Updated guidelines for gene nomenclature in wheat.

KEY MESSAGE: Here, we provide an updated set of guidelines for naming genes in wheat that has been endorsed by the wheat research community. The last decade has seen a proliferation in genomic resources for wheat, including reference- and pan-genome assemblies with gene annotations, which provide new opportunities to detect, characterise, and describe genes that influence traits of interest. The expansion of genetic information has supported growth of the wheat research community and catalysed strong interest in the genes that control agronomically important traits, such as yield, pathogen resistance, grain quality, and abiotic stress tolerance. To accommodate these developments, we present an updated set of guidelines for gene nomenclature in wheat. These guidelines can be used to describe loci identified based on morphological or phenotypic features or to name genes based on sequence information, such as similarity to genes characterised in other species or the biochemical properties of the encoded protein. The updated guidelines provide a flexible system that is not overly prescriptive but provides structure and a common framework for naming genes in wheat, which may be extended to related cereal species. We propose these guidelines be used henceforth by the wheat research community to facilitate integration of data from independent studies and allow broader and more efficient use of text and data mining approaches, which will ultimately help further accelerate wheat research and breeding.

Characterization of a DNA-binding protein implicated in transcription in wheat mitochondria.

To investigate the transcriptional apparatus in wheat mitochondria, mitochondrial extracts were subjected to column chromatography and protein fractions were analyzed by in vitro transcription and mobility shift assays. Fractions eluting from DEAE-Sephacel between 0.2 and 0.3 M KCl displayed DNA-binding activity and supported specific transcription initiated from a wheat cox2 promoter. The active DEAE-Sephacel pool was further resolved by chromatography on phosphocellulose. Fractions that exhibited DNA-binding activity and that stimulated both specific and nonspecific transcription in vitro were highly enriched in a 63-kDa protein (p63). From peptide sequence obtained from purified p63, a cDNA encoding the protein was assembled. The predicted amino acid sequence (612 amino acid residues, 69 kDa) contains a basic N-terminal targeting sequence expected to direct transport of the protein into mitochondria. The p63 sequence also features an acidic domain characteristic of transcriptional activation factors, as well as sequence blocks displaying limited similarity to positionally equivalent regions in sigma factors from eubacteria related to mitochondria. Recombinant p63 possesses DNA-binding activity, exhibiting an affinity for the core cox2 promoter element and upstream regions in gel shift assays and having the ability to enhance specific transcription in vitro. Transcripts encoding p63 are expressed at an early stage in the germination of isolated wheat embryos, in a temporal pattern parallelling that of newly synthesized precursors of cox2, a mitochondrial gene. Taken together, these data suggest a role for p63 in transcription in wheat mitochondria.

Identification of new low-molecular-weight glutenin subunit genes in wheat.

To clarify the composition of low-molecular-weight glutenin subunits (LMW-GSs) in a soft wheat cultivar, we cloned and characterized LMW-GS genes from a cDNA library and genomic DNA in Norin 61. Based on alignment of the conserved N- and C- terminal domains of the deduced amino-acid sequences, these genes are classified into 12 groups. One of these groups (group 5), the corresponding gene of which has not been reported previously, contains two additional hydrophobic amino-acid clusters interrupting the N-terminal repetitive domain. Other groups (groups 11 and 12), which were not identified in other cultivars as a protein product, showed all eight cysteines in the C-terminal conserved domain. With specific primer sets for these groups it was revealed that Glu-D3 and Glu-A3 encoded the former and the latter, respectively. Both groups of genes were expressed in immature seeds. The presence of these groups of LMW-GSs may affect the dough strength of soft wheat.

Characterization of low-molecular-weight glutenin subunit genes and their protein products in common wheats.

To characterize the low-molecular-weight glutenin subunit (LMW-GS), we developed specific PCR primer sets to distinguish 12 groups of LMW-GS genes of Norin 61 and to decide their loci with nullisomic-tetrasomic lines of Chinese Spring. Three, two, and ten groups were assigned to Glu-A3, Glu-B3, and Glu-D3 loci, respectively. To identify the proteins containing the corresponding amino acid sequences, we determined the N-terminal amino acid sequence of 12 spots of LMW-GSs of Norin 61 separated by two-dimensional gel electrophoresis (2DE). The N-terminal sequences of the LMW-GS spots showed that 10 of 12 groups of LMW-GSs were expressed as protein products, which included LMW-i, LMW-m, and LMW-s types. Four spots were encoded by Glu-A3 (LMW-i). Three spots were encoded by Glu-B3 (LMW-m and LMW-s). Five spots were encoded by Glu-D3 (LMW-m and LMW-s). A minor spot of LMW-m seemed to be encoded by the same Glu-B3 gene as a major spot of LMW-s, but processed at a different site. Comparing among various cultivars, there were polymorphic and non-polymorphic LMW-GSs. Glu-A3 was highly polymorphic, i.e., the a, b, and c alleles showed one spot, the d allele showed four spots, and the e allele had no spot. Insignia used as one of the Glu-A3 null standard cultivars had a LMW-GS encoded by Glu-A3. We also found that Cheyenne had a new Glu-D3 allele. Classification of LMW-GS by a combination of PCR and 2DE will be useful to identify individual LMW-GSs and to study their contribution to flour quality.

Deficiency of cox1 gene expression in wheat plants with Aegilops columnaris cytoplasm.

Wheat plants with Aegilops columnaris cytoplasm are characterized by growth inhibition and partial male sterility and show an impaired mitochondrial cytochrome c oxidase activity. We designed this study to clarify the functional relationship between this impaired cytochrome c oxidase activity and possible structural alterations to the mitochondrial genes cox1, cox2 and cox3 that encode three subunits of the cytochrome c oxidase. Using restriction mapping and DNA sequencing, we found major rearrangements in the flanking regions of the cox1 and cox3 genes. In Northern-blot analysis, we detected two different cox3 transcripts and a reduced level of a cox2 transcript, whereas we could not detect a transcript of cox1. These results suggest that rearrangements of the flanking regions of the cox1 gene may result in the severe suppression of its transcription, and subsequently, may lead to the impaired cytochrome c oxidase activity.

Identification and characterization of T3/T7 bacteriophage-like RNA polymerase sequences in wheat.

Using PCR-based methods, we assembled two wheat cDNA sequences, wheat-G and wheat-C, that encode T3/T7 bacteriophage-like RNA polymerases (RNAPs) sharing 45% amino acid identity. In phylogenetic analyses using maximum likelihood, parsimony and distance methods, the predicted protein sequence of wheat-G (1005 amino acids, 113 kDa) clusters with sequences of previously assigned mitochondrial RNAPs from dicotyledonous plants (Arabidopsis thaliana, Chenopodium album); likewise, in such analyses, the wheat-C sequence (949 amino acids, 107 kDa) affiliates specifically with the Arabidopsis sequence that encodes a phage-like RNAP thought to function in chloroplasts. To confirm biochemically the assignment of the gene encoding the putative wheat mitochondrial RNAP, we isolated a ca. 100 kDa wheat mitochondrial protein that is enriched in fractions displaying specific in vitro transcription activity and that reacts with an antibody raised against a recombinant maize phage-type RNAP. Internal peptide sequence information obtained from the 100-kDa polypeptide revealed that it corresponds to the predicted wheat-G cDNA sequence, providing direct evidence that the wheat-G gene (which we propose to call RpoTm) encodes the wheat mitochondrial RNAP.

Variations in chloroplast proteins and nucleotide sequences of three chloroplast genes in Triticum and Aegilops.

Two alloplasmic wheat lines having the same common wheat nucleus but the cytoplasms of Aegilops crassa and Ae. columnaris together with the corresponding normal line (control) were used in the two-dimensional gel electrophoresis of soluble and thylakoid membrane proteins of the chloroplast. Three chloroplast polypeptides: the Rubisco large subunit, the beta subunit of ATP synthase, and an unidentified 31 kDa protein, differed in the common wheat and two Aegilops cytoplasms. Three chloroplast genes, atpB, atpE and trnM, that respectively encode the beta and epsilon subunits of ATP synthase and tRNA(met), were sequenced. The atpB gene differed by two synonymous base substitutions, whereas the other two genes were identical in the two Aegilops cytoplasms. From the predicted amino acid sequences, the beta subunits of the ATP synthase in the Aegilops cytoplasms were assumed to have three amino acid substitutions: Ala by Val, Asp- by Ala, and Gln by Lys+, in contrast to the cytoplasm of common wheat. This accounts for the difference in pI values found for the common wheat and Aegilops cytoplasms. The two base substitutions for the atpE genes of common wheat and the Aegilops cytoplasms were synonymous. The differences detected in the genes encoding the two subunits of ATP synthase do not appear to be ascribable to the differences in phenotypic effects for the common wheat and Aegilops cytoplasms. The base substitution rate of the atpB-atpE-trnM gene cluster was similar to that of the rbcL gene. From the rate for the atpB gene alone, evolutionary divergence of the wheat-Aegilops complex is assumed to have begun ca. 3.0 x 10(6) years ago, as compared to ca. 8.0 x 10(6) years ago for the divergence of the wheat-Aegilops complex and barley.

Moderately repeated, dispersed, and highly variable (MRDHV) genomic sequences of common wheat usable for cultivar identification.

A 4.1-kb DNA clone (pTag546), which when used as a probe produces hypervariable DNA fingerprints in common wheat, was found among the genomic clones of Triticum aestivum cv 'Chinese Spring'. Nulli-tetrasomic analyses revealed that the sequences hybridizing to this clone were located at 12 loci on ten chromosomes of the A, B, and D genomes of common wheat. The complete nucleotide sequence of pTag546 was shown to have a transposable element-like structure within it, though no open reading frame was detected. The sequences located in the A and D genomes were assumed to have been derived from the B genome by transposition. Using this clone as a probe, we were able to identify 56 common wheat cultivars, some of which are closely related, by their DNA fingerprints. This suggests that pTag 546 will be useful for cultivar identification as well as for germ plasm evaluation in wheat.

Sequences homologous to yeast mitochondrial and bacteriophage T3 and T7 RNA polymerases are widespread throughout the eukaryotic lineage.

Although mitochondria and chloroplasts are considered to be descendants of eubacteria-like endo- symbionts, the mitochondrial RNA polymerase of yeast is a nucleus-encoded, single-subunit enzyme homologous to bacteriophage T3 and T7 RNA polymerases, rather than a multi-component, eubacterial-type alpha 2 beta beta' enzyme, as encoded in chloroplast DNA. To broaden our knowledge of the mitochondrial transcriptional apparatus, we have used a polymerase chain reaction (PCR) approach designed to amplify an internal portion of phage T3/T7-like RNA polymerase genes. Using this strategy, we have recovered sequences homologous to yeast mitochondrial and phage T3/T7 RNA polymerases from a phylogenetically broad range of multicellular and unicellular eukaryotes. These organisms display diverse patterns of mitochondrial genome organization and expression, and include species that separated from the main eukaryotic line early in the evolution of this lineage. In certain cases, we can deduce that PCR-amplified sequences, some of which contain small introns, are localized in nuclear DNA. We infer that the T3/T7-like RNA polymerase sequences reported here are likely derived from genes encoding the mitochondrial RNA polymerase in the organisms in which they occur, suggesting a phage T3/T7-like RNA polymerase was recruited to act in transcription in the mitochondrion at an early stage in the evolution of this organelle.

On the evolution of the single-subunit RNA polymerases.

Many eukaryotic nuclear genomes as well as mitochondrial plasmids contain genes displaying evident sequence similarity to those encoding the single-subunit RNA polymerase (ssRNAP) of bacteriophage T7 and its relatives. We have collected and aligned these ssRNAP sequences and have constructed unrooted phylogenetic trees that demonstrate the separation of ssRNAPs into three well-defined and nonoverlapping clusters (phage-encoded, nucleus-encoded, and plasmid-encoded). Our analyses indicate that these three subfamiles of T7-like RNAPs shared a common ancestor; however, the order in which the groups diverged cannot be inferred from available data. On the basis of structural similarities and mutational data, we suggest that the ancestral ssRNAP gene may have arisen via duplication and divergence of a DNA polymerase or reverse transcriptase gene. Considering the current phylogenetic distribution of ssRNAP sequences, we further suggest that the origin of the ancestral ssRNAP gene closely paralleled in time the introduction of mitochondria into eukaryotic cells through a eubacterial endosymbiosis.