Diploid Aegilops Species Research Articles

Development and characterization of Triticum turgidum - Aegilops comosa and T. turgidum - Ae. markgrafii amphidiploids.

Aegilops comosa and Ae. markgrafii are diploid progenitors of polyploidy species of Aegilops sharing M and C genomes, respectively. Transferring valuable genes/traits from Aegilops into wheat is an alternative strategy for wheat genetic improvement. The amphidiploids between diploid species of Aegilops and tetraploid wheat can act as bridges to overcome obstacles from direct hybridization and can be developed by the union of unreduced gametes. In this study, we developed seven Triticum turgidum - Ae. comosa and two T. turgidum - Ae. markgrafii amphidiploids. The unreduced gametes mechanisms, including first-division restitution (FDR) and single-division meiosis (SDM), were observed in triploid F1 hybrids of T. turgidum - Ae. comosa (STM) and T. turgidum - Ae. markgrafii (STC). Only FDR was observed in STC hybrids, whereas FDR or both FDR and SDM were detected in the STM hybrids. All seven pairs of M chromosomes of Ae. comosa and C chromosomes of Ae. markgrafii were distinguished by fluorescent in situ hybridization (FISH) probes pSc119.2 and pTa71 combinations with pTa-535 and (CTT)12/(ACT)7, respectively. Meanwhile, the chromosomes of tetraploid wheat and diploid Aegilops parents were distinguished by the same FISH probes. The amphidiploids possessed specific valuable traits such as multiple tillers, large seed size related traits, and stripe rust resistance that could be utilized in the genetic improvement of wheat.

Genome-wide polymorphisms from RNA sequencing assembly of leaf transcripts facilitate phylogenetic analysis and molecular marker development in wild einkorn wheat.

A survey of genome-wide polymorphisms between closely related species is required to understand the molecular basis of the evolutionary differentiation of their genomes. Two wild diploid wheat species, namely Triticum monococcum ssp. aegilopoides and T. urartu, are closely related and harbour the Am and A genomes, respectively. The A-genome donor of tetraploid and common wheat is T. urartu, and T. monococcum ssp. monococcum is the cultivated form derived from the wild einkorn wheat subspecies aegilopoides. Although subspecies aegilopoides has been a useful genetic resource in wheat breeding, genome-wide molecular markers for this subspecies have not been sufficiently developed. Here, we describe the detection of genome-wide polymorphisms such as single-nucleotide polymorphisms (SNPs) and insertions/deletions (indels) from RNA sequencing (RNA-seq) data of leaf transcripts in 15 accessions of the two diploid wheat species. The SNPs and indels, detected using the A genome of common wheat as the reference genome, covered the entire chromosomes of these species. The polymorphism information facilitated a comparison of the genetic diversity of einkorn wheat with that of two related diploid Aegilops species, namely, Ae. tauschii and Ae. umbellulata. Cleaved amplified polymorphic sequence (CAPS) markers converted from the SNP data were efficiently developed to confirm the addition of aegilopoides subspecies chromosomes to tetraploid wheat in nascent allohexaploid lines with AABBAmAm genomes. In addition, the CAPS markers permitted linkage map construction in mapping populations of aegilopoides subspecies accessions. Therefore, these RNA-seq data provide information for further breeding of closely related species with no reference genome sequence data.

Evolution of the S-Genomes in Triticum-Aegilops Alliance: Evidences From Chromosome Analysis.

Five diploid Aegilops species of the Sitopsis section: Ae. speltoides, Ae. longissima, Ae. sharonensis, Ae. searsii, and Ae. bicornis, two tetraploid species Ae. peregrina (= Ae. variabilis) and Ae. kotschyi (Aegilops section) and hexaploid Ae. vavilovii (Vertebrata section) carry the S-genomes. The B- and G-genomes of polyploid wheat are also the derivatives of the S-genome. Evolution of the S-genome species was studied using Giemsa C-banding and fluorescence in situ hybridization (FISH) with DNA probes representing 5S (pTa794) and 18S-5.8S-26S (pTa71) rDNAs as well as nine tandem repeats: pSc119.2, pAesp_SAT86, Spelt-1, Spelt-52, pAs1, pTa-535, and pTa-s53. To correlate the C-banding and FISH patterns we used the microsatellites (CTT)10 and (GTT)9, which are major components of the C-banding positive heterochromatin in wheat. According to the results obtained, diploid species split into two groups corresponding to Emarginata and Truncata sub-sections, which differ in the C-banding patterns, distribution of rDNA and other repeats. The B- and G-genomes of polyploid wheat are most closely related to the S-genome of Ae. speltoides. The genomes of allopolyploid wheat have been evolved as a result of different species-specific chromosome translocations, sequence amplification, elimination and re-patterning of repetitive DNA sequences. These events occurred independently in different wheat species and in Ae. speltoides. The 5S rDNA locus of chromosome 1S was probably lost in ancient Ae. speltoides prior to formation of Timopheevii wheat, but after the emergence of ancient emmer. Evolution of Emarginata species was associated with an increase of C-banding and (CTT)10-positive heterochromatin, amplification of Spelt-52, re-pattering of the pAesp_SAT86, and a gradual decrease in the amount of the D-genome-specific repeats pAs1, pTa-535, and pTa-s53. The emergence of Ae. peregrina and Ae. kotschyi did not lead to significant changes of the S*-genomes. However, partial elimination of 45S rDNA repeats from 5S* and 6S* chromosomes and alterations of C-banding and FISH-patterns have been detected. Similarity of the Sv-genome of Ae. vavilovii with the Ss genome of diploid Ae. searsii confirmed the origin of this hexaploid. A model of the S-genome evolution is suggested.

New genetic resources in wheat breeding for increased grain protein content

This review offers an overview of genetic research on grain protein content (GPC) in various Triticum L. and Aegilops L. species. Regularities in the geographic variability of GPC and the results of a longterm screening of accessions from the VIR collection for this trait are considered. Based on these assessments, a core collection of genetic sources with high GPC has been formed. It includes the diploid Aegilops species as donors of B, G, and D genomes for allopolyploid wheats, as well as accessions of di-, tetra-, and hexaploid wheat species. The use of high-protein sources in wheat breeding in the United States and Canada in the 1970s–1980s resulted in the GPC of bread wheat increasing by 0.5–3.0%; however, further attempts at increasing GPC by traditional breeding methods have failed. A breakthrough in increasing the total GPC has been achieved as a result of molecular genetics methods and development of molecular markers. For the first time, a functional locus, or the Gpc-B1 gene (chromosome 6BS) affecting the accumulation of protein, Zn, and Fe in grain, was identified in T. dicoccoides, cloned, and studied in detail. The application of molecular markers has revealed the active allele of this gene in some landraces and old cultivars of T. dicoccum, T. durum, T. spelta, and T. aestivum. Moreover, the Gpc-A1, Gpc-D1, and Gpc-2 wheat genes have been found in chromosomes 6A and 6D and homeologous group 2, respectively. All these genes have been identified as NAC transcription factors, which play an important role in the accelerated senescence of plants and the remobilization of nutrients from leaves to grain. The genes related to Gpc-B1 from T. dicoccoides were found in the G genome of T. timopheevii and B (=S) genome of different species of Aegilops sect. sitopsis. Functional Gpc-B1 alleles have been introduced into commercial tetra- and hexaploid wheat cultivars, which resulted in the creation of new high-protein and high-yield cultivars and series of nearly isogenic lines in different countries. They are promising sources for research and wheat breeding purposes.

New genetic resources in wheat breeding for an increased grain protein content

The present review offers an overview of genetic research on grain protein content (GPC) in various Triticum L. and Aegilops L. species. Regularities in geographic variability of GPC and the results of a longterm screening of accessions from the VIR collection for this trait are considered. On the basis of these assessments, a core-collection of genetic sources with high GPC has been formed. It includes the diploid Aegilops species as donors of B, G and D genomes for allopolyploid wheats, as well as accessions of di-, tetra- and hexaploid wheat species. The use of highprotein sources in wheat breeding in the United States and Canada in the 1970’s–1980’s resulted in the bread wheat GPC increase by 0.5–3.0 %; however, further purposeful attempts at increasing GPC by traditional breeding methods failed. A breakthrough in increasing the total GPC has been achieved as a result of molecular genetics methods and molecular markers development. For the first time, a functional locus, or the Gpc-B1 gene (chromosome 6BS) affecting the accumulation of protein, Zn and Fe in grain, was identified in T. dicoccoides, cloned and studied in detail. The application of molecular markers has revealed the active allele of this gene in some landraces and old cul-tivars of T. dicoccum, T. durum, T. spelta and T. aestivum. Moreover, Gpc-A1, Gpc-D1, and Gpc-2 wheat genes have been found in chromosomes 6A, 6D and homeologous group 2, respectively. All these genes have been identified as NAC transcription factors, which play an important role in the accelerated senescence of plants and remobilization of nutrients from leaves to grain. The genes related to Gpc-B1 from T. dicoccoides were found in the G genome of T. timopheevii and B (=S) genome of different species of Aegilops sect. sitopsis. Functional Gpc-B1 alleles have been introduced into commercial tetra- and hexaploid wheat cultivars, and it resulted in the creation of new highprotein and high-yield cultivars and series of nearly isogenic lines in different countries. They are promising sources for research and wheat breeding purposes.

Molecular-cytogenetic analysis of triticale and wheat lines with introgressions of the tribe Triticeae species genetic material

There are a number of problems in selection of cultivated cereals associated with the requirements to create forms with resistance to diseases, pests and unfavorable environmental conditions. The genetic diversity of genes for resistance to biotic and abiotic stresses can be increased by means of the gene pool of wild and cultivated wheat relatives. To improve agronomic traits in cereals, we have developed common wheat hybrid lines T. aestivum/ T. durum, T. aestivum/ T. dicoccoides and triticale lines by crossing hexaploid triticale with common wheat forms with the substitution of genome D for the geno­me of diploid Aegilops species. The aim of the study was to identify the lines of common wheat and hexaploid triticale with alien introgression using cytological and molecular-genetic analyses and evaluation of their cytological stability. Comparative analysis of the structure of chromosomes by GISH and FISH methods, microsatellite- and chromosome-specific markers revealed that hybridization of triticale with genome-substitution forms of wheat leads to the reorganization of the genome, including both the introgression of foreign material and wheat chromosome rearrangements, which lead to new combinations of genetic loci. The efficiency of wheat microsatellite markers to characterize of the T. aestivum/ T. durum, T. aestivum/ T. dicoccum interspecific hybrid lines was shown. From 4 to 12 translocations of different lengths from T. durum and T. dicoccum were identified in the chromosomes of A and В genomes in the hybrid lines. Meiotic stability of wheat and triticale hybrids was found. It creates prerequisites for preservation of alien genetic material in subsequent generations.

Characterization and sequence diversity of the Gsp-1 gene in diploid species of the Aegilops genus

Several studies have suggested a minor role in determining grain endosperm texture for the Gsp-1 gene, although appears related with the synthesis of the arabinogalactan-peptide. The current study evaluated the allelic variability of Gsp-1 genes in five diploid species of the Aegilops genus along with the molecular characterization of the main allelic variants found in each species and their phylogenetic relationships with the polyploid wheats. There was a high level of polymorphism in Gsp-1 genes – up to 19 alleles were obtained, of which 11 were novel. The processing of Gsp-1 amino acid sequences led to two main domains: AGP (arabinogalactan peptide) and GSP-1 (grain softness protein). Several substitutions were detected in both domains with respect to the bread wheat sequences, which could modify grain hardness. The sequences obtained showed relationship with the Gsp-B1 and Gsp-D1 genes of common wheat. These results showed that diploid Aegilops species are a rich resource of novel genetic variability for the Gsp-1 gene, whose hypothetical functionality should be tested in the genetic pool of modern wheat, valuing as the true role of these genes in grain hardness and their effective role as source of the AGP, a non-starch polysaccharide with health properties.

Celiac disease-specific prolamin peptide content of wheat relatives and wild species determined by ELISA assays and bioinformatics analyses

Enzyme-linked immunosorbent assays (ELISAs) are widely used to determine gluten contamination in gluten-free and low gluten food samples. ELISA assays developed using monoclonal antibodies against known toxic peptides have an advantage in the identification of toxic prolamin content in protein extracts of different food samples, as well as raw materials. R5 and G12 monoclonal antibodies specific for two known toxic peptides used in commercially available gluten ELISA assays were applied to test toxic peptide contents in wheat relatives and wild wheat species with different genome composition and complexity. Although the R5 peptide content showed some correlation with ploidy levels in Triticum species, there was a high variance among Aegilops species. Some of the analysed diploid Aegilops species showed extremely high R5 peptide contents. Based on the bioinformatics analyses, the R5 peptide was present in most of the sulphur rich prolamins in all the analysed species, whereas the G12 epitope was exclusively present in alpha gliadins. High variation was detected in the position and frequency of epitopes in sequences originating from the same species, thus highlighting the importance of genotypic variation within species. Identification of new prolamin alleles of wheat relatives and wild wheat species is of great importance in order to find germplasm for special end-use quality purposes as well as development of food with reduced toxicity.

Characterization of the Wx gene in diploid Aegilops species and its potential use in wheat breeding

Wx gene encodes for the granule-bound starch synthase I or waxy protein, which is the sole enzyme responsible for amylose synthesis in wheat seeds. The Aegilops species, which are related to wheat, could be important sources of variation in this gene. In addition to its role in starch quality, this gene has been used in phylogenetic studies of wheat. The current study evaluated the variability of Wx gene in seven diploid species of Aegilops genus and compared their nucleotide sequences with the wheat homeologous genes. Nineteen new Wx alleles were found in the seven species evaluated. The alleles detected in two species of the Sitopsis section, Ae. searsii and Ae. speltoides, were related to the Wx-B1 gene of wheat. Two more of the Sitopsis species did not appear to be associated with this genome, whereas the remaining species were related to the Wx-D1 gene of wheat. The results showed an important variation of the Wx gene present in the Aegilops genus, and the 19 new Wx alleles detected could enlarge the genetic pool of wheat.

Phylogenetic relationships among Triticum L. and Aegilops L. species as genome progenitors of bread wheat based on sequence diversity in trnT-F region of chloroplast DNA

Cultivated wheat, (Triticum aestivum L.), is one of the most important food crops in the world. The Aegilops L. genus is frequently utilized by plant breeders for improving the current wheat cultivars due to their close relationships. Therefore, understanding the phylogenetic relationships among the species of these genera is not only valuable for plant taxonomy, but also for plant breeding efforts. The presented phylogenetic analysis was based on the sequences of trnT-F chloroplast DNA containing three non-coding sub-regions. Twelve genotypes belonging to four species of Triticum L. genus and twenty-four genotypes belonging to eight species of Aegilops genus were used in the current study. The results postulated a close genetic relationship between diploid Aegilops species containing the BB genome and polyploid Triticum species. With the exception of Aegilops cylindrica Host (CCDD), all other Aegilops species having the CC genome were alienated from Aegilops speltoides Tausch (BB) and clustered together. These two clusters joined by a third cluster including the AA genome containing diploid Triticum species.

Variations and classification of toxic epitopes related to celiac disease among α-gliadin genes from four Aegilops genomes

The α-gliadins are associated with human celiac disease. A total of 23 noninterrupted full open reading frame α-gliadin genes and 19 pseudogenes were cloned and sequenced from C, M, N, and U genomes of four diploid Aegilops species. Sequence comparison of α-gliadin genes from Aegilops and Triticum species demonstrated an existence of extensive allelic variations in Gli-2 loci of the four Aegilops genomes. Specific structural features were found including the compositions and variations of two polyglutamine domains (QI and QII) and four T cell stimulatory toxic epitopes. The mean numbers of glutamine residues in the QI domain in C and N genomes and the QII domain in C, N, and U genomes were much higher than those in Triticum genomes, and the QI domain in C and N genomes and the QII domain in C, M, N, and U genomes displayed greater length variations. Interestingly, the types and numbers of four T cell stimulatory toxic epitopes in α-gliadins from the four Aegilops genomes were significantly less than those from Triticum A, B, D, and their progenitor genomes. Relationships between the structural variations of the two polyglutamine domains and the distributions of four T cell stimulatory toxic epitopes were found, resulting in the α-gliadin genes from the Aegilops and Triticum genomes to be classified into three groups.

Fate of Aegilops speltoides-Derived, Repetitive DNA Sequences in Diploid Aegilops Species, Wheat-Aegilops Amphiploids and Derived Chromosome Addition Lines

The present study reports the cloning and characterization of an Aegilopsspeltoides-derived subtelomeric repeat, designated as pSp1B16. Clone pSp1B16 has 98% sequence homology with the previously isolated Ae. speltoides repeat Spelt1. The distribution of pSp1B16 and another Ae. speltoides repeat, pGc1R1, was analyzed in diploid Aegilops species, tetra- and hexaploid wheats, wheat-Aegilops amphiploids and derived chromosome addition lines by fluorescence in situ hybridization (FISH). Clones pSp1B16 and pGc1R1 revealed FISH sites in Ae. speltoides, Ae. sharonensis and Triticumtimopheevii, whereas additional pGc1R1 FISH sites were observed in Ae. longissima and Ae. caudata. The pSp1B16 and pGc1R1 FISH patterns of the Aegilops chromosomes in the wheat-Aegilops amphiploids and chromosome addition lines are similar to those present in the Aegilops parent accession. We did not observe any evidence of pSp1B16 and pGc1R1 sequence elimination, which is in contrast to previous studies using similar hybrids and repeats. The presented data suggest that the genomic changes in synthetic amphiploids observed in previous studies might be caused by homoeologous recombination, which was suppressed in the amphiploid analyzed in this study.

Phylogenetic relationships among diploid Aegilops species inferred from 5S rDNA units

Relationships among the currently recognized 11 diploid species within the genus Aegilops have been investigated. Sequence similarity analysis, based upon 363 sequenced 5S rDNA clones from 44 accessions plus 15 sequences retrieved from GenBank, depicted two unit classes labeled the long AE1 and short AE1. Several different analytical methods were applied to infer relationships within haplomes, between haplomes and among the species, including maximum parsimony and maximum likelihood analyses of consensus sequences, “total evidence” phylogeny analysis and “matrix representation with parsimony” analysis. None were able to depict suites of markers or unit classes that could discern among the seven haplomes as is observed among established haplomes in other genera within the tribe Triticeae; however, most species could be separated when displayed on gene trees. These results suggest that the haplomes currently recognized are so refined that they may be relegated as sub-haplomes or haplome variants. Amblyopyrum shares the same 5S rDNA unit classes with the diploid Aegilops species suggesting that it belongs within the latter. Comparisons of the Aegilops sequences with those of Triticum showed that the long AE1 unit class of Ae. tauschii shared the clade with the equivalent long D1 unit class, i.e., the putative D haplome donor, but the short AE1 unit class did not. The long AE1 unit class but not the short, of Ae. speltoides and Ae. searsii both share the clade with the previously identified long {S1 and long G1 unit classes meaning that both Aegilops species can be equally considered putative B haplome donors to tetraploid Triticum species. The semiconserved nature of the nontranscribed spacer in Aegilops and in Triticeae in general is discussed in view that it may have originated by processes of incomplete gene conversion or biased gene conversion or birth-and-death evolution.

Molecular analysis of the phylogenetic relationships among the diploid Aegilops species of the section Sitopsis

RAPD analysis was used to study the intraspecific variation and phylogenetic relationships of S-genome diploid Aegilops species regarded as potential donors of the B genome of cultivated wheat. In total, 21 DNA specimens from six S-genome diploid species were examined. On a dendrogram, Ae. speltoides and Ae. aucheri formed the most isolated cluster. Among the other species, Ae. searsii was the most distant while Ae. longissima and Ae. sharonensis were the closest species. The maximum difference between individual accessions within one species was approximately the same (0.18-0.22) in Ae. bicornis, Ae. longissima. Ae. sharonensis, and Ae. searsii. The difference between the clusters of questionable species Ae. speltoides and Ae. aucheri corresponded to the intraspecific level; the difference between closely related Ae. longissima and Ae. sharonensis corresponded to the interspecific level. The section Sitopsis of the genus Aegilops includes six diploid species containing the S genome, which is regarded as an ancestor of the B genome of cultivated wheat. The species of the section are thought to be closest to the genus Triticum. Note that the taxonomic status of some forms of the section Sitopsis is questionable. For instance, Ae. speltoides and Ae. aucheri are variously considered as individual species or as a single species, Ae. speltoides. The situation with Ae. longissima and Ae. sharonensis is similar. Thus, although the group includes only diploid species and is well studied morphologically, its phylogeny and taxonomy are still questionable.

Novel puroindoline and grain softness protein alleles in Aegilops species with the C, D, S, M and U genomes

DNA from six hexaploid, tetraploid and diploid species of Aegilops with the C, D, S, M and U genomes was amplified with specific PCR primers to identify sequences encoding puroindolines (Pins) a and b and grain softness protein (GSP), all of which are encoded by genes at the Ha (hardness) locus, with Ae. tauschii (DD) and bread wheat (T. aestivum) (AABBDD) cv Hiline being studied as controls. Seven new allelic forms of Pin a and Pin b were identified, including forms with mutations within or close to the tryptophan motif. In addition, five new forms of GSP were detected. In all species both genomic DNA from leaves and cDNA from developing grain were analysed. This revealed the presence of both silent genes (with premature stop codons) and multiple genes, with the latter being confirmed by Southern blot analysis. Freeze fracture analysis demonstrated that all except one accession (Ae. sharonensis) were soft textured. However, this difference cannot be accounted for by the sequences of the Pin alleles present in this line.

Analysis of nucleotide sequence polymorphism of internal transcribed spacers of ribosomal genes in diploid Aegilops (L.) species

The nucleotide sequence of the fragment of the internal transcribed spacer (ITS) of rDNA comprising the full-length ITS1, the gene encoding 5.8S rRNA, and part of the ITS2 sequence was determined in 22 samples of five diploid Aegilops species. The full alignment length of compared sequences was 524 bp. Species-specific substitutions were found in the ITS nucleotide sequence of rDNA of different Aegilops species. Intraspecific differences in ITS structure in diploid Aegilops species were detected for the first time. Polymorphism of the ITS nucleotide sequence within the same sample was revealed, which might be due either to differences between the genomes of individual plants comprising the sample or to the presence of several types of ribosomal genes in the genome of one plant. In general, both interspecific and intraspecific variability of the ITS nucleotide sequences of rDNA is extremely low. In total, 26 variable sites, twelve of which were informative, were identified.

Phylogenetic analysis of complete 5′ external transcribed spacers of the 18S ribosomal RNA genes of diploid Aegilops and related species (Triticeae, Poaceae)

PCR systems were designed to amplify the entire 5′ external transcribed spacer (ETS) region of the 18S rRNA gene of all the diploid species of Aegilops and several other taxa closely related to domesticated wheat. Phylogenetic analysis was performed on the complete ETS sequences using the neighbor-joining, maximum parsimony, and maximum likelihood methods. Among the individual taxa studied, speciation in Secale is very recent. In the case of the A genome diploids, the results support the theory that the A genomes of wheat have experienced reticulate evolution owing to introgression. The B and G genomes of tetraploid domesticated wheats form a clade with Ae. speltoides in which the B genome diverged first and the G genome more recently. It was demonstrated that the complete ETS sequences of the Triticeae yield coherent phylogenetic information. The ETS is a useful tool for studying the phylogeny of closely related species.

A chromosome-specific sequence common to the B genome of polyploid wheat and Aegilops searsii

A low-copy, non-coding chromosome-specific DNA sequence, isolated from common wheat, was physically mapped to the distal 19% region of the long arm of chromosome 3B (3BL) of common wheat. This sequence, designated WPG118, was then characterized by Southern hybridization, PCR amplification and sequence comparison using a large collection of polyploid wheats and diploid Triticum and Aegilops species. The data show that the sequence exists in all polyploid wheats containing the B genome and absent from those containing the G genome. At the diploid level, it exists only in Ae. searsii, a diploid species of section Sitopsis, and not in other diploids including Ae. speltoides, the closest extant relative to the donor of the B genome of polyploid wheat. This finding may support the hypothesis that the B-genome of polyploid wheat is of a polyphyletic origin, i.e. it is a recombined genome derived from two or more diploid Aegilops species.

Cold-specific and light-stimulated expression of a wheat (Triticum aestivum L.) Cor gene Wcor15 encoding a chloroplast-targeted protein.

Wcor15, a member of the wheat cold-responsive (Cor) gene family, has been isolated and characterized. The deduced polypeptide WCOR15 (MW=14.7 kDa) showed high homology to the previously identified wheat and barley COR proteins. Southern blot analysis using diploid, tetraploid and hexaploid wheat and diploid Aegilops species showed that the wheat and related wild genomes possessed multiple copies of Wcor15 homologues. Five copies were assigned to the homologous group 2 chromosomes by nulli-tetrasomic analysis. Northern blot analysis showed that expression of Wcor15 was specifically induced by low-temperature. Homologous transcripts accumulated in leaves, and light markedly increased their steady-state levels. Bombardment-mediated transient expression analysis of a chimeric CaMV 35S::Wcor15-GFP construct showed protein-targeting to epidermal guard cell chloroplasts in excised spiderwort leaves. A promoter of Wcor15 contained at least three CRT/DRE-like sequence motifs found in Arabidopsis Cor genes and induced the reporter GUS gene expression in leaves of transgenic tobacco plants under low-temperature and light conditions. These results suggest that the functional Cor gene system involving the CRT/DRE cis-element is conserved in both monocotyledonous and dicotyledonous plants.

Analysis of HMW glutenin subunits and their coding sequences in two diploid Aegilops species.

Considerable progress has been made in understanding the structure, function and genetic regulation of high-molecular-weight (HMW) glutenin subunits in hexaploid wheat. In contrast, less is known about these types of proteins in wheat related species. In this paper, we report the analysis of HMW glutenin subunits and their coding sequences in two diploid Aegilops species, Aegilops umbellulata (UU) and Aegilops caudata (CC). SDS-PAGE analysis demonstrated that, for each of the four Ae. umbellulata accessions, there were two HMW glutenin subunits (designated here as 1Ux and 1Uy) with electrophoretic mobilities comparable to those of the x- and y-type subunits encoded by the Glu-D1 locus, respectively. In our previous study involving multiple accessions of Ae. caudata, two HMW glutenin subunits (designated as 1Cx and 1Cy) with electrophoretic mobilities similar to those of the subunits controlled by the Glu-D1 locus were also detected. These results indicate that the U genome of Ae. umbellulata and the C genome of Ae. caudata encode HMW glutenin subunits that may be structurally similar to those specified by the D genome. The complete open reading frames (ORFs) coding for x- and y-type HMW glutenin subunits in the two diploid species were cloned and sequenced. Analysis of deduced amino acid sequences revealed that the primary structures of the x- and y-type HMW glutenin subunits of the two Aegilops species were similar to those of previously published HMW glutenin subunits. Bacterial expression of modified ORFs, in which the coding sequence for the signal peptide was removed, gave rise to proteins with electrophoretic mobilities identical to those of HMW glutenin subunits extracted from seeds, indicating that upon seed maturation the signal peptide is removed from the HMW glutenin subunit in the two species. Phylogenetic analysis showed that 1Ux and 1Cx subunits were most closely related to the 1Dx type subunit encoded by the Glu-D1 locus. The 1Uy subunit possessed a higher level of homology to the 1Dy-type subunit compared with the 1Cy subunit. In conclusion, our study suggests that the Glu-U1 locus of Ae. umbellulata and the Glu-C1 locus of Ae. caudata specify the expression of HMW glutenin subunits in a manner similar to the Glu-D1 locus. Consequently, HMW glutenin subunits from the two diploid species may have potential value in improving the processing properties of hexaploid wheat varieties.