Relationships between the chromosomes of Aegilops umbellulata and wheat

 A comparative genetic map of Aegilops umbellulata with wheat was constructed using RFLP probes that detect homoeoloci previously mapped in hexaploid bread wheat. All seven Ae. umbellulata chromosomes display one or more rearrangements relative to wheat. These structural changes are consistent with the sub-terminal morphology of chromosomes 2 U, 3 U, 6 U and 7 U. Comparison of the chromosomal locations assigned by mapping and those obtained by hybridization to wheat/Ae. umbellulata single chromosome addition lines verified the composition of the added Ae. umbellulata chromosomes and indicated that no further cytological rearrangements had taken place during the production of the alien-wheat aneuploid lines. Relationships between Ae. umbellulata and wheat chromosomes were confirmed, based on homoeology of the centromeric regions, for 1 U, 2 U, 3 U, 5 U and 7 U. However, homoeology of the centromeric regions of 4 U with wheat group-6 chromosomes and of 6 U with wheat group-4 chromosomes was also confirmed, suggesting that a re-naming of these chromosomes may be pertinent. The consequences of the rearrangements of the Ae. umbellulata genome relative to wheat for gene introgression are discussed. Received: 10 July 1997 / Accepted: 19 September 1997     

Rat LINE1: The origin and evolution of a family of long interspersed middle repetitive DNA elements

Summary We present approximately 7.0 kb of composite DNA sequence of a long interspersed middle repetitive element (LINE1) present in high copy number in the rat genome. The family of these repeats, which includes transcribing members, is the rat homologue of the mouse MIF-Bam-R and human Kpn I LINEs. Sequence alignments between speciments from these three species define the length of a putative unidentified open reading frame, and document extensive recombination events that, in conjunction with retroposition, have generated this large family of pseudogenes and pseudogene fragments. Comparative mapping of truncated elements indicates that a specific endonucleolytic activity might bei involved in illegitimate (nonhomologous) recombination events. Sequence divergence analyses provide insights into the origin and molecular evolution of these elements.     

Assessment of a species-specific element (Brep 1) in banana

 The nuclear genome of wild-type banana accessions was investigated for repetitive elements. We report here the occurrence, in the banana genome, of a sequence family of species-specific repetitive elements: Brep 1. This sequence family is distributed throughout the Musaceae with various copy numbers. The two species Musa acuminata and M. schizocarpa carry the highest copy numbers in contrast to M. balbisiana and tested representatives of different other sections. PCR primers were defined in the core consensus sequence for specific amplifications, which allow representatives of this sequence family to be easily detected in wild and cultivated banana clones. Sequence data were analysed and hypotheses on the evolution of banana cultivars from the wild-type banana clones are discussed. Received: 17 January 1997 / Accepted : 7 March 1997     

Cloning and characterization of a centromere-specific repetitive DNA element from Sorghum bicolor

 A 823-bp Sau3AI fragment (pSau3A10) was subcloned from a sorghum bacterial artificial chromosome (BAC) clone, 13I16, that contains DNA sequences specific to the centromeres of grass species. Sequence analysis showed that pSau3A10 consists of six copies of an approximately 137-bp monomer. The six monomers were organized into three dimers. The monomers within the dimers shared 62–72% homology and the dimers were 79–82% homologous with each other. Fluorescence in situ hybridization (FISH) analysis indicated that the Sau3A10 family is present only in the centromeres of sorghum chromosomes. Sequencing, Southern hybridization, and Fiber-FISH analyses indicated that the Sau3A10 family is tandemly arranged and is present in uninterrupted stretches of up to at least 81 kb of DNA. Slot-blot analysis estimated that the Sau3A10 family constitutes 1.6–1.9% of the sorghum genome. The long stretches of Sau3A10 sequences were interrupted by other centromeric DNA elements. Southern analysis indicated that the Sau3A10 sequence is one of the most abundant DNA families located in sorghum centromeres and is conserved only in closely related sorghum species. Methylation experiments indicated that the cytosine of the CG sites in sorghum centromeric regions is generally methylated. The structure and organization of the Sau3A10 family shared similarities with centromeric DNA repeats in other eukaryotic species. It is suggested that the Sau3A10 family is probably an important part of sorghum centromeres. Received: 11 November 1997 / Accepted: 17 November 1997     

Evolutionary features of chondriome divergence in Triticum (wheat) and Aegilops shown by RFLP analysis of mitochondrial DNAs

The first comprehensive analysis was made of restriction fragment length polymorphism (RFLP) of the mitochondrial (mt) DNA of two related genera, Triticum (wheat) and Aegilops. This led to clarification of the nature of mtDNA variability and the inference of the phylogeny of the mitochondrial genomes (=chondriome). Forty-six alloplasmic lines and one euplasmic line of common wheat (2n = 42, genomes AABBDD) carrying plasmons (cytoplasmic genomes) of 47 accessions belonging to 33 species were used. This consisted of nearly all the Triticum and Aegilops species. RFLP analysis, carried out with seven mitochondrial gene probes (7.0 kb in total) in combination with three restriction endonucleases, found marked variation: Of the 168 bands detected, 165 were variable (98.2%), indicative that there is extremely high mtDNA variability in these genera. This high variability is attributed to the variation present in the intergenic regions. Most of the variation was between chondriomes of different plasmon types; only 8 bands (4.8%) between those of the same plasmon types were variable, evidence of clear chondriome divergence between different plasmon types. The first comprehensive phylogenetic trees of the chondriome were constructed on the basis of genetic distances. All but 1 of the polyploids had chondriomes closely related to those of 1 putative parent, indicative of uniparental chondriome transmission at the time of polyploid formation. The chondriome showed parallel evolutionary divergence to the plastome (chloroplast genome). Use of a minimum set of 3 mtDNA probe-enzyme combinations is proposed for tentative plasmon type identification and the screening of new plasmon types in those genera. Received: 20 March 1999 / Accepted: 22 June 1999     

Characterization of Aegilops uniaristata chromosomes by comparative DNA marker analysis and repetitive DNA sequence in situ hybridization

RFLP analyses were performed on wheat-Aegilops uniaristata Vis. addition and translocation lines to confirm the identity of added N-genome chromosomes. Complete 1N, 3N, 4N, 5N and 7N chromosome additions were identified, while the complete long arm and only part of the short arm was identified for chromosome 2N. There were no wheat-like 4/5 and 4/7 translocations in the Ae. uniaristata chromosomes. Chromosome 3N carried an asymmetric pericentric inversion, and the translocation line was a product of centric fusion between the long arms of chromosomes 3B and 3N. Chromosome-specific RAPD and microsatellite markers were also identified for all the added Ae. uniaristata chromosomes available in this set of addition lines. A new genomic in situ hybridization protocol combining pre-annealing of probe and blocking DNA and prehybridization with blocking DNA was developed to differentiate the very closely related genomes of Ae. uniaristata and wheat. Hybridization sites for the repetitive DNA sequences pAs1, pSc119.2 and pTa71 were identified on the N-genome chromosomes of Ae. uniaristata using the fluorescent in situ hybridization technique. Results showed deviation from the previously published ideogram of this species. A new ideogram, which shows the hybridization sites for the above sequences, was produced in which the chromosomes are arranged according to their homoeologous group. Received: 23 April 1999 / Accepted: 6 August 1999     

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

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

The retrotransposon RTip1 is integrated into a novel type of minisatellite, MiniSip1, in the genome of the common morning glory and carries another new type of minisatellite, MiniSip2

 Transposable elements have often been discovered as new insertion sequences in known genes, and minisatellites are often employed as molecular markers in diagnostic and mapping studies. We compared the genes for flower pigmentation in a line of the common morning glory bearing fully colored flowers with those in two anthocyanin ^flaked mutable lines producing variegated flowers and found RFLPs at the region of the ANS gene for anthocyanin biosynthesis. The DNA rearrangements detected by the RFLPs are due to integration of a novel type of minisatellite, MiniSip1, having a long LTR retrotransposon, RTip1, inserted in the mutable lines. The structural analysis of the rearranged region revealed that the 12.4-kb RTip1 element is flanked by 5-bp target duplications within the MiniSip1 sequence and contains two LTR sequences of about 590 bp, a primer binding site for tRNA^Lys, a typical polypurine tract and another new type of minisatellite, MiniSip2. Since no long open reading frame corresponding to the gag and pol genes was found, RTip1 appears to be a defective Ty3/gypsy-like element. Interestingly, the 269-bp-long MiniSip1 element comprises two alternating motifs of 41 bp and 19 bp, whereas the 962 bp long MiniSip2 element consists of two partially alternating motifs of 86 bp and 90 bp which are partially homologous to each other. Possible evolutionary processes that may have generated the rearranged structure at the ANS gene region are also discussed. Received: 25 April 1997 / Accepted: 16 May 1997     

Use of genome-specific repetitive DNA sequences to monitor chromatin introgression from Festuca mairei into Lolium perenne

Repetitive DNA sequences contribute considerably to an understanding of the genomes of higher plants. Repetitive DNA sequences tend to be genome-specific due to the rate of amplification and extent of divergence. Two genome-specific probes from the genomic DNA library of Festuca arundinacea var. genuina Schreb.were selected and characterized. TF521 was found to be P genome-specific since it was able to hybridize with Festuca pratensis Huds. (PP) and Festuca arundinacea var. genuina (PPG₁G₁G₂G₂), but not, or only weakly, with tetraploid Festuca species. TF521 hybridized only with the diploid Festuca and not with the Lolium species (LL). TF436 was specific to tetraploid species of Festuca, such as F. arundinacea var. glauces-cens Boiss. (G₁G₁G₂G₂) and Festuca mairei St. Yves (M₁M₁M₂M₂). By means of Southern hybridization, TF436 was used to detect chromatin introgression of F. mairei in the progenies of the hybrid F. mairei×Lolium perenne L. Potential addition and translocation lines were identified in the BC₁F₁ derivatives of F. mairei×L. perenne. In situ hybridization was used to confirm the genetic identity of these lines. Sequence analyses indicated that TF436 and TF521 were two novel DNA sequences as no homologous sequences were found in Genebank. Received: 22 June 2000 / Accepted: 3 November 2000     

Pstl repeat: a family of short interspersed nucleotide element (SINE)-like sequences in the genomes of cattle, goat, and buffalo.

The PstI family of elements are short, highly repetitive DNA sequences interspersed throughout the genome of the Bovidae. We have cloned and sequenced some members of the PstI family from cattle, goat, and buffalo. These elements are approximately 500 bp, have a copy number of 2 x 10(5) - 4 x 10(5), and comprise about 4% of the haploid genome. Studies of nucleotide sequence homology indicate that the buffalo and goat PstI repeats (type II) are similar types of short interspersed nucleotide element (SINE) sequences, but the cattle PstI repeat (type I) is considerably more divergent. Additionally, the goat PstI sequence showed significant sequence homology with bovine serine tRNA, and is therefore likely derived from serine tRNA. Interestingly, Southern hybridization suggests that both types of SINEs (I and II) are present in all the species of Bovidae. Dendrogram analysis indicates that cattle PstI SINE is similar to bovine Alu-like SINEs. Goat and buffalo SINEs formed a separate cluster, suggesting that these two types of SINEs evolved separately in the genome of the Bovidae.     

Role of a 461-bp G-rich repetitive element in H19 transgene imprinting

 The molecular mechanism leading to the imprinted expression of genes is poorly understood. While no conserved cis-acting elements have been identified within the known loci, many imprinted genes are located near directly repetitive sequence elements, suggesting that such repeats might play a role in imprinted gene expression. The maternally expressed mouse H19 gene is located approximately 1.5 kb downstream from a 461-bp G-rich repetitive element. We have used a transgenic model to investigate whether this element is essential for H19 imprinting. Previous results demonstrated that a transgene, which contains 14 kb of H19 sequence, exhibits parent-of-origin specific expression and methylation analogous to the endogenous H19 imprinting pattern. Here, we have generated transgenes lacking the G-rich repeat. One transgene, containing a deletion of the G-rich repetitive element but which includes an additional 1.7 kb of 5’H19 sequence, is imprinted similarly to the endogenous H19 gene. To determine whether the G-rich repeat is conserved in other imprinted mammalian H19 homologues, additional 5’ flanking sequences were cloned from the rat and human. This element is conserved in the rat but not in human DNA. These results suggest that the 461-bp G-rich repetitive element is not essential for H19 imprinting. Received: 26 August 1998 / Accepted: 14 December 1998     

Cloning and molecular characterization of a novel x-type glutenin gene Glu-D(t)1 from Aegilops tauschii

A novel gene encoding an x-type high molecular weight glutenin subunit (HMW-GS), designated 1Dx1.1 ^t , was isolated from Aegilops tauschii. It is the largest HMW-GS gene reported so far in this species and its product has a slower mobility than that of subunit 1Ax1 in SDS-PAGE. The open reading frame (ORF) of the gene was 2,628 bp, encoding a protein of 874 amino acid residues. Comparisons of amino acid sequences showed that subunit 1Dx1.1^t had high similarity with other 1Dx subunits but also had two unique characteristics. Firstly, a tripeptide of consensus LQE present in the N-terminal domains of other 1Dx subunits was absent from subunit Dx1.1^t. Secondly, three copies of tandem duplications of the tripeptide motif GQQ and a novel tripeptide sequence (GQL) were present in its central repetitive domain. Phylogenetic analysis showed that subunit 1Dx1.1^t clustered with other known 1Dx subunits.     

A cereal cyst nematode (Heterodera avenae Woll.) resistance gene transferred from Aegilops triuncialis to hexaploid wheat

 The cereal cyst nematode (Heterodera avenae) is an important root parasite of common wheat. A high level of resistance was transferred to wheat from Aegilops triuncialis (TR lines) using the cross [(T. turgidum×Ae. triuncialis)×T. aestivum]. Low fertility (3–5 viable kernels per plant) was observed during the process but the surviving hybrid plants were highly vigorous. To obtain stable resistant lines further crosses to T. aestivum were performed. The resistance in TR lines seems to be transferred from the C genome of Ae. triuncialis (genomes CCUU). Ae. triuncialis was highly resistant to the two Spanish populations of H. avenae tested, as well as to four French races and two Swedish populations. The histological analysis showed a hypersensitive reaction in the roots of a resistant TR line inoculated with the Ha71 pathotype of H. avenae, whereas well-formed syncytia were observed in the roots of the susceptible control. Resistance to the H. avenae Ha71 pathotype seemed to be inherited as determined by a single dominant factor in the crosses between resistant TR lines and susceptible cultivars. Received: 11 November 1997 / Accepted: 9 December 1997     

Amonabactin: a family of novel siderophores from a pathogenic bacterium

 Four peptide-based bis-catecholate siderophores, collectively known as the amonabactins, have been isolated from Aeromonas hydrophila. They have been fully characterized: tandem mass spectroscopy established the sequence of the amino acid components, chiral gas chromatographic mass spectra established the amino acid chirality, and two-dimensional NMR techniques determined the full connectivity and structure. Each of the amonabactins was synthesized and the synthetic material was compared to the natural product as a final proof of structure. These siderophores are bis-catecholates with the backbone composed of either tri- or tetrapeptides in the sequence (gly)-(L)-lys-(L)-lys-(D)-aro, where glycine is the optional amino acid attached to the N^ε of the N terminus lysine and aro is either tryptophan or phenylalanine. The ligand units, 2,3-dihydroxybenzamide groups, are attached to the N^ε amine of the C terminus lysine and either to glycine, if present, or to N^ε amines of the N terminus lysine. Each of the amonabactins supports growth of the organism under low-iron conditions in vitro and in serum. The architecture of these siderophores includes an inverted aromatic amino acid and unusual linkages which should prevent enzymatic hydrolysis of the peptide backbones. This, along with their ability to successfully compete for iron in serum, suggests a role in the pathogenicity of the organism. This paper is number 62 in the series Coordination Chemistry of Microbial Ion Transport. Details of the previous paper are given in [1]. Received: 6 June 1997 / Accepted: 9 September 1997     

The introgressed segment carrying rust resistance genes Yr17, Lr37 and Sr38 in wheat can be assayed by a cloned disease resistance gene-like sequence

A cloned gene sequence (Vrga1D), with features of the nucleotide-binding-site leucine-rich repeat class of disease resistance (R) gene sequence super family, was previously shown to belong to a family of five gene members derived from a Triticum ventricosum Ces. (syn. Aegilops ventricosa Tausch) segment in wheat (Triticum aestivum L.). This gene family was introgressed, together with the linked rust resistance genes Yr17, Lr37 and Sr38 from T. ventricosum, to wheat chromosome 2AS. An independently derived T. ventricosum segment carrying a leaf rust resistance gene in a French wheat cultivar, was shown to exhibit a rust resistance response equivalent to Lr37 as well as Yr17 and Sr38. DNA probes from different regions of the Vrga1D clone consistently detected the presence of RFLPs associated with the introgressed segment carrying the resistance genes Yr17, Lr37 and Sr38 present in diverse wheat genotypes from Australia, Canada, France and the UK. Our results showed that the transfer of the T. ventricosum- derived Vrga1 gene members and the rust resistance genes were always accompanied by the loss of a corresponding set of Vrga1-related gene members in recipient wheat cultivars presumed to be of homoeoallelic origin. A PCR assay, based on sequences from the 3"-untranslated region of a Vrga1 gene member isolated from the T. ventricosum donor line of the introgressed segment, was developed. The PCR assay detected the presence of the introgressed rust resistance genes across the diverse wheat backgrounds and should be useful in marker- assisted selection in wheat breeding. Received: 24 December 1999 / Accepted: 13 June 2000     

Gene flow from wheat (Triticum aestivum L.) to jointed goatgrass (Aegilops cylindrica Host.), as revealed by RAPD and microsatellite markers

In order to estimate the potential of gene flow between wheat (Triticum æstivum L.) and jointed goatgrass (Aegilops cylindrica Host.), we carried out mixed pollinations in experimental and natural conditions. A set of species-specific RAPD (random amplified polymorphic DNA) and microsatellite markers were used to detect the presence of parental markers in the progeny of the plants used in these experiments. No hybrids were found within the offsprings of the plants used for the greenhouse experiments, while 85 Ae. cylindrica×T. æstivum hybrids were found within 2400 analyzed F₁ plants resulting from the field pollinations. The hybridization rates for individuals of different populations of the wild species differed considerably: 1% for two populations known for more than 90 years versus 7% for a newly discovered population. Most of the hybrids were completely sterile, but five of them produced 13 seeds (BC₁) by backcross with Ae. cylindrica. Twelve seeds germinated and generated viable and partly fertile plants. About 25% of the wheat specific RAPD markers were found in the BC₁ plants, indicating that introgression of wheat DNA into Ae. cylindrica is possible. In addition, one microsatellite marker, known to be situated on the D genome (a genome shared by both species), was also found in the BC₁ plants.