Department of Molecular Genetics and Cell Biology, University of Chicago;
Department of Plant Pathology, Kansas State University
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Abstract |
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Introduction |
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All members of the Triticeae tribe, including wheat, barley, and rye, are presumed to have evolved from a common ancestor. Among them are polyploid wheat species such as the tetraploid durum (macaroni) wheat Triticum turgidum and the hexaploid bread wheat Triticum aestivum, which arose through processes of interspecific hybridization events among diverged ancestral wheats followed by spontaneous chromosome doubling. These polyploids contain entire genomes of two or three species in the homozygous condition. The major genomes of wheat and its relatives, named A, B, D, G, S, etc., have a basic chromosome number of seven and are homoeologous (reflecting residual homology of originally completely homologous chromosomes) to one another. The Triticeae tribe presents an interesting case because it includes species of different ploidy for which some key evolutionary steps are known.
Genetic studies have revealed that the polyploid wheat species constitute two evolutionary lineages (reviewed in Cox 1998
). Triticum turgidum (AABB genome) and T. aestivum (AABBDD) compose one lineage, and Triticum timopheevii (AAGG) and Triticum zhukovskyi (AAAAGG) compose the other lineage. The wild tetraploids T. turgidum ssp. dicoccoides (AABB) and T. timopheevii ssp. armeniacum (AAGG) arose as amphiploids from the hybridization between Triticum urartu (AA) (Dvorak et al. 1993
; Takumi et al. 1993
) and two different plasmon (the sum of extrachromosomal hereditary determinants) types of another wild diploid wheat which is thought to be Aegilops speltoides (SS) (Dvorak and Zhang 1990
; Wang, Miyashita, and Tsunewaki 1997
). Triticum aestivum (AABBDD) arose from spontaneous hybridization between T. turgidum (AABB) and the diploid goatgrass Aegilops tauschii (DD) (Kihara 1944
; McFadden and Sears 1946
; reviewed in Dvorak et al. 1998
). In T. zhukovskyi (AAAAGG), one set of A genomes was contributed by T. urartu and the other was contributed by Triticum monococcum, which is a close relative of T. urartu (Dvorak et al. 1993
). Therefore, T. zhukovskyi originated from the hybridization of T. timopheevii (AAGG) with T. monococcum (AA) to complete the second lineage (Upadhya and Swaminathan 1963
).
The homoeologous wheat chromosomes are, for the most part, colinear and composed of the same genes; e.g., homoeologous chromosomes 1A, 1B, and 1D are presumed to contain the same genes at equivalent positions along the chromosomes. This chromosome colinearity extends to some degree to the entire grass family (Bennetzen et al. 1998
; Gale and Devos 1998
). Polyploidization and large-scale chromosome rearrangements, evolutionary events, and processes occurring at lowcopy-number loci containing functional genes, as well as changes in intergenic regions work in parallel to shape the grass genomes. Gene duplications followed by evolution of new tissue and development specificity or subcellular targeting add plasticity to the system. Transposable elements play an important role in these processes. The contribution of these various phenomena to evolution of plant genomes and genes has recently been discussed (Gaut 1998
; Bennetzen 2000
; Fedoroff 2000
; Wendel 2000
).
A significant amount of information about ACCase and its genes and their structure and function is already available from other studies (Gornicki and Haselkorn 1993
; Gornicki et al. 1994, 1997
; Podkowinski et al. 1996
; unpublished results). The cytosolic isoform of ACCase present in plants provides malonyl-CoA for the synthesis of verylong-chain fatty acids and for secondary metabolism. It is a multidomain enzyme of eukaryotic origin, as indicated by its subunit structure and by amino acid sequence comparisons (Gornicki et al. 1994, 1997
; Konishi et al. 1996
; Podkowinski et al. 1996
; Incledon and Hall 1997
). A separate ACCase isozyme provides malonyl-CoA for de novo fatty acid biosynthesis in the plastid. The latter is a multisubunit enzyme of prokaryotic (endosymbiont) origin in most plants other than Poaceae and a multidomain enzyme in Poaceae (Egli et al. 1995
; Konishi et al. 1996
; Gornicki et al. 1997
; Schulte et al. 1997
; Christopher and Holtum 2000
). The genes encoding wheat cytosolic and plastid ACCase are
15 kb in size and have a large number of introns. The two proteins are 2,260 and 2,311 amino acids long, respectively, and their sequences are 67% identical. A putative plastid transit peptide is present at the N-terminus of the plastid isozyme (Gornicki et al. 1997
).
In this paper, we describe the chromosome localization of the cytosolic ACCase genes (Acc-2) and a related pseudogene (-Acc-2) in hexaploid wheat (T. aestivum), as well as phylogenetic relationships for the genes in wheat and some other representative species of the Triticeae tribe. The nature of evolutionary changes in different parts of the Acc-2 gene are also discussed.
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Materials and Methods |
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Cloning and Analysis of Acc-2 Genes
Identity of the 5' ends of genomic clones 191 (Acc-2,1, GenBank AF305204 and U39321) and 153 (Acc-2,2, GenBank AF305205), isolated previously (Podkowinski et al. 1996
), was confirmed in two steps by PCR using primers shown in table 1
. Fragments of three new genes, Acc-2,3 (GenBank AF305206), Acc-2,4 (GenBank AF305207), and Acc-2,5 (GenBank AF305208) were cloned by PCR using primers shown in table 1
. The fragment of Acc-2,3 included the first intron and short fragments of the flanking exons. The fragment of Acc-2,4 included the 5'-end fragment of the gene, the first intron, and a short fragment of exon 2. The fragment of Acc-2,5 included the first intron and a short fragment of exon 2. A 5-kb fragment of genomic clone 232 containing a putative pseudogene (
-Acc-2,1) isolated previously (Podkowinski et al. 1996
) was sequenced (GenBank AF305209). A 0.4-kb fragment of another copy of the pseudogene (
-Acc-2,2) was cloned by PCR using primers shown in table 1
and sequenced (GenBank AF362956). Wheat (T. aestivum cv. Tam 107) genomic DNA (Clontech) was used as PCR template. PCR products were cloned into the vector pC2.1 (Invitrogen) and sequenced.
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NT and dt lines of Chinese Spring wheat were used in this study to assign Acc-2 genes to individual chromosomes and chromosome arms, respectively. N2AT2B and N4BT4D plants were identified cytologically, as these stocks were maintained as monosomic-tetrasomic lines. Dt lines 2AL, 4AS, 5AS, 2BS, 4BL, 5BS, and 5DS were cytologically identified in the progeny of ditelo-monotelo lines.
To take the cytogenetic analysis one step further, genes and markers can be assigned to chromosome arm regions using the chromosome deletion lines. These lines have terminal chromosome deletions with breakpoints occurring at various positions along the chromosome. An approximate physical location of a molecular marker on a particular chromosome arm is indicated by the presence or absence of a restriction fragment detected by hybridization of a specific DNA probe to DNA from lines that each have different but known sizes of terminal chromosome deletions. Over 400 lines having terminal chromosome deletions of various sizes have been isolated (Endo and Gill 1996
) and used to construct physical maps of the wheat chromosomes (Gill, Gill, and Endo 1993
; Hohmann et al. 1994
; Delaney et al. 199
5a, 1995b; Mickelson-Young, Endo, and Gill 199
5; Gill et al. 199
6a, 1996b). For this study, chromosome deletion stocks having terminal deletions in the long arms of groups 3 and 5 were used.
The physical mapping experiments were complemented by placing Acc-2 genes on existing genetic linkage maps (GrainGenes, wheat.pw.usda.gov) using 135 recombinant inbred lines (RILs) derived from a cross between a synthetic hexaploid wheat, W-7984, and the common hexaploid wheat variety Opata 85 (Nelson et al. 1995
). The population was provided by Dr. M. E. Sorrells (Cornell University, Ithaca, N.Y.). Acc-2 genes were also placed on the genetic map of the D genome using a mapping population consisting of 56 F2 progeny derived from the cross of A. tauschii Coss. accessions TA1691, var. meyeri, and TA1704, var. typica (Kam-Morgan, Gill, and Muthukrishnan 1989
; Gill et al. 199
1). As described in the Introduction, A. tauschii is the D-genome progenitor of hexaploid bread wheat (T. aestivum), and its chromosomes are colinear with the corresponding D-genome chromosomes of hexaploid wheat.
Hybridization Probes
A series of cDNA hybridization probes was used for Southern analysis. First, cDNA probes ucg21 and ucg22, corresponding to the coding part of Acc-2 genes, were used to simultaneously detect multiple gene copies and to identify all chromosome loci which contained them. These probes were expected to hybridize with all cytosolic ACCase genes whose coding sequences were 98% identical (Gornicki et al. 1994
; Podkowinski et al. 1996
), as well as with the putative pseudogene (
-Acc-2), whose sequences (GenBank AF305209 and AF362956) were more than 90% identical to the Acc-2 coding sequence. Second, gene-specific probes derived from less-conserved regions of the Acc-2 genes, the 5'-end portion and the first intron, were used to identify and map individual genes. Probe ucg21 is a 7-kb SalI fragment, and ucg22 is a 3.4-kb BamHI fragment of the full-length wheat cytosolic ACCase cDNA described previously (Joachimiak et al. 1997
). 5'Fl2 is a 0.53-kb SalI-SacII fragment of the Acc-2,2 lambda genomic clone 153. Other probes were prepared by PCR using cloned fragments of genomic DNA as templates. PCR primers and probe sizes are shown in table 1
. The approximate positions of the target sites for these hybridization probes are shown in figure 1
.
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Acc-2 Gene Sequences for Phylogenetic Analysis
Fragments of the Acc-2 gene were cloned by High Fidelity PCR carried out according to the manufacturer's protocol (Roche) using different combinations of primers shown in table 1
. All components of the PCR except the DNA polymerase were incubated for 23 min at 94°C. The PCR was then initiated by the addition of polymerase. Amplification was for 35 cycles of 0.5 min at 94°C, 0.5 min at 52°C, and 46 min at 68°C using 0.51.0 µg of genomic DNA as template in a 50-µl reaction. DNA from plant material was extracted as described previously (Faris, Haen, and Gill 2000)
. The PCR products were cloned into the vector pGEM-T Easy (Promega) and sequenced (University of Chicago Cancer Center Sequencing Facility). Vector primers, as well as a series of universal gene-specific primers, were used for sequencing.
PCR-cloning-based sequence analysis of genes from species of different ploidy, some of which were genetically very closely related, required strict quality control to eliminate cloning and sequencing artifacts. The analysis became even more complex for multicopy gene families. First, for each gene, multiple clones were sequenced and compared to eliminate PCR related errors. High-quality sequence of both strands was needed to resolve sequencing-related problems. Second, consensus sequences generated for all of the genes were analyzed in pairwise and multiple-sequence comparisons to verify single-nucleotide differences and to exclude sequences of chimeric DNA molecules. Third, a large number of clones representing the gene family from each species, obtained in independent PCR experiments using different pairs of primers, were analyzed to overcome any bias of PCR amplification and to assess the maximum number of gene copies. Finally, multiple-sequence alignments were created and then inspected by eye at all polymorphic sites to verify substitutions found in a single taxon. PCR primer target sites were excluded from the alignments.
Only two single-clone sequences were included in the phylogenetic analysis: an Acc-2,2 sequence obtained from a clone isolated from a genomic library, and the barley (cyt2) sequence. The number of nucleotide differences between two barley sequences (cyt1 and cyt2) was much higher than could be accounted for by assuming only PCR errors, and there was no indication that the cyt2 clone was a chimera. The barley gene represented by cyt2 had two fewer introns than all other Acc-2 genes included in the analysis.
Sequence and Phylogenetic Analysis Software
Sequencher (Gene Codes Corporation, Ann Arbor, Mich.) was used to manage the sequencing project. ClustalX (Thompson, Higgins, and Gibson 1994
) was used to create multiple-sequence alignments. MacClade (W. P. Maddison and D. R. Maddison, University of Arizona, Tucson; phylogeny.arizona.edu/macclade/macclade) was used for analysis of multiple-sequence alignments. PAUP 4.0b5 (D. Swofford, Smithsonian Institution, Washington, D.C.; lms.si.edu/PAUP/) and MEGA (S. Kumar, K. Tamura, and M. Nei, Pennsylvania State University, University Park, Penn.; evolgen.biol.metro-u.ac.jp/MEGA) were used to calculate genetic distances and create phylogenetic trees.
Multiple-Sequence Alignment and Phylogenetic Analysis
After an initial phylogenetic analysis performed on all sequences at once to identify major clades, the final sequence alignment was created in four steps. First, all Triticum/Aegilops sequences of clade Acc-2I were aligned. Rye, barley, and wheat Acc-2,2 sequences were then added to the alignment. A short (CA)n repetitive sequence found in Acc-2,2 was treated as a single insertion rather than aligned with other C-rich sequences in this region, as suggested by the outcome of the ClustalX analysis. The six sequences of the clade Acc-2II were aligned separately. The two alignments were aligned with each other. At this stage, the alignment was adjusted by hand within variable segments of two introns. Finally, a Lolium rigidum sequence (cyt1, GenBank AF343454) was added to the alignment as an outgroup. Multiple alignment of all Acc-2 sequences at once did not provide any clear suggestions of possible further improvements. No attempts were made to improve the alignment of the Lolium sequence, which remained problematic at some intron sites. The alignment was 2,081 nt long, with 696 exon positions and 1,439 invariant positions. The gene sequences were deposited in GenBank under accession numbers AF306803AF306829. This alignment was separated into exon and intron parts. The intron-exon structure of the Acc-2 gene had previously been established (Podkowinski et al. 1996
). The alignment of exon sequences was unambiguous and without gaps. Multiple indels were found in introns. These alignments were used to create phylogenetic trees.
Phylogenetic trees were created in several different ways. First, phylogenetic trees based on intron sequences only were created by the Neighbor-Joining method excluding gaps from pairwise comparisons and without correction for multiple substitutions. This tree was characterized by very good support for major clades, indicated by bootstrap values >80% of 1,000 replicates. Second, Neighbor-Joining trees excluding gaps from pairwise comparisons but with correction for multiple substitutions by Jukes-Cantor methods and similar trees based on gene sequences (exons and introns) were generated. These trees had the same topology at most of those well-supported nodes. Finally, the same conclusion was reached for consensus trees generated by the heuristic maximum-parsimony search (equally weighted characters and nucleotide transformations, 1,000 random-addition replicates, tree bisection-reconnection branch swapping, gaps treated as missing data). Forty-two best trees (length 815) were found for the Acc-2 gene (exons and introns) based on 310 informative characters (consistency index [CI] = 0.896; retention index [RI] = 0.935). Sixty-three best trees (length 668) were found for the Acc-2 gene based on 242 informative characters in introns (CI = 0.910, RI = 0.941). Parsimony bootstrap analysis followed the same scheme, with 1,000 replicates, each with 10 random-addition replicates.
The alignment of the 5'-end portions of three Acc-2 genes (Acc-2,1, Acc-2,2, and Acc-2,4; GenBank accession numbers AF305204, AF305205, and AF305207) was 2,361 bp long with 1,879 invariant positions and included sequences from the conserved block X to the fifth codon of the ACCase open reading frame located in the second exon of the gene (fig. 1b
). The intron-exon structure of the 5'-end portion of the Acc-2 gene had previously been established (Podkowinski et al. 1996
).
Merged alignments of the coding region exon and intron sequences were used to calculate nucleotide substitution rates at synonymous positions and at all intron positions, respectively. In addition, substitution rates at all positions were calculated for individual introns, as well as some conserved segments of the 5'-end portion of the gene, based on their sequence alignments, described above. These rates were not corrected for multiple substitutions, and gaps were excluded only from pairwise comparisons.
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Results |
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To distinguish whether the Acc-2 locus on chromosome 5D arose before or after polyploidization, the Acc-2 gene was mapped in A. tauschii, the diploid D genome progenitor of hexaploid wheat. The genetic position of the Acc-2 genes was determined by hybridizing probe ucg21 to DNA of the segregating mapping populations (data not shown) and placing the detected loci on existing maps of A. tauschii. Linkage analysis of probe ucg21 in the diploid A. tauschii F2 population located the site distally on 3DL and 5DL (fig. 3 ). These results suggested that the 5D Acc-2 locus already existed in the diploid species.
Southern Analysis and Chromosome Mapping Using Gene-Specific Probes
NT and dt analysis using Acc-2,1-specific probes Pro1 and Int1 indicated that they both hybridize to common fragments on the long arms of homoeologous group 3 chromosomes (fig. 4a
), but they did not detect any hybridizing DNA on chromosome 5D that would correspond to the locus detected by ucg21 and ucg22. Genetic linkage mapping of Pro1 and Int1 indicated that they cosegregate with each other and with ucg21 on the long arm of chromosome 3A in the RIL population and on 3DL in the A. tauschii F2 population (fig. 3b
). It is most likely that Pro1 and Int1 detect homoeoloci on chromosomes 3A, 3B, and 3D in hexaploid wheat, but the 3B and 3D fragments were monomorphic in the RIL population and therefore could not be located on the genetic map. Based on phylogenetic analysis of the coding region described later in this paper, the Acc-2,1 gene was assigned to the A genome. All of this information indicates that a copy of the Acc-2,1 gene is present on each of the group 3 chromosomes of genomes A, B, and D.
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Hybridization of Pro4 to hexaploid wheats along with tetraploids (T. dicoccoides and T. turgidum, AABB genomes) and the A-genome diploid T. monococcum (fig. 4c ) suggested that an Acc-2,4like gene was present in the A-genome diploid but was absent from the A genome in the tetraploids and hexaploids. From NT analysis of hexaploid wheat, it was known that Pro4 detected fragments only on chromosomes 3B and 3D. The tetraploid wheats, lacking the D genome, had only the chromosome 3B fragment. Triticum monococcum lacks both the B and the D genomes, but Pro4 hybridized to a fragment approximately the same size as the chromosome 3B fragment in the tetraploids and hexaploids. Presumably, this is a chromosome 3A fragment in T. monococcum. Furthermore, we tested three additional accessions each of the A-genome diploids, T. monococcum ssp. monococcum and T. monococcum ssp. aegilopoides, and the putative donor of the hexaploid A genome, T. urartu. All the accessions were collected from various regions of the Middle East. For each accession, Pro4 hybridized intensely to a single restriction fragment, indicating the presence of an Acc-2,4like gene in the A genome. Based on phylogenetic analysis of the coding region described later in this paper, the Acc-2,4 gene cloned from hexaploid wheat was assigned to the D genome.
It is probable that the A-genome Acc-2,2 and Acc-2,4like genes are not found in hexaploid wheats because they were already absent from the tetraploid ancestor involved in the amphiploidization event that gave rise to hexaploid wheat. Acc-2,4, and probably Acc-2,2, was most likely eliminated from the A genome of tetraploids sometime after the formation of the ancestral tetraploid. This may reflect the evolutionary phenomenon of diploidization, for which it has been suggested that speciation through allopolyploidy may be accompanied by a rapid, nonrandom elimination of specific, low-copy DNA sequences at the early stages of allopolyploidization (Feldman et al. 1997
). It is also possible that some functional copies of the gene escaped detection with gene-specific probes because parts of the gene containing target sites for the probes were deleted or diverged beyond the point of detection by hybridization.
No gene-specific probes for Acc-2,3 were used in this study. The gene-specific probes for Acc-2,1, Acc-2,2, and Acc-2,4 all detected the same restriction fragments, except that probes for Acc-2,2 and Acc-2,4 detected no fragment on chromosome 3A. This result and the number of different genes detected by sequencing suggest that multiple copies of the cytosolic ACCase gene are present in the wheat genomes on the long arms of group 3 chromosomes, possibly in tandem repeats.
The gene-specific probes were targeted to the least conserved sites in the gene to eliminate cross-hybridization. Sequence comparison of probe Pro1 with the available sequence of gene Acc-2,2, and of Pro21 with the available sequence of gene Acc-2,1 reveals no significant sequence similarity to allow cross-hybridization. Short stretches of identical sequence existed in Pro21 and Acc-2,4 and in Pro4 and Acc-2,2, indicating the possibility of a weak signal from cross-hybridization. Such a weak signal, if scored as positive, could lead to a false estimate of gene copy number and assignment of individual genes to wrong genomes. This problem was overcome by using multiple gene-specific probes. All gene-specific probes except for Fl5'1 and Fl5'2 (fig. 1 ) lie within ACCase genes. Probes Fl5'1 and Fl5'2, which target the 5' flanking regions of the genes, as well as probe Int2, which targets the first intron of the gene, hybridized with multiple fragments, suggesting that they contain repetitive sequence elements. The 5' end of Acc-2,1 appears to contain a potentially multicopy retrotransposon-like element (fig. 1 ).
Unknown Character of the Acc-2Related Sequences Detected on Chromosome 5D
None of the gene-specific probes hybridized to the locus on chromosome 5D identified with probes ucg21 and ucg22. The 5D locus could contain either an active gene or yet another pseudogene. A functional cytosolic ACCase gene located on chromosome 5D would have to be significantly different in its sequence in the untranslated regions to escape detection by the gene-specific probes used in this study. The additional copy of a cytosolic ACCase gene with divergent promoter regions and specific chromosome localization suggested a gene duplication/translocation event in the D genome lineage. Such a gene may already have acquired an alternative function. Identifying this function will require cloning and sequencing of a genomic fragment containing the Acc-2related sequences from chromosome 5D.
Acc-2Related Pseudogene
An Acc-2 related pseudogene was identified by sequencing a 5-kb DNA fragment isolated from a hexaploid wheat genomic library (Podkowinski et al. 1996
). Its sequence revealed a partially processed condition. The pseudogene (
-Acc-2,1) contained only 2 out of the 13 introns expected based on the intron/exon structure of Acc-2 genes and had a 7-nt (TTTCAAC) repeat, creating a frameshift that would prevent synthesis of a full-length ACCase (fig. 1
), confirming the nonfunctional character of
-Acc-2. A fragment of a second copy of the pseudogene (
-Acc-2,2) was cloned by PCR. The two sequences differed by only 3% within a 350-bp fragment; however, the second copy did not have the 7-nt repeat. No other frameshifts or early stop codons were found in either copy of the pseudogene. The possibility of the pseudogene being located exclusively in the Acc-2 locus on chromosome 5D was excluded by the PCR experiment. A fragment of the pseudogene was amplified when genomic DNA from NT lines missing chromosome 5D was used as template. The identity of the DNA fragment was verified by sequencing cloned PCR products. The pseudogene was found in other Triticeae species (unpublished results). A copy of the pseudogene from the D-genome diploid A. tauschii ssp. meyeri (accession number 1691) was identical to
-Acc-2,1 from hexaploid wheat. A copy of the pseudogene from the A-genome diploid T. urartu (accession number 763) was similar to another pseudogene copy from hexaploid wheat (not shown). Therefore, we postulate that in hexaploid wheat, a copy of the pseudogene is located in the Xucg2 locus on each of the homoeologous group 3 chromosomes (A, B, and D). The intronless structure of the pseudogene can be explained by integration of a cDNA fragment formed by reverse transcription into one of the gene copies present in the Acc-2 locus. This scenario is consistent with the localization of the pseudogene in this locus and the presence of some introns at one end of the pseudogene. These remaining introns came from the active gene which was a target of the integration event.
Intron Loss
Intron loss or gain is another event detected in Acc genes. Two introns are missing from the entire cytosolic clade (Podkowinski et al. 1996
; Gornicki et al. 1997
). One barley Acc-2 gene (cyt2) has still two introns fewer in the gene region used for the phylogenetic analysis. Creation of partially processed pseudogenes with many fewer introns is another type of intron reduction event that adds complexity to the Acc-2 loci by creating nonfunctional genes.
Phylogenetic Analysis of Acc-2 Genes of the Triticeae Tribe
A segment of the ACCase gene (fig. 1
) with its ends anchored in highly conserved exons encoding the biotin carboxylase domain, which itself is the most conserved domain of ACCase, was selected for phylogenetic analysis of Acc genes because of universal applicability in Poaceae and because homology assessment of the nucleotide or amino acid positions was straightforward. Therefore, phylogenetic analysis can include the multidomain cytosolic and plastid ACCases from all plants. Acc-2specific primers, based on the available sequence information for plant ACCases, were relatively long (24-mer), and their target sites were offset relative to each other by several nucleotides. Various combinations of these primers worked effectively in PCR amplification at low annealing temperature even with some mismatches. Primer pairs yielding fewer nonspecific products were tested on multiple species from the Triticum/Aegilops complex, as well as on barley, rye, and Lolium (L. rigidum), for their ability to amplify fragments of multiple copies of the cytosolic ACCase gene, as well as the related pseudogene. These primers did not amplify the corresponding fragment of the plastid ACCase gene. The PCR-cloned Acc-2 gene fragment was about 1.8 kb long and included approximately the same number of nucleotides in six exons and five introns. The corresponding fragment of the pseudogene was about 0.7 kb long. Plant species included in the analysis are identified in figure 5 . All positions of the intron part of this alignment were used to calculate the neighbor-joining tree shown in figure 5 .
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How Many Copies of the Acc-2 Gene Are Present in Wheat Genomes?
The results of sequence analysis of genomic DNA and cDNA obtained from various cloning experiments suggest that there are more than three copies of the Acc-2 gene in hexaploid wheat (Gornicki et al. 1994
; Podkowinski et al. 1996
). Fragments of five genes, Acc-2,1Acc-2,5, were isolated from genomic library or cloned by PCR targeting the 5' end of the gene. Four copies of the Acc-2 gene from hexaploid wheat were included in the phylogenetic analysis described above: two sequences assigned to previously identified genes Acc-2,1 and Acc-2,4 by virtue of their identity with sequence of overlapping genomic and corresponding cDNA fragments, one genomic sequence which could not be assigned to any of the other genes mentioned above, and a sequence of gene Acc-2,2 which was cloned from a genomic library. Genes Acc-2,3 and Acc-2,5 could not be included in the phylogenetic analysis because the cloned fragments corresponded to a different part of the gene. Two copies of the Acc-2 gene were found in several diploid Triticeae species: T. urartu, T. monococcum, A. speltoides, and barley (fig. 5
). Three Acc-2 genes were found in diploid L. rigidum (GenBank accession numbers AF343454AF343456).
Identification of all copies of the Acc-2 gene family may prove difficult. On the one hand, the PCR-cloning experiment is expected to fail to detect all those copies of the gene that accumulated multiple mutations within the primer target sites. For example, primers designed for the amplification of the known cytosolic ACCase genes did not work for the plastid ACCase genes, although the latter were created by duplication of the former. The Acc-2related gene(s) identified by mapping experiments on chromosome 5D may represent another such case. This deficiency could probably be overcome by additional PCR-cloning experiments with new primers designed based on the much larger sequence database which is now available. Analysis of a large number of clones should overcome any bias in PCR amplification. On the other hand, recent gene duplication events that have not had enough time for mutations to accumulate would escape detection.
The phylogenetic analysis suggests that gene duplication was an important recurring event shaping the Acc-2 gene family. One duplication event led to the creation of the two major clades (Acc-2I and Acc-2II; fig. 5 ). It occurred before the divergence of the Triticeae species. The A genomes of diploids T. urartu and T. monococcum, and probably the A genome of the AG tetraploid T. armeniacum, have both copies of the gene. The topology of clade Acc-2II suggests that this copy of the gene is present in genomes B and G as well, although the A genome assignment of all the genes in clade Acc-2II cannot be strictly ruled out. It is then surprising that an Acc-2II clade gene was not found in the hexaploid wheat for which a close relative of T. urartu donated the A genome via an AABB tetraploid. The D genome was also not represented in clade Acc-2II.
It is possible that both copies of the gene, Acc-2I and Acc-2II, are retained only is some genomes: the A genome of A-genome diploids and AB-genome tetraploids, the B genome of AB-genome tetraploids, and the G genome of AG-genome tetraploids. The Acc-2II copy was probably lost from other diploid species and independently from the A genome of hexaploid wheat. The A-genome Acc-2I copy of the gene was not detected in AG tetraploids. Therefore, it is possible that this copy was lost from these species and, instead, the Acc-2II copy was retained in their A genomes (fig. 5 ). Some of these apparent gene loss events could be explained by a failure of our PCR-based experiment to amplify all existing gene copies despite a significant number of clones having been analyzed.
The phylogenetic analysis further suggests that independent Acc-2 gene duplication events occurred in some other lineages (fig. 5 ). Furthermore, none of the genes for which gene-specific probes were available mapped on chromosome 5D. The presence of the Acc-2II gene in A-genome diploids and the absence of this gene in D-genome diploids provides an argument against the possibility that the Acc-2 gene of chromosome 5D belongs to the Acc-2II clade. Therefore, it is likely that the chromosome 5D locus contains an additional copy or additional copies of the gene.
Assessment of Substitution Rate Variation in Different Segments of the Acc-2 Genes
Nucleotide substitution rates in introns and at synonymous positions among Acc-2I genes belonging to clades A, B, and D were compared for the gene fragment used in phylogenetic analysis (fig. 6a
). For all three pairwise comparisons, Acc-2IA and Acc-2IB, Acc-2IA and Acc-2ID, and Acc-2IB and Acc-2ID, substitution rates in introns were similar to each other, and synonymous substitution rates were approximately twice as high. Little variation was observed when rates for pairs of putative orthologs were compared (reflected in low standard deviations). Intron 6 in genes belonging to clade Acc-2IB is an exception. For an unknown reason, this intron is much more similar to the corresponding intron in Acc-2ID genes than the remaining four introns. This is reflected in the high standard deviation (introns 59, average for the Acc-2IB : Acc-2ID rate; fig. 6a
) and, because of the length of this intron, in an apparently shorter distance between taxa of clades B and D (fig. 5
). Synonymous substitution rates vary significantly from exon to exon, but most of this variability results from too few substitutions being counted in the shortest exons.
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These results point to potential problems that may be encountered in this type of phylogenetic analysis due to substitution rate variability between different gene segments leading to incongruent phylogenies. For example, nucleotide substitution rates between genes Acc-2,1 and Acc-2,2, and between clades Acc-2IB and Acc-2ID calculated based on intron sequences can vary significantly from intron to intron. Comparison of genes with different numbers of introns, such as barley genes cyt1 and cyt2 (fig. 5 ), could further skew the outcome of the analysis. Our system allows detection and evaluation of such phenomena. The final conclusions are based on sequence comparisons of multiple introns and exons.
Phylogenetic Inferences Based on Acc-2 Genes
The complex pattern of evolution, coupled with the limited set of sequences used in the analysis, made establishing orthology difficult. The fact that the genes seemed to form major clades consistent with their putative chromosome assignments (A, B, D, G) or taxonomic relationships (e.g., between Hordeum and Triticum) can only be taken as an indication of possible orthologous relationships.
Many of the branch points on the Neighbor-Joining tree shown in figure 5
were found with equally good statistical support on other neighbor-joining trees and on consensus maximum-parsimony trees. These well-supported clades reflect correctly known relationships among Triticum species (Cox 1998
), supporting orthologous relationships between these groups of genes. For example, the origins of A, B, and D genomes in hexaploid wheat can be traced to T. urartu, AB tetraploids, and A. tauschii, respectively. A, B, D, and G genomes diverged early in the evolution of the Triticum/Aegilops complex, whereas the polyploid species had a much more recent origin. Some of these relationships can be observed for both major Acc-2 clades (I and II).
The sequence of some other evolutionary events is ambiguous. This ambiguity is, at least in part, due to insufficient resolution of the method. It appears that the diploid progenitors of the Triticum species (donors of A, B, D, and G genomes) radiated at approximately the same time. Missing genes, lost from some genomes or simply not cloned in our experiments, could lead to paralogs being mistaken for orthologs. Finally, it is conceivable that a copy of a multicopy gene family is a pseudogene. This could be difficult to determine without sequencing the entire gene and may require analysis of gene expression. No such problems are evident in our phylogenetic analysis, but they could lead to erroneous phylogenetic inferences.
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Discussion |
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Our approach combined cytogenetic analysis with a molecular evolutionary study of this small gene family. First, multiple cytosolic ACCase genes from wheat were analyzed to reveal their structures. Second, the Acc-2 loci were mapped on wheat chromosomes. Finally, analysis of Acc-2 genes in wheat species of different ploidys using Southern hybridization, cloning, and sequencing enabled bridging of the gap between the cytogenetic and molecular approach by assigning gene sequences to specific loci on homoeologous chromosomes of known genomic affinity on the one hand, and by reconstructing phylogenetic relationships among them on the other hand. Nucleotide substitution rates in different segments of the gene were assessed.
Cytosolic ACCase is encoded by a small gene family with more than one copy of the gene present in at least some of the Triticeae genomes. In the reconstruction of the evolution of the Acc-2 gene, we documented gene duplication events that occurred in the polyploid species and/or their diploid ancestors and postulated that these duplications were followed by deletion or loss of function of some gene copies in different genomes and plant lineages. Gene loss due to deletion, homogenization, or formation of pseudogenes followed by accelerated mutation that occurred in parallel with gene duplication seems possible. All of these processes are likely to be more frequent in polyploid species. The end result of such recent evolutionary events would be a different set of active genes even in closely related species.
Evolutionary changes at the 5' end of the gene including promoter/regulatory elements, leader sequences, first exons, and introns are of special interest, as they may lead to important changes in gene function. The structure of promoter and regulatory elements and the activity of individual Acc genes and their tissue and development specificities are being investigated (unpublished results). These results indicate that multiple Acc-2 genes, including genes Acc-2,1, Acc-2,2, and Acc-2,4, discussed in this paper, are transcriptionally active and show distinct expression patterns in wheat plants. Evolution of the 5'-end region of the gene may be affected by events in the neighboring intergenic region, e.g., by insertion of transposable elements that occasionally invade the gene itself, affecting gene function. Higher substitution rates and the presence of a highly variable expansion elements may be signatures of such events. A better understanding of the evolution of the Acc-2 gene family makes a study of such border phenomena feasible.
Recurring duplication of the Acc gene in different plant lineages has played an important role in providing genetic material for the creation of new genes, such as the plastid ACCase gene, which occurred more then once during plant evolution: in Poaceae (Konishi et al. 1996
), in Brassicaceae (Schulte et al. 1997
), and in Geraniaceae (Christopher and Holtum 2000)
. This duplication event occurred at the onset of grass family evolution and was followed by a chromosome translocation. Additional copies of the Acc-2 gene that were created later during grass evolution may have or may eventually acquire a different function, e.g., different tissue or development specificity. Such duplication events, followed in some instances by translocation to a new locus on a different chromosome and/or potential deletion or inactivation of some gene copies accompanied by variable substitution rates in different gene parts, different genes, and different chromosome loci, add plasticity and shape the grass genome. These are probably very common locus/gene-specific events occurring in the context of large chromosomal rearrangements, frequent polyploidization, and rapid changes in the intergenic regions.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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1 Present address: USDA/ARS Cereal Crops Research Unit, Northern Crop Science Laboratory, Fargo, North Dakota.
1 Abbreviations: ACCase, acetyl-CoA carboxylase; Acc-2, gene encoding cytosolic ACCase; -Acc-2, Acc-2 related partially processed pseudogene; dt line, ditelosomic line; NT line, nullitetrasomic line; RIL, recombinant inbred line.
2 Keywords: Triticeae
Aegilops
pseudogene
gene sequence
grass
evolution
plant
3 Address for correspondence and research: Piotr Gornicki, Department of Molecular Genetics and Cell Biology, University of Chicago, 920 East 58th Street, Chicago, Illinois 60637. pg13{at}midway.uchicago.edu
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