Structural and transcriptional analysis of plant genes encoding the bifunctional lysine ketoglutarate reductase saccharopine dehydrogenase enzyme
© Anderson et al; licensee BioMed Central Ltd. 2010
Received: 13 November 2009
Accepted: 16 June 2010
Published: 16 June 2010
Among the dietary essential amino acids, the most severely limiting in the cereals is lysine. Since cereals make up half of the human diet, lysine limitation has quality/nutritional consequences. The breakdown of lysine is controlled mainly by the catabolic bifunctional enzyme lysine ketoglutarate reductase - saccharopine dehydrogenase (LKR/SDH). The LKR/SDH gene has been reported to produce transcripts for the bifunctional enzyme and separate monofunctional transcripts. In addition to lysine metabolism, this gene has been implicated in a number of metabolic and developmental pathways, which along with its production of multiple transcript types and complex exon/intron structure suggest an important node in plant metabolism. Understanding more about the LKR/SDH gene is thus interesting both from applied standpoint and for basic plant metabolism.
The current report describes a wheat genomic fragment containing an LKR/SDH gene and adjacent genes. The wheat LKR/SDH genomic segment was found to originate from the A-genome of wheat, and EST analysis indicates all three LKR/SDH genes in hexaploid wheat are transcriptionally active. A comparison of a set of plant LKR/SDH genes suggests regions of greater sequence conservation likely related to critical enzymatic functions and metabolic controls. Although most plants contain only a single LKR/SDH gene per genome, poplar contains at least two functional bifunctional genes in addition to a monofunctional LKR gene. Analysis of ESTs finds evidence for monofunctional LKR transcripts in switchgrass, and monofunctional SDH transcripts in wheat, Brachypodium, and poplar.
The analysis of a wheat LKR/SDH gene and comparative structural and functional analyses among available plant genes provides new information on this important gene. Both the structure of the LKR/SDH gene and the immediately adjacent genes show lineage-specific differences between monocots and dicots, and findings suggest variation in activity of LKR/SDH genes among plants. Although most plant genomes seem to contain a single conserved LKR/SDH gene per genome, poplar possesses multiple contiguous genes. A preponderance of SDH transcripts suggests the LKR region may be more rate-limiting. Only switchgrass has EST evidence for LKR monofunctional transcripts. Evidence for monofunctional SDH transcripts shows a novel intron in wheat, Brachypodium, and poplar.
Monogastric mammals, which include humans, depend on external dietary sources for half of the amino acids needed for protein synthesis. The aspartate-family pathway controls synthesis of the essential amino acids lysine, threonine, and methionine, with lysine feedback-inhibition and rates of lysine degradation being factors in this important pathway. Among the essential amino acids, lysine is the most severely limiting in the cereals - crops that make up half of the human diet . In contrast to animals, plants synthesize lysine and have evolved complex metabolic pathways to maintain lysine levels . To understand lysine metabolism, a thorough understanding of all aspects of these pathways is necessary. For the catabolic portion of lysine metabolism, the bifunctional enzyme lysine ketoglutarate reductase saccharopine dehydrogenase (LKR/SDH; synonym α-aminoadipic-δ-semialdehehyde synthase) converts lysine to glutamate and α-aminoadipic acid via a 2-step pathway; i.e., the LKR activity (E.C. 184.108.40.206) catalyzes the formation of saccharopine from lysine and α-ketoglutarate (2-oxoglutarate), and the SDH activity (E.C. 220.127.116.11) processes the saccharopine into glutamate and an α-aminoadipic-δ-semialdehehyde which is further catabolized to two glutamates [2, 3]. In both plants and animals, the LKR/SDH gene encodes an open reading frame composed of fused LKR and SDH domains - compared to yeast and fungi where the LKR and SDH activities are encoded by separate genes [4, 5]. In plant LKR/SDH genes, there is a linker, or interdomain, sequence not present in animals that separates the LKR and SDH encoding domains - leading to speculation that there are controls and functions unique to plants . Both LKR/SDH and monofunctional SDH mRNAs have been detected in mouse . Similarly in plants, an Arabidopsis SDH mRNA is reported that initiates transcription inside the 3' sequence of the interdomain , and a cotton LKR mRNA is reported that terminates at the 5' junction area of the linker . One question in the latter report was that the 3' noncoding sequence is not present in the LKR/SDH gene - leaving the origin of this sequence uncertain but attributed to a possible trans-splicing event. The function of such mono-functional mRNAs is not clear, but the SDH mRNA and protein levels were consistently higher than the LKR/SDH mRNA and protein levels in Arabidopsis tissues - leading to the proposal that the LKR activity was the rate limiting step and that the relative SDH excess assured rapid flux through the pathway for LKR/SDH .
The exact site of activity of LKR/SDH is considered to be the mitochondria , but evidence is not clear. The LKR/SDH enzyme has been localized to the cytosol in plants [6, 12], while lysine-α-ketoglutarate reductase and saccharopine dehydrogenase enzymatic activities were located only in the mitochondrial matrix in animal livers [13, 14]. Possible roles in transcription regulation include evidence of LKR/SDH being a co-factor involved in hormone-mediated transcription through regulation of H3 and H4 histone methylation  and the LKR/SDH gene is reported to be regulated by the Opaque2-type transcription factors that also control the expression of at least some classes of cereal seed proteins . In addition to a direct role in lysine metabolism, LKR/SDH has been reported to be regulated by a number of environmental and metabolic influences including osmotic balance, hormome levels, and salt and water stresses [10, 16, 17]. Suggestive evidence for more complex regulatory roles for LKR/SDH are that expression is enhanced in developing seeds of cereals and floral tissues known to contain limited amounts of lysine, and analyses suggesting LKR/SDH expression is not highly coordinated with other catabolic enzymes . Similarly, the finding of multiple transcripts from the same gene (encoding mono- and bifunctional enzymes) and a coding region composed of 25 exons in a dicot and 26 exons in a monocot  suggests complex regulation and roles in plant metabolism and development [2, 10].
The importance of lysine to animal/human nutrition and the role of LKR/SDH in lysine catabolism has lead to several approaches to increase plant seed lysine. These approaches include increasing seed lysine by transformation with feedback-insenstive versions of lysine anabolic genes , down-regulating the LKR/SDH gene , a combination of those two approaches , transgenic expression of a foreign protein high in lysine , and reducing synthesis of lysine-poor seed proteins .
Plant LKR/SDH genomic sequences have been formally reported only for Arabidopsis [8, 23] and maize . A comparison of these dicot and monocot genes found high conservation in exon size and sequence, with the maize gene having one additional exon in the 5' region . The dicot and moncot intron sequences have diverged completely and the maize introns are generally larger - from start to stop codons the maize LKR/SDH gene spans 9515 bp while the Arabidopsis gene spans 5590 bp. Additional plant LKR/SDH sequences are available (rice, poplar, grape, etc.), but have not been comparatively analyzed. The Triticeae crops (wheat, barley, rye, triticale) are, as a group, the largest direct fraction of the human diet worldwide, but no LKR/SDH gene has been reported for this important crop group.
The current report describes a BAC clone of a wheat genomic fragment containing an LKR/SDH gene, determines genome assignments of the BAC and EST contigs in hexaploid wheat, and compares relative homoeologue expression among the three hexaploid wheat genomes. Also described are a comparative analysis of a set of plant LKR/SDH genes including variant structures in the poplar and grape genomes. Wheat and other plant LKR/SDH ESTs are analyzed to determine splicing sites and evidence for alternative splicing. This analysis also finds EST evidence for both monofunctional LKR and SDH transcripts.
Results and Discussion
Wheat LKR genomic region
To isolate a wheat LKR/SDH gene, a durum wheat (Triticum turgidum) tetraploid 5× BAC library was screened. Six BACs were positive for LKR/SDH sequences and formed two contigs of four and two BACS respectively, as seen from Southern analysis and BAC fingerprinting (not shown). Each contig contained single LKR/SDH sequences - suggesting that there are single LKR/SDH genes in each of the wheat A and B genomes of tetraploid wheat. BAC 0006M07 was chosen for sequencing as having the LKR/SDH gene relatively centrally located in the BAC. This BAC was sequenced and found to be 161,506 bp in length. The sequence can be found as Genbank accession GU182251.
Structure of the a wheat LKR/SDH gene and protein
Analysis of the wheat LKR/SDH gene sequence indicates the structure shown in Figure 1B. Consensus exon/intron boundaries were determined using wheat EST sequences aligned to the genomic sequence. At least one wheat EST exists that overlaps all the coding sequence except for the region around exon 10 where maize and rice LKR/SDH coding sequences were used to estimate exon/intron boundaries. In regions with only 1-2 wheat ESTs, exon/intron boundaries matched rice and maize sequences in all cases. Similar to the previously reported maize LKR/SDH gene structure , the wheat LKR/SDH gene is comprised of 26 exons and 25 introns. The intron borders matched the canonical plant intron borders (GT...AG) for all 26 introns. The 5' portion of the sequence encodes the LKR activity of the bifunctional enzyme, which is encoded by eleven exons (blue boxes in Figure 1B); the 3' part of the sequence encodes for SDH activity and contains twelve exons (red boxes). The two regions are separated by an interdomain region composed of two exons (yellow boxes) and three introns, one of which (intron 14) is the longest intron in the LKR/SDH gene (1122 bp). This intron may include 5'-UTR/promoter sequences for monofunctional SDH transcripts (see below).
The coding regions of available LKR/SDH genes were also compared and formed a phylogenetic tree of the same form as in Figure 3 (not shown). A comparison of intron sequences found no significant conservation of intron sequences for available LKR/SDH sequences within both dicots and monocots - with the exception of the Brachypodium/wheat comparison where significant conservation is evident (Additional File 2). Further sequencing of LKR/SDH, and other genes from a larger panel of more closely related plant genera and species is needed to understand patterns of plant intron sequence divergence.
Chromosome and genome location of the wheat LKR/SDH gene
Southern analysis of hexaploid wheat found that wheat EST BE606591 hybridized to three genomic fragments http://wheat.pw.usda.gov/wEST. Two of the LKR/SDH-encoding fragments were mapped to the 6AL and 6BL chromosome arms but a third fragment could not be accurately mapped or assignment made to the 6DL chromosome (see Additional File 3). The same analysis localized the gene to the region of 0.4 to 0.55 of the wheat consensus group 6 chromosome long arm. The wheat group 6 chromosomes have most homology to rice chromosome 2  - consistent with the wheat LKR/SDH gene on the group 6 chromosomes since the rice LKR/SDH gene sequence is found on rice chromosome 2 http://www.ncbi.nlm.nih.gov.
The previous results supported LKR/SDH genes exist in each of the hexaploid wheat's A-, B-, and D-genomes. To determine if all three genes were actively transcribed, wheat LKR/SDH ESTs were identified (Additional File 5). These ESTs assembled into three contigs, one of which (contig 1) is identical over its 1574 bp to the sequenced BAC LKR/SDH coding region (not shown). The relationship of the three contigs to the same region in the BAC sequence is shown in Additional File 6. Contig 1 also contains all five T. monococcum (diploid grass related to the wheat A-genome donor) LKR/SDH ESTs, therefore confirming contig 1 and the BAC as originating from the A-genome. Contigs 1 and 2 contain, respectively, 7 and 17 ESTs from tetraploid wheats (A- and B-genomes). Contig 3 contains no tetraploid or T. monococcum ESTs. Therefore, contig 2 should represent the B-genome and contig 3 the D-genome. In support of these assignments, the sequences of contigs 1 and 3 are closer to each other than to contig 2 (Additional File 7), consistent with the previous reports that the A and D common ancestor diverged from the B-genome ancestor . Finally, the sequences amplified from specific genomes matched the three contigs and confirmed the genome assignments (not shown).
When the ESTs for the three hexaploid wheat LKR/SDH EST contigs are tallied, the distribution by genome for the A-, B-, and D-genomes is 54, 47, and 35 ESTs, respectively. A Chi-square goodness-of-fit test for departure from expected values yields P = 0.13. Thus, the number of ESTs from the three homoeologs is not considered statistically significantly different from the expected numbers. Further, more global, analyses are needed to understand whether differential homoeologue transcription has a role in polyploid plants.
Structure and expression of genes adjacent to the LKR/SDH locus
Three other genes are found near the LKR/SDH gene in this study (Figure 1). The first gene is for a pectinesterase (PE; a.k.a. pectin methylesterase). This class of enzymes catalyses the demethylesterification of cell wall polygalacturonans and produces de-esterified acidic pectins and methanol . The plant pectinesterases comprise a large family of enzymes with roles in a wide range of plant cell activities including cell adhesion, cell elongation, organelle formation, ribosome binding, and plant defense [31–33]. The pectinesterase gene in BAC 0006M07 contains one intron of 470 bp (not shown). No ESTs are found that exactly match the BAC A-genome pectinesterase gene, but Additional File 7 shows four similar wheat ESTs (BQ806129, CA717792, CJ525781, CJ634274) with 93-96% sequences matches to the BAC pectinesterase gene. These four ESTs are likely from one of the orthologous PE genes in either the B- or D-genomes. In addition, the best BLASTn match of these ESTs and the BAC PE sequence is to the rice pectinesterase gene adjacent to the LKR/SDH gene in the rice genome (not shown).
Finally, immediately 3' to the LKR/SDH gene is an apparent gene of unknown function. Similar genomic or EST sequences are found only in the Triticeae, but a unique 17 out of 18 bp sequence is found in the same relative position 3' to the LKR/SDH gene in rice. Although no wheat ESTs exactly match this unknown gene, a similar region is apparently transcribed in barley since two barley ESTs (BM099304 and BM372530) are close matches (Additional File 9). The alignment of the two barley ESTs to the wheat genomic region shows seven gaps with canonical intro/exon junctions at 14 of 15 positions. When the apparent exons are spliced together and the resulting DNA and derived amino acid sequences are used as queries in database searches, no significant match is found to any DNA (best match e = 0.1) or protein sequences (best match e = 0.28). The two barley ESTs are from pistil and embryo sac, respectively. These two tissues have not been commonly sampled for ESTs - which could account for the sequence not appearing in other plant EST collections. If this sequence is found only in Triticeae, then the sequence must have arisen after separation of the Triticeae from other grasses. Thus, although the intron/exon structure and ESTs argue for a functional gene, this remains to be further established.
Comparison to other genomes
The poplar monofunctional LKR gene in ends at about 200 bp 3' to the end of exon 11 when compared to the full-length genes (not shown). In addition, the sequence has a frameshift in exon 7 (not shown) caused by a TC to TCTC difference in sequence compared to the two full-length genes. Only five ESTs are available for the 5' end of the poplar LKR sequences and none match the monofunctional LKR gene sufficient to suggest any transcripts from that gene. However, these are too few ESTs to rule out gene activity, and the apparent frameshift would need to be confirmed.
Figure 6 compares adjacent gene organization among wheat and other plants (the unknown gene from wheat is not shown since no other plant had a similar gene). To find the location of the mTERF and PE genes, the relevant wheat DNA coding and derived amino acid sequences were used in BLAST searches to find the most similar sequences. If the best match was adjacent to the LKR/SDH gene, those matches are shown in Figure 6. The relative gene spacings are fairly conserved even with large differences in genome size, i.e., the PE, mTERF, and LKR/SDH genes share similar intergenic spacing despite as much as a ~30-fold difference in genome sizes, such as between Arabidopsis and one of the wheat genomes. Similar spacings occur in all other examined plant sequences which raises questions about the basis of genome size differences (gene-islands vs repetitive regions), and possible conserved functional clustering of genes. Figure 6 also shows that the conservation of the gene complement in this region with respect to the LKR/SDH gene, is not universal. All four monocot genomes contain the mTERF gene, but no dicot has an mTERF gene in this position of the genome. The PE gene is missing in Brachypodium but present in one copy in other available monocot sequences. Dicots show variation in the number of PE genes, with only one in this position in Arabidopsis, three each in Medicago and grape, and six in the current poplar assembly. Whether the difference in PE copy number is related to differential gene activity and function is unknown.
Multiple transcripts from single LKR/SDH genes
Alternative transcript production from individual genes is a mechanism to expand potential protein diversity. This strategy can include both differential splicing of exons and multiple promoter sites, sometimes with the two in concert. The most extensive analyses have been with mammalian systems - where estimates are that more than half of the genes are involved in alternative splicing and nearly half have alternative promoters [36, 37]. More limited analyses in plants indicate that upwards of 20% of plant genes are involved in alternative splicing [38, 39]. An analysis of the conservation of alternative splicing between a dicot (Arabidopsis) and monocot (rice) concluded that since there was little conservation between the two plant groupings, this implied a limited role for alternative splicing in expanding the plant proteome . However, even if specific alternative splicings are not conserved between dicots and monocots, this does not mean there are not important functional differences since such major differences in plant architecture, development, biochemistry, and genome organization are well-known.
The LKR/SDH gene, with its large number of exons, bifunctional nature, evidence of bi- and monofunctional transcripts, and diverse functional associations, would seem a good candidate for the study of multiple transcripts. The few reports on the relative abundance of monofunctional LKR or SDH mRNAs have not been consistent. It has been reported that the SDH mRNA is more abundant than LKR/SDH in Arabidopsis , a finding not evident in an earlier report . In comparison, in mouse the LKR/SDH form was found more abundant than the SDH form . The mouse study also failed to find evidence of a monofunctional LKR form. In plants, the only report of monofunctional LKR mRNAs is in cotton , although the authors speculate on the existence in other plants.
Besides wheat, only two other plant species' EST collection contained sequences consistent with transcript initiation within intron 14. One of those was Brachypodium as shown in Additional File 11A. Four Brachypodium ESTs (CCXG11317, CCXG13127, CCXO11098, CCXG8102) have identical 5' ends that begin immediately following the pyrimidine-rich region and may represent the actual start site for Brachypodium monofunctional SDH transcripts. Those four ESTs plus six other Brachypodium ESTs all match the junction of the intron 14-derived monofunctional SDH first exon to SDH exon 2 (LKR/SDH exon 15) as show in Additional File 11B - and match the same structure as with wheat (Figure 10). Finally, although there are only a few Poplar LKR/SDH ESTs, they also support the existence of both bifunctional and SDH monofunctional transcripts. ESTs DV465683 and DY800647 have 5' sequences reading from intron 12 sequence into exon 13, suggesting SDH monofunctional transcript. Poplar ESTs CX180963 and CN520125 read directly from exon 12 into exon 13 with no intervening intron 12 sequence, supporting a bifunctional LKR/SDH transcript (not shown).
The lack of EST support for monofunctional mRNAs in many systems does not mean they do not exist, but only that the EST resources do not support them. However, it does continue to support a preponderance of SDH transcripts, bi- and monofunctional, which suggests differential contributions of the LKR and SDH domains to plant cell metabolism.
Finally, the analysis of plant ESTs failed to convincing support for major multiple populations of alternatively spliced transcripts for the 25 dicot and 26 monocot LKR/SDH exons (exceptions being the monofunctional transcripts described above). Small numbers of differential splicing were found in several plants (not shown), but none in sufficient numbers to suggest differential roles in plant cell metabolism rather than examples of aberrant splicings with no functional roles. For example, a close examination of the wheat ESTs suggests a small number of such alternative splicings. The five wheat ESTs that cover the region that includes the bifunctional consensus start codon represent three sequences - presumably from the three hexaploid wheat genomes. ESTs BJ266925 and CJ702289 match the BAC A-genome sequence exactly, while CJ882974 is a second sequence and FL577869 plus BJ248520 represents the third sequence. Although EST BJ266925 spans the start codon region of the BAC sequences, it, unlike the other four ESTs, does not encode the same ATG codon - exon 2 is missing, with the splice going from the end of exon 1 to the beginning of exon 3. In another wheat example, three pairs of ESTs (reads from both ends) show multiple variant splicing at the 3' end of the SDH sequence: CJ965444+CJ953360; CJ950703+CJ962606; CJ567209+CJ6741282. These three different original cDNAs continue transcription into post-exon-26 genomic sequence, and differentially splice previous sequence after exon 23 (not shown). No obvious consensus splice site sequences are evident, but the three sequences use at least one different splice site from other cDNAs. More in-depth EST sequencing of more plants should clarify the existence and possible roles of specific alternative splicings.
The isolation and characterization of a segment of the wheat genome containing the LKR/SDH gene is shown. The wheat LKR/SDH genomic segment was found to originate from the A-genome of wheat, and EST analysis indicates all three LKR/SDH genes in hexaploid wheat are transcriptionally active, at least for monofunctional SDH transcripts. Comparative analyses with other plant LKR/SDH genes and ESTs shows conservation of the basic exon/intron organization between the wheat gene and previously analyzed genes from maize and Arabidopsis and previously unanalyzed genes from rice, Medicago, grape, poplar, sorghum, and Brachypodium. Relative conservation of exon+intron length, even in plants whose genome sizes differ by 30-fold or more, further supports the intergenic regions as sites of genome expansion. Exceptions to the general gene length conservation are Arabidopsis and grape, whose LKR/SDH genes are shorter and longer, respectively, due to shorter and longer intron lengths. For Arabidopsis, the smaller introns are consistent with the general compactness of the Arabidopsis genome. However, the basis and functionality of larger grape introns is not consistent with genes from plants with similarly-sized genomes. Both the structure of the LKR/SDH gene and the sets of immediately adjacent genes within the genome show lineage-specific differences between monocots and dicots, including different gene positionings and different copy numbers of an adjacent pectinesterase gene. Two findings suggest variation in structure and activity of LKR/SDH genes among plants. First, although most plants seem to contain a single conserved LKR/SDH gene, poplar possesses multiple genes. Second, there are differences among plants in evidence for bifunctional and monofunctional LKR and SDH transcripts among the available EST data. The analyses of ESTs provides some of the most detailed data for multiple transcripts from a single gene, particularly evidence for monofunctional LKR transcripts in switchgrass and monofunctional SDH transcripts in wheat and Brachypodium. There is also evidence in these plants that the monofunctional LKR transcripts read into an intron of the full-length sequence, and for an additional exon for SDH transcripts composed of a central portion of a full-length intron. The lack of similar EST evidence in other species may be due to sampling differences in EST production, but also may indicate fundamental differences in LKR/SDH control and function.
BAC isolation and sequencing
A BAC library of wheat tetraploid T. turgidum ssp. durum (2n - 4× = 28, AABB) cultivar Langdon  was screened using a mixed probe composed of two wheat EST clones encoding portions of the SDH domain (BE428366 and BE498116) and a maize full-length LKR/SDH cDNA clone (NM_001111403) obtained from P. Arruda . Twelve BACs were isolated and further characterized by Southern analysis and BAC fingerprinting to represent two distinct sequences. BAC 0006M07 was selected for sequencing based on its central position in one contig and apparent central location of the LKR/SDH sequence and was sized at about 160,000 bp. Sequencing of BAC 0006M07 was carried out to a depth of about 20× by procedures described in detail elsewhere . Briefly, randomly shear BAC DNA was blunt-ended with mung bean exonuclease (BioLab), dephosphyorylated with shrimp alkaline phosphatase (USB), single A-tailed with Taq polymerase, and the resulting DNA fractionated to 3-5 kb with agarose gels and the Qiagen Gel Extraction Kit. This DNA was used to generate shotgun libraries using the vector pCR4TOPO and transformed into DH10B electroMAX cells (Invitrogen). Randomly picked clones were sequenced at both insert ends with T3 and T7 primers and BigDye chemistry (Applied Biosystems) with an ABI3730×l sequencer.
Sequence analysis began with contig assembly using both Phrap http://www.phrap.org and the Lasergene SeqMan module http://www.dnastar.com. Gaps and uncertain sequences were resolved by comparing the assemblies from the two software packages and primer walking. Regions of less coverage or ambiguous reads were rechecked with primers designed to cover those regions.
Analysis of sequences
NCBI http://www.ncbi.nlm.nih.gov was used for annotation of the new wheat BAC sequence by BLAST analyses and total EST analyses by direct querying to NCBI. Exon/intron junctions are predicted by alignment with Triticeae EST sequences, when available, or with other monocot EST if no Triticeae ESTs covered those sequences.
Sources of genomic sequences were as follows: Arabidopsis thaliana LKR/SDH, Genbank ATU95759; Brachypodium distachyon, http://brachypodium.org; Medicago truncatula, http://www.tigr.org/tdb/e2k1/mta1/; poplar (Populus trichocarpa), http://genome.jgi-psf.org; sorghum (Sorghum bicolor), http://genome.jgi-psf.org; grape (Vitis vinifera), http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/; rice (Oryza sativa), http://gramene.org, MSU-TIGR pseudomolecule assembly release 5 of IRGSP (The International Rice Genome Sequencing Project) and Genbank AP004849. BAC sequences from Genbank were as follows: cotton (Glossypium hirsutum), AF264146; maize (Zea Mays), AF271636; poplar, AC209229. The Brachypodium sequence data were produced by the US Department of Energy Joint Genome Institute http://www.jgi.doe.gov/. For ease of reading, it will be understand that common names and genus names will be used unless referring to different species than noted above; e.g., Brachypodium instead of B. distachyon and rice instead of O. sativa. Plant ESTs were searched at Genbank, except for Brachypodium ESTs that were found at brachypodium.org. Determination of coding sequences and exon/intron junctions were accomplished by comparing genomic DNAs to ESTs and cDNA clones from the same plant, or where necessary, comparing to ESTs and cDNAs from closely related plants.
PCR primers for genome identification
The sequence of the rice LKR/SDH region from BAC AP004849 was compared to the wheat BAC 0006M07. Primer pairs were designed from conserved regions and tested against genomic DNA of a series of diploid, tetraploid, and hexaploid wheats and wheat ancestors. Primer pair F3 (AAAGAAGCATCTACCGTATATAGG) and R3 (TTCATGGTGGAGCAGTACCATATC) amplified the expected fragment size in all wheat DNAs including DNA from the A, AB, D, and ABD genomes. PCR products were sequenced from all these genomes and the sequences compared. Unique bases were used to design single genome-specific primers for the A, B, and D genomes: A genome, primer AF3 GCATTCAGTGTTATTTGCCAATGT; B genome, primer BF3 CTCCACATCTAACACAAAGATATAC; D genome, primer DF3 GGATTTTTCTCAATGACCTCCTTG.
Phylogenetic analysis of LKR/SDH proteins
A phylogenetic analysis of LKR/SDH proteins was carried out using the MEGA4 software package . A protein alignment used ClustalW and the evolutionary relationship inferred by the Neighbor-Joining method . A bootstrap test was used to determine the percentage of replicate trees in which the associated taxa clustered together . Evolutionary distances were computed using the Poisson correction method  and are in the units of the number of amino acid substitutions per site.
Thanks to Roger Thilmony and Kent McCue for reading the manuscript and making suggestions. This research was funded by USDA Agricultural Research Service CRIS 5325-21000-015-00D. Mention of a specific product name by the United States Department of Agriculture does not constitute an endorsement and does not imply a recommendation over other suitable products.
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