Genetics, Vol. 164, 673-683, June 2003, Copyright © 2003

A Large Rearrangement Involving Genes and Low-Copy DNA Interrupts the Microcollinearity Between Rice and Barley at the Rph7 Locus

S. Brunnera, B. Kellera, and C. Feuilleta
a Institute of Plant Biology, University of Zürich, CH-8008 Zürich, Switzerland

Corresponding author: C. Feuillet, University of Zürich, Zollikerstr. 107, CH-8008 Zürich, Switzerland., feuillet{at}botinst.unizh.ch (E-mail)

Communicating editor: A. PATERSON


*  ABSTRACT
*TOP
 *ABSTRACT
*MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
 *LITERATURE CITED

Grass genomes differ greatly in chromosome number, ploidy level, and size. Despite these differences, very good conservation of the marker order (collinearity) was found at the genetic map level between the different grass genomes. Collinearity is particularly good between rice chromosome 1 and the group 3 chromosomes in the Triticeae. We have used this collinearity to saturate the leaf rust resistance locus Rph7 on chromosome 3HS in barley with ESTs originating from rice chromosome 1S. Chromosome walking allowed the establishment of a contig of 212 kb spanning the Rph7 resistance gene. Sequencing of the contig showed an average gene density of one gene/20 kb with islands of higher density. Comparison with the orthologous rice sequence revealed the complete conservation of five members of the HGA gene family whereas intergenic regions differ greatly in size and composition. In rice, the five genes are closely associated whereas in barley intergenic regions are >38-fold larger. The size difference is due mainly to the presence of six additional genes as well as noncoding low-copy sequences. Our data suggest that a major rearrangement occurred in this region since the Triticeae and rice lineage diverged.

GRASSES evolved from a common ancestor ~50–80 million years ago (WOLFE et al. 1989 Down). Comparative mapping studies have demonstrated a good conservation of the marker order (collinearity) along the chromosomes in grass species although they differ greatly in genome size (MOORE et al. 1995 Down; GALE and DEVOS 1998 Down; KELLER and FEUILLET 2000 Down). Therefore, comparative genomics in grasses was suggested to be an excellent tool for map-based cloning of agronomically important genes from large and complex genomes using the small rice genome as a vehicle. However, several studies have shown that, although collinearity is generally good at the genetic map level, numerous small rearrangements at the gene level disrupt microcollinearity between rice and large genome species such as barley (KILIAN et al. 1997 Down; HAN et al. 1998 Down, HAN et al. 1999 Down; DRUKA et al. 2000 Down), wheat (LI and GILL 2002 Down; SANMIGUEL et al. 2002 Down), and maize (CHEN et al. 1997 Down, CHEN et al. 1998 Down; TARCHINI et al. 2000 Down; for recent reviews see BENNETZEN and RAMAKRISHNA 2002 Down; FEUILLET and KELLER 2002 Down). These data have demonstrated that rice can be very useful in providing numerous markers to saturate genetic maps in other cereals and that map-based cloning must be performed in the species of interest. As a consequence, large efforts were undertaken to develop efficient tools for gene isolation from barley and wheat. In barley, a number of detailed genetic maps (http://wheat.pw.usda.gov/ggpages/maps.shtml#barley), a bacterial artificial chromosome (BAC) library (YU et al. 2000 Down), >240,000 expressed sequence tags (ESTs) (http://www.ncbi.nlm.nih.gov/dbEST/dbEST_summary.html), and a gene tagging system (KOPREK et al. 2000 Down) were generated in the last years.

The development of BAC libraries allows the analysis of the gene organization in this large and complex genome as well as a comparison with the recently released rice genome sequences (http://rgp.dna.affrc.go.jp/; GOFF et al. 2002 Down; YU et al. 2002 Down) for a better understanding of genome evolution in grasses. Few detailed studies of large contiguous barley genomic sequences have been published. Two fragments of <100 kb have been analyzed at the Rar1 locus on chromosome 2H (SHIRASU et al. 2000 Down) and at the Mlo locus on chromosome 4H (PANSTRUGA et al. 1998 Down). Very recently, full sequence analyses of a 102.4-kb sequence on chromosome 5H (DUBCOVSKY et al. 2001 Down), 417.5 kb (ROSTOKS et al. 2002 Down) on four BAC clones from different chromosomal locations, and 261 kb at the Mla gene cluster on chromosome 1H (WEI et al. 2002 Down) were released. In these studies, the gene density was ~10-fold higher than expected from an equidistant gene distribution in the barley genome. Moreover, it was found that the genes were organized mostly in gene islands separated by large blocks of nested retrotransposons. In most cases, retrotransposons occupied ~70% of the intergenic regions, confirming data obtained in maize and wheat that indicate that they are the main cause for genome expansion during evolution (SANMIGUEL et al. 1996 Down; SANMIGUEL and BENNETZEN 1998 Down; BENNETZEN 2000B Down; DUBCOVSKY et al. 2001 Down; WICKER et al. 2001 Down). So far, a single study has compared large orthologous DNA stretches of barley (102.4 kb) and rice (50 kb) at the sequence level (DUBCOVSKY et al. 2001 Down). At the Xwg644 locus, four genes were completely conserved with one inversion and one duplication in barley compared to rice. Large size differences in the intergenic regions were associated with the insertion of different transposable elements (DUBCOVSKY et al. 2001 Down).

Here, we describe fine mapping of the barley leaf rust resistance gene Rph7 on chromosome 3HS and the establishment of a 212-kb barley BAC contig spanning the resistance gene locus. Sequence analysis revealed a high density of genes and an unusually low amount of repetitive DNA in this region. Sequence comparison with the orthologous rice region showed that microcollinearity is restricted to the conservation of one gene family and that a large rearrangement occurred at the Rph7 locus after the divergence of the Triticeae and rice genomes.


*  MATERIALS AND METHODS
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
*RESULTS
 *DISCUSSION
 *LITERATURE CITED

Plant material:
A mapping population of 1157 F2 plants was generated from a cross between the Rph7-containing barley cultivar Cebada Capa and the susceptible cultivar Bowman. Phenotypic characterization of each individual plant was performed as previously described (BRUNNER et al. 2000 Down). A subpopulation consisting of 105 plants showing recombination between the flanking markers S6246 and C10991 was selected and F3 seeds were produced for each of the recombinant plants. The phenotype of each recombinant F2 plant was confirmed by artificial infection of 15 F3 seedlings. Seeds of the barley cultivar Morex were kindly provided by Dr. A. Graner (IPK Gatersleben, Germany).

Restriction fragment length polymorphism (RFLP) analysis:
DNA isolation, Southern blotting, and labeling experiments were performed as described by GRANER et al. 1990 Down. Genomic DNA was digested with seven restriction endonucleases (BamHI, BglII, DraI, EcoRI, EcoRV, HindIII, and XbaI). Hybridization and washes (in 0.5x SSC, 0.1% SDS) were performed at 65°. The rice cDNA clones were kindly provided by Dr. T. Sasaki (National Institute of Agrobiological Resources, Tsukuba, Japan). The barley EST clone HY04A02T (AL506706) was kindly provided by Dr. A. Graner (IPK Gatersleben).

BAC library screening:
Filters of the barley Morex BAC library providing 6.3 haploid genome equivalents were screened by hybridization (YU et al. 2000 Down). DNA for fingerprint analysis and BAC end cloning was prepared by standard procedures (SAMBROOK et al. 1989 Down). Positive clones were analyzed by fingerprinting with the restriction enzymes HindIII and NotI. BAC end sequences were generated by inverse PCR, plasmid rescue (WOO et al. 1994 Down), or direct cycle sequencing (Thermo sequenase premixed cycle sequencing kit, Amersham Pharmacia) of 4 µg of pure BAC DNA [QIAGEN (Chatsworth, CA) plasmid midi kit] using an automated DNA Sequencer 4200 (Li-Cor).

Shotgun and low-pass sequencing:
Shotgun libraries were constructed for the barley BAC clones 211E24 and 252N19 as previously described (STEIN et al. 2000 Down). A total of 1256 shotgun clones were sequenced from both ends (average length: 696 bp), resulting in an eightfold coverage of a 211,664-bp sequence. The assembly of the shotgun clones resulted in 12 individual subcontigs. Gaps were due to either stretches of poly(G) or the presence of repetitive DNA. Gaps between the subcontigs were closed by PCR using 18- to 20-mer oligonucleotides. PCR fragments were cloned into the pGEM-T Easy vector system I (Promega, Madison, WI) and sequenced. The predicted error rate (1/1 kb) was based on the PHRED quality score and sequence coverage. The assembly was checked by comparison with the pattern of restriction enzyme digestion, and sequences corresponding to coding regions were resequenced to achieve an error rate of 1/10 kb.

Sequence analysis and phylogenetic analyis:
The 211,664-bp sequence was compared with National Center for Biotechnology Information dbEST and nonredundant databases using BLASTN, BLASTX, and TBLASTX algorithms (ALTSCHUL et al. 1997 Down) to identify putative genes and known repetitive elements. Repetitive elements were also detected by comparison with a local database of repetitive elements from wheat and barley of which an annotated version can be found at the Triticeae repeat database (TREP; http://wheat.pw.usda.gov/ITMI/Repeats/index.shtml). In addition, the sequence was analyzed using the rice Genomic Automated Annotation System (http://RiceGAAS.dna.affrc.go.jp/), which combines programs for coding region prediction, homology search analysis, repetitive DNA analysis, and protein motif prediction. Finally, comparative analysis of the barley sequence against itself and against the rice P1 artificial chromosome (PAC) sequence (AP002868) using the program Dotter (SONNHAMMER and DURBIN 1995 Down) allowed the identification of direct repeats, such as the long terminal repeats of retroelements, gene duplications, and deletions as well as miniature inverted repeat transposable elements (MITEs). The GCG software programs COMPOSITION, BLAST, BESTFIT, PILEUP, FIGURE, and MAP were also used to characterize the sequence. Cross-analysis of the information obtained by these different approaches was essential to validate the sequence analysis and annotations. Predicted genes were considered as putative genes only if significant homology was obtained with ESTs and/or proteins (including predicted ones) in the databases using BLASTn, tBLASTn, or BLASTx programs. Genes and proteins are named with the two first letters representing the initial letters of the Latin binomial followed by the original symbol.

Alignment of the amino acid sequences from the Arabidopsis thaliana, rice, and barley HGA genes was performed using the ClustalX program (THOMPSON et al. 1997 Down). A neighbor-joining (NJ) method was then applied to produce a phylogenetic tree. The optimal NJ tree was found using heuristic searches in PAUP (Phylogenetic Analysis Using Parsimony), version 4.04a, (Sinaur Associates, Sunderland, MA). The relative degree of branch support was determined within the NJ framework using the bootstrap procedure (FELSENSTEIN 1985 Down) in PAUP 4.04a. The original data set was resampled 1000 times.


*  RESULTS
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
*DISCUSSION
 *LITERATURE CITED

Fine mapping of the Rph7 leaf rust resistance gene in barley:
We have recently mapped the leaf rust resistance gene Rph7 on barley chromosome 3HS using a small segregating population (112 F2 plants) derived from a cross between the resistant cultivar Cebada Capa and the susceptible cultivar Bowman (BRUNNER et al. 2000 Down). Rph7 lies in a genetic interval of 19 cM between the barley cDNA ABC171 and the rice cDNA S1543 (Fig 1A). An additional rice cDNA C970 maps at 2.7 cM distal to S1543 (Fig 1A; BRUNNER et al. 2000 Down). The two rice cDNAs were located on rice chromosome 1 by the Rice Genome Project (http://rgp.dna.affrc.go.jp/). Thirty-four ESTs identified on rice yeast artificial chromosomes and PACs derived from this region were used as RFLP probes to saturate the Rph7 locus in barley. Twenty-nine of them (85%) gave a clear hybridization pattern on barley genomic DNA. Only three probes (S6246, C10991, and E1072), or 9%, identified polymorphic fragments between the parents of our mapping population. They all mapped close to Rph7 (Fig 1A). S6246, which is located 47 kb proximal to S1543 in rice (Fig 1C), revealed at least six bands on barley genomic DNA (data not shown). Only one was polymorphic and it was mapped at 0.4 cM distal to the Rph7 gene and 1.7 cM proximal to S1543 in barley (Fig 1A). In rice, the single-copy EST C10991 is present 365 kb proximal to S6246 (Fig 1C). It maps as a single locus 5 cM proximal to Rph7 in barley (Fig 1A). The single-copy EST E1072 is located between the ESTs S6246 and C10991 at a distance of 114 kb from S6246 in rice (Fig 1C). In barley, E1072 identifies a fragment that maps 3.6 cM proximal to C10991 (Fig 1A). Therefore, C10991 lies between E1072 and S6246, suggesting a local rearrangement (inversion or deletion) in barley compared to rice.



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  		Figure 1. Genetic and physical mapping of the Rph7 leaf rust resistance gene on barley chromosome 3HS using collinearity with rice chromosome 1S. (A) Genetic map of the Rph7 locus on barley chromosome 3HS (linkage analysis on 112 segregating F2 plants). The solid bar above the interval between XS6246 and Rph7 represents the interval that is described at high resolution at the genetic and physical levels in D and E, respectively. (B) Genetic map of the orthologous region on rice chromosome 1S. Rice ESTs marked with asterisks are mapped physically but not genetically in rice. (C) Physical map of the orthologous region of rice chromosome 1S. All rice ESTs used for mapping in barley are physically located on a rice PAC contig of 982 kb, which includes the PACs 439B06, 436F06, 698G03, 494A10, 698A04, 684C01, 445D12, and 37C04. Physical distances (in kilobases) are indicated between the rice markers. A possible rearrangement resulting from an inversion or a deletion at the XC10991 and XE1072 loci was observed. Probes S10623 and G107 were monomorphic between the parents of the barley mapping population and therefore could not be mapped. (D) High-density genetic map of the Rph7 locus based on a set of 105 F2 individuals showing recombination between the S6246 and C10991 markers. (E) Physical map at the Rph7 locus on barley chromosome 3HS. BACs 222G2 and 252N19 were isolated using the probes S6246 and HYO4A0T2, respectively. BAC 211E24 was identified with the low-pass sequencing probes HvA10, HvC11, and HvE6. Genetic distances are indicated in centimorgans.

For the map-based cloning of Rph7, a recombinant population consisting of 105 F2 plants showing recombination between the flanking markers S6246 and C10991 was selected out of 1157 F2 plants and was used for high-resolution mapping. A barley homolog to S6246 (62% identity), HY04A02T (AL506706), was identified by BLAST search against the barley EST database at IPK Gatersleben (http://hordeum.ipk-gatersleben.de/blast/blast_server.html). The HY04A02T sequence (2.2 kb) is longer than S6246 (750 bp), which corresponds to the very conserved 3' end region of HGA genes (see below). Therefore, Southern hybridization in barley showed more specific signals with HY04A02T than with S6246. Out of four strongly hybridizing fragments, one that was not detected by the probe S6246 was polymorphic and was completely linked to Rph7 (Fig 1D). Thus, the homologous cDNAs S6246 from rice and HY04A02T from barley identify different genetic loci, suggesting the presence of a gene family at the Rph7 locus in barley. Here, the use of rice ESTs allowed the saturation of the Rph7 locus and the delimitation of a genetic interval of 5.4 cM around the resistance gene.

Identification of a 212-kb physical contig spanning the Rph7 gene in barley:
The analysis of translocation breakpoints in the barley genome showed high recombination frequency in the telomeric region of 3HS with a physical-to-genetic distance ratio of 100 kb/cM (KUNZEL et al. 2000 Down). Our mapping data showed that the Rph7 locus lies in this region in an interval of 5.4 cM. Therefore, a physical distance of ~600 kb, which is compatible with map-based cloning, can be expected between the flanking markers. The barley BAC library of the susceptible cultivar Morex (YU et al. 2000 Down) was screened with the probes S6246 and HY04A02T (Fig 1D). Six and seven positive BAC clones were identified with the S6246 and HY04A02T probes, respectively. BAC-end and inverse-PCR fragment sequencing resulted only in repetitive sequences, which could not be used for further mapping. A low-pass sequencing strategy (STEIN et al. 2000 Down) was then performed on BAC 252N19 (Fig 1E). Of 29 nonrepetitive clones, 11 revealed polymorphisms between the parental lines and were mapped in the recombinant population. Nine of the nonrepetitive clones were completely linked with Rph7 whereas two (Hv283 and Hv480) mapped at one recombination event proximal to it (Fig 1D). A first step of chromosome walking was then performed by screening the barley BAC library with three of the linked markers (HvA10, HvC11, and HvE6; Fig 1E). They all identified the same six BAC clones, which overlap BAC 252N19 on 40 kb. BAC 211E24 (Fig 1E) was chosen for further analysis. The left BAC end (Hv211L) was nonrepetitive and mapped to the same locus as the marker S6246, i.e., at 0.3 cM (seven recombinants) distal to Rph7 (Fig 1D). A nonrepetitive low-pass clone, Hvgad1, which is a fragment of a putative glutamate decarboxylase gene, was mapped as a single locus at 0.09 cM (two recombinants) distal to the resistance gene (Fig 1D). Both the Hv211L and the Hvgad1 probes gave a hybridization signal on the BAC clone 222G2, which was isolated after screening the BAC library with S6246. The two BAC clones 211E24 and 222G2 share ~100 kb of sequence (Fig 1E). Thus, with one step of chromosome walking, a contiguous BAC contig of ~217 kb spanning the Rph7 resistance gene was established.

Sequencing of a 212-kb BAC contig in barley:
The BAC clones 252N19 and 211E24 were completely sequenced, providing 211,664 bp of contiguous sequence (AF521177) with an approximately eightfold coverage. The 212-kb contiguous sequence was annotated using a combination of comparative and prediction tools (see MATERIALS AND METHODS).

Repetitive elements:
Three full-length Ty1-copia-like retrotransposons were identified on the 212-kb contig (Fig 2). They correspond to a BARE-1 element (BARE-1_211E24_1) and two new retroelements, Katarina_211E24_1 and Bianca_252N19_1. Amino acid sequence comparison showed that the reverse transcriptase (RT) encoded by Katarina_211E24_1 is 47.5% similar to the one of Opie-1 in maize (SANMIGUEL et al. 1996 Down) and 46.7% similar to the BARE-1 RT. Bianca_252N19_1 shows less similarity to the BARE-1 RT (41%) than do the Katarina_211E24_1 and Opie elements. Interestingly, Katarina_211E24_1 is found inserted into a gene (HvHGA5) and not into another retroelement, as frequently found for the Opie-1 elements in maize (SANMIGUEL et al. 1996 Down). Seven additional incomplete retroelements that show homology to already characterized wheat and barley copia, gypsy, and TRIM retroelements (DUBCOVSKY et al. 2001 Down; WICKER et al. 2001 Down; http://wheat.pw.usda.gov/ITMI/Repeats/nrTREP_list.html) were detected on the sequence (Fig 2). A new transposon, Caspar_211E24_1, was also identified. It is a complete TNP2-like transposon of 9103 bp with a flanking CACTG sequence resembling the CACTA motif found in this type of transposon (NACKEN et al. 1991 Down) and a 3-bp target site duplication (TSD). It is putatively active as it contains an open reading frame [1105 amino acids (aa)] that shows >73% similarity to TNP2-like rice transposases such as RIM2 (HE et al. 2000 Down). Incomplete retroelements were found nested mainly around the Caspar_211E24_1 transposon and the BARE-1_211E24_1 and Bianca_252N19_1 retrotransposons (Fig 2). A total of 13 MITEs of the Stowaway family were identified. All except one (Ceres_211E24_1) are similar to already characterized wheat MITES (TREP database, http://wheat.pw.usda.gov/ITMI/Repeats/nrTREP_list.html). Seven of them are in close vicinity in genes or in introns (Fig 2). Interestingly, the Ceres_211E24_1 MITE is located at the 5' end of the HvHGA4 gene and is interrupted by a 638-bp element (XH_211E24) (Fig 2). XH_211E24 is flanked by a 9-bp TSD and is also found in the 5' untranslated region of two other barley genes in the databases. Three microsatellites of >15 (TA) repeats are located on the sequence. Two of them are associated with newly characterized elements of 370 bp (XI_211E24), which have TA target site duplications (Fig 2).



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  		Figure 2. Schematic representation of the physical contig of 212 kb at the Rph7 resistance locus. The genes are represented as colored boxes with an arrow indicating the transcription orientation. Asterisks indicate pseudogenes. Black/yellow patterned boxes represent full or partial retrotransposons whose names are indicated in the figure. Elements found as fragments are indicated only with the generic name of the element; full element names have been given according to the convention Name_BAC number_rank in the sequence. Red bars represent MITEs and transposons are indicated as blue striped white boxes. Microsatellites are represented as vertical blue arrowheads. Small horizontal black arrowheads indicate direct repeats or inverted repeats. New unknown elements (XH_211E24 and XI-211E24) are represented as orange bars. The duplication of a fragment of HvHGA5 preceding the insertion of the Katarina_211E24_1 element is indicated. The dotted line below the contig indicates the physical region spanning the Rph7 resistance gene. The red arrowheads under the contig indicate the positions of the shotgun clones used to assess the copy number of the uncharacterized fraction of the sequence. Annotations of the different elements can be found under accession no. AF521177.

Ten genes are present on the barley contig:
Ten genes were identified on the 212-kb contiguous barley sequence. Four of them, HvHGA1, HvHGA2, HvHGA4, and HvHGA5, belong to the same gene family (HGA). The six additional genes are all different and are located between the HvHGA4 and HvHGA2 genes (Fig 2). The length and the characteristics of the genes and the corresponding deduced proteins are given in Table 1.


 
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  		Table 1. Characteristics of the predicted genes found on the 212-kb contig spanning the Rph7 locus in barley

The intron/exon structure of the HvHGA genes has been deduced from predictions and comparisons with barley ESTs, including HY04A02T, which corresponds to a cDNA highly similar to HvHGA2. The HvHGA1 and HvHGA2 proteins are 80% similar and the intron/exon structure of the two genes is identical (Fig 3, Table 1). HvHGA4 shows ~60% similarity to HvHGA1 and HvHGA2. It has only four introns whose positions are not conserved with the HvHGA1 and HvHGA2 genes (Fig 3). The structure of HvHGA5 is more difficult to assess as the gene is interrupted by the insertion of the retrotransposon Katarina_211E24_1 into the last exon (Fig 3). In addition, HvHGA5 is probably truncated and is partially duplicated (Fig 2). The 2-kb duplicated fragment does not contain any sequence of the Katarina_211E24_1 retrotransposon, indicating that the duplication predated the insertion of the retroelement (Fig 2). A hypothetical gene sequence was reconstructed from the HvHGA5 sequence (Table 1). The predicted protein shows 54% similarity to the other members of the HvHGA gene family. In general, the C-terminal region of the putative HvHGA proteins is more conserved than the N-terminal region. One domain (GVHGAGLTN) is particularly well conserved and it is also found in several putative proteins from rice and A. thaliana. No known functional domain could be detected in the HvHGA proteins and no function so far has been assigned to any of the homologs of the HvHGA genes.



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  		Figure 3. Schematic representation of the HGA genes from barley and rice. The exon (solid)/intron (open) structure is represented for each orthologous pair of genes of the HGA gene family in barley (HvHGA) and rice (OsHGA). No barley ortholog of OsHGA3 was found on the 217-kb physical contig and the structure of the putative HvHGA3 gene is not known so far. The retrotransposon Katarina_211E24_1, which is inserted into the last exon of the HvHGA5 gene, is represented as a diagonally striped box. HvHGA5 is truncated and only the last exon can be compared with the predicted OsHGA5 orthologous rice gene. Vertical bars delimit the sequences, which are perfectly conserved between the different genes.

Out of the six other genes detected on the barley contig, three either showed homology to known proteins (Hvhel1, Hvgad1) or contained well-characterized motifs (Hvrh2; Table 1). Hvhel1 encodes a putative helicase and is a pseudogene because of three frameshift mutations and one stop codon. Hvgad1 codes for a putative glutamate decarboxylase and Hvrh2 is a pseudogene that would encode a protein containing a very conserved RING-H2 finger motif. HvRH2 shows ~60% similarity to the EL5 protein of rice (TAKAI et al. 2001 Down), which is induced by a biotic elicitor and was recently shown to be a ubiquitin ligase (TAKAI et al. 2002 Down). The three other genes (Hvpg1, Hvpg3, Hvpg4) are putative genes that do not have any identified function in any of the organisms in which homologs were found. Interestingly, two of them (Hvpg1 and Hvpg4) are well conserved in animal species such as Caenorhabditis elegans, Homo sapiens, and/or Drosophila melanogaster. For all of the predicted genes, at least two ESTs were found to confirm the gene prediction.

With 10 genes within 212 kb, the gene density found at the Rph7 locus is one gene every 20 kb. However, the genes are not homogeneously distributed along the contig. Most of them are present in two clusters—cluster 1 (Hvrh2, Hvpg3, Hvgad1) and cluster 2 (Hvpg4, Hvhel1, HvHGA2, HvHGA1)—with a higher gene density of one gene every 6–9 kb. In contrast, the intergenic regions located between HvHGA4 and Hvrh2 as well as between Hvgad1 and Hvpg1 are larger than 34 and 43 kb, respectively.

Ratio of physical-to-genetic distances:
All the RFLP probes used for mapping showed an identical hybridization pattern for the susceptible parent Bowman and Morex, the variety used to construct the BAC library. This allowed the estimation of the correlation between physical and genetic distances by comparing the RFLP fragments with the BAC sequence of Morex. From S6246 to Hv480, an overall physical-to-genetic distance ratio of 588 kb/cM was estimated (Fig 1D and Fig E). This is over five times less than 3.7–4.2 Mb/cM, which was estimated for the barley genome (GRANER et al. 1991 Down; KLEINHOFS et al. 1993 Down). The distribution of the recombination frequency was analyzed in detail. From Hv211L to Hvgad1, 88 kb represents a genetic distance of 0.21 cM, resulting in a ratio of 419 (±214) kb/cM. A similar ratio was estimated in the interval between Hvgad1 and the HvC11 (Fig 1D and Fig E). In contrast, around the Rph7 gene, no recombination was found over a distance of ~110 kb (>2.5 Mb/cM). On the proximal side of Rph7, a ratio of 75 (±37.5) kb/cM, which is very similar to the 100 kb/cM estimated by KUNZEL et al. 2000 Down for this region of chromosome 3H, was found.

Regions without genes or repetitive elements:
After extensive sequence analysis, ~130 kb of the total sequence did not show homology to any known genes or repetitive elements and remained uncharacterized. This represents ~60% of the sequence while the coding regions and the identified repetitive elements represent 15 and 25%, respectively. This contrasts with previous analysis of large genomic fragments in barley, wheat, and maize where ~70% of the sequence was repetitive and composed mainly of retrotransposons (SANMIGUEL et al. 1996 Down, SANMIGUEL et al. 2002 Down; TIKHONOV et al. 1999 Down; DUBCOVSKY et al. 2001 Down; WICKER et al. 2001 Down). Thus, the nonannotated regions correspond to either uncharacterized or extremely degenerated repetitive elements or low-copy regions without any identified equivalent in the large grass genomes. To test these hypotheses, 15 randomly chosen shotgun clones corresponding to unidentified regions (Fig 2) were used as probes on Southern blots of barley genomic DNA. Four were highly repetitive whereas the others showed 2–10 discrete bands with little background signal (data not shown). On the basis of these results, we estimate that ~25% of the nonannotated 130-kb sequence still corresponds to uncharacterized repetitive elements. Consequently, ~45% of the 212-kb contig might correspond to noncoding, low-copy sequence, suggesting that the Rph7 locus is unusually rich in noncoding low-copy sequence.

All the unidentified sequences were assembled into a single stretch of 130 kb and compared to the 907 kb (eight BACs) of barley genomic sequences currently available in the public databases. Four stretches of 301–1282 bp (for positions see AF521177) showing 75–86% of identity were identified in at least another BAC sequence. Three of them are present at the Mla locus in predicted gene regions (WEI et al. 2002 Down) as well as in other BACs in nonannotated regions. The frequency of these sequences in the small subset of the barley genome covered by these BACs strongly suggests that they belong to the pool of repetitive sequences, which remain to be identified. This will require further sequencing and comparison of barley genomic sequences.

The HvHGA gene family is conserved in rice and A. thaliana:
Five predicted genes (P0698A04.6, P0698A04.8, P0698A04.9, P0698A04.11, P0698A04.13) showing homology to the HvHGA genes are present in a tandem array spanning 35.8 kb on rice chromosome 1 (AP002868). To simplify the analysis, P0698A04.6, P0698A04.8, P0698A04.9, P0698A04.11, and P0698A04.13 were renamed OsHGA6, OsHGA5, OsHGA4, OsHGA1, and OsHGA3, respectively. OsHGA6 corresponds to the rice EST S6246, which mapped 0.3 cM distal to Rph7 (Fig 1). Phylogenetic analysis showed that each barley HvHGA gene is more similar to one rice OsHGA homolog than to one another (Fig 4), indicating orthologous relationships between the rice and barley genes. The only exception concerns HvHGA1 and HvHGA2, which are more similar to each other than to the rice ortholog OsHGA1 (Fig 4). The intron/exon structure of the barley and rice HGA orthologs is generally well conserved, in particular for the last large exon, which contains highly conserved domains of the gene family (Fig 3).



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  		Figure 4. Unrooted phylogenetic tree of the HGA genes from A. thaliana (atxg), rice (OsHGA), and barley (HvHGA). ClustalX amino acid sequence alignment was used to produce the best tree after neighbor-joining analysis. Branches that had bootstrap values <100 are indicated.

No orthologs were found on the contiguous 212-kb barley sequence for the two other rice genes, OsHGA6 (EST S6246) and OsHGA3, which are present in the rice gene cluster (Fig 5A). However, a sequence strongly hybridizing with S6246 was detected on BAC 222G2 (Fig 1), suggesting the presence of a gene homologous to OsHGA6 (S6246) on this BAC. A HindIII fragment of 5 kb hybridizing with S6246 was subcloned from BAC 222G2 and sequenced. It contains a gene of 2689 bp, which we named HvHGA6 (Fig 3, AF525024). The predicted 637-aa protein shows the highest similarity (65%) to OsHGA6 (Fig 4), confirming that HvHGA6 is orthologous to OsHGA6 (Fig 5A). The estimated physical distance between HvHGA5 and HvHGA6 is ~25 kb (Fig 5A). Thus, our data show that each barley HvHGA gene has an ortholog in rice. The order of the genes is completely conserved and the transcription orientation is identical except for HvHGA4, which is inverted compared to OsHGA4 (Fig 5A).



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  		Figure 5. Comparison of the gene organization at the HGA gene loci in rice, barley, and A. thaliana. (A) Microcollinearity is retained only for the barley HvHGA genes and the orthologous rice OsHGA genes. A large rearrangement interrupting the microcollinearity between rice and barley occurred in the HGA5/HGA4 and the HGA4/HGA2 intergenic regions. (B) Organization of the HGA gene homologs in A. thaliana. Seven genes are distributed on chromosomes 2 and 3 in the A. thaliana genome. Two pairs of paralogs, highlighted with a red circle, are found on each chromosome. The genes At2g41640 and At3g57380 are located in chromosomal regions known to have originated from a large duplication (interrupted circle). The HGA genes are presented as green boxes whereas the additional genes are shown in different colors. Arrows indicate the direction of transcription except for HvHGA6, which is located on BAC 222G2 and whose orientation respective to the 212-kb contig is not known.

In A. thaliana, BLASTp and BLASTx searches identified seven HvHGA gene homologs. They are located on chromosome 2 (atg2g03360, at2g03370, at2g41640) and chromosome 3 (at3g10320, at3g57380, at3g18170, at3g18180; Fig 5B). at3g18170 and at3g18180 as well as atg2g03360 and at2g03370 are arranged in tandem on chromosome 3 and 2, respectively (Fig 5B). This and their close relationships in the phylogenetic analysis (Fig 4) suggest that they originate from recent duplications. The at2g41640 and at3g57380 genes are located in regions of chromosome 2 and 3, which are known to result from a ancient genome duplication (VISION et al. 2000 Down), and they are closely related in the phylogenetic analysis (Fig 4). Although the intron numbers and positions differ among the HvHGA homologs in barley, rice, and A. thaliana, the position and the length of the last exon is highly conserved (data not shown). Phylogenetic analysis indicated that the at3g18170 and at3g18180 gene products have the highest similarity (~53%) to the HvHGA protein sequences of rice and barley (Fig 4). This suggests that a single gene, which resembled the at3g18170/80 genes, was present in a common ancestor of the monocots and dicots.

Microcollinearity is lost in the HGA intergenic regions:
In addition to the insertion of a retrotransposon in HvHGA5 and the inversion of HvHGA4, very few rearrangements affected individual members of the HGA gene family during evolution in rice and barley. In contrast, major changes were observed in the intergenic regions. The distance between the barley genes HvHGA5 and HvHGA4 (30 kb) as well as between the HvHGA4 and HvHGA2 genes (153 kb) is >38-fold greater than the distance between the orthologous rice genes (700 bp and 4 kb, respectively; Fig 5A). In the HvHGA4/HvHGA2 interval, two full-length retrotransposons, six truncated retrotransposons, two transposons, and six MITEs contribute to ~30% (45 kb) of this size difference. However, the most striking difference resides in the presence of six additional single genes (Hvrh2, Hvpg3, Hvgad1, Hvpg1, Hvpg4, Hvhel1) and of a substantial proportion of noncoding low-copy sequence in barley compared to rice (Fig 5A). Southern hybridization and BLAST search against the recently published rice genome drafts of Syngenta (http://www.tmri.org) and the Beijing rice genome project (http://210.83.138.53/rice/; GOFF et al. 2002 Down; YU et al. 2002 Down) showed that, except for the helicase-like gene that belongs to a multigene family, one to four copies of each of the additional barley genes are present elsewhere in the rice genome. Their chromosomal location was estimated on the basis of the results of BLAST search against rice BAC and PAC sequences in public databases (Table 1). None of these genes were found on PACs located at the distal end of chromosome 1 in the vicinity of the OsHGA genes. In some cases, homologs were found on the same chromosome (i.e., Hvpg3, Hvgad1, and Hvrh2 on chromosome 2) but never on the same BAC/PAC clones. These data indicate that the gene cluster found in barley between the HvHGA4 and HvHGA2 genes has no equivalent in rice and that a large rearrangement occurred at the Rph7 locus since the divergence of the two species. We have used the HvHGA1, HvHGA2, Hvpg1, Hvpg3, Hvpg4, and Hvgad1 genes as probes for hybridization on wheat genomic DNA of the nulli-tetrasomic lines of the variety Chinese Spring. All the genes hybridized to single fragments on each homeologous chromosome 3 in hexaploid wheat (data not shown), suggesting that the additional genes found in barley are also present in the orthologous region in wheat. Thus, the rearrangement likely predates the Bambusoideae/Poideae divergence.


*  DISCUSSION
*TOP
 *ABSTRACT
 *MATERIALS AND METHODS
 *RESULTS
 *DISCUSSION
*LITERATURE CITED

Characterization of the genetic and physical interval spanning the Rph7 locus in barley:
Mapping of ESTs originating from rice chromosome 1S allowed the development of a high-density genetic map and the initiation of chromosome walking at the Rph7 resistance locus on barley chromosome 3HS. Our data confirmed the good synteny between these chromosomes (SMILDE et al. 2001 Down) and the usefulness of rice as a source of new probes for saturating genetic regions in other grasses. A physical contig of ~217 kb spanning the Rph7 resistance gene was established in a single step of chromosome walking. The average physical-to-genetic distance ratio of 588 kb/cM that was estimated at this locus is higher than the 100 kb/cM that was previously estimated by cytogenetic analysis (KUNZEL et al. 2000 Down). This is probably due to different levels of resolution in the two techniques. Nevertheless, it confirms that in this region of chromosome 3H, the ratio of physical-to-genetic distance is lower than that expected from a genome-wide average (3–4 Mb/cM) and that gene-rich regions are associated with a higher recombination frequency (FARIS et al. 2000 Down; ROSTOKS et al. 2002 Down; SANDHU and GILL 2002A Down). The recombination frequency was unevenly distributed (from 75 kb/cM to >2.5 Mb/cM). It is known that the relationship between physical and genetic distance varies throughout the genome but very few studies have reported on the variation of this ratio at the molecular level in wheat and barley. In wheat, local variations from 600 kb/cM to 12 Mb/cM were found at the Lr10 locus (STEIN et al. 2000 Down), and in barley, variations from 176 kb/cM to 5 Mb/cM were observed in eight intervals spanning the Mla cluster (WEI et al. 1999 Down). Thus, large local variations in recombination frequencies might be a feature of these large genomes. As in the Lr10 and Mla disease resistance loci, suppression of recombination was observed at the Rph7 resistance locus. In these cases, suppressed recombination was postulated to be due to extreme polymorphism associated with the origin of the resistant region. Here, although a high level of polymorphim was observed between Cebada Capa and the susceptible cultivars Bowman and Morex, no clear correlation could be established between the degree of polymorphism and the recombination rate.

High gene density and diversity is found at the Rph7 locus in barley:
Together with the 212 kb of genomic sequence analyzed in this study, >1.1 Mb of barley genomic sequence distributed over nearly all the chromosomes has been studied by now. General patterns of genome organization have started to emerge from the analysis of these sequences. For example, the average gene density found at the Rph7 locus (one gene/20 kb) seems to be very representative of the gene density in the gene-containing regions of the barley genome (PANSTRUGA et al. 1998 Down; SHIRASU et al. 2000 Down; DUBCOVSKY et al. 2001 Down; ROSTOKS et al. 2002 Down). It is also commonly observed that the average gene density value does not reflect the unhomogeneous distribution of the genes. At the Rph7 locus, two clusters of three and four genes with a density of one gene/6–9 kb are present, whereas the Hvpg1 is the only gene present in a 60-kb region rich in repetitive elements. Similar gene clustering has been observed at different loci in barley, wheat, and maize and it has become clear that high-gene-density islands and single genes separated by large regions of repetitive elements are both found in gene-rich regions of the grass genomes (FEUILLET and KELLER 1999 Down, FEUILLET and KELLER 2002 Down; ROSTOKS et al. 2002 Down; SANDHU and GILL 2002B Down; WEI et al. 2002 Down).

Seven different types of gene were found at the Rph7 locus. Except for the glutamate decarboxylase (Hvgad1), the helicase (Hvhel1), and the ring-H2 (Hvrh2)-like genes, no function based on similarity with known proteins could be assigned to the different genes. Linkage analysis indicates that five genes (Hvpg1, Hvpg4, Hvhel1, HvHGA2, and HvHGA1) are putative candidates for Rph7. Remarkably, none shows similarity to already characterized disease resistance genes (HULBERT et al. 2001 Down). For each candidate gene detected in the susceptible variety, an allele was detected in the resistant cultivar Cebada Capa. However, we cannot exclude the possibility that an additional gene is present in the resistant haplotype. We are currently constructing a genomic library from Cebada Capa to test this possibility and to identify the Rph7 resistance gene.

Microcollinearity at the Rph7 locus in barley and rice is interrupted by a large genic rearrangement:
Each member of the barley HGA gene family has a clear ortholog in rice, suggesting that this gene family arose through tandem duplications before the Bambusoideae/Poideae divergence, i.e., ~40–50 million years ago. In barley, two genes, HvHGA1 and HvHGA2, are orthologous to the rice gene OsHGA1. This suggests that either gene duplication occurred in barley or one paralog was deleted in rice after the barley-rice divergence. The fact that HvHGA1 and HvHGA2 are more similar to each other than to OsHGA1 favors the first hypothesis. A similar local gene duplication in barley compared to rice has also been found at the Xwg644 locus (DUBCOVSKY et al. 2001 Down). Thus, the HGA genes did not undergo massive rearrangements during evolution, resulting in an apparently good microcollinearity between barley and rice in this region. In contrast, considerable differences in size and composition were found in the intervals between the HGA5/HGA4 and the HGA4/HGA2 genes in barley compared to rice. In the HGA4/HGA2 intergenic region, collinearity is interrupted by the presence of six additional genes and a large proportion of noncoding low-copy sequences in barley. Microcollinearity studies at orthologous loci between rice and other grass genomes have already shown many small rearrangements (deletions, duplications, translocations, inversions; for review see BENNETZEN 2000A Down; BENNETZEN and RAMAKRISHNA 2002 Down; FEUILLET and KELLER 2002 Down). However, they generally involved one to three genes and the rearrangement of a segment with six genes has not been reported so far.

Three different scenarios can explain such a rearrangement. In one scenario, it is possible that the current gene organization in barley reflects the ancestral locus organization. In this case, the organization observed in rice could result from the deletion of a large fragment containing the six genes or from their relocation to nonorthologous loci during evolution. In an alternative scenario, the ancestral locus was similar to the modern rice locus in only the HGA gene family, and additional sequences including the six genes were inserted during barley genome evolution.

The presence of homologs of the six additional barley genes in low-copy numbers at nonorthologous loci in the rice genome supports the hypothesis of rearrangements in rice. It is not possible to discriminate between the deletion or relocation hypothesis by comparing the degree of divergence between the six additional genes and the HGA orthologs in rice and barley. The similarity at the amino acid level is comparable between the two groups of genes. The deletion scenario would imply that more than one copy of each gene was originally present in the ancestral genome and that only the copies located at the Rph7 locus were deleted in rice. Large deletions of repetitive regions caused by unequal intrastrand recombination or illegitimate recombination have been found to partially counteract genome expansion in plants (BENNETZEN 2002 Down; DEVOS et al. 2002 Down). However, such a mechanism has not yet been shown to affect large genic regions. The alternative mechanism of rearrangement requires the relocation of six rice genes to different nonhomeologous positions. Recent studies suggest that single genes (BENNETZEN and RAMAKRISHNA 2002 Down) or small blocks of adjacent genes (LI and GILL 2002 Down) have moved to new locations during evolution in grass genomes.

Finally, as a third scenario, we cannot exclude that a new and not-yet-discovered mechanism might have been involved in shaping this genomic region in barley. The presence of a large amount of noncoding, low-copy DNA beside the six additional genes is intriguing and might result from such a new mechanism. Further comparative analysis of the Rph7 locus in other members of the Poaceae family, such as maize, will be needed to address the question of the origin of this rearrangement and its molecular basis.

The amount of low-copy sequence strongly contrasts with previous analysis of large genomic fragments in grasses where ~70% of the sequences corresponded to repetitive elements (SANMIGUEL et al. 1996 Down, SANMIGUEL et al. 2002 Down; CHEN et al. 1997 Down; TIKHONOV et al. 1999 Down; DUBCOVSKY et al. 2001 Down; WICKER et al. 2001 Down). Here, 60% of the sequence could not be annotated. Recently, ROSTOKS et al. 2002 Down found on average 37% of uncharacterized sequence but they did not indicate if this was potentially repetitive. We have estimated that 25% of the nonannotated sequence likely corresponds to uncharacterized repetitive elements and that 45% of the total sequence is low copy. It is well established that the insertion of repetitive elements has largely contributed to genome expansion in grasses (SANMIGUEL et al. 1996 Down; SANMIGUEL and BENNETZEN 1998 Down; BENNETZEN 2000B Down; WICKER et al. 2001 Down). Our findings raise the question of whether the rearrangement of genes and low-copy sequences can also participate in local genome expansion.

We conclude that multiple rearrangements involving not only repetitive elements but also coding regions and low-copy sequences have reshaped the barley and rice genomes since their divergence. Thus, grass genome evolution is certainly more complex than expected on the basis of collinearity assessments, and microcollinearity between rice and the Triticeae genome is possibly not very high. Our results also support the idea that the development of efficient structural and functional genomic tools should be pursued in the Triticeae to achieve isolation of genes of agronomic importance.


*  ACKNOWLEDGMENTS

The authors thank Dr. Sasaki and Dr. Yano from the Rice Genome Project for kindly providing the EST sequences and unpublished mapping information and for mapping of the ABC 171 RFLP probe in rice. We thank Dr. N. Yahiaoui and Dr. C. Ringli for critical review of the manuscript. This project was supported by the Swiss National Science Foundation grant no. 31-53597.98.

Manuscript received November 1, 2002; Accepted for publication February 19, 2003.


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