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.     

Independent wheat B and G genome origins in outcrossing Aegilops progenitor haplotypes

The origin of modern wheats involved alloploidization among related genomes. To determine if Aegilops speltoides was the donor of the B and G genomes in AABB and AAGG tetraploids, we used a 3-tiered approach. Using 70 amplified fragment length polymorphism (AFLP) loci, we sampled molecular diversity among 480 wheat lines from their natural habitats encompassing all S genome Aegilops, the putative progenitors of wheat B and G genomes. Fifty-nine Aegilops representatives for S genome diversity were compared at 375 AFLP loci with diploid, tetraploid, and 11 nulli-tetrasomic Triticum aestivum wheat lines. B genome-specific markers allowed pinning the origin of the B genome to S chromosomes of A. speltoides, while excluding other lineages. The outbreeding nature of A. speltoides influences its molecular diversity and bears upon inferences of B and G genome origins. Haplotypes at nuclear and chloroplast loci ACC1, G6PDH, GPT, PGK1, Q, VRN1, and ndhF for approximately 70 Aegilops and Triticum lines (0.73 Mb sequenced) reveal both B and G genomes of polyploid wheats as unique samples of A. speltoides haplotype diversity. These have been sequestered by the AABB Triticum dicoccoides and AAGG Triticum araraticum lineages during their independent origins.     

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.     

Coevolution of A and B genomes in allotetraploid Triticum dicoccoides.

Data is presented on the coevolution of A and B genomes in allotetraploid wheat Triticum dicoccoides (2n = 4x = 28, genome AABB) obtained by genomic in situ hybridization (GISH). Probing chromosomes of T. dicoccoides with DNA from the proposed A/B diploid genome ancestors shows evidence of enriching A-genome with repetitive sequences of B-genome type. Thus, ancestral S-genome sequences have spread throughout the AB polyploid genome to a greater extent than have ancestral A-genome sequences. The substitution of part of the A-genome heterochromatin clusters by satellite DNA of the B genome is detected by using the molecular banding technique. The cause may be interlocus concerted evolution and (or) colonization. We propose that the detected high level of intergenomic invasion in old polyploids might reflect general tendencies in speciation and stabilization of the allopolyploid genome.     

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.     

Genetic Relationship and Genomic in situ Hybridization Analysis of the Three Genomes in Triticum aestivum

Common wheat ( Triticum　aestivum L.) is an allohexaploid, consisting of three different genomes (Au, B and D ) which are genetically closely related. Genomic DNA of the three possible genome donors, T. urartu Thum., Aegilops speltoides Tausch and Ae. tauschii Coss.,were employed as probes to hybridize with the diploid genomic DNA digested by Eco RⅠand Hin dⅢ respectively. Both the hybridization strength and band patterns among the genomes would be good indicators of genome relationships. Combining distr ibution data of some repetitive DNA sequences cloned from T. urartu in the three genomes, the authors draw a conclusion that Au and D are more closely related to each other than either one to the B genome. Genomic in situ hybridization (GISH) of T. aestivum cv. Chinese Spring with genomic DNA probes of the three diploid progenitors respectively indicated that the three genomes could be discriminated clearly via GISH. The signals on the chromosomes of Au and D genomes were even. However, when Ae. speltoides DNA was used as probe, there were very strong cross hybridization and the signals condensed on some areas of the metaphasic chromosomes. In the interphase nucleus, the chromatin of B genome dispersed on the same region and the signals on the homologous chromosomes distributed symmetrically. Rich repetitive DNA sequences in B genome, especially the tandem repetitives, perhaps take an important role for the formation of the special hybridization pattern. The main difference between B and the other two genomes probably is in the repetitive DNA sequences.     

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。     

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.     

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.     

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.     

RAPD markers linked to the nuclear gene from Triticum timopheevii that confers compatibility with Aegilops squarrosa cytoplasm on alloplasmic durum wheat.

Alien cytoplasms cause a wide range of phenotypic alterations in the nucleus-cytoplasm (NC) hybrids in the Triticeae. Nuclear genomes of timopheevii wheat (Triticum timopheevii and Triticum araraticum) are fully compatible with the cytoplasm of Aegilops squarrosa, while those of a majority of emmer or durum wheat cultivars and more than half the wild emmer wheats are incompatible, and a maternal 1D chromosome is required to restore seed viability and male fertility in the NC hybrids. A euploid NC hybrid of Triticum durum cv. Langdon with Ae. squarrosa cytoplasm produced by introgressing the NC compatibility (Ncc) gene from T. timopheevii was used to identify random amplified polymorphic DNA (RAPD) markers linked to it. After a survey of 200 random decamer primers, four markers were selected, all of which were completely linked in 64 individuals of a SB8 mapping population. One marker was derived from a single locus, while three others were from interspersed repetitive sequences. Also, the hybrid chromosomes and those of the parental T. durum had identical C-banding patterns. RAPD-PCR analysis of 65 accessions from wild and cultivated tetraploid wheat species showed the exclusive presence of the markers in timopheevii wheat. In conclusion, the chromosomal region flanking Ncc of T. timopheevii is highly conserved in the genome of this group of tetraploid wheats.     

Origin, dispersal and genomic structure of a low-copy-number hypervariable RFLP clone in Triticum and Aegilops species

The genome of common wheat has evolved through allopolyploidization of three ancestral diploid genomes. A previously identified restriction fragment length polymorphism (RFLP) marker, pTag546, has the unique feature of showing hypervariability among closely related common wheat cultivars. To understand the origin and the mode of dispersal of this hypervariable sequence in the wheat genome, the distribution and structure of the homologous sequences were studied using ancestral diploid species, tetraploid disomic substitution lines and synthetic hexaploid lines. Comparative Southern blot and PCR analyses suggested that pTag546 homologs in the tetraploid and hexaploid wheat were derived from the S genome of Aegilops speltoides. Some pTag546 homologs were found to have transposed to A and D genomes in polyploid wheat. Evidence of transposition and elimination in some synthetic hexaploid lines was also obtained by comparing their copy numbers with those in the parental lines. Southern blot analysis of a genomic clone using a contiguous subset of sequences as probes revealed a core region of hypervariability that coincided with the region containing pTag546. No obvious structural characteristics that could explain the hypervariability, however, were found around the pTag546 sequence, except for accumulation of small repetitive sequences at one border. It was concluded that pTag546 increased its copy number through yet unknown mechanism(s) of transposition to various chromosomal locations over the period of allopolyploid evolution and during the artificial genome manipulation in wheat.     

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 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.     

USE OF REPEATED DNA SEQUENCES AS CYTOLOGICAL MARKERS

The majority of DNA that is found in most of the flowering plants appears to be non-coding DNA. Much of this excess DNA consists of nucleotide sequences which exist as multiple copies throughout the genome and are designated as repetitive sequences. Those sequences which are found in moderately high to high numbers of copies are observed to be of the greatest value as cytological markers. Moderately high copies may exist as sequences which are dispersed throughout the chromosomes of some species and not dispersed in other more distantly related species. By taking advantage of this characteristic and the technique of in situ hybridization with biotinylated probes, breakpoints of chromosomal translocations may be observed between species such as wheat and rye. Many of the high copy number repetitive sequences are organized in a tandem fashion in specific loci in the chromosome. Chromosomal identification may be accomplished by using the in situ hybridization technique. Upon in situ hybridization with a repetitive sequence isolated from Aegilops squarrosa, the patterns of the sites of hybridization allowed the D-genome chromosomes to be identified. The sequence was also observed only on the D-genome chromosomes of several polyploid species indicating its usefulness as a genome specific marker. Using this genome specificity, assessment of the orientation of the D-genome chromosomal segments of hexaploid wheat carrying the sequence during interphase and prophase of mitotic root tip cells was possible. Repetitive DNA sequences, therefore, provide cytological markers necessary for studies of chromosomal identification, genome allocation, and genome orientation. The use of biotin-labeled DNA probes allows the technique of in situ hybridization to be performed much more rapidly and with a greater degree of safety and reliability.     

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。     

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.     

小麦染色体组的起源与进化探讨

对小麦染色体组的起源及其进化进行了全面综述后,提出了一个新的小麦进化途径,并认为：（１）Ｔｒｉｔｉｃｕｍｍｏｎｏｃｏｃｕｍｖａｒｕｒａｒｔｕ是多倍体小麦Ａ组的原初供体,在Ａ组进入多倍体小麦后有Ｔｍｏｎｏｖａｒｂｏｅｏｔｉｃｕｍ的基因渗入;（２）Ｂ和Ｇ组的原初供体是Ｔｓｐｅｌｔｏｉｄｅｓ的Ｓ组,在该Ｓ组进入多倍体小麦后有两个进化方向,即Ｓ组结构分化形成Ｇ组和Ｓ组经外源染色体代换及重组等而进化成Ｂ组;（３）Ｔｔｕｒｇｉｄｕｍ和Ｔｔｉｍｏｐｈｅｖｉ都是来自Ｔｓｐｅｌｔｏｉｄｅｓ为母本与Ｔｍｏｎｏｖａｒｕｒａｒｔｕ杂交后并双二倍化而形成的原初四倍体小麦（ＳＳＡＡ）,并由它分别经遗传渗入和结构分化而成;（４）Ｔｚｈｕｋｏｖｓｋｙｉ是Ｔｔｉｍｏｐｈｅｖｉ作母本与Ｔｍｏｎｏｖａｒｂｏｅｏｔｉｃｕｍ杂交并双二倍化而形成,故它具有分别来自Ｔｍｏｎｏｖａｒｕｒａｒｔｕ和Ｔｍｏｎｏｖａｒｂｏｅｏｔｉｃｕｍ的两类Ａ组;（５）Ｔａｅｓｔｉｖｕｍ的Ｄ组来自Ｔｔａｕｓｃｈｉ;（６）无论Ａ组、Ｂ组、Ｄ组、Ｇ组在进入多倍体小麦后均有相当分化,同时在其供体种中也有一定分化。