Download A Large Rearrangement Involving Genes and Low-Copy DNA

Copyright  2003 by the Genetics Society of America
A Large Rearrangement Involving Genes and Low-Copy DNA Interrupts the
Microcollinearity Between Rice and Barley at the Rph7 Locus
S. Brunner, B. Keller and C. Feuillet1
Institute of Plant Biology, University of Zürich, CH-8008 Zürich, Switzerland
Manuscript received November 1, 2002
Accepted for publication February 19, 2003
ABSTRACT
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.
RASSES evolved from a common ancestor ⵑ50–80
million years ago (Wolfe et al. 1989). 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; Gale and Devos
1998; Keller and Feuillet 2000). 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; Han et al. 1998, 1999; Druka et al. 2000), wheat
(Li and Gill 2002; SanMiguel et al. 2002), and maize
(Chen et al. 1997, 1998; Tarchini et al. 2000; for recent
reviews see Bennetzen and Ramakrishna 2002; Feuillet and Keller 2002). These data have demonstrated
that rice can be very useful in providing numerous markers to saturate genetic maps in other cereals and that mapbased 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.
G
1
Corresponding author: Institute of Plant Biology, University of Zürich,
Zollikerstr. 107, CH-8008 Zürich, Switzerland.
E-mail: [email protected]
Genetics 164: 673–683 ( June 2003)
2000), ⬎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) 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; Yu et al. 2002) 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) and at the Mlo locus on chromosome 4H
(Panstruga et al. 1998). Very recently, full sequence
analyses of a 102.4-kb sequence on chromosome 5H
(Dubcovsky et al. 2001), 417.5 kb (Rostoks et al. 2002)
on four BAC clones from different chromosomal locations, and 261 kb at the Mla gene cluster on chromosome 1H (Wei et al. 2002) were released. In these studies, the gene density was ⵑ10-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 ⵑ70% 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; SanMiguel
and Bennetzen 1998; Bennetzen 2000b; Dubcovsky
et al. 2001; Wicker et al. 2001). So far, a single study has
compared large orthologous DNA stretches of barley
674
S. Brunner, B. Keller and C. Feuillet
(102.4 kb) and rice (50 kb) at the sequence level (Dubcovsky et al. 2001). 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).
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
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). 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). Genomic DNA was digested with seven restriction endonucleases
(BamHI, BglII, DraI, EcoRI, EcoRV, HindIII, and XbaI). Hybridization and washes (in 0.5⫻ 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). DNA for fingerprint
analysis and BAC end cloning was prepared by standard procedures (Sambrook et al. 1989). 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), or direct cycle sequencing
(Thermo sequenase premixed cycle sequencing kit, Amersham Pharmacia) of 4 ␮g 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). 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 211,664bp sequence was compared with National Center for Biotechnology Information dbEST and nonredundant databases using BLASTN, BLASTX, and TBLASTX algorithms (Altschul
et al. 1997) 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) 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). 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) in PAUP 4.04a.
The original data set was resampled 1000 times.
RESULTS
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).
Rph7 lies in a genetic interval of 19 cM between the
barley cDNA ABC171 and the rice cDNA S1543 (Figure
1A). An additional rice cDNA C970 maps at 2.7 cM distal
to S1543 (Figure 1A; Brunner et al. 2000). The two rice
cDNAs were located on rice chromosome 1 by the Rice
Genome Project (http://rgp.dna.affrc.go.jp/). Thirtyfour ESTs identified on rice yeast artificial chromosomes
and PACs derived from this region were used as RFLP
Genome Rearrangement at the Rph7 Locus
675
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 lowpass sequencing probes HvA10, HvC11, and HvE6. Genetic distances are indicated in centimorgans.
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 (Figure 1A). S6246, which is
located 47 kb proximal to S1543 in rice (Figure 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 (Figure 1A). In rice, the single-copy
EST C10991 is present 365 kb proximal to S6246 (Figure
1C). It maps as a single locus 5 cM proximal to Rph7
in barley (Figure 1A). The single-copy EST E1072 is
located between the ESTs S6246 and C10991 at a distance of 114 kb from S6246 in rice (Figure 1C). In
barley, E1072 identifies a fragment that maps 3.6 cM
proximal to C10991 (Figure 1A). Therefore, C10991
lies between E1072 and S6246, suggesting a local rearrangement (inversion or deletion) in barley compared to rice.
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 highresolution 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 (Figure 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). Our mapping data showed that the
Rph7 locus lies in this region in an interval of 5.4 cM.
Therefore, a physical distance of ⵑ600 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) was
676
S. Brunner, B. Keller and C. Feuillet
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 XI211E24) 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.
screened with the probes S6246 and HY04A02T (Figure
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) was then performed on BAC
252N19 (Figure 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 (Figure 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; Figure 1E). They
all identified the same six BAC clones, which overlap
BAC 252N19 on 40 kb. BAC 211E24 (Figure 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 (Figure 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
(Figure 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
ⵑ100 kb of sequence (Figure 1E). Thus, with one step
of chromosome walking, a contiguous BAC contig of
ⵑ217 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
(Figure 2). They correspond to a BARE-1 element (BARE1_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) 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). Seven additional incomplete retroelements that show homology to already characterized
wheat and barley copia, gypsy, and TRIM retroelements
(Dubcovsky et al. 2001; Wicker et al. 2001; http://
Genome Rearrangement at the Rph7 Locus
677
TABLE 1
Characteristics of the predicted genes found on the 212-kb contig spanning the Rph7 locus in barley
Name
Length (bp)
HvHGA1
HvHGA2
HvHGA4
HvHGA5
Hvgad1
Hvhel1
3362
2990
2235
2002
3151
4452
Hvrh2
Hvpg1
Hvpg3
Hvpg4
783
4893
5421
2510
Structurea
5E,
5E,
4E,
2E,
7E,
2E,
4I
4I
3I
1I
6I
1I
1E
8E, 7I
14E, 13I
5E, 4I
Proteinb
Predicted function
505
536
525
427
490
1423
Unknown
Unknown, pseudogene
Unknown
Unknown, disrupted
Glutamate decarboxylase
Helicase, pseudogene
210
1049
689
226
Ring-H2 finger motif, pseudogene
Unknown, Gly-Cys rich domain
Unknown, DNA repair domain
Unknown
Rice 1 orthologsc
698A04.11
698A04.11
698A04.9
698A04.8
No
No
No
No
No
No
Rice homologsd
Chromosomes
1, 2, 3, 4, 5, 6, 10
C terminus only e
Chromosomes 2, 3, 4
Chromosomes 1, 2, 3,
4, 5, 6, 8, 10
Chromosome 2
Chromosome 10
Chromosomes 2, 4
Chromosome 3
a
The number of exons (E) and introns (I) deduced either from comparison with cDNAs or from prediction programs.
The number of amino acid residues.
c
For each barley gene, orthologs found on rice chromosome 1 are indicated with the names given in the GenBank annotation.
d
Homologs to the 10 barley genes identified in the rice databases are indicated with their chromosomal location as annotated
in the database.
e
Homology was found with rice genes other than the OsHGA orthologs but was limited to the very conserved C-terminal region
of the HGA genes.
b
wheat.pw.usda.gov/ITMI/Repeats/nrTREP_list.html)
were detected on the sequence (Figure 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)
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). Incomplete
retroelements were found nested mainly around the
Caspar_211E24_1 transposon and the BARE-1_211E24_1
and Bianca_252N19_1 retrotransposons (Figure 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
(Figure 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) (Figure 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
(Figure 2).
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 (Figure 2).
The length and the characteristics of the genes and the
corresponding deduced proteins are given in Table 1.
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 (Figure 3, Table
1). HvHGA4 shows ⵑ60% similarity to HvHGA1 and
HvHGA2. It has only four introns whose positions are
not conserved with the HvHGA1 and HvHGA2 genes
(Figure 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 (Figure 3). In addition, HvHGA5 is probably truncated and is partially duplicated (Figure 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 (Figure 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.
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
678
S. Brunner, B. Keller and C. Feuillet
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.
(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 ⵑ60% similarity
to the EL5 protein of rice (Takai et al. 2001), which is
induced by a biotic elicitor and was recently shown to
be a ubiquitin ligase (Takai et al. 2002). 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 (Figure 1, D and 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;
Kleinhofs et al. 1993). 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 (Figure 1, D and E). In contrast, around the
Rph7 gene, no recombination was found over a distance
of ⵑ110 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) for
this region of chromosome 3H, was found.
Regions without genes or repetitive elements: After
extensive sequence analysis, ⵑ130 kb of the total sequence did not show homology to any known genes or
repetitive elements and remained uncharacterized. This
represents ⵑ60% 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 ⵑ70% of the sequence was repetitive
and composed mainly of retrotransposons (SanMiguel
et al. 1996, 2002; Tikhonov et al. 1999; Dubcovsky et
al. 2001; Wicker et al. 2001). 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
(Figure 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 ⵑ25% of the nonannotated
130-kb sequence still corresponds to uncharacterized
repetitive elements. Consequently, ⵑ45% of the 212kb 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
Genome Rearrangement at the Rph7 Locus
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.
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) 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 (Figure 1). Phylogenetic analysis showed that each barley HvHGA gene is
more similar to one rice OsHGA homolog than to one
another (Figure 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
(Figure 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 (Figure 3).
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 (Figure 5A). However, a sequence strongly
hybridizing with S6246 was detected on BAC 222G2
679
(Figure 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 (Figure 3, AF525024).
The predicted 637-aa protein shows the highest similarity
(65%) to OsHGA6 (Figure 4), confirming that HvHGA6
is orthologous to OsHGA6 (Figure 5A). The estimated
physical distance between HvHGA5 and HvHGA6 is ⵑ25
kb (Figure 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 (Figure 5A).
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; Figure 5B). at3g18170 and at3g18180 as well
as atg2g03360 and at2g03370 are arranged in tandem
on chromosome 3 and 2, respectively (Figure 5B). This
and their close relationships in the phylogenetic analysis
(Figure 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), and they are closely related in the phylogenetic analysis (Figure 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
(ⵑ53%) to the HvHGA protein sequences of rice and
barley (Figure 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; Figure 5A). In the HvHGA4/
HvHGA2 interval, two full-length retrotransposons, six
truncated retrotransposons, two transposons, and six
MITEs contribute to ⵑ30% (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 (Figure 5A). Southern hybridization and BLAST search against the recently published rice
680
S. Brunner, B. Keller and C. Feuillet
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.
genome drafts of Syngenta (http://www.tmri.org) and the
Beijing rice genome project (http://210.83.138.53/rice/;
Goff et al. 2002; Yu et al. 2002) 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
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) and the usefulness of rice as a source of new
probes for saturating genetic regions in other grasses. A
physical contig of ⵑ217 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). 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
Genome Rearrangement at the Rph7 Locus
associated with a higher recombination frequency
(Faris et al. 2000; Rostoks et al. 2002; Sandhu and
Gill 2002a). 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), 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). 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; Shirasu et al. 2000; Dubcovsky et
al. 2001; Rostoks et al. 2002). 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, 2002;
Rostoks et al. 2002; Sandhu and Gill 2002b; Wei et
al. 2002).
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 char-
681
acterized disease resistance genes (Hulbert et al. 2001).
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., ⵑ40–50 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). 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; Bennetzen and Ramakrishna 2002; Feuillet and Keller
2002). 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
682
S. Brunner, B. Keller and C. Feuillet
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; Devos et al. 2002). 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) or small blocks of adjacent genes (Li
and Gill 2002) 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 ⵑ70% of the sequences corresponded to
repetitive elements (SanMiguel et al. 1996, 2002; Chen
et al. 1997; Tikhonov et al. 1999; Dubcovsky et al. 2001;
Wicker et al. 2001). Here, 60% of the sequence could
not be annotated. Recently, Rostoks et al. (2002) 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; SanMiguel and Bennetzen 1998; Bennetzen 2000b; Wicker et al. 2001).
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.
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.
LITERATURE CITED
Altschul, S. F., T. L. Madden, A. A. Schaffer, J. H. Zhang, Z. Zhang
et al., 1997 Gapped BLAST and PSI-BLAST: a new generation of
protein database search programs. Nucleic Acids Res. 25: 3389–
3402.
Bennetzen, J. L., 2000a Comparative sequence analysis of plant
nuclear genomes: microcolinearity and its many exceptions. Plant
Cell 12: 1021–1029.
Bennetzen, J. L., 2000b Transposable element contributions to
plant gene and genome evolution. Plant Mol. Biol. 42: 251–269.
Bennetzen, J. L., 2002 Mechanisms and rates of genome expansion
and contraction in flowering plants. Genetica 115: 29–36.
Bennetzen, J. L., and W. Ramakrishna, 2002 Numerous small rearrangements of gene content, order and orientation differentiate grass genomes. Plant Mol. Biol. 48: 821–827.
Brunner, S., B. Keller and C. Feuillet, 2000 Molecular mapping
of the Rph7.g leaf rust resistance gene in barley (Hordeum vulgare
L.). Theor. Appl. Genet. 101: 783–788.
Chen, M., P. SanMiguel, A. C. deOliveira, S. S. Woo, H. Zhang et
al., 1997 Microcolinearity in sh2 homologous regions of the
maize, rice, and sorghum genomes. Proc. Natl. Acad. Sci. USA
94: 3431–3435.
Chen, M. S., P. SanMiguel and J. L. Bennetzen, 1998 Sequence
organization and conservation in sh2/a1 homologous regions of
sorghum and rice. Genetics 148: 435–443.
Devos, K. M., J. K. M. Brown and J. L. Bennetzen, 2002 Genome
size reduction through illegitimate recombination counteracts
genome expansion in Arabidopsis. Genome Res. 12: 1075–1079.
Druka, A., D. Kudrna, F. Han, A. Kilian, B. Steffenson et al., 2000
Physical mapping of the barley stem rust resistance gene rpg4.
Mol. Gen. Genet. 264: 283–290.
Dubcovsky, J., W. Ramakrishna, P. J. SanMiguel, C. S. Busso, L. L.
Yan et al., 2001 Comparative sequence analysis of colinear barley
and rice bacterial artificial chromosomes. Plant Physiol. 125:
1342–1353.
Faris, J. D., K. M. Haen and B. S. Gill, 2000 Saturation mapping
of a gene-rich recombination hot spot region in wheat. Genetics
154: 823–835.
Felsenstein, J., 1985 Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791.
Feuillet, C., and B. Keller, 1999 High gene density is conserved
at syntenic loci of small and large grass genomes. Proc. Natl.
Acad. Sci. USA 96: 8265–8270.
Feuillet, C., and B. Keller, 2002 Comparative genomics in the
grass family: molecular characterization of grass genome structure and evolution. Ann. Bot. 89: 3–10.
Gale, M. D., and K. M. Devos, 1998 Comparative genetics in the
grasses. Proc. Natl. Acad. Sci. USA 95: 1971–1974.
Goff, S. A., D. Ricke, T. H. Lan, G. Presting, R. L. Wang et al.,
2002 A draft sequence of the rice genome (Oryza sativa L. ssp
japonica). Science 296: 92–100.
Graner, A., H. Siedler, A. Jahoor, R. G. Herrmann and G. Wenzel,
1990 Assessment of the degree and the type of restriction fragment length polymorphism in barley (Hordeum vulgare). Theor.
Appl. Genet. 80: 826–832.
Graner, A., A. Jahoor, J. Schondelmaier, H. Siedler, K. Pillen et
al., 1991 Construction of an RFLP map of barley. Theor. Appl.
Genet. 83: 250–256.
Han, F., A. Kleinhofs, S. E. Ullrich, A. Kilian, M. Yano et al., 1998
Synteny with rice: analysis of barley malting quality QTLs and
rpg4 chromosome regions. Genome 41: 373–380.
Han, F., A. Kilian, J. P. Chen, D. Kudrna, B. Steffenson et al., 1999
Genome Rearrangement at the Rph7 Locus
Sequence analysis of a rice BAC covering the syntenous barley
Rpg1 region. Genome 42: 1071–1076.
He, Z. H., H. T. Dong, J. X. Dong, D. B. Li and P. C. Ronald, 2000
The rice Rim2 transcript accumulates in response to Magnaporthe
grisea and its predicted protein product shares similarity with
TNP2-like proteins encoded by CACTA transposons. Mol. Gen.
Genet. 264: 2–10.
Hulbert, S. H., C. A. Webb, S. M. Smith and Q. Sun, 2001 Resistance gene complexes: Evolution and utilization. Annu. Rev. Phytopathol. 39: 285–312.
Keller, B., and C. Feuillet, 2000 Colinearity and gene density in
grass genomes. Trends Plant Sci. 5: 246–251.
Kilian, A., J. Chen, F. Han, B. Steffenson and A. Kleinhofs, 1997
Towards map-based cloning of the barley stem rust resistance
genes Rpg1 and rpg4 using rice as an intergenomic cloning vehicle.
Plant Mol. Biol. 35: 187–195.
Kleinhofs, A., A. Kilian, M. A. S. Maroof, R. M. Biyashev, P. Hayes
et al., 1993 A molecular, isozyme and morphological map of
the barley (Hordeum vulgare) genome. Theor. Appl. Genet. 86:
705–712.
Koprek, T., D. McElroy, J. Louwerse, R. Williams-Carrier and
P. G. Lemaux, 2000 An efficient method for dispersing Ds elements in the barley genome as a tool for determining gene
function. Plant J. 24: 253–263.
Kunzel, G., L. Korzun and A. Meister, 2000 Cytologically integrated physical restriction fragment length polymorphism maps
for the barley genome based on translocation breakpoints. Genetics 154: 397–412.
Li, W. L., and B. S. Gill, 2002 The colinearity of the Sh2/A1 orthologous region in rice, sorghum and maize is interrupted and accompanied by genome expansion in the Triticeae. Genetics 160:
1153–1162.
Moore, G., K. M. Devos, Z. Wang and M. D. Gale, 1995 Cereal
genome evolution: grasses, line up and form a circle. Curr. Biol.
5: 737–739.
Nacken, W. K. F., R. Piotrowiak, H. Saedler and H. Sommer, 1991
The transposable element Tam1 from Antirrhinum majus shows
structural homology to the maize transposon En/Spm and has no
sequence specificity of insertion. Mol. Gen. Genet. 228: 201–208.
Panstruga, R., R. Buschges, P. Piffanelli and P. Schulze-Lefert,
1998 A contiguous 60 kb genomic stretch from barley reveals
molecular evidence for gene islands in a monocot genome. Nucleic Acids Res. 26: 1056–1062.
Rostoks, N., Y. Park, W. Ramakrishna, J. Ma, A. Druka et al., 2002
Genomic sequencing reveals gene content, genomic organization, and recombination relationships in barley. Funct. Integr.
Genomics 2: 51–59.
Sambrook, J., E. F. Fritsch and T. Maniatis, 1989 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY.
Sandhu, D., and K. S. Gill, 2002a Gene containing regions of wheat
and the other grass genomes. Plant Physiol. 128: 803–811.
Sandhu, D., and K. S. Gill, 2002b Structural and functional organization of the ‘1S0.8 gene-rich region’ in the Triticeae. Plant Mol.
Biol. 48: 791–804.
SanMiguel, P., and J. L. Bennetzen, 1998 Evidence that a recent
increase in maize genome size was caused by the massive amplification of intergene retrotransposons. Ann. Bot. 82: 37–44.
SanMiguel, P., A. Tikhonov, Y. K. Jin, N. Motchoulskaia, D. Zakharov et al., 1996 Nested retrotransposons in the intergenic regions of the maize genome. Science 274: 765–768.
SanMiguel, P. J., W. Ramakrishna, J. L. Bennetzen, C. S. Busso
and J. Dubcovsky, 2002 Transposable elements, genes and recombination in a 215-kb contig from wheat chromosome 5Am.
Funct. Integr. Genomics 2: 70–80.
Shirasu, K., A. H. Schulman, T. Lahaye and P. Schulze-Lefert,
683
2000 A contiguous 66 kb barley DNA sequence provides evidence for reversible genome expansion. Genome Res. 10: 908–
915.
Smilde, W. D., J. Halukova, T. Sasaki and A. Graner, 2001 New
evidence for the synteny of rice chromosome 1 and barley chromosome 3H from rice expressed sequence tags. Genome 44:
361–367.
Sonnhammer, E. L. L., and R. Durbin, 1995 A dot matrix program
with dynamic threshold control suited for genomic DNA and
protein sequence analysis. Gene 167: 1–10.
Stein, N., C. Feuillet, T. Wicker, E. Schlagenhauf and B. Keller,
2000 Subgenome chromosome walking in wheat: a 450 kb physical contig in Triticum monococcum L. spans the Lr10 resistance
locus in hexaploid wheat (Triticum aestivum L.). Proc. Natl. Acad.
Sci. USA 97: 13436–13441.
Takai, R., K. Hasegawa, H. Kaku, N. Shibuya and E. Minami, 2001
Isolation and analysis of expression mechanisms of a rice gene,
EL5, which shows structural similarity to ATL family from Arabidopsis, in response to N-acetylchitooligosaccharide elicitor. Plant
Sci. 160: 577–583.
Takai, R., N. Matsuda, A. Nakano, K. Hasegawa, C. Akimoto et
al., 2002 EL5, a rice N-acetylchitooligosaccharide elicitorresponsive RING-H2 finger protein, is a ubiquitin ligase which
functions in vitro in co-operation with an elicitor-responsive ubiquitin-conjugating enzyme, OsUBC5b. Plant J. 30: 447–455.
Tarchini, R., P. Biddle, R. Wineland, S. Tingey and A. Rafalski,
2000 The complete sequence of 340 kb of DNA around the
rice Adh1-Adh2 region reveals interrupted colinearity with maize
chromosome 4. Plant Cell 12: 381–391.
Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin and D. G.
Higgins, 1997 The CLUSTAL_X windows interface: flexible
strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25: 4876–4882.
Tikhonov, A. P., P. J. SanMiguel, Y. Nakajima, N. M. Gorenstein,
J. L. Bennetzen et al., 1999 Colinearity and its exceptions in
orthologous adh regions of maize and sorghum. Proc. Natl. Acad.
Sci. USA 96: 7409–7414.
Vision, T. J., D. G. Brown and S. D. Tanksley, 2000 The origins
of genomic duplications in Arabidopsis. Science 290: 2114–2117.
Wei, F. S., K. Gobelman-Werner, S. M. Morroll, J. Kurth, L. Mao
et al., 1999 The Mla (powdery mildew) resistance cluster is associated with three NBS-LRR gene families and suppressed recombination within a 240-kb DNA interval on chromosome 5S (1HS)
of barley. Genetics 153: 1929–1948.
Wei, F. S., R. A. Wing and R. P. Wise, 2002 Genome dynamics and
evolution of the Mla (powdery mildew) resistance locus in barley.
Plant Cell 14: 1903–1917.
Wicker, T., N. Stein, L. Albar, C. Feuillet, E. Schlagenhauf et
al., 2001 Analysis of a contiguous 211 kb sequence in diploid
wheat (Triticum monococcum L.) reveals multiple mechanisms of
genome evolution. Plant J. 26: 307–316.
Wolfe, K. H., M. L. Gouy, Y. W. Yang, P. M. Sharp and W. H. Li,
1989 Date of the monocot dicot divergence estimated from
chloroplast DNA sequence data. Proc. Natl. Acad. Sci. USA 86:
6201–6205.
Woo, S. S., J. M. Jiang, B. S. Gill, A. H. Paterson and R. A. Wing,
1994 Construction and characterization of a bacterial artificial
chromosome library of Sorghum bicolor. Nucleic Acids Res. 22:
4922–4931.
Yu, J., S. N. Hu, J. Wang, G. K. S. Wong, S. G. Li et al., 2002 A draft
sequence of the rice genome (Oryza sativa L. ssp indica). Science
296: 79–92.
Yu, Y., J. P. Tomkins, R. Waugh, D. A. Frisch, D. Kudrna et al., 2000
A bacterial artificial chromosome library for barley (Hordeum
vulgare L.) and the identification of clones containing putative
resistance genes. Theor. Appl. Genet. 101: 1093–1099.
Communicating editor: A. Paterson