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.     

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.     

Intraspecific divergence in wheats of the Timopheevi group as revealed by in situ hybridization with tandem repeats of the Spelt1 and Spelt52 families

Fluorescent in situ hybridization (FISH) was used to study the distribution of the Spelt1 and Spelt52 repetitive DNA sequences on chromosomes of ten accessions representing three polyploid wheat species of the Timopheevi group: Triticum araraticum (7), T. timopheevii (2), and T. kiharae (1). Sequences of both families were found mostly in the subtelomeric chromosome regions of the G genome. The total number of Spelt1 sites varied from 8 to 14 in the karyotypes of the species under study; their number, location, and size differed among the seven T. araraticum accessions and were the same in the two T. timopheevii accessions and T. kiharae, an amphidiploid T. timopheevii-Aegilops tauschii hybrid. The Spelt52 tandem repeat was detected in the subtelomeric regions of chromosomes 1-4; its sites did not coincide with the Spelt1 sites. The chromosome distribution and signal intensity of the Spelt52 repeats varied in T. araraticum and were the same in T. timopheevii and T. kiharae. The chromosome distributions of the Spelt1 and Spelt52 repeats were compared for the polyploid wheats of the Timopheevi group and diploid Ae. speltoides, a putative donor of the G genome. The comparison revealed a decrease in hybridization level: both the number of sites per genome and the size of sites were lower. The decrease was assumed to result from repeat elimination during polyploidization and subsequent evolution of wheat and from the founder effect, since the origin of Timopheevi wheats might involve the genotype of Ae. speltoides, which is highly polymorphic for the distribution of Spelt1 and Spelt52 sequences and is similar in the chromosome location of the repeats to modern wheat.     

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 mechanisms of variation of subtelomeric repeats Speltl in the progeny of introgressive line Triticum aestivum L. x Aegilops speltoides Tausch

Quantitative variation of species-specific subtelomeric repeat Speltl was studied in the progeny of an individual accession from the introgressive line Triticum aestivum x Aegilops speltoides. In the progeny, no cases of the Speltl increased content were observed. On the contrary, in some cases statistically significant decrease of the repeat copy number was detected. It seems likely that the mechanisms of the Spelt1 elimination involve either the selection at the gamete level versus the increase of the satellite DNA content in the telomeres, or intramolecular (within one chromatid) homologous recombination.     

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.     

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。     

Resistance genes in wild accessions of Triticeae--inoculation test and STS marker analyses

Sequence tagged site (STS) markers have been developed recently to identify resistance genes in wheat. A number of wild relatives have been used to transfer resistance genes into wheat cultivars. Accessions of wild species of Triticeae: Aegilops longissima (4), Ae. speltoides (6), Ae. tauschii (8), Ae. umbellulata (3), Ae. ventricosa (3), Triticum spelta (2), T. timopheevi (3), T. boeoticum (4) and T. monococcum (1), 34 in total, were examined using PCR-STS markers for resistance genes against Puccinia recondita f.sp. tritici (Lr) and Erysiphe graminis (Pm). Additionally, a set of cv. Thatcher near-isogenic lines conferring resistance genes Lr 1, Lr 9, Lr 10, Lr 24, Lr 28, Lr 35 and Lr 37 were examined with the same procedure. Twenty-two accessions were tested using the inoculation test for resistance to Erysiphe graminis, Puccinia recondita, P. striiformis and P. graminis. The most resistant entries were those of Aegilops speltoides and Triticum timopheevi and among T. boeoticum accession #5353. Markers of all mentioned Lr resistance genes were identified in all corresponding cv. Thatcher near-isogenic lines (except Lr 35 gene marker). The following resistance gene markers were identified in wild Triticeae accessions: Lr 1 in two accessions of Ae. tauschii and one accession of Ae. umbellulata, Lr 9 in one accession of Ae. umbellulata, Lr 10 in one accession of T. spelta, Lr 28 in 11 accessions: Ae. speltoides (4), Ae. umbellulata (2), T. spelta (2) and T. timopheevi (3), Lr 37 in 3 accessions of Ae. ventricosa, Pm 1 in all 34 accessions, Pm 2 in 28 accessions, Pm 3 in all 4 accessions of T. boeoticum, 1 accession of T. spelta and 1 of T. timopheevi, and Pm 13 in 5 out of 6 accessions of Ae. speltoides. Reliability and usefulness of STS markers is discussed.     

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.     

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.     

Pairing affinities of the B- and G-genome chromosomes of polyploid wheats with those of Aegilops speltoides.

Chromosome pairing at metaphase I was studied in different interspecific hybrids involving Aegilops speltoides (SS) and polyploid wheats Triticum timopheevii (AtAtGG), T. turgidum (AABB), and T. aestivum (AABBDD) to study the relationships between the S, G, and B genomes. Individual chromosomes and their arms were identified by means of C-banding. Pairing between chromosomes of the G and S genomes in T. timopheevii x Ae. speltoides (AtGS) hybrids reached a frequency much higher than pairing between chromosomes of the B and S genomes in T. turgidum x Ae. speltoides (ABS) hybrids and T. aestivum x Ae. speltoides (ABDS) hybrids, and pairing between B- and G-genome chromosomes in T. turgidum x T. timopheevii (AAtBG) hybrids or T. aestivum x T. timopheevii (AAtBGD) hybrids. These results support a higher degree of closeness of the G and S genomes to each other than to the B genome. Such relationships are consistent with independent origins of tetraploid wheats T. turgidum and T. timopheevii and with a more recent formation of the timopheevi lineage.     

Construction and study of leaf rust-resistant common wheat lines with translocations of Aegilops speltoides Tausch

Genotyping was performed for the leaf rust-resistant line 73/00i (Triticum aestivum x 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 x 5BL-5SL translocation was highly resistant to leaf rust, and the lines carrying the T6BS x 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 x 5BL-5SL translocation was preliminarily designated as LrAsp5.     

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。     

Alterations in subtelomeric tandem repeats during early stages of allopolyploidy in wheat.

The genomic content of the subtelomeric repeated sequences Spelt1 and Spelt52 was studied by dot, Southern, and in situ hybridization in 11 newly synthesized amphiploids of Aegilops and Triticum, and data were compared with the parental plants. Spelt1 had reduced copy numbers in the first generation of three synthetic amphiploids, but two others did not change; Spelt52 was amplified in nine amphiploids and did not change in two. In the second allopolyploid generation, Spelt1 copy number did not change, whereas there was amplification of Spelt52 in some allopolyploids and decreases in others. Neither allopolyploidy level nor the direction of the cross affected the patterns of change in the newly synthesized amphiploids. Changes did not result from intergenomic recombination because similar alterations were noticed in allopolyploids with and without Ph1, a gene that suppresses homoeologous pairing. No differences in Spelt1 and Spelt52 tandem organization were found by Southern hybridization. The significance of these data are discussed in relation to the establishment of newly formed allopolyploids.     

The mechanisms of variation of subtelomeric repeats Spelt1 in the progeny of introgressive line <Emphasis Type="Italic">Triticum aestivum</Emphasis> L. × <Emphasis Type="Italic">Aegilops speltoides</Emphasis> Tausch.

Quantitative variation of species-specific subtelomeric repeat Spelt1 was studied in the progeny of an individual plant from the introgressive line Triticum aestivum × Aegilops speltoides. In the progeny, no cases of the Spelt1 increased content were observed. On the contrary, in some cases statistically significant decrease of the repeat copy number was detected. It seems likely that the mechanisms of the Spelt1 elimination involve either the selection at the gamete level versus the increase of the satellite DNA content in the telomeres, or intramolecular (within one chromatid) homologous recombination.     

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.     

Elimination of a tandem repeat of telomeric heterochromatin during the evolution of wheat

 An analysis of accessions of Triticum and Aegilops species (86 diploid, 91 tetraploid and 109 hexaploid) was performed using squash-dot hybridization with the tandem repeat Spelt1 sequence as a probe. The Spelt1 sequence is a highly species-specific repeat associated with the telomeric heterochromatin of Aegilops speltoides Boiss. in which its copy numbers vary from 1.5×10⁵ to 5.3×10⁵. The amounts of Spelt1 are sharply decreased in tetraploid and hexaploid species and vary widely from less than 10² to 1.2×10⁴. Two tetraploid wheats, Triticum timopheevii Zhuk. and T. carthlicum Nevski, are exceptional endemic species and within their restricted geographical distributions maintain the amounts of Spelt1 unaltered. The Spelt1 repetitive sequence was localized on the 6BL chromosome of tetraploid wheat Triticum durum Desf. cv ‘Langdon’ by dot-hybridization using D-genome disomic substitution lines. The possible causes of the loss of the telomere-associated tandem repeat Spelt1 in the process of wheat evolution and polyploidization are discussed. Received: 5 March 1998 / Accepted: 28 May 1998     

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.     

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.     

Atrazine-resistant cytoplasmic male-sterile-nigra broccoli obtained by protoplast fusion between cytoplasmic male-sterile Brassica oleracea and atrazine-resistant Brassica campestris

Summary By using restriction endonuclease digestion patterns, the degree of intraspecific polymorphism of mitochondrial DNA in four diploid species of wheat and Aegilops, Ae. speltoides, Ae. longissima, Ae. squarrosa, and Triticum monococcum, was assessed. The outbreeding Ae. speltoides was found to possess the highest degree of variability, the mean number of nucleotide substitutions among conspecific individuals being 0.027 substitutions per nucleotide site. A very low degree of mtDNA variation was detected among Ae. longissima accessions, with most of the enzyme-probe combinations exhibiting uniform hybridization patterns. The mean number of substitutions among Ae. longissima individuals was 0.001 substitutions per nucleotide site. The domesticated diploid wheat T. monococcum var. monococcum and its conspecific variant T. monococcum var. boeoticum seem to lack mitochondrial DNA variability altogether. Thus, the restriction fragment pattern can be used as a characteristic identifier of the T. monococcum cytoplasmic genome. Similarly, Ae. squarrosa accessions were found to be genetically uniform. A higher degree of variation among accessions is observed when noncoding sequences are used as probes then when adjacent coding regions are used. Thus, while noncoding regions may contain regulatory functions, they are subject to less stringent functional constraints than protein-coding regions. Intraspecific variation in mitochondrial DNA correlates perfectly with the nuclear variability detected by using protein electrophoretic characters. This correlation indicates that both types of variation are selectively neutral and are affected only by the effective population size.