The Colinearity of the Sh2/A1 Orthologous Region in Rice, Sorghum and Maize Is Interrupted and Accompanied by Genome Expansion in the Triticeae

Corresponding author: Bikram S. Gill, 4024 Throckmorton Hall, Kansas State University, Manhattan, KS 66506-5502., bsg{at}ksu.edu (E-mail)

Communicating editor: J. A. BIRCHLER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Sh2/A1 orthologous region of maize, rice, and sorghum contains five genes in the order Sh2, X1, X2, and two A1 homologs in tandem duplication. The Sh2 and A1 homologs are separated by 20 kb in rice and sorghum and by 140 kb in maize. We analyzed the fate of the Sh2/A1 region in large-genome species of the Triticeae (wheat, barley, and rye). In the Triticeae, synteny in the Sh2/A1 region was interrupted by a break between the X1 and X2 genes. The A1 and X2 genes remained colinear in homeologous chromosomes as in other grasses. The Sh2 and X1 orthologs also remained colinear but were translocated to a nonhomeologous chromosome. Gene X1 was duplicated on two nonhomeologous chromosomes, and surprisingly, a paralog shared homology much higher than that of the orthologous copy to the X1 gene of other grasses. No tandem duplication of A1 homologs was detected but duplication of A1 on a nonhomeologous barley chromosome 6H was observed. Intergenic distances expanded greatly in wheat compared to rice. Wheat and barley diverged from each other 12 million years ago and both show similar changes in the Sh2/A1 region, suggesting that the break in colinearity as well as X1 duplications and genome expansion occurred in a common ancestor of the Triticeae species.

WHEAT (Triticum aestivum L., 2n = 6x, AABBDD; T. turgidum L., 2n = 4x, AABB; and T. monococcum L., 2n = 2x, A^mA^m), barley (Hordeum vulgare L. 2n = 2x, HH), rye (Secale cereale L. 2n = 2x, RR), rice (Oryza sativa L.), sorghum [Sorghum bicolor (L.) Moench], and maize (Zea mays L.) are important food crops of the grass family (Gramineae or Poaceae). Despite 55 million years of coevolution (KELLOGG 2001 ) and the 40-fold variation in genome size among these taxa (ARUMUGANATHAN and EARLE 1991 ), their gene content and gene order are conserved as demonstrated by comparative, albeit low resolution, mapping (HULBERT et al. 1990 ; AHN and TANKSLEY 1993 ; AHN et al. 1993 ; MOORE et al. 1995 ). With advances in DNA cloning and sequencing technology, comparative genetics can be employed on a finer scale using large cloned fragments or long stretches of genomic sequences. Comparative sequence analysis of orthologous regions of rice, sorghum, and maize has provided important information on grass genome evolution, colinearity, and small rearrangements at the gene level (CHEN et al. 1997 ; MESSING and LLACA 1998 ; TIKHONOV et al. 1999 ; TARCHINI et al. 2000 ).

One genomic region analyzed by comparative sequence analysis in rice and sorghum is the Sh2/A1, a region initially investigated by maize geneticists. Sh2 (shrunken2) codes for the large subunit of ADP-glucose pyrophosphorylase and A1 (anthocyaninless1) encodes dihydroflavonol-4-reductase. These two genes are separated by 140 kb in maize (CIVARDI et al. 1994 ). The Sh2 and A1 are only 20 kb apart in rice and sorghum. Two putative transcription-factor genes X1 and X2 lie between Sh2 and A1 (CHEN et al. 1997 ; BENNETZEN and RAMAKRISHNA 2002 ; GenBank accession no. AF101045). Sequence characterization detected a direct tandem duplication of A1 in this region of rice and sorghum and several miniature inverted repeat transposable elements in the intergenic regions and introns (CHEN et al. 1997 , CHEN et al. 1998 ). Although gene colinearity was maintained between maize, sorghum, and rice, intergenic regions expanded greatly in maize, which is consistent with its large genome (2500 Mb) compared to the smaller genomes of rice (430 Mb) and sorghum (750 Mb; ARUMUGANATHAN and EARLE 1991 ).

The Triticeae species, including wheat and barley, have genomes much larger than those of the other grasses. For example, the barley (4873 Mb) and diploid wheat (5751 Mb) genomes are 11- to 13-fold larger than the rice genome. The amplification of retrotransposons is the major cause of genome obesity in maize (SANMIGUEL et al. 1996 ; MESSING and LLACA 1998 ; SANMIGUEL and BENNETZEN 1998 ; TIKHONOV et al. 1999 ). A similar picture of genome organization in the Triticeae has emerged from the contiguous sequences of mlo (PANSTRUGA et al. 1998 ) and rar1 regions (SHIRASU et al. 2000 ) of barley and the Lr10 region of T. monococcum (WICKER et al. 2001 ). A further increase in genome size was brought about by polyploidy; bread wheat (T. aestivum) is hexaploid and has a genome of 15,966 Mb. Theoretically, the process of genome inflation could affect the gene order and content. Because the Sh2/A1 interval has been used as a meter of genomic obesity in other cereals, it would be instructive to explore the fate of this region in terms of synteny and intergenic distances after genome expansion in the Triticeae. Here, we present the genome organization of the Sh2/A1 gene region in the Triticeae.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Clones and primers:
A HindIII bacterial artificial chromosome (BAC) library of diploid wheat T. monococcum cv. DV92 (LIJAVETZKY et al. 2000 ) arrayed in high density on filters was used in this study. The BAC filters were probed with the wheat cDNA clone Aga7 (GenBank accession no. X14350) homologous to Sh2 (provided by Dr. P. Sharp, Sydney University, Australia), the rice cDNA clone DFR (dihydroflavonol-4-reductase, GenBank accession no. AB003496) homologous to A1 (provided by Dr. Y. Inagaki, National Institute for Basic Biology, Myodaiji, Okazaki, Japan), and the rice cDNA clone R2277 (GenBank accession no. D24626) homologous to the X1 gene (supplied by Dr. T. Sasaki, Institute of Agricultural Resources, Japan). Primer pairs X2-1 (5'-ATTCATCAGCTTGGTGGC-3' and 5'-GAGTTCTGAATCATCTGCCC-3') and X2-2 (5'-ACCGTCAAGTACGCCTTCCC-3' and 5'-CGCACACGTCGTGGCTGCAC-3') were designed for PCR amplification of the last and the first exons of the X2 gene from rice (O. sativa subsp. japonica cv. Nipponbare). Subclones X1-532 (550-bp insert) and X1-554 (420-bp insert) are homologous to the X1 gene. X2-611 (300-bp fragment) is homologous to the last exon of the rice X2 gene. The inserts were amplified by PCR from the subclones and used as probes for mapping.

Plant materials:
For mapping the genes on chromosomes, the following cytogenetic stocks of T. aestivum cv. Chinese Spring (CS) were used: nullitetrasomic (NT) lines, in which a missing pair of chromosomes is compensated by four doses of its homeolog (SEARS 1966 ); ditelosomic lines, where one chromosome arm is missing (SEARS and SEARS 1978 ); deletion lines arising from single breaks and loss of distal acentric segments (ENDO and GILL 1996 ); and wheat-alien addition lines, where an alien chromosome pair is added to wheat. CS-Imperial rye and CS-Betzes barley addition lines were obtained from Dr. T. E. Miller, John Innes Centre, United Kingdom, and Dr. A. K. M. R. Islam, University of Adelaide, Australia, respectively. For genetic mapping, a population of recombinant inbred lines (RILs) derived from a cross between the common wheat cv. Opata 85 and the synthetic hexaploid wheat W-7984 as described in NELSON et al. 1995 was used (provided by Dr. M. E. Sorrells, Cornell University, Ithaca, NY).

Filter hybridization:
Plant DNA was isolated following the protocol described in FARIS et al. 2000 . BAC plasmids were isolated according to SAMBROOK et al. 1989 . BAC and plant genomic DNA were digested with restriction endonucleases (Promega, Madision, WI; New England BioLabs, Beverly, MA), separated by agarose gel electrophoresis, and blotted onto N⁺ Hybond membrane (Amersham Biosciences, Piscataway, NJ) following the manufacturer's instructions. BAC plasmids, digested with rare cutters BglI, BssHII, NotI, PmeI, SfiI, SgrAI, and SwaI (New England BioLabs) were separated by pulse field gel electrophoresis using a CHEF-DRII System (Bio-Rad, Emeryville, CA) at a field strength of 6 V/cm for 16 hr at 12° with an initial pulse time of 5 sec and final pulse time of 15 sec. The size of the BAC inserts was determined using a ladder (New England BioLabs) as reference. Hybridization, probe labeling, and filter washing were performed as described previously (FARIS et al. 2000 ).

Subcloning and sequence analysis:
Based on Southern hybridization, specific bands homologous to A1, X1, and X2 were purified from an agarose gel, ligated in pUC18, and transformed in competent cells of the Escherichia coli strain DH10B by electroporation. White (recombinant) colonies were inoculated into 96-well plates. Colony-blot hybridization was performed to select positive clones and grown in a Luria-Bertani broth medium containing 100 µg/ml carbenicillin. Plasmids were purified and used as templates for sequencing from both directions. Ligation, colony-blot hybridization, plasmid isolation, and purification were done using standard protocols described in SAMBROOK et al. 1989 .

Deduction of open reading frames (ORFs) and amino acid sequences, prediction of protein secondary structure, and multiple sequence alignments were performed using the Baylor College of Medicine (BCM) Search Launcher (www site: http://www.dot.imgen.bcm.tmc.edu). Multiple sequence alignment results were output by using the BOXSHADE program (version 3.2) with fraction of sequences set at 0.5 (http://www.ch.embnet.org/software/BOX_form.html). Homology searches were made using the BLAST 2.0 program of the National Center of Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov).

Genetic mapping:
The mapping population, used extensively by investigators of the International Triticeae Mapping Initiative (ITMI), consists of 114 RILs. Mapping data were obtained from the GrainGenes database (http://genome.cornell.edu/cgi-bin/WebAce/webace?db=graingenes). The first 60 RILs were used for genetic mapping in this study. Linkage analysis and genetic distances were estimated with MAPMAKER software (LANDER et al. 1987 ). Recombination frequencies were converted into map distances using the Haldane mapping function (HALDANE 1919 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

BAC library screening:
Four high-density filters containing 73,728 BAC clones (1.15 genome equivalents) from diploid wheat were screened by hybridization to candidate clones. Two BACs each for Sh2 and A1 and three for X1 were isolated. The BAC insert size ranged from 45 to 155 kb (Table 1). An agarose gel electrophoresis of the HindIII-digested DNA of seven BAC clones showed an overlap between the two BACs containing Sh2 or A1, but no overlap among the three containing X1. Southern hybridization analysis with the probes Aga7 (Sh2), DFR (A1), and R2277 (X1) further confirmed the above results.

Using the first and last exons of the X2 gene as probes, Southern hybridization of HindIII- and NotI-digested BACs showed that the X2 homolog was present in BACs 611L12 and 683A21, which also contained the A1 gene homolog. BACs 611L12 and 683A21 contain identical copies of the A1 and X2 homologs because hybridization patterns resulting from HindIII digestion were identical. Southern analysis, using the last exon of X2 and the 5' portion of A1 as probes, showed that X2 and A1 are present in a NotI fragment of 50 kb in both BACs (data not shown).

Sh2 homologs were detected in BACs 655N4 and 692D11. A 7-kb fragment and two smaller ones were seen in BAC 655N4 but only the 7-kb fragment was present in BAC 692D11. Sh2 is a large gene with 15 exons (CHEN et al. 1998 ), and it is likely that two overlapping pieces of the gene were cloned into independent BACs during library construction.

For the X1 gene, Southern blot analysis of BACs 532J13, 539B21, and 554G10 probed with R2277 showed different-sized fragments of varying intensities (Fig 1), suggesting that T. monococcum carries at least three copies of X1 with variable sequence homologies.

View larger version (37K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Southern blot of HindIII digestion of BAC 532J13 (lane 1), BAC 539B21 (lane 2), and BAC 554G10 (lane 3) probed with rice cDNA R2277.

Homology confirmation:
Several cDNA clones coding for the large subunit of ADP-glucose pyrophosphorylase (the product of the Sh2 gene) have been isolated from wheat (GenBank accession nos. AF026539, U61178, U61179, X14349, X14350, and Z21969) and barley (GenBank accession nos. U66876, X62242, and X67151). All of these cDNA clones, among which Aga7 (X14349) was used as a probe in this study, showed >80% identity at the nucleotide level to the protein-coding region of the Sh2 gene of maize, Sh2 homologs of rice and sorghum, and an identity/similarity of >70%/>85% at the amino acid sequence level.

We subcloned and sequenced the homologs of genes A1, X1, and X2 from the corresponding BACs of T. monococcum, because no sequences were available for these genes in wheat.

The wheat A1 homolog (A1-683) was cloned from BAC 683A21 and is predicted to encode a protein of 374 amino acids. As expected, A1-683 showed the highest (90%) sequence identity to the barley A1 gene homolog from the promoter through the 5' untranslated region (UTR), the protein-coding region, and the 3' UTR to the 3' downstream region beyond the poly(A) signal except for introns. A1-683 showed >80% sequence identity to the A1 homologs of sorghum, rice, and maize but only in the protein-coding regions. The inferred amino acid sequence of A1-683 was similar to those of the dihydroflavanol-4-reductases from diverse plant species. Alignment against the deduced A1 protein sequences of maize, rice, sorghum, and barley revealed an insertion of 20-amino-acid residues in A1-683 of wheat between positions 97 and 116 (data not shown).

Three X1 homologs, X1-532, X1-539, and X1-554, were subcloned from three separate BAC clones (532J13, 539B21, and 554G10). The X1-539 subclone had the highest (75–89%) identity to all six exons of the X1 gene of rice (AF101045) and sorghum (AF010283). A protein of 639 amino acids was deduced from the nucleotide sequence of X1-539 and showed 79 and 82% sequence similarity to the X1 gene products of rice and sorghum, respectively. Comparison with the amino acid sequences of the X1 proteins of rice and sorghum detected an insertion of 10-amino-acid residues at positions 97–106 in the wheat X1 protein encoded by X1-539 (Fig 2). In addition, the deduced protein product of X1-539 showed high similarity to the X1 gene product of rice and sorghum in secondary structure and is predicted to possess coiled-coil domains (CHEN and BENNETZEN 1996 ).

View larger version (129K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Alignment of the amino acid sequences of proteins encoded by the X1 genes of rice and sorghum and wheat X1 homolog X1-539.

X1-532 and X1-554 showed sequence similarity only to the first exon, not to the other five exons of the X1 gene of rice and sorghum. At the nucleotide sequence level, these two clones showed sequence identity of >71% (126 and 143 bp of X1-554 and 316 bp of X1-554) to the rice X1 gene. BLASTX (translation alignment) detected sequence similarity of >55% in clone X1-532 spanning 200-amino-acid residues (600 bp in nucleotide sequence) and in clone X1-554 spanning 130-amino-acid residues (400 bp) to the X1 gene of rice and sorghum.

Using the last exon of the rice X2 gene as a probe, a subclone (X2-611) was isolated from BAC 611L12 containing the X2 homolog. Sequence analysis showed 85% identity to the last exon of the predicted X2 gene of rice and sorghum.

Using the coding sequences of the wheat homologs as queries, a BLAST search was performed against a wheat expressed sequence tag (EST) database at The Institute of Genome Research (TIGR) Gene Indices (http://www.tigr.org/tdb/tgi.shtml). Two A1 homologs were found in the wheat cDNA library made from spikes at 5–15 days after pollination (DAP). Many X1 homologs were found in cDNA libraries made from wheat tissues including root, leaf, seedling, spikelet, preanthesis spike, 5–15 DAP spike, and endosperm, indicating that A1 and X1 (X1-539) are actively transcribed in wheat. No EST match was found for the X2 gene.

Chromosomal localization:
To investigate the colinearity between genomes of the Triticeae species and those of rice, sorghum, and maize in the Sh2/A1 region, the wheat clones were mapped using the CS NT, ditelosomic, deletion lines, wheat-alien addition lines, and the ITMI mapping population.

On the basis of restriction fragment length polymorphism (RFLP) analyses of NT lines and CS-alien addition lines, Aga7 was located on wheat chromosomes 1A, 1B, and 1D (Fig 3); rye chromosome 1R (Fig 4); and the long arm of barley chromosome 1H (Fig 4). Aga7 detected a polymorphic fragment between the parents of the ITMI mapping population, Opata 85 and Synthetic wheat W-7984, and was genetically mapped to the distal region on the long arm of chromosome 1D (Fig 5).

View larger version (98K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Chromosome localization of the wheat homologs of genes A1, X1, and Sh2 by Southern blot hybridization using NT lines. The 21 NT lines are indicated on the top of the lanes of Southern blots. The genes (clones) are listed to the left and the chromosome locations of the bands are indicated on the right.

View larger version (104K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Chromosome localization of wheat homologs of the A1, X1, and Sh2 genes by Southern blot hybridization using CS-alien addition lines. The 14 CS-alien disomic addition (DA) lines and their alien parents are indicated on the top, genes (clones) are listed to the left, and the alien chromosomes on which the genes are located are indicated to the right. The remaining bands are from CS wheat.

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Linkage map of wheat chromosome arm 1DL. The telemere is toward the bottom. Centimorgan (cM) distances are indicated to the left of the chromosome and marker loci to the right. The positions of clones mapped in this experiment are indicated in boldface type. The symbol for the marker locus detected by clones X1-532 is Xksu935.

The wheat A1 homolog (A1-683) was mapped to chromosomes 3A, 3B, 3D (Fig 3), and 3R (Fig 4). Ditelosomic and deletion line analysis indicated that the A1 gene homologs are located in the proximal region of long arms of the group 3 chromosomes, between breakpoints FL0.26 and FL0.42 (FL, fraction length of distance from centromere). Two copies of the A1 homologs exist in the barley genome on chromosomes 3H and 6H (Fig 4). No polymorphism for A1-683 was detected between Opata 85 and W-7984 even though 15 restriction enzymes were used.

T. monococcum has three copies of X1 homologs, X1-532, X1-539, and X1-554. Both 3' and 5' regions of X1-539 were located on chromosomes 7A, 7B, 7D (Fig 3), 7H (Fig 4), and 6R (Fig 4). A specific fragment missing in N7D-T7B also was missing in N1A-T1D (Fig 3); in the latter the distal region of chromosome 7D was deleted during its development (DEVOS et al. 1993 ). Similarly, X1-539 is present on 6R and not on 7R (Fig 4), because the distal region of the long arm of the original 7R chromosome was translocated to the long arm of 6R (DEVOS et al. 1993 ). Therefore, X1-539 can be localized to the distal region of the long arms of group 7 chromosomes of the Triticeae. The 7A band was missing from all of the CS-Betzes addition lines (Fig 3 and Fig 4), suggesting that a deletion proximal to the X1 locus occurred on chromosome 7A of the CS used before or during development of the addition lines. X1-539 was monomorphic between Opata 85 and W-7984.

X1-532 was located on wheat chromosomes 1A and 1D (Fig 3) and on the long arm of barley chromosome 1H (Fig 4). No homolog was detected in the rye genome. A polymorphic band was mapped to chromosome 1D, 1.5 cM proximal to the Sh2 homolog Aga7 (Fig 5).

X1-554 was mapped to chromosomes 3A, 3B, and 3D of wheat. Deletion line analysis localized the 3A fragment to the proximal region of the short arm (data not shown).

As expected, a wheat fragment (300 bp) homologous to the last exon of the X2 gene was localized to wheat group 3 chromosomes (data not shown).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Intergenic expansion:
Initial analysis of the genomic sequences of the orthologous Sh2/A1 region of rice and sorghum identified three genes, Sh2, X, and A1, which span 30 kb. A direct tandem duplication of A1 was found in sorghum and rice (CHEN et al. 1997 , CHEN et al. 1998 ). Subsequent annotation concluded that the original X "gene" consists of two separate genes, i.e., X1 and X2 (GenBank accession no. AF101045; BENNETZEN and RAMAKRISHNA 2002 ). We sequenced the rice cDNA clone R2277; it showed 100% identity to rice X1 and 0% identity to X2. On the basis of current knowledge, five genes (Sh2, X1, and X2) and two tandem A1 homologs exist in this region of the rice and sorghum genomes. The same scenario also has been revealed in maize by sequencing of the orthologous region (see BENNETZEN and RAMAKRISHNA 2002 ).

In the T. monococcum BAC library, we identified two BACs containing Sh2 homologs, two containing A1 homologs, and three containing X1 homologs. Homologs of A1 and X2 exist in the same BACs separated by 50 kb. They are separated by 11.9 kb in rice and by 7.4 kb in sorghum. These data predict an expansion of approximately fourfold in the A1/X2 interval in wheat compared to rice.

No overlap was found among BACs containing Sh2 and X1 in wheat, suggesting that their physical distance may be >115 kb, the average insert size of the BAC library. Tight genetic linkage of 1.5 cM, however, was observed between X1-532 and Sh2 (Aga7) in the distal region of wheat chromosome arm 1DL. Triticeae species have large genomes and low recombination frequency, overall 4.4 Mb cM^-1. In the gene-rich regions, recombination can be very high, ranging from 20 to 270 kb cM^-1 in 1DS of Aegilops tauschii (SPIELMEYER et al. 2000 ), 50 kb cM^-1 in 1A^mS (WICKER et al. 2001 ), and 260 kb cM^-1 in 5A^mS of T. monococcum (TRANQUILLI et al. 1999 ) and in 5AL of T. aestivum (J. D. FARIS and B. S. GILL, unpublished data). On the basis of the above data, Sh2 and X1 orthologs may be separated by a physical distance between 115 and 390 kb, the latter estimate derived by multiplying the estimated 260 kb cM^-1 value by 1.5 cM genetic distance. However, the physical distance between Sh2 and X1 may be even greater because in a DNA fiber fluorescent in situ hybridization (FISH) experiment, the Sh2 and X1 orthologs did not hybridize to the same DNA fibers of Ae. tauschii (P. ZHANG, W. LI, B. FRIEBE and B. S. GILL, unpublished data). This technique can usually measure distances as far as 660 kb (FRANSZ et al. 1996 ). Sh2 and X1 are separated by 2 kb in rice and 8.2 kb in sorghum. Therefore, the estimated expansion in the Sh2/X1 interval of wheat is 195-fold of that of rice.

Colinearity interruption:
The Sh2 and A1 orthologs map to chromosome 1 of rice (A. REDDY and J. L. BENNETZEN, personal communication) and chromosome 3 of maize (DAVIS et al. 1999 ). These chromosomes are homeologous with the group 3 chromosomes of the Triticeae (AHN et al. 1993 ; MOORE et al. 1995 ). Several markers (bcd134, cdo455, and cdo118) flanking Sh2/A1 on chromosome arm 3L of maize (DAVIS et al. 1999 ) also were mapped to homeologous chromosomes: chromosomes 1 of rice (AHN et al. 1993 ) and 3L of wheat (ANDERSON et al. 1992 ; GrainGenes database). The markers bcd134 and cdo118 were mapped genetically to the proximal region of chromosome arm 3L of wheat (NELSON et al. 1995 ). On the basis of these data, Sh2, X1, X2, and A1 should be located on group 3 chromosomes of the Triticeae.

As expected, wheat homologs of A1 (A1-638) and X2 (X2-611) mapped to the proximal region of the 3L arm of the Triticeae. Each detected a single copy in the A, B, and D genomes of wheat. Therefore, the A1 and X2 genes constitute an orthologous set and have maintained a syntenic position on homeologous chromosomes in wheat, maize, sorghum, and rice even after 55 million years of coevolution.

However, contrary to the expected synteny, a wheat homolog of Sh2 (Aga7) has been mapped to the distal regions of the long arms of group 1 chromosomes: 1A^mL of T. monococcum (DUBCOVSKY et al. 1996 ); 1DL of Ae. tauschii (LAGUDAH et al. 1991 ); 1HL of barley (KLEINHOFS et al. 1993 ); and 1AL, 1BL, and 1DL of T. aestivum (AINSWORTH et al. 1995 ). Our results confirmed these locations of Sh2 in the wheat genome. Because Aga7 detected a single copy in the A, B, and D genomes of bread wheat, it should be orthologous to the gene Sh2 of maize. We show that Aga7 is closely linked to X1-532, a presumed ortholog of the X1 gene of rice (see later section). Thus, it appears that although these two genes are syntenic, they are located on nonhomeologous chromosomes in the Triticeae compared to those in maize, sorghum, and rice.

As discussed earlier, Sh2, X1, X2, and A1 genes are syntenic in maize, sorghum, and rice. However, Sh2 and X1 were mapped on group 1, and X2 and A1 mapped on group 3 chromosomes in the Triticeae. Therefore, colinearity in the Sh2/A1 region was interrupted by a break between the X1 and X2 genes and another break between Sh2 and bcd134 in the Triticeae. Next, the Sh2-X1 segment was translocated or transposed at an interstitial position in group 1 chromosomes in the Triticeae. This scheme is consistent with that from wheat-rice comparative mapping, where most markers flanking but excluding Aga7 on the consensus map of chromosome arm 1L in the Triticeae align to their counterparts on chromosome 5 of rice (VAN DEYNZE et al. 1995 ).

Wheat and barley diverged from the same ancestor 12 million years ago (HUANG et al. 2002 ). Both wheat and barley share a break in colinearity in the Sh2/A1 region and associated microrearrangements in relation to rice, sorghum, and maize. Wheat, barley, and all other species of the Triticeae also have large genomes organized into a basic set of seven chromosomes irrespective of the ploidy, which ranges from 2x to 10x. Therefore, we postulate that a break in colinearity accompanied by genome expansion occurred 12 million years ago in an ancestral species of the Triticeae.

Tandem duplication of A1:
Nearly 50 years ago, LAUGHNAN 1952 demonstrated the tandem duplication of functional A1 genes in maize. Genomic sequencing of the Sh2/A1 homologous region revealed a tandem duplication of A1 homologs 10 kb apart in sorghum and 5 kb apart in rice (CHEN et al. 1997 , CHEN et al. 1998 ). In the Triticeae, however, there is no evidence for tandem duplication of A1 homologs. A1 homologs are present as a single copy in the proximal region of the long arms of chromosomes 3A, 3B, and 3D in common wheat and in the diploid species T. monococcum, T. urartu, Ae. tauschii, and rye. Ae. speltoides showed two hybridizing fragments that were caused by heterozygosity rather than duplication of the A1 locus because it is an outcrossing species. A nontandem duplication of the A1 homolog was found in barley. The A1 paralogue was located on chromosome 6H (Fig 4). Unequal recombination was probably responsible for the tandem duplication of the A1 gene (CHEN et al. 1998 ). The A1 tandem duplication might have occurred in an ancestor of maize, sorghum, and rice (CHEN et al. 1998 ), but not in the Triticeae ancestor. Alternatively, an A1 tandem duplication occurred in the common ancestor of cereals and one copy was lost in the Triticeae ancestor.

Orthology vs. homology:
Based on its chromosome location, X1-532 is syntenic with Sh2 in wheat, maize, sorghum, and rice. On the basis of synteny, we conclude that X1-532 constitutes part of an orthologous set of genes in these grasses. We observed additional copies of X1, i.e., X1-539 on 7L and X1-554 on 3S of the Triticeae. On the basis of these data, X1-539 and X1-554 should be considered paralogous to the X1 gene of rice, sorghum, and X1-532 of wheat. However, sequencing showed that it is the paralog (X1-539) rather than the orthologous copy (X1-532) that has maintained the highest homology to the X1 gene of rice and sorghum. X1-532 and X1-554 underwent extensive degeneration, showed only limited homology in the first exon, and have lost the other five exons of the X1 gene. Our results indicate that an ortholog based on map position is not always the functional or the most homologous copy in a genome. The discrepancy between orthology and homology may cause misleading results in comparative mapping involving distantly related genomes.

We propose the following hypothesis to explain these results (see also Fig 6). First, an X1 ortholog on 3L was duplicated on 7L (X1-539) early during the evolution of the Triticeae. Next Sh2/X1 was translocated or transposed to 1L followed by another round of X1 duplication and homology degradation in the current Triticeae. The loss of the 3' portion of X1-532 in the current Triticeae might be associated with the Sh2/X1 translocation/transposition and low selection pressure because an intact paralog X1-539 existed somewhere else in the genome (on 7L). Both X1-532 (on 1L) and X1-554 (on 3S) lack the 3' portion of the X1 gene compared with X1-539 and X1-554 is more divergent than X1-532 in relation to the X1 gene of rice and sorghum. This suggests that X1-554 was derived from X1-532 and evolved independently after the Sh2/X1 translocation/transposition event.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 6. A tentative scheme of the evolution of Sh2/A1 counterparts in the Triticeae. X1' and X1" represent the deleted X1 fragments, detected by X1-532 and X1-554, respectively.

The unusual evolutionary pattern of X1 homologs implies a mechanism of colinearity breakage by duplication-deletion events. As discussed above, the ortholog X1-532 underwent extensive homology degradation and lost the five exons in the 3' region, but the paralog X1-539 maintains a high degree of homology to the X1 gene. An extreme situation was observed in rye where no homology was detected to X1-532, whereas a single-copy homolog was detected by X1-539 in the distal region of 7RL, which was translocated to 6RL (Fig 4). If only rye is compared with rice and sorghum, one would conclude that colinearity of the Sh2/A1 homologous interval was interrupted by two single-gene translocations. The X1 ortholog (X1-532) also was lost in the B genome of T. aestivum (Fig 3), but is present in Ae. speltoides, the putative B-genome donor species (data not shown), indicating that the deletion event occurred following polyploidization.

Use of a model genome:
The Triticeae species have large genomes, 80% of which are composed of repeated DNA sequences. The use of a small genome as a reference is a natural choice for positional cloning of agriculturally important genes from these species. On the basis of results of comparative mapping, rice has been proposed as a model for grass biology because it has the smallest genome among the grasses, conserved gene content, and gene colinearity with other cereal crops (HAVUKKALA 1996 ). The entire rice genome is being sequenced. However, microrearrangements (small translocations, deletions, and duplications) pose a major difficulty for the application of rice as a surrogate for large cereal genomes. The situation might be more severe in polyploid species, where rapid genome restructuring can occur during speciation (see review by PATERSON et al. 2000 ), and the resulting structural variation is buffered by the duplicated genomes and fixed during subsequent evolution. Our results support other recent reports documenting frequent microrearrangements between Triticeae and rice (FOOTE et al. 1997 ; FEUILLET and KELLER 1999 ). We further demonstrate that colinearity may break, even in orthologous regions as small as 7.2 kb in rice that are perfectly colinear in other grass species. Furthermore, a gene cloned by map position in fact may not even be a functional copy, and a gene cloned on the basis of sequence homology may not be an ortholog, an outcome of gene amplification and gene homology degradation events, as demonstrated for the X1 gene of the Triticeae.

	FOOTNOTES

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AF434703 (A1-683), AF434704 (X1-532), AF434705 (X1-539), AF434706 (X1-554), and AF434707 (X2-611).

	ACKNOWLEDGMENTS

We thank Dr. J. L. Bennetzen for constructive suggestions; Drs. P. Sharp, Y. Inagaki, and T. Sasaki for providing cDNA clones; and Drs. M. E. Sorrells, T. Miller, and A. K. M. R. Islam for supplying plant materials. This study is contribution No. 02-87J from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan, Kansas.

Manuscript received September 6, 2001; Accepted for publication December 17, 2001.

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