RAMAGE 1985 . For example, the two chromosomes involved in translocation T1-2 are designated T12 and T21. The first numeral refers to the chromosome providing the centromere-carrying segment, the second to the chromosome supplying the acentric segment. The TBs were assigned by chromosome measurements and in relation to Giemsa N bands according to MARTHE and KUNZEL 1994 .

Chromosome terminology and measurements:
Except for T lines, the designation of chromosomes follows the Triticeae system (LINDE-LAURSEN et al. 1997 ). To avoid confusion with the designation of T lines, in tables and figures the old barley enumeration is given in parentheses: 1H(5), 2H(2), 3H(3), 4H(4), 5H(7), 6H(6), and 7H(1).

Arm lengths and TB positions were defined on the basis of milliGeNome units (1 mGN = 1/1000 mitotic metaphase genome length) according to JENSEN and LINDE-LAURSEN 1992 . The positions of TBs are given as the distances in mGN from centromeres (= position 0) within short (S) and long (L) arms, respectively. Usually, TB positions correspond to midpoints of refined mGN intervals (for refinement of TB positions, see Fig 8) to which the TBs were assigned (see Tables S1–S7 online at http://www.genetics.org/cgi/content/full/154/1/397/DC1/.) and were calculated as fraction lengths of chromosome arms (FL, length of the nontranslocated arm segment relative to the whole arm) in Fig 1 Fig 2 Fig 3 Fig 4 Fig 5 Fig 6 Fig 7.

View larger version (34K): In this window In a new window Download PPT slide   Figure 1. Chromosome 1H(5). Comparison of physical and genetic maps. The idiograms and RFLP maps are aligned at approximate centromere positions (Cen). Subregions of similar recombination rates are shown in the same color. As compared to the genome-wide average (4.4 Mb/cM), blue indicates suppressed (>4.4 Mb/cM), green increased (1.0–4.4), and red strongly increased (1.0 Mb/cM) recombination. Black regions of the physical maps mark Giemsa N bands. Positions of N bands, nucleolus organizer regions (NOR) and translocation breakpoints (TB) are given in fraction-length (FL) estimates. If several TBs were mapped between the same loci, the Mb distance between the two physically most distant TBs was divided by the cM distance of the two flanking markers; such estimates are marked by the preceding symbol . Special cases are denoted by superscript a–c. (a) Corresponding genetic distance is not definable since the TBs are located between cosegregating markers, as indicated to the right of the genetic maps. (b) Physical subregions are arbitrarily assigned to 0.01-FL intervals because TBs of the same FL estimate are located in different regions of the genetic map. (c) cM positions of both the NOR and Cen are included by inference on the basis of the Steptoe/Morex linkage map according to KLEINHOFS et al. 1993 .

View larger version (43K): In this window In a new window Download PPT slide   Figure 2. Chromosome 2H(2). See legend to Fig 1.

View larger version (40K): In this window In a new window Download PPT slide   Figure 3. Chromosome 3H(3). See legend to Fig 1.

View larger version (36K): In this window In a new window Download PPT slide   Figure 4. Chromosome 4H(4). See legend to Fig 1.

View larger version (43K): In this window In a new window Download PPT slide   Figure 5. Chromosome 5H(7). See legend to Fig 1.

View larger version (62K): In this window In a new window Download PPT slide   Figure 6. Chromosome 6H(6). See legend to Fig 1.

View larger version (41K): In this window In a new window Download PPT slide   Figure 7. Chromosome 7H(1). See legend to Fig 1.

View larger version (27K): In this window In a new window Download PPT slide   Figure 8. Refinement of TB positions by mapping between markers of linkage maps. Example for the long arm of chromosome 6H: 24 informative TBs, karyologically assigned to chromosomal segments of varying size, were integrated into the genetic map. The possible refinements of TB positions are indicated by arrowed lines. For example, MWG984.a must be 23 mGN apart from the centromere because its distance from the centromere cannot be smaller than that of the proximal breakpoint T67as. All TBs distal to MWG984.a should, therefore, have this same or larger distance to the centromere (marked in red). The same works also in the opposite direction. Since MW-G820 must be less distant from the centromere than the more distal breakpoint T62as, its physical position is 64 mGN apart from the centromere. All TBs proximal to MWG820 should, therefore, be located at this same or shorter distance from the centromere (marked in blue).

Chromosome preparations and microdissection:
Procedures for chromosome preparations and microscopically controlled chromosome dissections were as described previously (SOROKIN et al. 1994 ), with a few modifications. The number of prophase nuclei and microisolated chromosomes used per PCR run was reduced from 10 to 5 and 20 to 5, respectively. The nuclei and chromosomes (~1 µl per sample) were collected in a solution containing 10 mM Tris-HCl (pH 9.0), 10 mM NaCl, and 0.1% SDS, overlaid with mineral oil to avoid evaporation.

RFLP maps and sequencing of RFLP probes:
The present study is based on the Igri x Franka molecular linkage map (I/F map; GRANER et al. 1991 , GRANER et al. 1993 ), with updated centimorgan positions available from the Triticeae database GrainGenes (http://www.probe.nalusda.gov:8300/cgi-bin/browse/graingenes; "Map, Hordeum-Graner1"). The origin of probes is as indicated in GRANER et al. 1993 . Tables S1–S7 (online at http://www.genetics.org/cgi/content/full/154/1/397/DC1/.) list the 301 markers of the I/F map that were included in our study after at least partial sequencing according to standard procedures using an automated laser fluorescence (ALF) DNA sequencer (Pharmacia, Uppsala, Sweden). All the sequences are available from the database GrainGenes. For MWG probes, the sequence data have also been deposited in the EMBL/GenBank/DBBJ nucleotide sequence databases under the accession numbers AJ234400–AJ234900.

PCR analyses:
Primers were designed to convert RFLP markers into PCR markers for sequence-tagged sites (STS). To minimize the generation of polymorphic amplification fragments, the primers were arranged to yield relatively short products of ~150–500 bp.

PCR was performed in volumes of 50 µl for 35–45 cycles. Five microisolated chromosomes or prophase nuclei, pooled in the collection drop, were transferred into PCR tubes. The final reaction mixture contained 100 mM Tris-HCl (pH 8.8), 500 mM KCl, 0.8% Nonidet P40, 0.2 µM of each primer, 0.2 mM of each dNTP, 2.0 mM MgCl₂, and 1.5 units Taq polymerase (MBI Fermentas). PCR conditions were optimized for each specific primer set. Amplified DNA fragments were electrophoresed in 1.2–1.6% agarose gels and stained with ethidium bromide. Fragment sizes were estimated using a DNA length marker VI (Boehringer Mannheim, Mannheim, Germany) or a pUC marker (MBI Fermentas).

Locating markers in chromosomal subregions:
For assignment to physical subregions, primer pairs for each marker were used for PCR with prophase nuclei as a positive control, the two interchanged chromosomes of a T line as the critical "present/absent" count (all microdissected from the same cytological preparation), and a negative control without template DNA. Thus, markers mapped to linkage groups corresponding to one of the two T chromosomes could be reliably assigned to one of the corresponding T segments (for demonstration, see SOROKIN et al. 1994 ). PCR analyses were performed until the two markers most closely flanking a TB were identified. For chromosomes 2H, 3H, 4H, and 7H, T lines involving one of the SAT chromosomes (5H or 6H) were used preferentially, since in such cases, one of the two interchanged chromosomes could be recognized easily by its satellite. Then, two to three markers on either side of the TB were checked by PCR in addition to the TB-flanking markers to ensure that the TBs were correctly localized onto the genetic maps.

Megabase/centimorgan relationships:
Assuming a constant DNA density along chromosomes, megabase/centimorgan ratios (Mb/cM) were calculated on the basis of a DNA content of 5350 Mb/haploid barley genome (BENNETT and SMITH 1976 ) and relative chromosome measurements of MARTHE and KUNZEL 1994 . Considering the entire genetic length of the I/F map of 1214.2 cM, an average value of 4.4 Mb/cM is assumed for the barley genome. For individual subregions, the Mb/cM estimates were calculated on the basis of the midpoints of both physical segments to which the flanking TBs were assigned and the cM intervals within which the TBs were mapped. Occasional deviations from this procedure are explained in the legends of the corresponding figures.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

After PCR mapping, all TBs individually flanked by two genetic markers (or by one genetic marker and a centromere or nuclear organizer region [NOR]) were used to divide the corresponding chromosome arms into subregions. According to their recombinative activity, these subregions were categorized into those of suppressed recombination (>4.4 Mb/cM), high recombination (1.0–4.4 Mb/cM), or very high (hot spots) recombination (<1.0 Mb/cM). Immediately adjacent subregions of high/very high recombination frequency were composed and denoted as recombinogenic areas.

Chromosome 1H:
The 35 TBs integrated into this linkage map were predominantly located (20 out of 35) within an extended, marker-free region around the centromere spanning ~46% of the S- and 44% of the L-arm lengths (Fig 1). On the basis of the positions of 13 TBs, 6 subregions of the S arm and 9 of the L arm could be compared to corresponding regions of the genetic map.

The scarcity of markers on the I/F map seems to be a reason for the high Mb/cM estimate (~19) obtained for the distal region of the L arm. High and very high recombination rates were confined to four areas comprising 10 subregions that are relatively small in size. Two of the highly recombinogenic interstitial regions, one each in the S and L arms, were found to be embedded in large physical segments that are poor in markers. In the L arm, three hot spots for recombination were identified, with Mb/cM estimates ranging from 0.3 to 0.6. In total, the recombinogenic areas comprise ~30% of the chromosome length and contain 85% of the 47 markers mapped on this chromosome (Table 1).

Chromosome 2H:
Of the 31 TBs localized within this linkage group, 9 were found within a large median chromosome segment comprising 56% of the S and 37% of the L arm lengths (Fig 2). These areas of highly suppressed recombination were devoid of markers. With 19 TBs as landmarks, 18 subregions (7 of the S arm and 11 of the L arm) could be compared with their corresponding linkage segments.

Six TBs were localized between the cosegregating markers at the position of 82.3, indicating that this cluster of markers is spread over a region comprising ~29% of the S-arm length, of which a large part (FL interval 0.56–0.81) separates the markers cMWG663 and MWG874. Further cosegregating markers at the cM positions 19.0, 83.7, 106.2, and 191.0 were physically split by inserted TBs. Since no TBs resolve the marker cluster at the position of 191.0 cM, the high estimate of 6.3 Mb/cM is probably not representative for the end segment of the L arm. Five hot spots for recombination with Mb/cM estimates ranging from 0.2 to 0.8 were identified. High and very high recombination is confined to 11 subregions of 5 areas spanning ~24% of the chromosome length. Nearly 81% of the 68 markers mapped in this chromosome reside in these recombinogenic areas (Table 1).

Chromosome 3H:
Out of 20 TBs localized on this linkage map, 15 were used to divide the chromosome into 17 subregions (Fig 3). The genetic map of this chromosome is characterized by several clusters of cosegregating markers that were partly split by inserted TBs. The cluster of markers at cM position 55.6 includes the centromere that is flanked by MWG852 in the S arm and ABG462 in the L arm. These two cosegregating markers are spaced by ~45% of the entire chromosome length representing a region with apparently no recombination in the I/F map.

In contrast, three hot spots for recombination were identified which are characterized by Mb/cM estimates ranging from 0.1 to 0.4. The recombinogenic areas, which are sometimes sharply demarcated from regions of suppressed recombination, amount to nearly 16% of the total chromosome length and contain ~79% of the 82 markers mapped in this chromosome (Table 1).

Chromosome 4H:
Out of 28 TBs localized onto this linkage map, 16 were used to divide the chromosome into 18 subarm intervals (Fig 4). The centromere is flanked by MWG2036 and a cluster of cosegregating markers at the position of 61.1 cM in the L arm. A large region surrounding the centromere, including half of each of the S- and L-arm lengths, contains no markers. The two clusters of cosegregating markers in the L arm, genetically close to the centromere (one of them split by 2 TBs), were physically allocated to the FL interval 0.50–0.62.

Two terminal and three interstitial areas consisting of nine subregions revealed high or very high recombination frequencies, respectively. The mean Mb/cM estimates for the five recombination hot spots range from 0.2 to 0.8. The areas expressing high and very high recombination rates comprise ~24% of the total chromosome length; >60% of the 37 markers reside in these regions (Table 1).

Chromosome 5H:
Out of 58 TBs inserted into this linkage map, 24 were used to divide the chromosome into 23 subregions (Fig 5). Half of the TBs (29) occurred between the NOR of the S arm and the middle of the L arm. This region amounts to ~56% of the chromosome length and is free of markers. The TBs outside this region tend to be clustered at certain regions of the genetic map. On the S arm, the 3 TBs localized distally to MWG502 indicate that this outermost locus on the I/F map does not represent the terminus of this arm.

Very high recombination rates in the S arm are restricted to a short proximal part of the satellite that probably includes the NOR. Recombination within this chromosome is found to be confined nearly exclusively to five areas that correspond to ~19% of the chromosome length containing ~98% of the 63 markers mapped in this chromosome in the I/F map (Table 1).

Chromosome 6H:
A total of 33 out of 45 TBs inserted into this genetic map define 31 chromosomal subregions (Fig 6). Besides a surplus around the centromere, the TBs were distributed more evenly on this genetic map than on those of other chromosomes. The centromere was located within a marker-free region of highly suppressed recombination comprising about one-third each of the S- and L-arm lengths. A number of cosegregating markers could be physically resolved by TBs.

Contrary to the satellite of chromosome 5H, high and very high recombination rates were found within the distal 40% of the satellite. Despite covering more subregions, the results are comparable to those obtained for other chromosomes. High recombination was mainly found within the distal regions of both arms. In most cases, the recombination frequency switched abruptly between the subregions, as is evident for small subregions of high and very high recombination at interstitial positions. Five subregions were identified as hot spots of recombination, with Mb/cM estimates ranging from 0.1 to 0.8. In total, the recombinogenic areas correspond to ~21% of the chromosome length and contain ~82% of the 51 markers mapped in this chromosome (Table 1).

Chromosome 7H:
Out of 24 TBs localized within this linkage map, 15 divide the chromosome into 16 subregions (Fig 7). The linkage map of this chromosome is characterized by several clusters of cosegregating markers. As with chromosome 3H, the centromere was localized within a large cluster of cosegregating markers at cM position 123.7, flanked by cMWG649.a in the S arm and PBI21.b in the L arm. In contrast to all other chromosomes, no TBs were found between the centromere and these two markers. Therefore, the positions of cMWG649.a and PBI21.b, relative to the centromere, were identified by PCR with telosomes microisolated from barley telotrisomic stocks. Since the PCR products differed between barley and wheat, the arm assignment of cMWG649.a and PBI21.b could be confirmed with DNA from barley-wheat telosome addition lines. A median chromosome region, including the proximal 37% of the S arm and the proximal 25% of the L arm, showed no recombination within the I/F map.

Each chromosome arm is characterized by two areas of high and very high recombination frequency positioned within the distal 9% and an interstitial area of the S arm (FL 0.37–0.57) and within the distal 14% and an interstitial area of the L arm (FL 0.40–0.50). These recombinogenic areas of ~27% of the chromosome length contain ~94% of the 81 markers mapped in this chromosome (Table 1).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Quality of results:
Precise physical positions of the TBs and their correct integration into the genetic maps are crucial for the reliability of the results obtained in this work. In the present study, >300 STS markers were developed from RFLP probes to integrate the physical and genetic maps of the barley genome. The conversion of RFLP into STS markers clearly bears one risk for fallacies, since the PCR products may not necessarily be derived from the target locus (TRAGOONRUNG et al. 1992 ; BLAKE et al. 1996 ; ERPELDING et al. 1996 ). About 20% locus duplication was reported for the I/F map (GRANER et al. 1993 ). This is in fact a minimal estimation because nonpolymorphic multiple fragments escape detection. The distribution of duplicated loci between and within chromosomes may be affected by a number of modifying parameters (DUBCOVSKY et al. 1996 ). Nevertheless, in our experiments, possible mistakes from locus duplication are reduced considerably because each case considered only two (the translocated ones) of the seven chromosomes. Since the primers were designed from RFLP probes derived from different genotypes, their functionality was tested with genomic DNA of the varieties from which the T lines originated. In ~25% of all cases, it was necessary to design new primers to obtain a single distinct PCR product. Primers derived from single-copy marker sequences that yielded a distinct PCR product of the expected size were considered to be locus specific. To identify mapped multiple loci detected by low-copy probes in additional maps, data-base searches (GrainGenes) covering all Triticeae species were performed. In a number of cases, the primer sets were also checked for arm specificity of their PCR products, either with microisolated telosomes of barley telotrisomic stocks (SINGH and TSUCHIYA 1982 ) or with DNA of barley-wheat telosome addition lines (ISLAM 1983 ), if the amplified fragments were polymorphic between wheat (cv. Chinese Spring) and barley (cv. Betzes). Markers mapped to more than one locus in the same chromosome or in both chromosomes involved in a T line were excluded from the studies if unequivocal results could not be expected.

Another crucial point is the reliability of physical TB positions. The strategy applied for the iterative delimitation of TB positions is depicted in Fig 8. The physical range to which a TB was originally assigned by chromosome measurement became gradually decreased as more TBs were mapped correctly between markers of a defined genetic distance, because of the mutual determination of the respective genetic and physical positions. Considering all linkage groups, further mutual improvements resulted from interconnections of the different chromosomes combined by various translocations. Since this complex system of logical interrelationships required computer analysis, we created a software, which proved to be crucial for the level of physical resolution, as well as the degree of reliability of the mapping results that were attained. A consequence of this specific feature of TB mapping was that the highest degree of physical resolution and the highest degree of reliability of the mapping results could not be reached before all suitable TBs were mapped into all linkage groups. This procedure also revealed individual T lines of which one of the two TBs was incorrectly located on the genetic map. This is the case when improved physical positions of the two TBs of a T line (deduced from their map positions) do not complement each other, as expected for a reciprocal translocation. A few such translocations were identified and omitted from the final evaluations. Most probably, unrecognized structural changes are responsible for the observed discrepancies that, for example, can be caused by association of translocated segments with inversions. This strategy rendered data free of internal contradictions. The extent of refinements for the TB positions becomes evident when the extension of segments to which the TBs were karyologically assigned (23.6 mGN length on average) are compared with those obtained after refinement by PCR mapping (5.1 mGN length on average).

Any cytological measurement, even from excellent chromosome preparations, is affected by an uncertainty. From our experience in TB karyotyping, deviations of ~±2 mGN are to be expected on average. Since the FL values are based on the midpoints of physical segments within which the TBs were located after positional refinements, the TB positions given in Fig 1 Fig 2 Fig 3 Fig 4 Fig 5 Fig 6 Fig 7 might still deviate from the real ones. This seems to be negligible, however, since the data finally obtained are in excellent agreement with the results obtained by other authors after in situ hybridization in barley or by deletion mapping in wheat (see below).

Comparison between physical and genetic distances:
First hints of suppressed rates of meiotic recombination within the middle regions of barley chromosomes came from early observations on inverted and translocated chromosomes more than 30 years ago (HOLM 1960 ; NILAN et al. 1968 ; HAGBERG and HAGBERG 1969 ; KREFT 1969 ). Further studies on barley (e.g., KUNZEL 1982 ; LINDE-LAURSEN 1982 ; LEHFER et al. 1993 ; LEITCH and HESLOP-HARRISON 1993 ; BUSCH et al. 1994 ; FUKUI et al. 1994 ), wheat (DVORAK and CHEN 1984 ; SNAPE et al. 1985 ; CURTIS and LUKASZEWSKI 1991 ; for review see GILL and GILL 1994 ), and rye (for review see HESLOP-HARRISON 1991 ; WANG et al. 1992 ) have shown that reduced recombination frequency is a characteristic of pericentric regions of the large Triticeae chromosomes.

In barley, 10 physical anchor sites distributed over 7 of the 14 chromosome arms have been located by in situ hybridization so far. Suppressed recombination in proximal and high recombination rates in distal regions was demonstrated by PEDERSEN et al. 1995 . This, however, identified only rather large regions. In all cases where the results of in situ hybridization can be compared with those obtained in this study, the respective markers are found at nearly the same physical positions. This confirms the accuracy of results obtained with both methods.

The cytogenetically integrated linkage maps resulting from TB mapping provide a much more differentiated insight into the physical size and recombination frequency of defined subchromosomal regions. Surprisingly, most recombination was found to be confined to only a few distinct chromosomal areas. These areas occur mostly at the arm ends, but were also found at interstitial positions specific for each of the individual chromosome arms. As indicated by suitable TBs, the recombinogenically active areas often alternate abruptly with regions of severely suppressed recombination. For example, on the S arms of chromosomes 2H and 3H, large areas of 110 and 88 Mb in size are located between cosegregating markers at the 82.3- and 42.7-cM positions, which are flanked by small subregions of high and very high recombination, respectively. Moreover, one to three small regions per chromosome arm were identified as hot spots for recombination, except for 1HS. The relationship between genetical and physical distance in these areas was found to range from 0.1 to 0.9 Mb/cM on average. The physical density of markers corresponds closely to the distribution of recombination events. The recombination hot spot regions (1 Mb/cM) amount to only 4.9% of the total barley genome, but they harbor 47.3% of 429 markers from the I/F map, i.e., their marker density is ~20 times that of the remaining genome (Table 1).

Finally, TBs in chromosome segments without markers provide a prospective tool for further refinement of physically integrated genetic maps when new markers that map within these regions become available.

Homoeology relationships with wheat:
Except for a few translocations and inversions (DEVOS et al. 1995 ; DUBCOVSKY et al. 1996 ; LINDE-LAURSEN et al. 1997 for review), close collinearity between molecular markers in barley and the three wheat genomes is well established (e.g., DEVOS and GALE 1993 ; DEVOS et al. 1993 ; NAMUTH et al. 1994 ; VAN DEYNZE et al. 1995 ; DUBCOVSKY et al. 1996 ). With the present results, for the first time, a detailed comparison of the chromosomal distribution of recombination events between all chromosomes of barley and wheat, on the basis of results obtained directly for both species, is also possible. Taken together, the findings for barley correspond well with those derived from deletion mapping for chromosomes in group 1 (KOTA et al. 1993 ; GILL et al. 1996B ), group 2 (DELANEY et al. 1995A ), group 3 (DELANEY et al. 1995B ), group 4 (MICKELSON-YOUNG et al. 1995 ), group 5 (GILL et al. 1996A ), group 6 (GILL and GILL 1994 ), and group 7 (WERNER et al. 1992 ; HOHMANN et al. 1994 ) in wheat.

For example, in wheat group-1 chromosomes (GILL and GILL 1996b), ~86% of 305 markers were found to be present in five major clusters that are spaced by large, marker-poor chromosomal regions. Most of the recombination events occurred within these marker clusters, which span only ~10% of the consensus chromosome length. Although the terminal segments could not be resolved sufficiently by TBs and the corresponding I/F map contains only 47 markers, the similarity of results described in our study for barley compared to those of wheat is striking. Four areas of high and very high recombination rates, which are spaced by large chromosomal segments of suppressed recombination, were detected in barley. Even the relative physical positions for some of the highly recombinogenic regions are nearly identical, e.g., in the L arm of the barley chromosome 1H at FL 0.44–0.47, FL 0.81–0.86, and FL 0.92–0.93 as compared to FL 0.40, FL 0.85, and FL 0.98 in wheat group-1 chromosomes.

On the basis of the gene collinearity among Triticeae, HOHMANN et al. 1995B mapped many RFLP markers of the I/F map 7H to a set of deletion lines of group-7 chromosomes. Although the cytogenetic ladder maps of wheat were based on consensus physical maps that combine deletions from the A, B, and D genomes that differ in size and in the amount and distribution of heterochromatin, the results obtained by TB mapping in barley are in surprisingly good agreement with those derived for wheat. In general, there are no deviations in the relative physical positions of markers in barley chromosome 7H and the wheat group-7 chromosomes. The only difference goes back to tightly linked markers around the centromere, since the conclusions in wheat were based on the cM values originally published (GRANER et al. 1991 , GRANER et al. 1993 ), while in our study, cM values of an updated version were used (GrainGenes: "Map, Hordeum-Graner 1"). For instance, the markers PBI-21.b and MWG957 were assigned to the S arm of the physical consensus map of wheat, but were reliably located in the L arm of the barley chromosome by TB mapping. Also, the heterogeneous distribution of recombination events and the pronounced accumulation of markers in recombinogenically active regions of this chromosome become more evident by TB mapping than by comparison to deletion mapping in wheat. The results thus confirm the accuracy of the two methods, and they show that both techniques lead to the same conclusions with respect to the chromosomal position of hot spots for recombination and marker density. This indicates an evolutionary conservation of subchromosomal organization between barley and wheat and probably all large-sized Triticeae genomes.

Conclusions for marker applications:
Besides ~20% of known-function and anonymous cDNA markers, the remaining markers of the I/F map are genomic PstI probes. From similarity searches against DNA and protein sequence databases, MICHALEK et al. 1999 inferred that a significant portion of these PstI probes represents genes. Therefore, the high correlation between marker density and recombination frequency implies that most recombination events occur in gene-rich regions and that these correspond to only small chromosomal areas. This is in line with the view that genes are generally considered as recombination hot spots (SCHNABLE et al. 1998 ), and emphasizes conclusions drawn from the comprehensive work based on deletion-mapping in wheat. For marker-rich areas, the average marker distance was calculated to be 244 kb (corresponding to 118 kb/cM) in wheat (GILL et al. 1996A , GILL et al. 1996B ). This is comparable to similar regions in tomato (GANAL et al. 1989 ; SEGAL et al. 1992 ; ZHANG et al. 1994 ; TANKSLEY et al. 1995 ) and rice (UMEHARA et al. 1995 ), and suggests that the gene densities and recombination frequencies are similar for gene-rich regions among all plants, irrespective of their genome sizes. Therefore, these regions should be equally accessible to map-based cloning (GILL et al. 1996B ). The results obtained by TB mapping in barley strongly support these conclusions.

From TB mapping in barley and deletion mapping in wheat, it is obvious that the smaller a physical region that can be compared to its corresponding linkage segment, the more the heterogeneous distribution of recombination becomes revealed. For barley, this is in line with results from high-resolution genetic mapping combined with electrophoretic analysis of high-molecular-weight DNA. For instance, in the vicinity of the Mla locus for mildew resistance, two segments of 0.45 and 2.35 Mb were characterized by ratios of 0.46 and 2.5 Mb/cM, respectively (DESCENZO and WISE 1996 ). A similar ratio of ~0.6 Mb/cM was estimated for a yeast artificial chromosome (YAC) clone that includes the Mla locus by SCHWARZ et al. 1999 . The Mla region is positioned in the terminal segment of the S arm of chromosome 1H, which is characterized by a mean estimate of 1.3 Mb/cM in this study. Molecular data are also available for the Mlo locus, of which the recessive alleles confer a durable type of resistance to barley powdery mildew. In this case, estimates of 0.1 and 0.3 Mb/cM were reported for two short DNA segments, including this locus (SIMONS et al. 1997 ). The Mlo locus is located in the end segment of chromosome 4HL, which was found to be a region of high recombination (~2.6 Mb/cM) in this study. Another case is the Rar1 locus, which encodes a putative signaling component of race-specific resistance to barley mildew. On the basis of a YAC contig encompassing the locus, LAHAYE et al. 1998 estimated 0.63 Mb for the 0.05-cM target interval. The Rar1 locus is included in the chromosome segment between FL 0.67 and FL 0.79 on the L arm of chromosome 2H that was defined as a highly recombinogenic subregion, with a mean estimate of 1.1 Mb/cM in this study. Differences like the latter ones require caution in interpreting data that represent averages, since significant fluctuations in recombination frequencies may occur even within short stretches of DNA. For example, in maize, within a genetic interval of 0.01 cM corresponding to ~140 kb, recombination frequencies were found to vary by about sevenfold (CIVARDI et al. 1994 ).

	ACKNOWLEDGMENTS

We thank Andreas Graner for providing the RFLP probes, Ingo Schubert for many helpful suggestions in preparing and critical reading of the manuscript, Susanne König for sequencing of RFLP clones and Katrin Kumke, Ines Walde, and Elke Höpfner for technical assistance in chromosome microisolation and PCR analyses. This work was supported by the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (grant no. 0311002) and by the Deutsche Forschungsgemeinschaft (Ku 1129/1-1) to Gottfried Künzel and the Fonds der Chemischen Industrie to Armin Meister.

Manuscript received May 4, 1999; Accepted for publication September 9, 1999.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

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