Gliadin polymorphism in wild and cultivated einkorn wheats

To study the relationships between different species of the Einkorn group, 408 accessions of Triticum monococcum, T. boeoticum, T. boeoticum ssp. thauodar and T. urartu were analyzed electrophoretically for their protein composition at the Gli-1 and Gli-2 loci. In all the species the range of allelic variation at the loci examined is remarkable. The gliadin patterns of T. monococcum and T. boeoticum were very similar to one another but differed substantially from those of T. urartu. Several accessions of T. boeoticum and T. monococcum were shown to share the same alleles at the Gli-1 and Gli-2 loci, confirming the recent nomenclature that considers these wheats as different subspecies of the same species, T. monococcum. The gliadin composition of T. urartu resembled that of the A genome of polyploid wheats more than did T. boeoticum or T. monococcum, supporting the hypothesis that T. urartu, rather than T. boeoticum, is the donor of the A genome in cultivated wheats. Because of their high degree of polymorphism the gliadin markers may help in selecting breeding parents from diploid wheat germ plasm collections and can be used both to search for valuable genes linked to the gliadin-coding loci and to monitor the transfer of alien genes into cultivated polyploid wheats. Received: 8 July 1996 / Accepted: 12 July 1996     

Exploring the diploid wheat ancestral A genome through sequence comparison at the high-molecular-weight glutenin locus region

The polyploid nature of hexaploid wheat (T. aestivum, AABBDD) often represents a great challenge in various aspects of research including genetic mapping, map-based cloning of important genes, and sequencing and accurately assembly of its genome. To explore the utility of ancestral diploid species of polyploid wheat, sequence variation of T. urartu (A^uA^u) was analyzed by comparing its 277-kb large genomic region carrying the important Glu-1 locus with the homologous regions from the A genomes of the diploid T. monococcum (A^mA^m), tetraploid T. turgidum (AABB), and hexaploid T. aestivum (AABBDD). Our results revealed that in addition to a high degree of the gene collinearity, nested retroelement structures were also considerably conserved among the A^u genome and the A genomes in polyploid wheats, suggesting that the majority of the repetitive sequences in the A genomes of polyploid wheats originated from the diploid A^u genome. The difference in the compared region between A^u and A is mainly caused by four differential TE insertion and two deletion events between these genomes. The estimated divergence time of A genomes calculated on nucleotide substitution rate in both shared TEs and collinear genes further supports the closer evolutionary relationship of A to A^u than to A^m. The structure conservation in the repetitive regions promoted us to develop repeat junction markers based on the A^u sequence for mapping the A genome in hexaploid wheat. Eighty percent of these repeat junction markers were successfully mapped to the corresponding region in hexaploid wheat, suggesting that T. urartu could serve as a useful resource for developing molecular markers for genetic and breeding studies in hexaploid wheat.     

High molecular weight glutenin subunit variation in wild and cultivated einkorn wheats (Triticum spp.,Poaceae)

Variation in high molecular weight (HMW) glutenin subunit composition among wild and cultivated einkorn wheats (2n = 2x = 14, AA) was investigated using one- (SDS-PAGE and urea/SDS-PAGE) and two-dimensional (IEF × SDS-PAGE) electrophoretic analyses. The material comprised 150 accessions ofTriticum urartu, 160 accessions ofT. boeoticum, 24 accessions ofT. boeoticum subsp.thaoudar and 74 accessions of primitive domesticatedT. monococcum from many different germplasm collections. The biochemical characteristics of HMW-glutenin subunits ofT. boeoticum andT. monococcum were highly similar to one another but distinctly different from those ofT. urartu. All the species analysed were characterised by large intraspecific variation and only three HMW-glutenin subunit patterns were identical betweenT. boeoticum andT. monococcum. Consistent with the distinct nature ofT. urartu, all its HMW-glutenin patterns were different from those found inT. boeoticum andT. monococcum. The differences detected between these species might reflect their reproductive isolation and are consistent with recent nomenclatural and biosystematic treatments that recogniseT. urartu as separate species fromT. boeoticum andT. monococcum. The presence of three distinct glutenin components in some accessions of the species studied seems to be evidence for the existence of at least three active genes controlling the synthesis of the HMW-glutenin subunits in the A genome of wild and primitive domesticated diploid wheats. Results indicate also that HMW-glutenin subunits could represent useful markers for the evaluation of genetic variability present in different wild diploid wheat collections and subsequently for their conservation and future utilisation.     

Phylogeny of the a genomes of wild and cultivated wheat species

Diploid species of the genus Triticum L. are its most ancient representatives and have the A genome, which was more recently inherited by all polyploid species. Studies of the phylogenetic relationships among diploid and polyploid wheat species help to identify the donors of elementary genomes and to examine the species specificity of genomes. In this study, molecular analysis of the variable sequences of three nuclear genes (Acc-1, Pgk-1, and Vrn-1) was performed for wild and cultivated wheat species, including both diploids and polyploids. Based on the sequence variations found in the genes, clear differences were observed among elementary genomes, but almost no polymorphism was detected within each genome in polyploids. At the same time, the regions of the three genes proved to be rather heterogeneous in the diploid species Triticum boeoticum Boiss., T. urartu Thum. ex Gandil., and T. monococcum L., thus representing mixed populations. A genome variant identical to the A genome of polyploid species was observed only in T. urartu. Species-specific molecular markers discriminating the diploid species were not found. Analysis of the inheritance of morphological characters also failed to identify a species-specific character for the three diploid wheat species apart from the hairy leaf blade type, described previously.     

Transferable bread wheat EST-SSRs can be useful for phylogenetic studies among the Triticeae species

The genetic similarity between 150 accessions, representing 14 diploidand polyploid species of the Triticeae tribe, was investigated following the UPGMA clustering method. Seventy-three common wheat EST-derived SSR markers (EST-SSRs) that were demonstrated to be transferable across several wheat-related species were used. When diploid species only are concerned, all the accessions bearing the same genome were clustered together without ambiguity while the separation between the different sub-species of tetraploid as well as hexaploid wheats was less clear. Dendrograms reconstructed based on data of 16 EST-SSRs mapped on the A genome confirmed that Triticum aestivum and Triticum durum had closer relationships with Triticum urartu than with Triticum monococcum and Triticum boeoticum, supporting the evidence that T. urartu is the A-genome ancestor of polyploid wheats. Similarly, another tree reconstructed based on data of ten EST-SSRs mapped on the B genome showed that Aegilops speltoides had the closest relationship with T. aestivum and T. durum, suggesting that it was the main contributor of the B genome of polyploid wheats. All these results were expected and demonstrate thus that EST-SSR markers are powerful enough for phylogenetic analysis among the Triticeae tribe.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

TRITICUM URARTU AND GENOME EVOLUTION IN THE TETRAPLOID WHEATS

The A genome of the tetraploid wheats (AABB, 2n = 28) shows 5-6 bivalents in crosses with Triticum boeoticum (2n = 14) and various Aegilops diploids (2n = 14). The B genome has never been similarly identified with any species, and is commonly thought to have been modified at the tetraploid level. Triticum boeoticum was presumably accepted as the A-genome donor because of its morphological similarity to the wild tetraploids and because it was formerly the only known wild diploid wheat. The B donor has been thought to be Ae. speltoides or another species of the Sitopsis section of Aegilops, but these diploids show pairing affinity with A rather than B. More recently, another diploid wheat, T. urartu, was found to be sympatric with T. boeoticum throughout the natural range of the tetraploids. The synthetic boeoticum-urartu amphiploid was virtually identical morphologically with the wild tetraploid wheats, whereas various boeoticum-Sitopsis amphiploids were markedly different. But the urartu genome, like those of T. boeoticum and Sitopsis, paired with A and not with B. However, cytological evidence also shows (1) that the genomes of any plausible parental combination pair with one another, (2) that the A and B genomes of the tetraploid wheats pair with one another in the absence of the gene Ph, and (3) that homoeologous chromosomes of the tetraploids have differentiated further, presumably as a result of diploidization. Consequently, chromosome pairing at Meiosis I can be expected to give ambiguous evidence regarding the identity of the tetraploid genomes with their parental prototypes. A hypothesis regarding the expected pairing affinities between tetraploid homoeologues that have differentiated from closely related parental chromosomes is advanced to explain the anomalous pairing behavior of the A and B genomes. Triticum boeoticum and T. urartu are inferred to be the parents of the tetraploid wheats.     

Morphological and molecular characterisation confirm that Triticum monococcum s.s. is resistant to wheat leaf rust

The three diploid wheat species Triticum monococcum, Triticum boeoticum and Triticum urartu differ in their reaction to wheat leaf rust, Puccinia triticina. In general, T. monococcum is resistant while T. boeoticum and T. urartu are susceptible. However, upon screening a large collection of diploid wheat accessions, 1% resistant T. boeoticum accessions and 16% susceptible T. monococcum accessions were found. In the present study these atypical accessions were compared with 49 typical T. monococcum, T. boeoticum and T. urartu accessions to gain insight into the host-status of the diploid wheat species for wheat leaf rust. Cluster analysis of morphological data and AFLP fingerprints of the typical accessions clearly discriminated the three diploid species. T. monococcum and T. boeoticum had rather-similar AFLP fingerprints while T. urartu had a very different fingerprint. The clustering of most atypical accessions was not consistent with the species they were assigned to, but intermediate between T. boeoticum and T. monococcum. Only four susceptible T. monococcum accessions were morphologically and moleculary similar to the typical T. monococcum accessions. Results confirmed that T. boeoticum and T. monococcum are closely related but indicate a clear difference in host-status for the wheat leaf rust fungus in these two species. Received: 7 November 2000 / Accepted: 31 March 2001     

The presence of the enhanced K/Na discrimination trait in diploid Triticum species

Summary A number of accessions of the three species of diploid wheat, Triticum boeoticum, T. monococcum, and T. urartu, were grown in 50 mol m^-3 NaCl+2.5 mol m^-3 CaCl₂. Sodium accumulation in the leaves was low and potassium concentrations remained high. This was not the case in T. durum grown under the same conditions, and indicates the presence in diploid wheats of the enhanced K/Na discrimination character which has previously been found in Aegilops squarrosa and hexaploid wheat. None of the accessions of diploid wheat showed poor K/Na discrimination, which suggests that if the A genome of modern tetraploid wheats was derived from a diploid Triticum species, then the enhanced K/Na discrimination character became altered after the formation of the original allopolyploid. Another possibility is that a diploid wheat that did not have the enhanced K/Na discrimination character was involved in the hybridization event which produced tetraploid wheat, and that this diploid is now extinct or has not yet been discovered.     

BAC libraries of Triticum urartu, Aegilops speltoides and Ae. tauschii, the diploid ancestors of polyploid wheat

Triticum urartu, Aegilops speltoides and Ae. tauschii are respectively the immediate diploid sources, or their closest relatives, of the A, B and D genomes of polyploid wheats. Here we report the construction and characterization of arrayed large-insert libraries in a bacterial artificial chromosome (BAC) vector, one for each of these diploid species. The libraries are equivalent to 3.7, 5.4 and 4.1 of the T. urartu, Ae. speltoides, Ae. tauschii genomes, respectively. The predicted levels of genome coverage were confirmed by library hybridization with single-copy genes. The libraries were used to estimate the proportion of known repeated nucleotide sequences and gene content in each genome by BAC-end sequencing. Repeated sequence families previously detected in Triticeae accounted for 57, 61 and 57% of the T. urartu, Ae. speltoides and Ae. tauschii genomes, and coding regions accounted for 5.8, 4.5 and 4.8%, respectively.     

Additional evidence implicating Triticum searsii as the B-genome donor to wheat

In vitro DNA:DNA hybridizations and hydroxyapatite thermal-elution chromatography were employed to identify the diploid wheat species ancestral to the B genome of Triticum turgidum. ³H-T. turgidum DNA was hybridized to the unlabeled DNAs of T. urartu, T. speltoides, T. sharonensis, T. bicorne, T. longissimum, and T. searsii. ³H-Labeled DNAs of T. monococcum and a synthetic tetraploid AADD were hybridized with unlabeled DNAs of T. urartu and T. searsii to determine the relationship of the A genome of polyploid wheat and T. urartu. The heteroduplex thermal stabilities indicated that T. searsii was most closely related to the B genome of T. turgidum (AB) and that the genome of T. urartu and the A genome have a great deal of base-sequence homology. Thus, it appears that T. searsii is the B-genome donor to polyploid wheat or a major chromosome donor if the B genome is polyphyletic in origin.Published with the approval of the Director of The West Virginia Agricultural Experiment Station as Scientific Paper No. 1837.     

NADP-dependent aromatic alcohol dehydrogenase in polyploid wheats and their diploid relatives. On the origin and phylogeny of polyploid wheats

Summary The three major isoenzymes of the NADP-dependent aromatic alcohol dehydrogenase (ADH-B), distinguished in polyploid wheats by means of polyacrylamide gel electrophoresis, are shown to be coded by homoeoalleles of the locus Adh-2 on short arms of chromosomes of the fifth homoeologous group. Essentially codominant expression of the Adh-2 homoeolleles of composite genomes was observed in young seedlings of hexaploid wheats (T. aestivum s.l.) and tetraploid wheats of the emmer group (T. turgidum s.l.), whereas only the isoenzyme characteristic of the A genome is present in the seedlings of the timopheevii-group tetraploids (T. timopheevii s.str. and T. araraticum).The slowest-moving B³ isoenzyme of polyploid wheats, coded by the homoeoallele of the B genome, is characteristic of the diploid species Aegilops speltoides S.l., including both its awned and awnless forms, but was not encountered in Ae. bicornis, Ae. sharonensis and Ae. longissima. The last two diploids, as well as Ae. tauschii, Ae. caudata, Triticum monococcum s.str., T. boeoticum s.l. (incl. T. thaoudar) and T. urartu all shared a common isoenzyme coinciding electrophoretically with the band B² controlled by the A and D genome homoeoalleles in polyploid wheats. Ae. bicomis is characterized by the slowest isoenzyme, B⁴, not found in wheats and in the other diploid Aegilops species studied.Two electrophoretic variants of ADH-B, B¹ and B², considered to be alloenzymes of the A genome homoeoallele, were observed in T. dicoccoides, T. dicoccon, T. turgidum. s.str. and T. spelta, whereas B² was characteristic of T. timopheevii s.l. and only B¹ was found in the remaining taxa of polyploid wheats. The isoenzyme B¹, not encountered among diploid species, is considered to be a mutational derivative which arose on the tetraploid level from its more ancestral form B² characteristic of diploid wheats.The implication of the ADH-B isoenzyme data to the problems of wheat phylogeny and gene evolution is discussed.     

Comparative and evolutionary analysis of new variants of ω-gliadin genes from three A-genome diploid wheats

A genomic polymerase chain reaction (PCR) cloning strategy was applied to isolate ω-gliadin sequences from three A-genome diploid wheats (Triticum monococcum, T. boeoticum and T. urartu). Amplicon lengths varied from 744 and 1,044 bp, and those of the corresponding deduced mature proteins from 248 to 348 residues. The primary structure of the deduced polypeptides comprised a short N- and C-terminal conserved domain, and a long, variable repetitive domain. A phylogenetic analysis recognised several clades: the first consisted of three T. aestivum sequences; the second and the third two T. boeoticum and six T. monococcum sequences; and the rest four T. urartu and three T. aestivum sequences. Among the functional (non-pseudogene) ARQ/E-type ω-gliadin sequences, two were derived from T. boeoticum and three from T. monococcum; one of the latter sequences appeared to be a chimera originating via illegitimate recombination between the other two T. monococcum sequences. None of the 12 intact ω-gliadin sequences contained any cysteine or methionine residues. We discussed the variation and evolution of A-genome ω-gliadin genes.     

Genetic diversity in wild diploid wheats Triticum monococcum var. boeoticum and T. urartu (Poaceae)

Summary The genetic diversity of two wild diploid wheat species, Triticum monococcum var. boeoticum and T. urartu, was assessed using starch gel electrophoresis. Genetic diversity is uniformly low in both species. Number of alleles per locus was very low with a mean of 1.22 for T. monococcum var. boeoticum and 1.19 in T. urartu. Percentage of polymorphic loci was also low, with a mean of 19.71 for T. monococcum var. boeoticum and a mean of 18.35 for T. urartu. Mean gene diversity was low with a mean of 0.052 in populations of T. monococcum var. boeoticum and a mean of 0.040 in populations of T. urartu. Genetic affinities of the species and of populations were computed using Nei's identity index (NI). Overall genetic affinities of the two species are NI=0.697. The genetic affinities of different populations of a species are uniformly high with NIs ranging from 0.894 to 1.000 in T. monococcum var. boeoticum and from 0.898 to 1.000 in T. urartu.Research supported by the California Agricultural Experiment Station and the International Board of Plant Genetic Resources     

Polyphenol oxidase (PPO) in wheat and wild relatives: molecular evidence for a multigene family

Wheat polyphenol oxidase (PPO) is the major cause of browning reactions that discolor Asian noodles and other wheat products. It has been hypothesized that genes encoding wheat PPOs may have evolved by gene duplication into a multigene family. Here we characterized PPO genomic sequences from diploid (Triticum monococcum, T. urartu, Aegilops tauschii, and Ae. speltoides), tetraploid (T. turgidum, subspecies dicoccoides and durum) and hexaploid (T. aestivum cultivars Klasic and ID377s) wheat species to gain a better understanding of the structure and organization of PPO genes. DNA fragments were amplified from a highly polymorphic and phylogenetic informative region of the gene. As a result, we obtained highly discriminative sequences. Three distinct PPOs, obtained from the A genome of T. monococcum, provided evidence for gene duplication events (paralogous loci). Furthermore, the number of sequences obtained for bread and durum wheat was higher than the expected number of orthologous loci. Sequence comparison revealed nucleotide and structural diversity, and detected five sequence intron types, all with a common insertion position. This was hypothesized to be homologous to that of intron 2 of previously reported wheat PPOs. A MITE of the Stowaway family accounted for the major difference between the five intervening sequences, and was unique to T. aestivum cv. Klasic. Nucleotide and structural diversity, together with well-resolved phylogenetic trees, provided molecular evidence to support the hypothesis of a PPO multigene family structure and organization. Mention of trademark or proprietary products does not constitute a guarantee or warranty of a product by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. This article is in the public domain and not copyrightable.     

New 18S·26S ribosomal RNA gene loci: chromosomal landmarks for the evolution of polyploid wheats

Three new 18S·26S rRNA gene loci were identified in common wheat by sequential N-banding and in situ hybridization (ISH) analysis. Locus Nor-A7 is located at the terminal area of the long arm of 5A in both diploid and polyploid wheats. Locus Nor-B6 is located in N-band 1BL2.5 of the long arm of chromosome 1B in Triticum turgidum and Triticum aestivum. ISH sites, similar to Nor-B6, were also detected on the long arms of chromosomes 1G in Triticum timopheevii and 1S in Aegilops speltoides, but their locations on the chromosomes were different from that of Nor-B6, indicating possible chromosome rearrangements in 1GL and 1BL during evolution. The third new locus, Nor-D8, was only found on the short arm of chromosome 3D in the common wheat Wichita. The loss of rRNA gene locus Nor-A3 and gain of repetitive DNA sequence pSc119 on the terminal part of 5AS suggest a structural modification of 5AS. Comparative studies of the location of the 18S·26S rRNA gene loci in polyploid wheats and putative A and B (G) genome progenitor species support the idea that: (1) Triticum monococcum subsp. urartu is the donor of both the A and A^t genome of polyploid wheats. (2) Ae. speltoides is closer to the B and G genome of polyploid wheats than Aegilops longissima and is the most probable progenitor of these two genomes.     

Length variation of i-type low-molecular-weight glutenin subunit genes in diploid wheats

Allelic variation of the low-molecular-weight glutenin subunit (LMW-GS) is associated with the significant differences of dough quality in bread and durum wheat, and has been widely evaluated at protein level in wheat and its relatives. In this study, a PCR primer set, targeting the high variable repetitive domains, was employed to assay the length variation of i-type LMW-GS genes in the A-genomes of diploid wheats, the diploid progenitors of tetraploid and hexaploid wheat. A total of 71 accessions of diploid wheats, belonging to two wild and one cultivated species, were investigated. The higher variations of repetitive length in i-type LMW-GS genes were found in diploid wheats with Nei’s genetic variation index (H) of 0.834. The two wild species, T. boeoticum and T. urartu, were found to possess the similar degree of variability, with the Nei’s genetic variation index of 0.806 and 0.783, respectively. Less variation was detected in T. monococcum (H = 0.680), a cultivated species domesticated from T. boeoticum. The sufficient variation found in this study could be used as valuable source for the enrichment of genetic variations and the alteration of flour-processing properties of the cultivated wheat. To our knowledge, it was the first time that an analysis of length variation targeting a particular group of genes of LMW-GS complex multigene families was conducted. This article was submitted by the authors in English.     

Quantification and organization of WIS2-1A and BARE-1 retrotransposons in different genomes of <Emphasis Type="Italic">Triticum</Emphasis> and <Emphasis Type="Italic">Aegilops</Emphasis> species

A real-time PCR approach was adopted and optimized to estimate and compare, through a relative quantification, the copy number of WIS2-1A and BARE-1 retrotransposons. The aim of this approach was to identify and quantify the presence of these retrotransposons in Triticum and Aegilops species, and to understand better the genome organization of these retroelements. The species were selected to assess and compare the evolution of the different types of genomes between the more recent species such as the diploid Triticum monococcum, tetraploid T. dicoccon and hexaploid T. spelta, and the corresponding genome donors of the ancient diploids Aegilops (Ae. speltoides, Ae. tauschii, Ae. sharonensis and Ae. bicornis) and T. urartu. The results of this study indicated the presence of great variation in copy number both within and among species, and the existence of a non-linear relationship between retrotransposon copy number and ploidy level. For WIS2-1A, as expected, T. monococcum showed the lowest copy number which instead was similar in T. dicoccon and T. spelta; also T. urartu (AA), Ae. speltoides (BB) and Ae. tauschii (DD) showed a higher WIS2-1A copy number. Similar results were observed for BARE-1 retroelements except for Ae. tauschii which as in T. monococcum showed lower retroelements content; a similar content for T. dicoccon and T. urartu, whereas a higher number was found in T. spelta and Ae. speltoides. The results presented here are in accord with previous studies and contribute to unravelling the structure and evolution of polyploidy and repetitive genomes.     

Chromosomal location of genes for Rubisco small subunit and Rubisco-binding protein in common wheat

Summary The genes coding for the Rubisco small subunit (SSU) and for the -subunit of the Rubisco-binding protein were located to chromosome arms of common wheat. HindIII-digested total DNA from the hexaploid cultivar Chinese Spring and from ditelosomic and nullisomic-tetrasomic lines was probed with these two genes, whose chromosomal location was deduced from the disappearance of or from changes in the relative intensity of the relevant band(s). The Rubisco SSU pattern consisted of 14 bands, containing at least 21 different types of DNA fragments, which were allocated to two homoeologous groups: 15 to the short arm of group 2 chromosomes (4 to 2AS, 7 to 2BS, and 4 to 2DS) and 6 to the long arm of group 5 chromosomes (2 on each of arms 5AL, 5BL, and 5DL). The pattern of the Rubisco-binding protein consisted of three bands, each containing one type of fragment. These fragments were located to be on the short arm of group 2 chromosomes. The restriction fragment length polymorphism (RFLP) patterns of several hexaploid and tetraploid lines were highly conserved, whereas the patterns of several of their diploid progenitors were more variable. The variations found in the polyploid species were mainly confined to the B genome. The patterns of the diploids T. monococcum var. urartu and Ae. squarrosa were similar to those of the A and D genome, respectively, in polyploid wheats. The pattern of T. monococcum var. boeoticum was different from the patterns of the A genome, and the patterns of the diploids Ae. speltoides, Ae. longissima, and Ae. Searsii differed from that of the B genome.