用ITS序列确定小麦B基因组的可能供体间的关系

对小麦B基因组的可能供体山羊草属Aegilops sect．Sitopsis的5个种的核糖体DNA的内部转 录区(ITS)进行了PCR扩增和克隆,井测定ITSl和ITS2的序列,用ITSl+ITS2的序列重建了 Aegilops sect．Sitopsis中5个种的系统发育关系。结果表明,斯卑尔脱山羊草Ae．speltoides是sect． Sitopsis中特殊的一个种,它与该组其余4种间的平均遗传距离是后者彼此间平均遗传距离的3倍,Ae． speltoides与同组其余4个种的分离要比后者相互间的分离早得多;在拟斯卑尔脱组Sect．Sitopsis的5 个种中,长柱山羊草Ae．longissima与沙融山羊草Ae．sharonensis的关系最近。ITS序列可以进一步用来作为确定B基因组起源的分子标记。     

Transferability of wheat microsatellites to diploid Aegilops species and determination of chromosomal localizations of microsatellites in the S genome.

Overall, 253 genomic wheat (Triticum aestivum) microsatellite markers were studied for their transferability to the diploid species Aegilops speltoides, Aegilops longissima, and Aegilops searsii, representing the S genome. In total, 88% of all the analyzed primer pairs of markers derived from the B genome of hexaploid wheat amplified DNA fragments in the genomes of the studied species. The transferability of simple sequence repeat (SSR) markers of the T. aestivum A and D genomes totaled 74%. Triticum aestivum-Ae. speltoides, T. aestivum-Ae. longissima, and T. aestivum-Ae. searsii chromosome addition lines allowed us to determine the chromosomal localizations of 103 microsatellite markers in the Aegilops genomes. The majority of them were localized to homoeologous chromosomes in the genome of Aegilops. Several instances of nonhomoeologous localization of T. aestivum SSR markers in the Aegilops genome were considered to be either amplification of other loci or putative translocations. The results of microsatellite analysis were used to study phylogenetic relationships among the 3 species of the Sitopsis section (Ae. speltoides, Ae. longissima, and Ae. searsii) and T. aestivum. The dendrogram obtained generally reflects the current views on phylogenetic relationships among these species.     

The phylogenetic relationships among the possible donors of B-genome of common wheat based on internal transcribed spacer (ITS) sequences

The ITS regions of 5 species in Aegilops sect. Sitopsis, the possible donors of Bgenome of common wheat, were amplified by PCR, cloned and sequenced. The phylogenetic relationships among 5 species in Aegilops sect. Sitopsis were constructed based on ITS1 + ITS2 sequences. The results demonstrated that Ae. speltoides was a distinct species in Aegilops sect. Sitopsis. The average of the pairwise distances between Ae. speltoides and the other four species was three times as high as that among the latter four. Ae. speltoides was the earliest lineage of the section under question. Relationship between Ae. longissima and Ae. sharonensis was the closest in Aegilops sect. Sitopsis. Sequence of ITS regioncould be used as a molecular marker to identify origin of B-genome in polyploid wheats.     

Diversity of long terminal repeat retrotransposon genome distribution in natural populations of the wild diploid wheat Aegilops speltoides

The environment can have a decisive influence on the structure of the genome, changing it in a certain direction. Therefore, the genomic distribution of environmentally sensitive transposable elements may vary measurably across a species area. In the present research, we aimed to detect and evaluate the level of LTR retrotransposon intraspecific variability in Aegilops speltoides (2n = 2x = 14), a wild cross-pollinated relative of cultivated wheat. The interretrotransposon amplified polymorphism (IRAP) protocol was applied to detect and evaluate the level of retrotransposon intraspecific variability in Ae. speltoides and closely related species. IRAP analysis revealed significant diversity in TE distribution. Various genotypes from the 13 explored populations significantly differ with respect to the patterns of the four explored LTR retrotransposons (WIS2, Wilma, Daniela, and Fatima). This diversity points to a constant ongoing process of LTR retrotransposon fraction restructuring in populations of Ae. speltoides throughout the species' range and within single populations in time. Maximum changes were recorded in genotypes from small stressed populations. Principal component analysis showed that the dynamics of the Fatima element significantly differ from those of WIS2, Wilma, and Daniela. In terms of relationships between Sitopsis species, IRAP analysis revealed a grouping with Ae. sharonensis and Ae. longissima forming a separate unit, Ae. speltoides appearing as a dispersed group, and Ae. bicornis being in an intermediate position. IRAP display data revealed dynamic changes in LTR retrotransposon fractions in the genome of Ae. speltoides. The process is permanent and population specific, ultimately leading to the separation of small stressed populations from the main group.     

Phylogenetic reconstruction of Aegilops section Sitopsis and the evolution of tandem repeats in the diploids and derived wheat polyploids.

The evolution of 2 tandemly repeated sequences Spelt1 and Spelt52 was studied in Triticum species representing 2 evolutionary lineages of wheat and in Aegilops sect. Sitopsis, putative donors of their B/G genomes. Using fluorescence in situ hybridization we observed considerable polymorphisms in the hybridization patterns of Spelt1 and Spelt52 repeats between and within Triticum and Aegilops species. Between 2 and 28 subtelomeric sites of Spelt1 probe were detected in Ae. speltoidies, depending on accession. From 8 to 12 Spelt1 subtelomeric sites were observed in species of Timopheevi group (GAt genome), whereas the number of signals in emmer/aestivum accessions was significantly less (from 0 to 6). Hybridization patterns of Spelt52 in Ae. speltoides, Ae. longissima, and Ae. sharonensis were species specific. Subtelomeric sites of Spelt52 repeat were detected only in T. araraticum (T. timopheevii), and their number and chromosomal location varied between accessions. Superimposing copy number data onto our phylogenetic scheme constructed from RAPD data suggests 2 major independent amplifications of Spelt52 and 1 of Spelt1 repeats in Aegilops divergence. It is likely that the Spelt1 amplification took place in the ancient Ae. speltoides before the divergence of polyploid wheats. The Spelt52 repeat was probably amplified in the lineage of Ae. speltoides prior to divergence of the allopolyploid T. timopheevii but after the divergence of T. durum. In a separate amplification event, Spelt52 copy number expanded in the common ancestor of Ae. longissima and Ae. sharonensis.     

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.     

Repetitive DNas of wild emmer wheat (Triticum dicoccoides) and their relation to S-genome species: molecular cytogenetic analysis.

We have analyzed the chromosomal GISH molecular banding patterns of three populations of the wild allopolyploid wheat Triticum dicoccoides in an attempt to unravel the evolutionary relationships between highly repetitive DNA fractions of T. dicoccoides and proposed diploid progenitors of the B genome. Aegilops speltoides showed almost complete affinity of its repetitive DNA to C-heterochromatin of T. dicoccoides, whereas other S-genome species demonstrated relatedness only to distal heterochromatin. This substantiates the priority of Ae. speltoides as the most similar to the wheat B-genome donor in comparison with other Sitopsis species. Using molecular banding technique with DNA of different Aegilops species as a probe permits tracing of the origin of each heterochromatin cluster. Molecular banding analysis reveals polymorphism between three wild emmer wheat populations. Comparison of molecular banding patterns with chromosomal distribution of the Ty1-copia retrotransposons, which constitute a large share of T. dicoccoides genome, makes it possible to propose that the activity of transposable elements may lie in the background of observed intraspecific polymorphism.     

Genetic Diversity of the Cytoplasm in Triticum and Aegilops. VIII. Fraction I Protein of 39 Cytoplasms

The electrophoretic characteristics of the cytoplasmically controlled large subunit of the Fraction I protein of 36 alloplasmic and three euplasmic control lines are reported. These lines, representing the cytoplasms of 32 Triticum and Aegilops species, had either H- or L-type large subunits in their Fraction I protein; the diploid Triticum and most Aegilops species, including Ae. bicornis and Ae. sharonensis, had the L-type subunits; whereas, all the polyploid Triticum species (emmer, timopheevi, common wheats), Ae. speltoides, Ae. aucheri, and Ae. longissima had H-type subunits. Therefore, section Sitopsis of Aegilops exhibits interspecific heterogeneity. The H-type is believed to have originated in the Sitopsis section from an L-type subunit because of the prevalence of the latter among the diploid species.     

Isolation and characterization of S genome specific sequences from Aegilops sect. sitopsis species.

Three S genome specific sequences were isolated from Aegilops sect. sitopsis species using different experimental approaches. Two clones, UTV86 and UTV39, were isolated from a partial genomic library obtained from DNA of Aegilops sharonensis, whereas a third clone, UTV5, was isolated from Aegilops speltoides. The three clones were characterized by sequencing, analysis of methylation, and sequence organization and abundance in some Aegilops and Triticum species. The clones UTV39 and UTV5 belong to the same family of tandem repeated sequences and showed high homology with a sequence already present in nucleotide databases. The UTV86 clone from Ae. sharonensis corresponded to an interspersed low frequency repeated sequence and did not show any significant homology with reported sequences. Southern hybridization experiments, using the cloned sequences as probes, detected polymorphism in the restriction patterns of all the five Aegilops species in section sitopsis. Aegilops speltoides showed the most divergent hybridization pattern. A close relationship was detected between the S genome of Ae. speltoides and the G genome of the wild Triticum timopheevii. In situ hybridization revealed a telomeric and (or) subtelomeric location of the sequences UTV39 and UTV5.     

Intra- and interspecific phylogenetic relationships among diploid Triticum-Aegilops species (Poaceae) based on base-pair substitutions, indels, and microsatellites in chloroplast noncoding sequences

This study analyzes intra- and interspecific variation in chloroplast DNA (cpDNA) in diploid Triticum-Aegilops species. This analysis focused on DNA sequence variation in noncoding regions of cpDNA, which included base-pair substitutions, insertion/deletions (indels, 50 loci pooled), microsatellites (7 loci pooled), and inversions. Nine of 13 Triticum-Aegilops species were successfully identified and genotyped using these data. Sixty-two haplotypes were detected in 115 accessions of 13 diploid species. Because of the large number of characters examined, novel deep relationships within and among Triticum-Aegilops species could be identified and evaluated. Phylogenetic trees for the genus Triticum-Aegilops were constructed with Hordeum vulgare and Dasypyrum villosum as outgroups, and the results were compared to previous studies. These data support the following inferences: (1) Aegilops species should be included in Triticum; (2) groups D, T, M, N, U, and section Sitopsis (except Ae. speltoides) underwent speciation concurrently, but most diploid species evolved independently; (3) Ae. mutica does not occupy a basal position in Triticum-Aegilops; (4) Ae. speltoides is in a basal position and differs significantly from other Sitopsis species; (5) Ae. caudata is polyphyletic in all trees; (6) the genus Aegilops is paraphyletic with Secale.     

Complex genome rearrangements reveal evolutionary dynamics of pericentromeric regions in the Triticeae.

Most pericentromeric regions of eukaryotic chromosomes are heterochromatic and are the most rapidly evolving regions of complex genomes. The closely related genomes within hexaploid wheat (Triticum aestivum L., 2n=6x=42, AABBDD), as well as in the related Triticeae taxa, share large conserved chromosome segments and provide a good model for the study of the evolution of pericentromeric regions. Here we report on the comparative analysis of pericentric inversions in the Triticeae, including Triticum aestivum, Aegilops speltoides, Ae. longissima, Ae. searsii, Hordeum vulgare, Secale cereale, and Agropyron elongatum. Previously, 4 pericentric inversions were identified in the hexaploid wheat cultivar 'Chinese Spring' ('CS') involving chromosomes 2B, 4A, 4B, and 5A. In the present study, 2 additional pericentric inversions were detected in chromosomes 3B and 6B of 'CS' wheat. Only the 3B inversion pre-existed in chromosome 3S, 3Sl, and 3Ss of Aegilops species of the Sitopsis section, the remaining inversions occurring after wheat polyploidization. The translocation T2BS/6BS previously reported in 'CS' was detected in the hexaploid variety 'Wichita' but not in other species of the Triticeae. It appears that the B genome is more prone to genome rearrangements than are the A and D genomes. Five different pericentric inversions were detected in rye chromosomes 3R and 4R, 4Sl of Ae. longissima, 4H of barley, and 6E of Ag. elongatum. This indicates that pericentric regions in the Triticeae, especially those of group 4 chromosomes, are undergoing rapid and recurrent rearrangements.     

The origin of the B-genome of bread wheat (Triticum aestivum L.)

Understanding the origin of cultivated wheats would further their genetic improvement. The hexaploid bread wheat (Triticum aestivum L., AABBDD) is believed to have originated through one or more rare hybridization events between Aegilops tauschii (DD) and the tetraploid T. turgidum (AABB). Progenitor, of the A-genome of the tetraploid and hexaploid wheats has generally been accepted to be T. urartu. In spite of the large number of attempts and published reports about the origin of the B-genome in cultivated wheats, the donor of the B-genome is still relatively unknown and controversial and, hence, remains open. This genome has been found to be closely related to the S-genome of the Sitopsis section (Ae. speltoides, Ae. longissima, Ae. sharonensis, Ae. searsii, and Ae. bicornis) of the genus Aegilops L. Among Sitopsis species, the most positive evidence has been accumulated for Ae. speltoides as the progenitor of the B-genome. Therefore, one or more of the Sitopsis species were proposed frequently as the B-genome donor. Although several reviews have been written on the origin of the genomes of wheat over the years, this paper will attempt for the first time to review the immense literature on the subject, with a particular emphasis on the B-genome which has attracted a huge attention over some 100 years. The ambiguity and conflicting results in most of the methods employed in deducing the precise B-genome donor/s to bread wheat are also discussed.     

The Molecular Basis of Genetic Diversity among Cytoplasms of TRITICUM and AEGILOPS Species. II. on the Origin of Polyploid Wheat Cytoplasms as Suggested by Chloroplast DNA Restriction Fragment Patterns

In attempts to identify the phylogenetic donors of cytoplasm to Emmer-Dinkel and Timopheevi groups of wheat (Triticum), and the Aegilops kotschyi-Ae. variabilis complex, the restriction fragment patterns of chloroplast DNAs of representative species were compared with those of their putative diploid ancestors. The following seven restriction enzymes were used; BamHI, EcoRI, HindIII, KpnI, PstI, SmaI and XhoI. The restriction fragment patterns of an Emmer and a Dinkel (common) wheat were identical with those of Ae. longissima , and different from those of Ae. aucheri, Ae. bicornis, Ae. searsii, Ae. sharonensis, Ae. speltoides, and T. urartu by 4 to 12 fragments. The restriction fragment patterns of a Timopheevi wheat were identical with those of Ae. aucheri, and different from those of all other diploids by four to nine fragments. The restriction fragment patterns of Ae. variabilis were identical to those of Ae. bicornis and Ae. searsii , and different from those of all other species. Thus, we have concluded that Ae. longissima, Ae. aucheri and Ae. bicornis (or Ae. searsii) were the cytoplasm donors to the Emmer-Dinkel and the Timopheevi groups, and the Ae. kotschyi-Ae. variabilis complex, respectively. A diphyletic origin of Emmer and Timopheevi groups is supported by the present results.     

ITS regions in diploids of Aegilops (Poaceae) and their phylogenetic implications

The nucleotide sequences of the internal transcribed spacer (ITS) of nuclear ribosomal DNA in nine diploid species representing six sections of Aegilops were determined by direct sequencing of PCR-amplified DNA fragments. These sequences were aligned with two ITS sequences of additional species from Genbank. Sequence divergences were estimated using Kimura two-parameter model, and the phylogenetic analyses were performed using the maximum parsimony (MP) and the neighbor-joining (NJ) methods with PAUP and PHYLIP, respectively. The sequence divergences between the diploid species varied from 0.5% to 4.68%. The resulting MP tree and NJ tree showed relatively congruent phylogenetic relationships among these species, except Ae. caudata. Particularly, Ae. speltoides was basal within the two trees. The paraphyletic relationships between Ae. speltoides and two species of Sect. Sitopsis, and between Ae. uniaristata and two species of Sect. Comopyrum were supported strongly. The ITS data suggest that currently recognized sections within Aegilops should be reconsidered.     

山羊草属核型分析及其与小麦属的进化关系

作者研究了山羊草属(Aegilops)中的新疆节节麦(Ae.squarrosa)、拟斯卑尔脱山羊草(Ae.speltoides)、沙融山羊草(Ae.sharonensis)、尾状山羊草(Ae.caudata)、卵圆山羊草(Ae.ovata)、偏凸山车草(Ae.ventricosa),钩状山羊草(Ae.triuncialis)、三芒山羊草(Me.triaristata)、欧山羊草(Ae.biuncialis)、柱穗山羊草(Ae.cylindrica)、可兹山羊草(Ae.kotschyi)和肥厚山羊草(Ae.crassa)的核型和部分材料的Giemsa N-带,结果表明山羊草属的C组核型为:4sm+3st;D组核型为:6m+1sm;S组的核型为:6m+1sm;M组的核型为:4m+1sm+2t。在四倍体、六倍体中,各染色体组保持着相对稳定。山羊草属S、D染色体组的核型与带型表明它们是小麦B、D染色体组的可能供体,C、M染色体组的一部分染色体带型亦与小麦B组带型相似。     

Genome differentiation in Aegilops. 1. Distribution of highly repetitive DNA sequences on chromosomes of diploid species.

Genome differentiation in 12 diploid Aegilops species was analyzed using in situ hybridization with the highly repetitive DNA sequences pSc119 and pAs1 and C-banding. Chromosomes of all these diploid Aegilops species hybridized with the pSc119 probe; however, the level of hybridization and labeling patterns differed among genomes. Only four species (Ae. squarrosa, Ae. comosa, Ae. heldreichii, and Ae. uniaristata) showed distinct hybridization with pAs1. The labeling patterns were species-specific and chromosome-specific. Differences in in situ hybridization (ISH) patterns, also observed by C-banding, exist between the karyotypes of Ae. comosa and Ae. heldreichii, suggesting that they are separate, although closely related, subspecies. The S genome of Ae. spelioides was most similar to the B and G genomes of polyploid wheats on the basis of both C-banding and ISH patterns, but was different from other species of section Sitopsis. These species had different C-banding patterns but they were similar to each other and to Ae. mutica in the distribution of pSc119 hybridization sites. Two types of labeling were detected in Ae. squarrosa with the pAs1 probe. The first resembled that of the D-genome chromosomes of bread wheat, Triticum aestivum L. em. Thell., while the second was similar to the D genome of some of the polyploid Aegilops species. Relationships among diploid Aegilops species and the possible mechanisms of genome differentiation are discussed. Key words : wheat, Triticum, Aegilops, in situ hybridization, C-banding, evolution.     

Electrophoretic survey of seedling esterases in wheats in relation to their phylogeny

Summary Evolutionary and ontogenetic variation of six seedling esterases of independent genetic control is studied in polyploid wheats and their diploid relatives by means of polyacrylamide gel electrophoresis. Four of them are shown to be controlled by homoeoallelic genes in chromosomes of third, sixth and seventh homoeologous groups.The isoesterase electrophoretic data are considered supporting a monophyletic origin of both the primitive tetraploid and the primitive hexaploid wheat from which contemporary taxa of polyploid wheats have emerged polyphyletically and polytopically through recurrent introgressive hybridization and accumulation of mutations. Ancestral diploids belonging or closely related to Triticum boeoticum, T. urartu, Aegilops speltoides and Ae. tauschii ssp. strangulata are genetically the most suitable genome donors of polyploid wheats. Diploids of the Emarginata subsection of the section Sitopsis, Aegilops longissima s.str., Ae. sharonensis, Ae. searsii and Ae. bicornis, are unsuitable for the role of the wheat B genome donors, being all fixed for the esterase B and D electromorphs different from those of tetraploid wheats.     

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.     

Studies on the origin and evolution of tetraploid wheats based on the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA

In this study, the internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA in the tetraploid wheats, Triticum turgidum (AABB) and Triticum timopheevii (AAGG), their possible diploid donors, i.e., Triticum monococcum (AA), Triticum urartu (AA), and five species in Aegilops sect. Sitopsis (SS genome), and a related species Aegilops tauschii were cloned and sequenced. ITS1 and ITS2 regions of 24 clones from the above species were compared. Phylogenetic analysis demonstrated that Aegilops speltoides was distinct from other species in Aegilops sect. Sitopsis and was the most-likely donor of the B and G genomes to tetraploid wheats. Two types of ITS repeats were cloned from Triticum turgidum ssp. dicoccoides, one markedly similar to that from T. monococcum ssp. boeoticum (AA), and the other to that from Ae. speltoides (SS). The former might have resulted from a recent integression event. The results also indicated that T. turgidum and T. timopheevii might have simultaneously originated from a common ancestral tetraploid species or be derived from two hybridization events but within a very short interval time. ITS paralogues in tetraploid wheats have not been uniformly homogenized by concerted evolution, and high heterogeneity has been found among repeats within individuals of tetraploid wheats. In some tetraploid wheats, the observed heterogeneity originated from the same genome (B or G). Three kinds of ITS repeats from the G genome of an individual of T. timopheevii ssp. araraticum were more divergent than that from inter-specific taxa. This study also demonstrated that hybridization and polyploidization might accelerate the evolution rate of ITS repeats in tetraploid wheats.     

Molecular Variation in Chloroplast DNA Regions in Ancestral Species of Wheat

Restriction map variation in two 5-6-kb chloroplast DNA regions of five diploid Aegilops species in the section Sitopsis and two wild tetraploid wheats, Triticum dicoccoides and Triticum araraticum, was investigated with a battery of four-cutter restriction enzymes. A single accession each of Triticum durum, Triticum timopheevi and Triticum aestivum was included as a reference. More than 250 restriction sites were scored, of which only seven sites were found polymorphic in Aegilops speltoides. No restriction site polymorphisms were detected in all of the other diploid and tetraploid species. In addition, six insertion/deletion polymorphisms were detected, but they were mostly unique or species-specific. Estimated nucleotide diversity was 0.0006 for A. speltoides, and 0.0000 for all the other investigated species. In A. speltoides, none of Tajima's D values was significant, indicating no clear deviation from the neutrality of molecular polymorphisms. Significant non-random association was detected for three combinations out of 10 possible pairs between polymorphic restriction sites in A. speltoides. Phylogenetic relationship among all the plastotypes (plastid genotype) suggested the diphyletic origin of T. dicoccoides and T. araraticum. A plastotype of one A. speltoides accession was identical to the major type of T. araraticum (T. timopheevi inclusively). Three of the plastotypes found in the Sitopsis species are very similar, but not identical, to that of T. dicoccoides, T. durum and T. aestivum.