Isolation and characterization of genome-specific DNA sequences in Triticeae species

Two contrasting genome-specific DNA sequences were isolated from Aegilops speltoides (wild goat grass) and Hordeum chilense (wild barley), each representing more than 1 % of the genomes. These repetitive DNA fragments were identified as being genome-specific before cloning by genomic Southern hybridization (using total genomic DNA as a probe), and hence extensive screening of clones was not required. For each fragment, up to six recombinant plasmid clones were screened and about half were genome-specific. Clone pAesKB52 from Ae. speltoides was a 763 by EcoRI fragment, physically organized in simple tandem repeats and shown to localize to sub-telomerec chromosome regions of species with the Triticeae S-genome by in situ hybridization to chromosomes. The sequence data showed an internal duplication of some 280 bp, which presumably occurred before sequence amplification and dispersion, perhaps by unequal crossing-over or reciprocal translocation. In situ hybridization showed that the sequence distribution varied between closely related (S-genome) species. Clone pHcKB6 was a 339 by DraI fragment from H. chilense, also tandemly repeated but more variable; loss of the DraI site resulting in a ladder pattern in Southern blots which had little background smear. In situ hybridization showed that the tandem repeats were present as small clusters dispersed along all chromosome arms except at a few discrete regions including the centromeres and telomeres. The clone hybridized essentially specifically to the H-genome of H. chilense and hence was able to identify the origin of chromosomes in a H. chilense x Secale africanum hybrid by in situ hybridization. It has a high A + T content (66%), small internal duplications, and a 50 by degenerate inverted repeat. We speculate that it has dispersed by retrotransposition in association with other sequences carrying coding domains. The organization and evolution of such sequences are important in understanding long-range genome organization and the types of change that can occur on evolutionary and plant breeding timescales. Genome-specific sequences are also useful as markers for alien chromatin in plant breeding.     

Hordeum chilense repetitive sequences. Genome characterization using biotinylated probes

Summary A library of random DNA fragment clones of wild barley Hordeum chilense was screened for clones of repeated nucleotide sequences. Five clones were isolated that gave a stronger hybridization signal in colony and dot blot hybridization with total H. chilense DNA in comparison to Triticum aestivum DNA. Clones labelled with biotinylated nucleotides were used as probes to investigate the repeated sequences organization in the H. chilense genome. Tandemly arranged and interspersed sequences have been found, together with homology differences with related sequences present in T. Aestivum, which could allow the differentiation of H. chilense DNA when it is present in wheat. We show that biotin can replace the use of ³²P in preparing repeated sequence probes for Southern and DNA dot blot analyses.     

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.     

Detection of 5S rDNA and other repeated DNA on supernumerary B chromosomes ofTriticum species (Poaceae)

The supernumerary B chromosomes ofTriticum speltoides andT. tripsacoides were analyzed in mitotic metaphases of spike primordium cells by C-banding and in situ hybridization (ISH) analyzes. B chromosomes ofT. speltoides have larger telomeric and interstitial C-bands, whereas those ofT. tripsacoides are almost completely devoid of C-bands. A prominent ISH site of rye related DNA sequences (using probe pSc119) was detected on B chromosomes ofT. tripsacoides and only a minor ISH site was observed on theT. speltoides B chromosomes. However, two ISH sites of 5S rRNA loci were detected at a terminal and an interstitial location of theT. speltoides B chromosomes. These sites were absent on B chromosomes ofT. tripsacoides. The results are discussed with respect to the phylogenetic origin of these B chromosomes.     

Molecular and cytological characterization of repetitive DNA sequences in Brassica

Summary We isolated three different repetitive DNA sequences from B. campestris and determined their nucleotide sequences. In order to analyze organization of these repetitive sequences in Brassica, Southern blot hybridization and in situ hybridization with metaphase chromosomes were performed. The sequence cloned in the plasmid pCS1 represented a middle repetitive sequence present only in B. campestris and not detected in closely related B. Oleracea. This sequence was localized at centromeric regions of six specific chromosomes of B. campestris. The second plasmid, pBT4, contained a part of the 25S ribosomal RNA gene, and its copy number was estimated to be 1,590 and 1,300 per haploid genome for B. campestris and B. oleracea, respectively. In situ hybridization with this sequence showed a clear signal at the NOR region found in the second largest chromosome of B. Campestris. The third plasmid, pBT11, contained a 175-bp insert that belongs to a major family of tandem repeats found in all the Brassica species. This sequence was detected at centromeric regions of all the B. campestris chromosomes. Our study indicates that in situ hybridization with various types of repetitive sequences should give important information on the evolution of repetitive DNA in Brassica species.     

Relationships betweenNor-loci from differentTriticeae species

TheNor-loci of polyploid wheats and their putative diploid progenitor species were assayed by probing isolated nuclear DNA with ribosomal DNA spacer sequences (spacer rDNA sequences, isolated by cloning), from theNor-loci of genomes B (Triticum aestivum), G (T. timopheevi), B (syn. S,T. speltoides), A (T. monococcum) and V (Dasypyrum villosum). DNA samples for analysis were digested with the restriction endonuclease Taq 1 and assayed by DNA-DNA hybridization under standard (37°C) and high stringency (64°C) conditions. The assay procedure emphasized differences between the divergent spacer sequences of the polyploid species and allowed relative homologies to the respective sequences in diploid species to be established. — The studies indicated thatT. timopheevi andT. speltoides contain different sets of spacer rDNA sequences which were readily distinguishable and, in the case ofT. timopheevi, assigned toNor-loci on different chromosomes. This contrast with the spacer rDNA sequences of the majorNor-loci on chromosomes 1 B and 6 B inT. aestivum, which were difficult to distinguish and were deduced to contain very similar sequences. Among the diploid progenitor species only the spacer rDNA fromT. speltoides shared close homology with polyploid wheat species. OneNor-locus inT. timopheevi (on chromosome 6 G) did not show close homology with any of the rDNA spacer probes available. — The data suggestsT. speltoides was the origin of someNor-loci for both theT. timopheevi andT. turgidum lines of tetraploid wheats. The possibility that the 6GNor-locus inT. timopheevi may have derived from an unknown diploid species by introgressive hybridization is discussed. The spacer rDNA sequence probe fromT. monococcum shared good homology with some accessions ofD. villosum and a line ofT. dicoccoides; the implications of this finding for evolution of present-day wheats are discussed.     

Genome-specific repetitive sequences in the genus Oryza

Summary Repetitive DNA sequences are useful molecular markers for studying plant genome evolution and species divergence. In this paper, we report the isolation and characterization of four genome-type specific repetitive DNA sequences in the genus Oryza. Sequences specific to the AA, CC, EE or FF genome types are described. These genome-type specific repetitive sequences will be useful in classifying unknown species of wild or domestic rice, and in studying genome evolution at the molecular level. Using an AA genome-specific repetitive DNA sequence (pOs48) as a hybridization probe, considerable differences in its copy number were found among different varieties of Asian-cultivated rice (O. sativa) and other related species within the AA genome type. Thus, the relationship among some of the members of AA genome type can be deduced based on the degree of DNA sequence similarity of this repetitive sequence.     

A structural and evolutionary analysis of a dispersed repetitive sequence

A family of dispersed repetitive sequences (Hch1) which is present in the genome of the wild barley Hordeum chilense was studied in detail. Hch1 sequences are found both as part of short tandem arrays and dispersed throughout the H. chilense chromosomes. Subcloning of sections of the sequence reveals that it is composed of unrelated classes of sequences which can also be found separately in other genomic locations. Analysis of these sequences in the genomes of wheat and two other wild barley species strongly suggests that specific amplifications and arrangements of the repeated sequences have taken place during speciation. Nucleotide sequence analysis fails to detect, in their entirity, the features shown by plant transposons.     

Characterization of species-specific repeated DNA sequences from B. nigra

Summary The construction and characterization of two genome-specific recombinant DNA clones from B. nigra are described. Southern analysis showed that the two clones belong to a dispersed repeat family. They differ from each other in their length, distribution and sequence, though the average GC content is nearly the same (45%). These B genome-specific repeats have been used to analyse the phylogenetic relationships between cultivated and wild species of the family Brassicaceae.     

A molecular and cytogenetic survey of major repeated DNA sequences in tomato (Lycopersicon esculentum)

Summary The major families of repeated DNA sequences in the genome of tomato (Lycopersicon esculentum) were isolated from a sheared DNA library. One thousand clones, representing one million base pairs, or 0.15% of the genome, were surveyed for repeated DNA sequences by hybridization to total nuclear DNA. Four major repeat classes were identified and characterized with respect to copy number, chromosomal localization by in situ hybridization, and evolution in the family Solanaceae. The most highly repeated sequence, with approximately 77000 copies, consists of a 162 bp tandemly repeated satellite DNA. This repeat is clustered at or near the telomeres of most chromosomes and also at the centromeres and interstitial sites of a few chromosomes. Another family of tandemly repeated sequences consists of the genes coding for the 45 S ribosomal RNA. The 9.1 kb repeating unit in L. esculentum was estimated to be present in approximately 2300 copies. The single locus, previously mapped using restriction fragment length polymorphisms, was shown by in situ hybridization as a very intense signal at the end of chromosome 2. The third family of repeated sequences was interspersed throughout nearly all chromosomes with an average of 133 kb between elements. The total copy number in the genome is approximately 4200. The fourth class consists of another interspersed repeat showing clustering at or near the centromeres in several chromosomes. This repeat had a copy number of approximately 2100. Sequences homologous to the 45 S ribosomal DNA showed cross-hybridization to DNA from all solanaceous species examined including potato, Datura, Petunia, tobacco and pepper. In contrast, with the exception of one class of interspersed repeats which is present in potato, all other repetitive sequences appear to be limited to the crossing-range of tomato. These results, along with those from a companion paper (Zamir and Tanksley 1988), indicate that tomato possesses few highly repetitive DNA sequences and those that do exist are evolving at a rate higher than most other genomic sequences.     

Involvement of transposition in dispersion of tandem repeat sequences (TrsA) in rice genomes

We describe a method to identify and characterize DNA fragments containing the junction of AA genome-specific tandem repeat sequences (here called TrsA) with adjacent chromosomal sequences of rice by the polymerase chain reaction (PCR) using a pair of primers that hybridize with TrsAs and a flanking non-TrsA sequence. With this method, we obtained results suggesting that TrsA sequences present at two loci (here called trsA1 and trsA2) are flanked by direct repeats of chromosomal sequences of 172 by and about 440 by in length, respectively. These results support the idea that the TrsA sequences have been inserted into each locus by transposition, resulting in duplication of the chromosomal sequence used as target. We also describe a method to identify and characterize TrsA sequences repeated in only a few copies in the rice genome by PCR, using a pair of primers that hybridize with two different portions in the TrsA sequence, and demonstrate that TrsA sequences are present not only in rice strains with the AA genome, but also in those with non-AA genomes. The TrsA sequences were present at the trsA1 locus in all the rice strains examined, indicating that TrsA was inserted and amplified at the locus before the divergence of the various species of rice in the Oryza genus. TrsA sequences were present at the trsA2 locus, however, only in an O. sativa IR36 strain, indicating that TrsA was inserted and amplified at this locus during divergence of rice strains with the AA genome.     

The origin of transfer (oriT) of the conjugative plasmid R46: characterization by deletion analysis and DNA sequencing

Summary The origin of transfer (oriT) is the sequence within which conjugal transfer of plasmid DNA is initiated, and is absolutely required in cis for plasmid mobilization. We have cloned oriT from the 52 kb IncN plasmid R46 on a 600 bp fragment, and mapped the limits of the relevant sequence by deletion analysis and transposon mutagenesis. The nucleotide sequence of the oriT region contains 13 direct repeats of an 11 bp consensus sequence, 3 different pairs of 10 bp inverted repeats, and a segment that is extremely A-T rich. The direct repeats are within a region required for high frequency transfer and their sequence is such that their periodic alignment along the helix may induce curvature of the DNA. Analysis of Tn1725 insertions within the sequenced fragment of R46 revealed that, unlike most other transposons, transposition of Tn1725 can cause target sequence duplications of three different sizes.     

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.     

Molecular and cytogenetic characterization of repetitive DNA sequences from Lolium and Festuca: applications in the analysis of Festulolium hybrids

Summary A set of species-specific repetitive DNA sequences was isolated from Lolium multiflorum and Festuca arundinacea. The degree of their species specificity as well as possible homologies among them were determined by dot-blot hybridization analysis. In order to understand the genomic organization of representative Lolium and Festuca-specific repetitive DNA sequences, we performed Southern blot hybridization and in situ hybridization to metaphase chromosomes.Southern blot hybridization analysis of eight different repetitive DNA sequences of L. multiflorum and one of F. arundinacea indicated either tandem and clustered arrangements of partially dispersed localization in their respective genomes. Some of these sequences, e.g. LMB3, showed a similar genomic organization in F. arundinacea and F. pratensis, but a slightly different organization and degree of redundancy in L. multiflorum. Clones sequences varied in size between 100 bp and 1.2 kb. Estimated copy number in the corresponding haploid genomes varied between 300 and 2×10⁴. Sequence analysis of the highly species-specific sequences from plasmids pLMH2 and pLMB4 (L. multiflorum specific) and from pFAH1 (F. arundinacea specific) revealed some internal repeats without higher order. No homologies between the sequences or to other repetitive sequences were observed. In situ hybridization with these latter sequences to metaphase chromosomes from L. multiflorum, F. arundinacea and from symmetric sexual Festulolium hybrid revealed their relatively even distribution in the corresponding genomes. The in situ hybridization thus also allowed a clearcut simple identification of parental chromosomes in the Festulolium hybrid.The potential use of these species-specific clones as hybridization probes in quantitative dot-blot analysis of the genomic make-up of Festulolium (sexual and somatic) hybrids is also demonstrated.Abbreviations bp Base pair (s) - CMA chromomycin A₃ - DAPI 4,6-diamidino-2-phenylindole - IPTG isopropyl -D-thio-galactopyranoside - kb kilobase pair(s) - NBT nitroblue tetrazolium chloride - X-gal 5-bromo-4-chloro-3-inonyl -D-galactopyranoside     

Isolation and identification of a non-specific tandemly repeated DNA sequence in Oryza species

A tandemly repeated DNA sequence (RRS7) was isolated from Oryza alta (CCDD). RRS7-related sequences were also found tandemly arrayed in genomes AA, BB, BBCC, CC, and EE, and a small amount of RRS7-related sequences were detected in genome FF and the Oryza species with unknown genomes. DNA sequence analysis of the 1844-bp insert of RRS7 revealed that it contained six tandemly repeated units, of which five were 155 bp in length and one was 194 bp in length and contained an imperfect internal 39-bp duplication. Southern blot analysis showed that the boundary sequence contained in RRS7 is a single-copy sequence. A 155-bp consensus sequence derived from the six monomeric repeats contained no internal repeat and showed no significant homology to other currently known sequences. The results of Southern blot and sequence analysis revealed that there are at least two subfamilies present in the RRS7 family; these are represented by the DraI site and the MspI site, respectively. Restriction digestion with two pairs of isoschizomers MboI/Sau3A and MspI/HpaII demonstrated that most of the C residues in the GATC sites and the internal C in the CCGG sites of the RRS7 family in O. Alta were methylated. The usefulness of the RRS7 family in determining the evolutionary relationship of the genome DD and other Oryza genomes is discussed.     

Characterization of a chloroplast DNA sequence from Chlorella ellipsoidea that promotes autonomous replication in yeast

Summary An EcoRI 2.7 kbp fragment from Chlorella ellipsoidea chloroplast DNA (cpDNA) cloned in YIp5 was shown to promote autonomous replication in Saccharomyces cerevisiae. The fragment was localized in the small single copy region close to the inverted repeat. The ARS activity (autonomously replicating sequences in yeast) was found to be confined within a subclone of a ca. 300 bp HindIII fragment. Sequence analysis of this fragment revealed its high AT content and the presence of several direct and inverted repeats and a few elements that were related to the yeast ARS consensus sequence. Electron microscopic studies revealed that this sequence did not coincide with the primary replication origin of chloroplast DNA. The functioning of this sequence as a possible origin of plasmid replication in vivo is discussed. This is the first report on Chlorella cpDNA sequence. re]19850821 rv]19851211 ac]19851216     

Comparisons of the hybrids Hordeum chilensexH. vulgare,H. chilensexH. bulbosum,H. chilensexSecale cereale and the amphidiploid of H. chilensexH. vulgare

Summary Diploid hybrids between Hordeum chilense and three other species, namely H. vulgare, H. bulbosum and Secale cereale, are described together with the amphidiploid of H. chilensexH. vulgare. Both the diploid hybrid and the amphidiploid of H. chilensexH. vulgare were chromosomally unstable, H. chilensexH. bulbosum was less so, while H. chilensexS. cereale was stable. Differential amphiplasty was found in all combinations. No homoeologous pairing was found in the Hordeum hybrids but in H. chilensexS. cereale there was chromosome pairing both within the two genomes and between the genomes.     

Analysis of DNA from a Beta procumbens chromosome fragment in sugar beet carrying a gene for nematode resistance

Summary We have begun to apply techniques for the preparation and anaylsis of large DNA segments from sugar beet (Beta vulgaris) addition lines carrying a mitotically stable chromosome fragment from B. procumbens that confers monogenic resistance to the nematode Heterodera schachtii, with a view towards isolating the resistance gene. DNA probes specific for this chromosome fragment were selected, and various methods for cloning genome-specific fragments, including probes from megabase DNA separated in pulsed-field slab gels, are compared. Probes that display high homology to B. procumbens have been used for hybridization of a representative genomic library and for initial step in mapping the chromosome fragment via pulsed-field gel electrophoresis after restriction with infrequently cutting enzymes. Our data indicate that DNA molecules from the entire chomosome fragment can be separated from protoplast DNA lysates.