Localization of repetitive DNA in cereals byin situ hybridization: Cross hybridization among wheat,rye, barley,and oat

³H-RNA, complementary to repetitive DNA of wheat, rye, barley, and oat, was hybridizedin situ to root tip or pollen mother cells of the species mentioned. The cRNAs hybridized best with the DNA in cell nuclei of the species from which they were prepared. Cross hybridization with cells of the other related species resulted in a significant but diminished labelling. Wheat, rye, and barley hybridized better to each other than to oat, andvice versa, in agreement with the usual taxonomical classification. Over the interphase nuclei the label was distributed unevenly; not all regions of dense chromatin were labelled, and little label was found over the nucleoli. On chromosomes, the repetitive DNA was located somewhere along the chromosome arms or near the centromers in wheat, barley, and oat. Only in rye, most of the label was located near the telomers, probably over the large heterochromatin areas.     

Host range,mating type and population structure of Magnaporthe sp. of a single barley field in São Paulo state,Brazil

Brazil is blast disease hot spot because severe epidemics have occurred among wheat, triticale, rye, barley and oat crops. Although the first outbreak of barley blast appeared in 1998, little information is available. Therefore, this study aimed to examine host range, mating type composition and population structure of Magnaporthe sp. from a single barley field in São Paulo, Brazil. To examine pathogenicity, 25 Magnaporthe isolates were inoculated on five, three, two and two cultivars of barley, wheat, oat and rice, respectively, and one cultivar each of rye, corn, sorghum, triticale and certain weeds (Cenchrus echinatus, Setaria geniculata, Brachiaria plantaginea and Eleusine indica). Mating type distribution of 33 isolates was investigated by molecular tools. The genotypic divergence of 41 barley and five wheat isolates was investigated by 15 random amplified polymorphic DNA primers and unweighted pair group method with arithmetic mean. The host range of the barley blast pathogen included wheat, oat, rye and triticale but not rice and weeds. Sexual reproduction appeared to not be involved in the high genotypic diversity because only a single isolate, MAT1‐2, was identified. The majority of barley isolates clustered together with wheat blast, except for four, suggesting a different origin.     

The development of sequence-tagged sites (STSs) in Lolium perenne L.: the application of primer sets derived from other genera

Genetic analysis, particularly the development of genetic linkage maps in forage grass species, lags well behind other members of the Poaceae. Comparative mapping within this family has revealed extensive conservation in gene and marker synteny among chromosomes of diverse genera. Recently, the ability to transfer mapped STS markers between barley and wheat has been demonstrated. The transfer of mapped STS markers between cereals and forage grasses could provide PCR-based markers for comparative mapping in these species providing they amplify homologous sequences. In this study, primers derived from three barley genes of defined function and a gene from Phalaris coerulescens were used to amplify homologous fragments in Lolium perenne. Primers derived from two barley and two oat cDNA clones were also tested along with eight barley and two Triticum tauchii STS markers. Twenty one primer pairs derived from 18 loci were tested. Eleven primer pairs (52%) amplified homologous sequences in L. perenne from ten (55%) of the loci targetted. Thirteen new STS markers were generated in L. perenne, of which ten have been mapped in barley or rye and amplify homologous sequences in L. perenne. Received: 20 October 2000 / Accepted: 13 January 2001     

Molecular and chromosomal organization of DNA sequences coding for the ribosomal RNAs in cereals

The chromosomal locations of ribosomal DNA in wheat, rye and barley have been determined by in situ hybridization using high specific activity ¹²⁵I-rRNA. The 18S-5.8S-26S rRNA gene repeat units in hexaploid wheat (cv. Chinese Spring) are on chromosomes 1B, 6B and 5D. In rye (cv. Imperial) the repeat units occur at a single site on chromosome 1R(E), while in barley (cv. Clipper) they are on both the chromosomes (6 and 7) which show secondary constrictions. In wheat and rye the major 5S RNA gene sites are close to the cytological secondary constrictions where the 18S-5.8S-26S repeating units are found, but in barley the site is on a chromosome not carrying the other rDNA sequences. — Restriction enzyme and R-loop analyses showed the 18S-5.8S-26S repeating units to be approximately 9.5 kb long in wheat, 9.0 kb in rye and barley to have two repeat lengths of 9.5 kb and 10 kb. Electron microscopic and restriction enzyme data suggest that the two barley forms may not be interpersed. Digestion with EcoR1 gave similar patterns in the three species, with a single site in the 26S gene. Bam H1 digestion detected heterogeneity in the spacer regions of the two different repeats in barley, while in rye and wheat heterogeneity was shown within the 26S coding sequence by an absence of an effective Bam H1 site in some repeat units. EcoR1 and Bam H1 restriction sites have been mapped in each species. — The repeat unit of the 5S RNA genes was approximately 0.5 kb in wheat and rye and heterogeneity was evident. The analysis of the 5S RNA genes emphasizes the homoeology between chromosomes 1B of wheat and 1R of rye since both have these genes in the same position relative to the secondary constriction. In barley we did not find a dominant monomer repeat unit for the 5S genes.     

Structural and functional organization of the '1S0.8 gene-rich region' in the Triticeae

Wheat genes are present in physically small, gene-rich regions, interspersed by gene-poor blocks of retrotransposon-like repetitive sequences. One of the largest gene-rich regions is present around fraction length (FL) 0.8 of the short arm of wheat homoeologous group 1 chromosomes and is called `1S0.8 region'. The objective of this study was to reveal the structural and functional organization of the `1S0.8 region' in various Triticeae and other Poaceae species. Consensus genetic linkage maps of the `1S0.8 region' were constructed for wheat, barley, and rye by combining mapping information from 16, 11, and 12 genetic linkage maps, respectively. The consensus genetic linkage maps were compared with each other and with a consensus physical map of wheat homoeologous group 1. Comparative analyses localized 75 agronomically important genes to the `1S0.8 region'. This high-resolution comparison revealed exceptions to the rule of conserved gene synteny, established using low-resolution marker comparisons. Small rearrangements such as duplications, deletions, and inversions were observed among species. Proportion of chromosomal recombination occurring in the `1S0.8 region' was very similar among species. Within the gene-rich region, the extent of recombination was highly variable but the pattern was similar among species. Relative recombination among markers was similar except for a few loci where drastic differences were observed among species. Chromosomal rearrangements did not always change the extent of recombination for the region. Differences in gene order and relative recombination were the least between wheat and barley, and were the highest between wheat and oat.     

PHYLOGENETIC ANALYSIS OF 32 STRAINS OF VAUCHERIA (XANTHOPHYCEAE) USING THE rbcL GENE AND ITS TWO FLANKING SPACER REGIONS1

The complete rbcL gene was sequenced for 21 species and 32 strains of Vaucheria and for five other Xanthophyceae (Asterosiphon dichotomus (Kützing) Rieth, Botrydium becharianum Vischer, B. cystosum Vischer, B. stoloniferum Mitra, Tribonema intermixtum Pascher). The psbA‐rbcL spacer, upstream of the rbcL gene, and the RUBISCO spacer between the rbcL and rbcS genes were also completely sequenced for the Vaucheria strains and Asterosiphon. The psbA‐rbcL spacer was the most variable region that was sequenced, and only the 3′ end of the spacer could be aligned. Phylogenetic analyses (maximum parsimony, neighbor joining, and maximum likelihood) were conducted using the DNA sequence and the amino acid sequence for the rbcL gene, and a second analysis was conducted using a portion of the psbA‐rbcL spacer +rbcL gene + RUBISCO spacer. All analyses showed that Vaucheria species formed monophyletic clades that corresponded with morphologically based subgeneric sections, including the section Racemosae. Species producing a gametophore (= fruiting branch, bearing both an antheridium and oogonium) formed a monophyletic clade in all analyses. The nongametophore species sometimes formed a monophyletic clade but other times formed a basal grade. Pair‐wise comparisons of nucleotides and amino acids showed that for some species, numerous nucleotide changes resulted in relatively few amino acid changes. Consequently, phylogenetic analysis of the amino acids produced numerous trees, which in a strict consensus tree resulted in numerous polychotomies. An original strain of V. terrestris that was deposited in two culture collections over 25 years ago had identical sequences, suggesting no rapid change was occurring in the sequenced regions. Two strains of V. prona, isolated from Europe and North America, had identical sequences. Other species, for which two or more strains were examined, had different sequences. These results suggest that cryptic species complexes exist within Vaucheria because the rbcL gene is a conservative gene that is identical in other protists.     

II. Cereal disease: the interplay between resistance and pathogenicity: Inheritance of pathogenicity in Gaeumannomyces graminis var. tritici

Variation in host response of isolates of the eyespot pathogen from different sources was examined over a number of years. Pathogen types were found in intensively-cropped couch-infested cereal sites that were almost as virulent on Agropyron repens (couch) as on wheat or barley. The commonly occurring wheat (W) type isolates from couch-free cereal crops were virulent on wheat and barley but avirulent on couch. Couch (C) types were isolated not only from couch but also from wheat, barley and oat crops with couch infestation. In pathogenicity tests on rye, C. types did not differ in virulence from the more commonly occurring W types. Aegilops ventricosa was equally resistant to both types. W type isolates from wheat and barley were examined to assess differential pathogenicity on wheat and barley. Sequential cropping with single cereal crops was used to separate out possible specific types. Isolates from fourth wheat and fourth barley crops were more pathogenic on the original than on the alternative host. When comparisons were made between isolates from third and fifth consecutive wheat and barley crops only those from barley showed a preference for the original host. An experiment comparing isolates from third and seventh consecutive wheat and barley crops showed a decline in virulence from the short to the longer sequences on the alternative but not on the original host.     

Utility of rbcS gene as a novel target DNA region for brown algal molecular systematics

The usefulness of molecular phylogenetic studies has increased remarkably as the quantity and quality of available DNA sequences has increased. When compared with the progress that has occurred in angiosperms and animals, there have been relatively few target DNA regions identified for use in taxonomic studies of brown algae. Therefore, in this study, we developed a new set of primers to amplify Rubisco small subunit (rbcS) gene sequences and determined the rbcS gene sequences of various species of brown algae including those belonging to Dictyotales, Ectocarpales, Fucales and Sphacelariales. The level of sequence variations in the rbcS gene varied according to the brown algal lineages. When focusing on the relationship of species within the genus Sargassum, the rbcS gene sequences provided useful information regarding the phylogenetic relationship among sections of the subgenus Bactrophycus. Based on the broad applicability and phylogenetic utility of the rbcS gene, we suggest that the sequence be used as a new target region for the molecular systematics of brown algae.     

Updating of transposable element annotations from large wheat genomic sequences reveals diverse activities and gene associations

Triticeae species (including wheat, barley and rye) have huge and complex genomes due to polyploidization and a high content of transposable elements (TEs). TEs are known to play a major role in the structure and evolutionary dynamics of Triticeae genomes. During the last 5 years, substantial stretches of contiguous genomic sequence from various species of Triticeae have been generated, making it necessary to update and standardize TE annotations and nomenclature. In this study we propose standard procedures for these tasks, based on structure, nucleic acid and protein sequence homologies. We report statistical analyses of TE composition and distribution in large blocks of genomic sequences from wheat and barley. Altogether, 3.8 Mb of wheat sequence available in the databases was analyzed or re-analyzed, and compared with 1.3 Mb of re-annotated genomic sequences from barley. The wheat sequences were relatively gene-rich (one gene per 23.9 kb), although wheat gene-derived sequences represented only 7.8% (159 elements) of the total, while the remainder mainly comprised coding sequences found in TEs (54.7%, 751 elements). Class I elements [mainly long terminal repeat (LTR) retrotransposons] accounted for the major proportion of TEs, in terms of sequence length as well as element number (83.6% and 498, respectively). In addition, we show that the gene-rich sequences of wheat genome A seem to have a higher TE content than those of genomes B and D, or of barley gene-rich sequences. Moreover, among the various TE groups, MITEs were most often associated with genes: 43.1% of MITEs fell into this category. Finally, the TRIM and copia elements were shown to be the most active TEs in the wheat genome. The implications of these results for the evolution of diploid and polyploid wheat species are discussed. Electronic Supplementary Material Supplementary material is available for this article at     

Abundance, variability and chromosomal location of microsatellites in wheat

The potential of microsatellite sequences as genetic markers in hexaploid wheat (Triticum aestivum) was investigated with respect to their abundance, variability, chromosomal location and usefulness in related species. By screening a lambda phage library, the total number of (GA)_n blocks was estimated to be 3.6 x 10⁴ and the number of (GT)_n blocks to be 2.3 x 10⁴ per haploid wheat genome. This results in an average distance of approximately 270 kb between these two microsatellite types combined. Based on sequence analysis data from 70 isolated microsatellites, it was found that wheat microsatellites are relatively long containing up to 40 dinucleotide repeats. Of the tested primer pairs, 36% resulted in fragments with a size corresponding to the expected length of the sequenced microsatellite clone. The variability of 15 microsatellite markers was investigated on 18 wheat accessions. Significantly, more variation was detected with the microsatellite markers than with RFLP markers with, on average, 4.6 different alleles per microsatellite. The 15 PCR-amplified microsatellites were further localized on chromosome arms using cytogenetic stocks of Chinese Spring. Finally, the primers for the 15 wheat microsatellites were used for PCR amplification with rye (Secale cereale) and barley accessions (Hordeum vulgare, H. spontaneum). Amplified fragments were observed for ten primer pairs with barley DNA and for nine primer pairs with rye DNA as template. A microsatellite was found by dot blot analysis in the PCR products of barley and rye DNA for only one primer pair.     

Higher-level systematics of Acanthaceae determined by chloroplast DNA sequences

Parsimony analyses of ndhF chloroplast gene sequences were undertaken for 15 species of Acanthaceae and nine representative outgroup species. In addition, parsimony analyses of rbcL sequences were undertaken for 12 species of Acanthaceae and the same nine outgroup species as for ndhF. The results indicate that ndhF provides more informative characters and greater systematic resolution at this hierarchical level than rbcL. The ndhF analyses demonstrate that Elytraria and Thunbergia are successive sister taxa to all Acanthaceae taxa that have retinacula and explosive fruits. These data also demonstrate that taxa with both retinacula and explosive fruits can be subdivided further into two monophyletic groups that correspond to taxa with and without cystoliths. Within the group with cystoliths three putatively monophyletic groups correspond to taxa possessing quincuncial, left contort, and ascending-cochlear corolla aestivation patterns. The results of the rbcL analysis provide less systematic resolution than ndhF but do contain several congruent arrangements of taxa within Acanthaceae.     

Tissue specificity of the sugarcane bacilliform virus promoter in oat,barley and wheat

The tissue-specificity of the sugarcane bacilliform virus (SCBV) promoter was investigated in oat, barley, and wheat to determine whether its expression pattern in one species was predictive of promoter specificity in the other closely related Gramineae species. Progeny of transgenic plants produced using constructs containing the SCBV promoter driving gusA were sampled at different stages of plant development and stained for GUS activity using a histochemical assay. Overall, the GUS staining patterns were most similar between oat and barley. In all three species, similar GUS staining patterns were observed in mature endosperms, leaves, and floral bracts of developing infloresences. No GUS staining was detected in oat embryos whereas the entire barley embryo was stained, and GUS staining was confined to the scutellum of wheat embryos. Oat and barley stems exhibited GUS staining whereas no GUS staining was observed in stems of the transgenic wheat plants. The SCBV promoter conferred strong GUS staining intensity in most tissues of oat and barley but was generally weaker in wheat. These differences in SCBV promoter specificity indicate that promoter evaluation should be conducted in the target species of interest rather than by extrapolation from expression patterns in other species.     

Identification of Homologous Globulins from Embryos of Wheat, Barley, Rye and Oats

Total globulins from embryos and endosperms of barley, wheat,rye, and oats were separated by SDS-PAGE under reducing andnon-reducing conditions. The preparations from embryos of allfour cereals contained major groups of bands with M_r's of 50-60000,which were not affected by reduction. These have been characterizedpreviously from oats and shown to correspond to subunits ofthe 7S storage globulin. Immunochemical relationships betweenthese bands (and others with M_r's between 40000 and 70000) weredemonstrated by immunodiffusion and ‘Western Blotting’using antiserum raised against the major subunits of the oat7S globulins. The 7S globulins were also prepared from hand-dissectedembryos of the four cereals using sucrose density ultracentrifugation.Their amino acid compositions were broadly similar, but differedfrom those of the 7S vicilins of legumes. It is concluded thatstructurally-related 7S globulins are present in the embryosof the four species of cereals. Key words: Homologous globulins, embryo, wheat, barley, rye, oats     

Molecular phylogeny of the genus Ceanothus (Rhamnaceae) using rbc L and ndh F sequences

 Intrageneric phylogeny among ten representative Ceanothus species was investigated using DNA sequences of the chloroplast encoded ndhF and rbcL genes. Parsimony analysis of the ndhF sequences identified two main clades corresponding to two subgenera Ceanothus and Cerastes. The phylogenetic results suggest that three monophyletic clades within the subgenus Ceanothus can be delimited on the basis of (1) evergreen or (2) deciduous leaves and (3) thorn presence within the evergreen clade. The estimated divergence time based on rbcL sequences suggests that the two subgenera diverged 18–39 million years ago whereas species within each subgenus diverged more recently. Taken together, the results support the division of Ceanothus into two monophyletic subgenera and are consistent with the postulated recent divergence of many species within each subgenus. Received: 25 September 1996/Accepted: 8 November 1996     

A Fragment of the rpoC2 Chloroplast RNA Polymerase Gene in Perennial and Annual Rye

A 1397-bp fragment corresponding to the rpoC2 chloroplast RNA polymerase gene was obtained by direct rye DNA amplification. Two rye species, Secale montanum Guss. and S. cereale L., did not practically differ in the structure of this DNA fragment (the nucleotide sequences were 99% identical). The corresponding nucleotide sequences in rye and wheat (Triticum aestivum L., Genbank accession no. AB027572) were 97–98% similar. The extent of the homology of various stretches of the rpoC2 rye gene with the corresponding sequences in maize and rice was 81–95%, whereas the deduced amino acid sequences of rpoC2 in rye, wheat, maize, and rice were considerably identical (96–97% of homology). The rye fragment of the rpoC2 gene differed from the corresponding sequences in three other grass species primarily by a short (49 bp) insert into the region of numerous short repeats corresponding to nucleotides 15750/15751, 28728/28729, and 27472/27473 in wheat, maize, and rice, respectively.     

Phylogenetic analysis of the Plagiotheciaceae (Musci) and its relatives based on rbcL gene sequences

Phylogenetic relationships are investigated using nucleotide sequences of the chloroplast gene, rbcL, for members of the family Plagiotheciaceae and its relatives. Nucleotide sequences of the rbcL were determined in 32 species (38 samples). In some species small differences in the nucleotide sequences of rbcL were recognized between the materials from different localities. The phylogenetic tree deduced from the sequences of the rbcL loci indicates the following: (1) pleurocarpous mosses form a monophyletic clade; (2) Plagiothecium is monophyletic; (3) Taxiphyllum is not closely related to Plagiothecium; (4) the family Hypnaceae is paraphyletic; (5) Pylaisiella is heterogeneous.     

Specific 5S ribosomal RNA primers for plant species i dentification in admixtures 3

The DNA sequences of the spacers between the 5S ribosomal RNA genes were determined for the cereals maize, barley, soghum, rye, rice, oat, and wheat. Species-specific primers were designed from the spacer region. PCR with these primers and a common primer from the conserved 5S ribosomal RNA gene sequence was investigated as a method for detection of the seven cereal species. DNA from these species could be specifically detected in mixtures. This technique could find application in the determination of the composition of admixtures or processed cereal products. The protocol described has potential for general application in the identification of plant species.     

The invasion of root tips of cereals by the cereal cyst nematode Heterodera avenue

Newly germinated seedlings of susceptible cultivars of oats, wheat, barley and rye were inoculated with second-stage juveniles of Heterodera avenae in pots of sand. Subsequent examination showed oat root tips to be more commonly invaded, and by a greater range of nematode numbers than the other cereals. A comparison of oats and barley showed that lower nematode numbers in barley were not due to a higher emigration from barley; invasion, establishment and emigration by nematodes all being greater in oats. Second-stage juveniles were more likely to migrate prior to establishment in barley than in oats.     

The occurrence of Ds-like sequences in cereal genomes

Summary The occurrence of DNA sequences similar to the Ds-element of sh-m5933 maize (Ds-like sequences) was studied in other representatives of the Gramineae. The approximate number of copies of such sequences found under gentle and stringent conditions of washing was determined by dot-hybridization. It was shown that in the maize genome the number of copies of Ds-like sequences exceeds about ten-fold the content of such sequences found in wheat, rye and barley genomes. Quantitative differences in Ds-like sequences between wheat species with various genomes and ploidies (when estimated per genome) as well as between different H. vulgare varieties was not determined. The various melting points (T_m) of DNA-duplexes formed when the Ds-element is hybridized with wheat, rye and barley DNA respectively do not show significant differences and are essentially lower than the T_m of the Ds-element (by 8°–9°C). Thus, these duplexes have 9–11% of nucleotide substitutions in comparison to Ds sh-m5933. The data obtained permit one to suppose the presence of a series of Ds-like sequences heterogenous for the length and degree of homology to the Ds-element isolated from the shrunken locus (sh-m5933) of maize DNA.