Amylase protein inhibitors and the role of Aegilops species in polyploid wheat speciation

Protein inhibitors extracted with water from seeds of Triticum and genetically related species were characterized according to their apparent molecular weights, electrophoretic mobilities and their specificities in inhibiting α-amylases from human saliva and Tenebrio molitor L. larvae. No detectable amylase inhibition activity was found in extracts from diploid wheats, whereas in all tetraploid and hexaploid wheats as well as in the Aegilops species tested we found several amylase inhibitor groups of different molecular weights. In each group, several inhibitor components slightly different in their electrophoretic mobilities, but identical in their inhibition behaviour toward amylases from different origins have been shown. Both from the qualitative and quantitative standpoints, amylase protein inhibitors from hexaploid wheats were the summation of those from tetraploid wheats plus the ones from Aegilops squarrosa. Amylase inhibitors from Aegilops speltoides largely differed from those extracted from tetraploid wheats as well as from all the amylase inhibitors described in plant seeds up to now. These results indicate a relevant homology between the amylase inhibitor coding genes of the D wheat genome and those of the D Aegilops genome and confirm that Ae. squarrosa is the donor of the whole D genome to hexaploid wheats. They also suggest that Ae. speltoides is not the donor of the B genome to polyploid wheats, although a not yet identified Aegilops species might be such a donor.     

High-resolution organellar genome analysis of Triticum and Aegilops sheds new light on cytoplasm evolution in wheat

We have utilised polymorphic chloroplast microsatellites to analyse cytoplasmic relationships between accessions in the genera Triticum and Aegilops. Sequencing of PCR products revealed point mutations and insertions/deletions in addition to the standard repeat length expansion/contraction which most likely represent ancient synapomorphies. Phylogenetic analyses revealed three distinct groups of accessions. One of these contained all the non-Aegilops speltoides S-type cytoplasm species, another comprised almost exclusively A, C, D, M, N, T and U cytoplasm-type accessions and the third contained the polyploid Triticum species and all the Ae. speltoides accessions, further confirming that Ae. speltoides or a closely related but now extinct species was the original B-genome donor of cultivated polyploid wheat. Successive decreases in levels of genetic diversity due to domestication were also observed. Finally, we highlight the importance of elucidating longer-term evolutionary processes operating at microsatellite repeat loci.Communicated by J.S. Heslop-Harrison     

Molecular phylogeny of the genus Triticum L

The genus Triticum L. includes the major cereal crop, common or bread wheat (hexaploid Triticum aestivum L.), and other important cultivated species. Here, we conducted a phylogenetic analysis of all known wheat species and the closely related Aegilops species. This analysis was based on chloroplast matK gene comparison along with trnL intron sequences of some species. Polyploid wheat species are successfully divided only into two groups – Emmer (sections Dicoccoides and Triticum) and Timopheevii (section Timopheevii). Results reveal strictly maternal plastid inheritance of synthetic wheat amphiploids included in the study. A concordance of chloroplast origin with the definite nuclear genomes of polyploid species that were inherited at the last hybridization events was found. Our analysis suggests that there were two ancestral representatives of Aegilops speltoides Tausch that participated in the speciation of polyploid wheats with B and G genome in their genome composition. However, G genome species are younger in evolution than ones with B genome. B genome-specific PCR primers were developed for amplification of Acc-1 gene.     

Phylogenetic analysis of the dimeric alpha-amylase inhibitor sequences from an orthologous region in 21 different genomes of the tribe Triticeae (Poaceae)

Six hundred and thirty gene sequences from 21 different genomes in Triticeae tribe were obtained and subjected to phylogenetic analysis. The sequences showed high homology in both nucleotide sequences and length variation, and had a common conserved cysteine skeleton C–Xn–C–Xn–C–Xn–CC–Xn–C–X–C–Xn–C–Xn–C–Xn–C. The sequences from common wheat formed three clusters; two were close to Aegilops tauschii and Aegilops speltoides sequences, respectively, and the third cluster was complex with sequences from Ae. speltoides, Aegilops searsii, and Aegilops bicornis. Different S genome(s) of Aegilops contributed α-amylase inhibitor loci to polyploid wheat by gene introgression in interspecific hybridizations. No sequence from common wheat was similar to that from einkorn wheat. We conclude that the occurrence of multiple chromosomal translocations or inversions in the different genomes of Triticeae had not dramatically affected the primary structure of dimeric α-amylase inhibitors. The results revealed important information on genome shaping events and processes occurring at the dimeric α-amylase inhibitor genes loci and their bearing on the phylogenetic relationships in the tribe Triticeae (Poaceae).     

Ecological distribution and species diversity of Aegilops L. genus in Bulgaria

The genus Aegilops has an important potential utilization in wheat improvement because of its resistance to different biotic and abiotic stresses and close relation with the cultivated wheat. Therefore, a better knowledge of the eco-geographical distribution of Aegilops species and their collection and conservation are required. A total of 297 Aegilops accessions representing nine (five tetraploid and four diploid) species were collected in different regions of Bulgaria, and the ecological characteristics of the 154 explored sites were recorded. The distribution of the diploid species (Ae. caudata L., Ae. speltoides Tausch, Ae. umbellulata Zhuk. and Ae. comosa Sibth. and Sm.) was limited to specific environments in south-central Bulgaria. Tetraploid species were present in harsher environments than diploid species and showed wider adaptation and distribution. Species–environment relationships were analysed by considering the worldwide distribution of the species and their physiological resistance to abiotic stress. Aegilops cylindrica Host was more frequently found in northern Bulgaria and at high altitudes. Its distribution was closely related to its tolerance to low temperatures. Aegilops geniculata Roth and Ae. neglecta Req. ex Bertol. were absent in the north of Bulgaria, but widely distributed in low rainfall areas. Aegilops neglecta, more frost resistant than Ae. geniculata, was present at higher altitude. Aegilops biuncialis Vis. and Ae. triuncialis L. showed adaptation to a wide range of climatic conditions. The study of Aegilops species ecology and distribution in Bulgaria provided useful information for the future collection and for the genetic resource management in this region.     

A comparative analysis of the composition and organization of two subtelomeric repeat families in <Emphasis Type="Italic">Aegilops speltoides</Emphasis>Tausch. and related species

The structural organization and evolution of two tandemly repeated families, Spelt1 and Spelt52, located in the subtelomeric regions of Aegilops speltoides chromosomes were studied. The Spelt1 family of sequences with a monomer length of 178 bp was characterized by cloning and sequence analysis of polymerase chain reaction (PCR) products. Members of the Spelt1 family revealed sequence similarities exceeding 95\%. This conservation has remained despite divergence of species in Aegilops section Sitopsis and after independent multiple amplification events in the genome of Ae. speltoides. Sequences representing the Spelt52 family were cloned, sequenced and compared with other sequences in databases. The Spelt52 repeat family contains monomers of two types, Spelt52.1 and Spelt52.2. The two monomers share a homologous stretch of 280 bp and have two regions without sequence similarity of 96 bp and 110 bp, respectively. PCR analysis was conducted to 15 lines in Ae. speltoidesTausch., Ae. longissimaSchw.&Mushc.,Ae. sharonensisEig.,Ae. bicornis(Forssk)Jaub.&Sp., andAe. searsii Feld.&Kis. using primers to the homologous and non- homologous regions of Spelt52 family. Intraspecies and interspecies differences in the occurrence and abundance of combinations of Spelt52.1 and Spelt52.2 monomers were detected. The use of primers to telomeric and subtelomeric repeats followed by Southern hybridization, cloning, and sequence analysis demonstrated that Spelt1 and Spelt52 are localized close to each other and to telomeric repeats. The efficiency of a PCR approach for the analysis of telomeric/subtelomeric junction regions of chromosomes is discussed.     

Aegilops (Poaceae) in Italy: taxonomy,geographical distribution,ecology, vulnerability and conservation

ABSTRACT

Aegilops L. includes wild species from which, over millennia, man has cultivated forms of Triticum L. Ten species of Aegilops occur in Italy. Three species are allochthonous and eight out of ten species are recorded in the Apulia region. Five out of the ten species have been included in Red Lists. Each taxon is presented and discussed, citing old and new sites of occurrence, by examining specimens from many different herbaria, and describing their ecology and habitats, according to the Directive 92/43 EEC. A new taxonomic key, for the identification of all Aegilops species growing in Italy, is provided. The occurrence of Aegilops caudata L., A. peregrina (Hack. in J. Fraser) Maire & Weiller and A. speltoides Tausch in Italy is doubtful.     

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.     

Restriction Fragment Length Polymorphism (RFLP) for protein disulfide isomerase (PDI) gene sequences in Triticum and Aegilops species

RFLP variation revealed by protein disulfide isomerase (PDI) coding gene sequences was assessed in 170 accessions belonging to 23 species of Triticum and Aegilops. PDI restriction fragments were highly conserved within each species and confirmed that plant PDI is encoded either by single-copy sequences or by small gene families. The wheat PDI probe hybridized to single EcoRI or HindIII fragments in different diploid species and to one or two fragments per genome in polyploids. Four Aegilops species in the Sitopsis section showed complex patterns and high levels of intraspecific variation, whereas Ae. searsii possessed single monomorphic fragments. T. urartu and Ae. squarrosa showed fragments with the same mobility as those in the A and D genomes of Triticum polyploid species, respectively, whereas differences were observed between the hybridization patterns of T. monococcum and T. boeoticum and that of the A genome. The single fragment detected in Ae. squarrosa was also conserved in most accessions of polyploid Aegilops species carrying the D genome. The five species of the Sitopsis section showed variation for the PDI hybridization fragments and differed from those of the B and G genomes of emmer and timopheevi groups of wheat, although one of the Ae. speltoides EcoRI fragments was similar to those located on the 4B and 4G chromosomes. The similarity between the EcoRI fragment located on the 1B chromosome of common and emmer wheats and one with a lower hybridization intensity in Ae. longissima, Ae. bicornis and Ae. sharonensis support the hypothesis of a polyphyletic origin of the B genome. Received: 25 June 1999 / Accepted: 14 September 1999     

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.     

Construction and study of leaf rust-resistant common wheat lines with translocations of <Emphasis Type="Italic">Aegilops speltoides</Emphasis> Tausch. Genetic material

Genotyping was performed for the leaf rust-resistant line 73/00i (Triticum aestivum × Aegilops speltoides). Fluorescence in situ hybridization (FISH) with probes Spelt1 and pSc119.2 in combination with microsatellite analysis were used to determine the locations and sizes of the Ae. speltoides genetic fragments integrated into the line genome. Translocations were identified in the long arms of chromosomes 5B and 6B and in the short arm of chromosome 1B. The Spelt1 and pSc119.2 molecular cytological markers made it possible to rapidly establish lines with single translocation in the long arms of chromosomes 5B and 6B. The line carrying the T5BS · 5BL-5SL translocation was highly resistant to leaf rust, and the lines carrying the T6BS · 6BL-6SL translocation displayed moderate resistance. The translocations differed in chromosomal location from known leaf resistance genes transferred into common wheat from Ae. speltoides. Hence, it was assumed that new genes were introduced into the common wheat genome from Ae. speltoides. The locus that determined high resistance to leaf rust and was transferred into the common wheat genome from the long arm of Ae. speltoides chromosome 5S by the T5BS · 5BL-5SL translocation was preliminarily designated as LrAsp5.     

CHy-banding patterns and chromatin organization inAegilops andTriticum species (Poaceae)

The distribution of CHy-banded heterochromatin was studied in the chromosomes ofAegilops longissima, Ae. speltoides, Triticum monococcum, andT. turgidum. Interphase nuclei were measured after Feulgen staining at different thresholds of optical density; the curves so obtained indicated the relationship among the species with respect to the different fractions of the genomic DNA. The karyological and cytophotometric analyses indicate differences betweenAe. speltoides andAe. longissima, the latter species being enriched in heterochromatin. Similar results were demonstrated for the genusTriticum, in whichT. turgidum showed more heterochromatin when compared withT. monococcum. The results suggest that the B genome of the cultivated wheats possesses a type of heterochromatin that resembles the type present inAe. longissima.     

RAPD analysis of the intraspecific and interspecific variation and phylogenetic relationships of <Emphasis Type="Italic">Aegilops</Emphasis> L. species with the U genome

RAPD analysis was used to study the genetic variation and phylogenetic relationships of polyploid Aegilops species with the U genome. In total, 115 DNA samples of eight polyploid species containing the U genome and the diploid species Ae. umbellulata (U) were examined. Substantial interspecific polymorphism was observed for the majority of the polyploid species with the U genome (interspecific differences, 0.01–0,2; proportion of polymorphic loci, 56.6–88.2%). Aegilops triuncialis was identified as the only alloploid species with low interspecific polymorphism (interspecific differences, 0–0.01, P = 50%) in the U-genome group. The U-genome Aegilops species proved to be separated from other species of the genus. The phylogenetic relationships were established for the U-genome species. The greatest separation within the U-genome group was observed for the US-genome species Ae. kotschyi and Ae. variabilis. The tetraploid species Ae. triaristata and Ae. columnaris, which had the UX genome, and the hexaploid species Ae. recta (UXN) were found to be related to each other and separate from the UM-genome species. A similarity was observed between the UM-genome species Ae. ovata and Ae. biuncialis, which had the UM genome, and the ancestral diploid U-genome species Ae. umbellulata. The UC-genome species Ae. triuncialis was rather separate and slightly similar to the UX-genome species.     

Homoeologous relationships of Aegilops speltoides chromosomes to bread wheat

 Homoeologous pairing at metaphase I was analysed in the standard-type, ph2b and ph1b hybrids of Triticum aestivum (AABBDD) and Aegilops speltoides (SS). Data from relative pairing affinities were used to predict homoeologous relationships of Ae. speltoides chromosomes to wheat. Chromosomes of both species, and their arms, were identified by C-banding. The Ae. speltoides genotype carried genes that induced a high level of homoeologous pairing in the three types of hybrids analyzed. All arms of the seven chromosomes of the S genome showed normal homoeologous pairing, which implies that no apparent chromosome rearrangements occurred in the evolution of Ae. speltoides relative to wheat. A pattern of preferential pairing of two types, A-D and B-S, confirmed that the S genome is very closely related to the B genome of wheat. Although this pairing pattern was also reported in hybrids of wheat with Ae. longissima and Ae. sharonensis, a different behaviour was found in group 5 chromosomes. In the hybrids of Ae. speltoides, chromosome 5B-5S pairing was much more frequent than 5D-5S, while these chromosome associations reached similar frequencies in the hybrids of Ae. longissima and Ae. sharonensis. These results are in agreement with the hypothesis that the B genome of wheat is derived from Ae. speltoides. Received: 8 January 1998 / Accepted: 4 February 1998     

Syntenic Relationships between the U and M Genomes of Aegilops,Wheat and the Model Species Brachypodium and Rice as Revealed by COS Markers

Diploid Aegilops umbellulata and Ae. comosa and their natural allotetraploid hybrids Ae. biuncialis and Ae. geniculata are important wild gene sources for wheat. With the aim of assisting in alien gene transfer, this study provides gene-based conserved orthologous set (COS) markers for the U and M genome chromosomes. Out of the 140 markers tested on a series of wheat-Aegilops chromosome introgression lines and flow-sorted subgenomic chromosome fractions, 100 were assigned to Aegilops chromosomes and six and seven duplications were identified in the U and M genomes, respectively. The marker-specific EST sequences were BLAST-ed to Brachypodium and rice genomic sequences to investigate macrosyntenic relationships between the U and M genomes of Aegilops, wheat and the model species. Five syntenic regions of Brachypodium identified genome rearrangements differentiating the U genome from the M genome and from the D genome of wheat. All of them seem to have evolved at the diploid level and to have been modified differentially in the polyploid species Ae. biuncialis and Ae. geniculata. A certain level of wheat–Aegilops homology was detected for group 1, 2, 3 and 5 chromosomes, while a clearly rearranged structure was showed for the group 4, 6 and 7 Aegilops chromosomes relative to wheat. The conserved orthologous set markers assigned to Aegilops chromosomes promise to accelerate gene introgression by facilitating the identification of alien chromatin. The syntenic relationships between the Aegilops species, wheat and model species will facilitate the targeted development of new markers specific for U and M genomic regions and will contribute to the understanding of molecular processes related to allopolyploidization.     

Electrophoretic and molecular analysis of alpha-gliadins inAegilops species (Poaceae) belonging to the D genome cluster and in their putative progenitors

The D genome cluster includes six allopolyploidAegilops species having as pivotal genome that ofAegilops squarrosa. Alpha-gliadins, endosperm proteins coded by multigenic families, have been analyzed in the D genome species cluster and in their putative progenitors. They can be present or weakly expressed when analyzed in acid polyacrylamide gel electrophoresis. Molecular analysis has shown the possibility to distinguish subsp.strangulata from subsp.eusquarrosa and to confirm the presence ofAe. caudata and ofAe. umbellulata in the polyploidsAe. cylindrica andAe. juvenalis, respectively. Finally, introgression fromAe. longissima orAe. searsii in tetraploid and hexaploidAe. crassa, Ae. juvenalis, andAe. vavilovii is supposed.     

Molecular analysis of the phylogenetic relationships among the diploid <Emphasis Type="Italic">Aegilops</Emphasis> species of the section <Emphasis Type="Italic">Sitopsis</Emphasis>

RAPD analysis was used to study the intraspecific variation and phylogenetic relationships of Sgenome 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.     

Species relationships and genetic variation in the diploid wheats (Triticum,Aegilops) as revealed by starch gel electrophoresis

Twenty enzyme loci were examined in the diploid species ofTriticum andAegilops for allelic variation by starch gel electrophoresis. SectionSitopsis, including the five species,Ae. speltoides, Ae. lingissima, Ae. sharonensis, Ae. bicornis andAe. searsii form a close subgroup withAe. speltoides slightly removed from the others.T. monococcum s. lat., was found to be closest to the species of theSitopsis group.Ae. comosa, Ae. umbellulata andAe. uniaristata form a second subgroup withAe. caudata most closely related to these species.Ae. squarrosa appears almost equally related to all of the species, showing no special affinity for any one species group. Nineteen out of twenty loci examined were polymorphic with a mean of 6.7 alleles per locus. Species could be, for most loci, characterized by the presence of predominant alleles. A conspicious genetic characteristic ofTriticum-Aegilops is the sharing of these predominant alleles between species. Within species variation is characterized by a diffuse distribution of secondary alleles.     

Comparative genetic maps reveal extreme crossover localization in the Aegilops speltoides chromosomes

A total of 137 loci were mapped in Aegilops speltoides, the closest extant relative of the wheat B genome, using two F₂ mapping populations and a set of wheat-Ae. speltoides disomic addition (DA) lines. Comparisons of Ae. speltoides genetic maps with those of Triticum monococcum indicated that Ae. speltoides conserved the gross chromosome structure observed across the tribe Triticeae. A putative inversion involving the short arm of chromosome 2 was detected in Ae. speltoides. A translocation between chromosomes 2 and 6, present in the wheat B genome, was absent. The ligustica/aucheri spike dimorphism behaved as allelic variation at a single locus, which was mapped in the centromeric region of chromosome 3. The genetic length of each chromosome arm was about 50 cM, irrespective of its physical length. Compared to T. monococcum genetic maps, recombination was virtually eliminated from the proximal 50–100 cM and was localized in short distal regions, which were often expanded compared to the T. monococcum maps. The wheat B genome and the genome of Ae. longissima, a close relative of Ae. speltoides, do not show the extreme localization of crossovers observed in Ae. speltoides.     

Morphological Variability of Wheat Powdery Mildew in the Context of Its Parasitic Adaptation to Wheat–Aegilops Lines with Different Resistance

The interaction between the pathogen and wheat–Aegilops lines with different resistance as well as their parental forms in the course of powdery mildew infection was studied using scanning electron microscopy. The course of infection in the line 51/99i and its parental form, the Rodina variety, proved to be similar. The plants of both genotypes featured pronounced adhesion of the primary infection structures to the surface of plant epidermal cells and the formation of large, well-developed colonies of the fungus, which was evidence for parasite–host compatibility. The development of powdery mildew on the line 135/99i was similar to that on the parental form Aegilops speltoides K-389. The pathogen–host interaction was characterized by a longer incubation period. Sparse fungal colonies were formed from mycelial hyphae with multiple hyphal lobes, and their adhesion to the surface of the epidermal cells was disturbed in most cases. Such a pattern of pathogen development indicated that the host plants had some resistance factors operating mainly at the level of pathogen penetration. The types of resistance in lines 95/99i and 56/99i were not characteristic of the parental form Ae. speltoides K-389, but they were described for Ae. speltoides samples from other natural ranges (Ryabchenko et al., 2002). This fact suggests that the immune potential of Ae. speltoides as a species is polygenic, and its elements can be transmitted to hybrids irrespective of concrete plants used as the donors of resistance.