Search

In wheat-rye hybrids the nucleolus organizer regions (NORs), the sites of ribosomal RNA genes, from rye are suppressed. Wheat and wheat-rye hybrid genetic stocks containing different numbers of wheat and rye nucleolus organizers, as well as addition lines and rye-barley hybrids, were used in Southern hybridization experiments to determine the cause of nucleolar dominance and suppression in cereal hybrids. Based on the use of restriction endonucleases that cleave near the ends of the spacer unit and an additional, methylation-sensitive enzyme, HpaII, which does not recognize the CCGG restriction site if the internal C is methylated, an indirect method of assaying NOR expression was established. The results indicated that cleavage by the HpaII enzyme of the rye NOR sequences, is reduced when major NORs from other cereals were present. The reduction in the number of rye rRNA genes containing an unmethylated CCGG site in the promoter was associated with the suppression of the rye nucleolus. These results are consistent with a model in which promoter and upstream regulatory repeats of ribosomal RNA genes compete for limited concentrations of regulatory proteins, and genes that are methylated at key binding sites fail to engage these regulatory proteins and thus remain inactive.

WIS 2-1A, the first retrotransposon found in wheat, has been recently studied and characterized. Southern hybridization experiments utilizing several species from the Triticeae revealed the presence of homologous sequences in all the taxa tested, showing high levels of interspecific variability and almost no intraspecific differentiation. Further experiments using in situ hybridization in several species showed that the retroposon was almost completely dispersed throughout the genomes tested. These results suggested that WIS 2-1A is an ancient element that probably was present in the unknown common ancestor of the Triticeae and that only under rare circumstances does it become active. DNA fragments homologous to the WIS 2-1A reverse transcriptase gene were isolated from most of the Triticeae species using PCR. The fragments obtained were sequenced and analyzed. Even though the sequence alignment was consistent with the phylogenetic studies made in the past, the genus Thinopyrum showed new evidence for a possible horizontal propagation of the retroelement.Mention of a trade name or proprietary product does not constitute a guarantee, warranty or recommendation of the product by the U.S. Department of Agriculture or the University of Missouri and does not imply its approval to the exclusion of other products that may be suitableAll programs and services of the U.S. Department of Agriculture are offered on a non-discriminatory basis without regard to race, colour, national origin, religion, sex, age, marital status, or handicap.

Sequences homologous to the retro-element BIS-1 and the stem-loop repeat Hi-10 are present in the genomes of a number of cereal species. A detailed characterization of these elements indicated that they are non-randomly organized in the genomes of at least two of these species, namely barley and rye. In contrast to the BIS-1 retro-elements, the stem-loop repeats are also non-randomly organized into discrete domains in interphase nuclei from barley and rye. Features of the organization of these repeats along chromosomes and within interphase nuclei of rye, barley and rice are discussed.

The actin gene family of Nicotiana tabacum has been partially characterised by Southern hybridisation and by isolating lambda EMBL4 recombinants from a genomic library having homology to the soybean actin gene, Sac3. The number of actin genes with homology to Sac3 is estimated at between 20 to 30, based on Southern hybridisation and library screening, though the total gene family may be larger. Twenty-four recombinant lambda clones were isolated, 18 had unique restriction profiles and from these, 2 clones, Tac9 and Tac25, were selected for further study. The region of Tac25 hybridizing to Sac3 was sequenced and shown to contain an open reading frame (ORF) with homology to actin. Partial sequencing of Tac9 revealed a sequence with homology to the third exon of Tac25 and Sac3. The two tobacco actin sequences were compared to other reported actin gene sequences; Tac25 was closely related to the allelic potato actins, Pac58 and Pac85, while Tac9 was more related to Pac79 than to other plant actins. Northern hybridisation analysis showed that while Tac9 detected actin transcripts in RNA from root, leaf, stigma and pollen, Tac25 transcripts were only detected in pollen RNA.

WIS-2-1A, a 8624 bp insertion in the Glu-1A-2 locus of chromosome 1A of wheat, consists of two 1755 bp long terminal repeats enclosing a 5114 bp internal region. No long open reading frames could be found, but inspection of the predicted amino acid sequence showed regions with homology to retrotransposon structures, including a methionine tRNA initiator binding site, a nucleotide binding domain, a protease, an integrase and a polymerase. DNA replication errors have resulted in frame-shifts in the protein coding region, suggesting that retrotransposition of WIS-2-1A, if it occurs, must be mediated by trans-acting factors.

Mitochondrial DNAs have been examined in accessions of 25 Mexican races of maize and compared with the mitochondrial DNAs previously found in inbred lines from the USA. Many variants were found. Low molecular weight DNA components, not previously found in US lines, were found in many of the accessions. Accessions classified as belonging to the same race, and plants from a single accession, sometimes had different mitochondrial genomes. Mitochondrial genomes similar to those in T and S cytoplasms were found in Mexican accessions.A low molecular weight linear DNA species has partial homology with a sequence in the high molecular weight mitochondrial genome. All plants with a shorter version of the linear molecule had a correspondingly altered region of homology in the high molecular weight genome.There is evidence that the geographical distribution of mitochondrial DNA types within Mexico is not random. One type, found in the oldest races, appears to be widely dispersed but another less common type appears to be confined largely to coastal regions. The potential value of these findings in maize breeding and for evolutionary studies is discussed.

Restriction enzyme digestion of the ribosomal RNA genes of the nucleolar organisers of wheat has revealed fragment length polymorphisms for the nucleolar organiser on chromosome 1B and the nucleolar organiser on 6B. Variation between genotypes for these regions has also been demonstrated. This variation has been exploited to determine the recombination frequency between the physically defined nucleolar organiser on 1B (designatedNor1) and other markers; two loci,Glu-B1 andGli-B1 which code for endosperm storage proteins andRf3, a locus restoring fertility to male sterility conditioned byT. timopheevi cytoplasm.Gli-B1 andRf3 were located on the short-arm satellite but recombine with the nucleolar organiser giving a gene order ofNor1 - Rf3 - Gli-B1. Glu-B1 is located on the long arm of 1B but shows relatively little recombination withNor1, which is, in physical distance, distal on the short arm. This illustrates the discrepancy between map distance and physical distance on wheat chromosomes due to the distal localisation of chiasmata. The recombination betweenNor1 andRf3 indicates that, contrary to previous suggestions, fertility restoration is not a property of the nucleolar organiser but of a separate locus.

Subunits of wheat endosperm proteins have been fractionated by two-dimensional electrophoresis. To determine which subunits in the two-dimensional electrophoretic pattern belong to gliadin or glutenin the endosperm proteins have also been fractionated by a modified Osborne procedure and by gel filtration on Sephadex G-100 and Sepharose CL-4B prior to separation by two-dimensional electrophoresis.The control of production of five major grain protein subunits is shown to be determined by chromosomes 6A, 6B and 6D by comparing two-dimensional electrophoretic protein subunit patterns of aneuploid lines of the variety 'Chinese Spring'. From these and previous studies it is concluded that some α, β and γ gliadins (molecular weights by SDS-PAGE 30,000 to 40,000) are specified by genes on the short arms of homoeologous Group 6 chromosomes, the ω gliadins (molecular weights by SDS-PAGE 50,000 to 70,000) are specified by genes on the short arms of homoeologous Group 1 chromosomes and the glutenin subunits (molecular weights by SDS-PAGE85,000) are specified by genes on the long arms of homoeologous Group 1 chromosomes.No major gliadins or glutenin subunits were absent when any of the chromosomes in homoeologous Groups 2, 3, 4, 5 or 7 were deleted. However two gliadins whose presumed structural genes are on chromosome 6D were absent in aneuploid stocks of 'Chinese Spring' carrying two additional doses of chromosome 2A. Two out of thirty-three intervarietal or interspecific chromosome substitution lines examined, involving homoeologous Group 2 chromosomes, lacked the same two gliadins. All the subunits in the other thirty-one chromosome substitution lines were indistinguishable from those in 'Chinese Spring'. It is therefore concluded that the major variation affecting gliadin and glutenins in wheat is concentrated on the chromosomes of homoeologous Groups 1 and 6 but Group 2 chromosomes are candidates for further study.An endosperm protein controlled by chromosome 4D in 'Chinese Spring' is shown to be a high molecular weight globulin.

The presence of ribosomal RNA genes on rye supernumerary B chromosomes from three different rye populations has been investigated by ribosomal RNA/DNA hybridisation. DNAs were purified from wheat plants containing zero, two, three or four rye B chromosomes and from individual plants from two rye populations containing different numbers of B chromosomes. The number of ribosomal RNA genes was highly correlated with rye B chromosome number in the wheat plants but not in the rye plants. It is suggested, therefore, that not all rye B chromosomes are the same and that those transferred into the wheat genetic background each carry approximately 650 ribosomal RNA genes.

A simple repeating unit of the rye genome, previously known in tandem arrays, has now been shown to occur also in more complex repeating units. One such unit has been cloned and found to contain some sequences unrelated to the simple repeat. It is proposed that tandem arrays of complex repeats are often produced by amplification of DNA segments formed by recombination of different repeats and possibly unique sequences. Some complex variants of repeats are not in high copy number on all rye chromosomes.

The relationship between the chromosomal location of heterochromatin C-bands and of four non-homologous repeated sequence families constituting 8 to 12% of total rye DNA has been investigated in chromosomes of rye (Secale cereale) by in situ hybridisation. Three rye varieties, a set of rye disomic additions to wheat and a triticale were studied. Only centromeric and nucleolar organizer region (NOR) associated C-bands failed to display hybridisation to at least one of the sequences and many telomeric blocks of heterochromatin contained all four repeated sequence families. Both between-variety differences in the chromosomal distribution of repeated sequences, and intravarietal heterozygosities were frequently noted and are probably widespread. — Previously reported deletions of heterochromatin from King II rye chromosomes added to the Holdfast wheat complement were correlated with deletions of some, but not all, of the highly repeated sequence families. A previously unreported loss of some families from King II rye chromosome 4R/7R in a Holdfast wheat genetic background was detected. This loss was not associated with complete deletion of a C-band. A deletion has also probably occurred from the short arm telomere of 4R/7R in the triticale variety Rosner. It is suggested that the families of repeats in rye telomeric heterochromatin which are absent from wheat are selected against in the wheat genetic background.

Two pairs of chromosomes (1U and 5U) in Aegilops umbellulata possess ribosomal RNA genes. This has been proven by studying wheat plants into which 1U and 5U chromosomes have been introduced separately. These plants have more ribosomal RNA genes than the recipient wheat plants and additional clusters of rDNA when examined by in situ hybridisation. The repeating rDNA unit in Aegilops umbellulata is longer than most of the units in the wheat variety Chinese Spring, the additional DNA probably being in the non-transcribed spacer. This was determined from restriction endonuclease maps of rDNA. In Chinese Spring plants possessing 1U or 5U chromosomes, the largest nucleoli are formed on 1U or 5U chromosomes and the wheat nucleolus organisers form micronucleoli. This is not because the nucleolus organisers on chromosomes 1U and 5U have many more rRNA genes than wheat nucleolus organisers. It is suggested that the Aegilops umbellulata nucleolus organisers are dominant over those of wheat because they compete more effectively for some limiting factor. — The partial inactivation of the wheat nucleolus organisers by chromosomes 1U or 5U does not result in a reduced total nucleolus volume in root tip or pollen mother cells, because of the compensation by the nucleolus organisers of chromosomes 1U or 5U. The amount of RNA in seedlings is not markedly affected by the partial inactivation of the wheat nucleolus organisers.

The technique of in situ hybridisation has been used to demonstrate that some wheat varieties have very different numbers of ribosomal RNA genes clustered in homologous nucleolus organisers. In these experiments the labelled probe was RNA synthesized in vitro using wheat ribosomal DNA cloned in a bacterial plasmid as template. Homologous nucleolus organisers were identified by the use of intervarietal chromosome substitution lines. A variety of rye has been shown to be heterozygous for the number of rRNA genes clustered in its major pair of nucleolus organisers. The in situ hybridisation technique has also been used to demonstrate an anomalous deletion of rDNA in an aneuploid derivative of Chinese Spring wheat.

cDNA clones encoding wheat HMW glutenin subunits have been isolated from a cDNA bank made to poly A(+) RNA from developing wheat endosperm var. Chinese Spring. One such clone, pTag 1290, has enabled us to identify the HMW glutenin mRNA species. The DNA sequence of this clone has been partially determined and it contains several tandem DNA repeats. The sequence is discussed in relation to the generation of the HMW glutenin subunit gene family. Analysis of the organization of the HMW glutenin sequences in the wheat genome revealed that the genes encoding HMW glutenin subunits exist in low copy number and are located on the long arm of each of the homoeologous group 1 chromosomes.

For successful bread-making, dough made from wheat flour needs to have considerable elasticity. The elasticity is provided in part by seed storage proteins called high molecular weight (HMW) glutenin subunits. Two particular HMW subunits, numbers 12 and 10 have been found previously to confer different degrees of elasticity to dough. We compare here the amino acid sequences of these closely related proteins. They differ in their central regions, which consist of arrays of repeating hexamer and nonamer amino acid units. HMW glutenin 10 has a higher proportion of repeats of the consensus type than 12 and we postulate that this produces a more regular pattern of repetitive β turns in the protein, contributing to dough elasticity. A cysteine residue, likely to become involved in intra or intermolecular linkages may also be in a different configuration in the two subunits.

The structure, copy number and chromosomal location of arrays of four families of highly repeated sequences have been investigated in representative species of the genus Secale. The four unrelated families, previously characterised in Secale cereale, have repeating units of 480, 610, 630 and 120 base pairs respectively. The following general conclusions can be drawn in addition to detailed knowledge of the sequence content of heterochromatin in each accession studied: (1) Every species is unique in its complement or chromosomal distribution or both of the four highly repeated sequence families. S. montanum and S. cereale accessions studied here show the same complement of repeated sequences, but they differ substantially in the amounts they contain of the 610 and 630 base pair (bp) families, and in the distribution over the chromosomes of the 480 bp family. The structure of the repeating unit is also different in many members of the 480 bp family in S. montanum. — (2) The substantial differences between species in the amounts of the most highly repeated DNA sequences exist in the absence of any such conspicuous differences in most other repeated sequences which were detected as fluorescent bands after restriction enzyme digestion and gel electrophoresis. — (3) Each of the different highly repeated families can exist independently of the other families, though all the families have telomeric sites. Also, in the outbreeding species, heteromorphisms are frequent, and are particularly conspicuous in hybridisation detecting the 480 bp sequence family. — (4) The association of the highly repeated sequences with heterochromatin, discussed in the accompanying paper is generally true for other species in the genus, and the lower amounts of heterochromatin in other Secale species compared to S. cereale are associated with lower amounts of specific families of highly repeated DNA sequences. — (5) Analysis of highly repeated sequence families is likely to provide an easy method of identification of new accessions of Secale.

Mitochondria isolated from etiolated shoots of a range of maize genotypes with the "Texas" cytoplasm conferring cytoplasmically-inherited male sterility, are sensitive to a pathotoxin isolated from Helmintho-sporium maydis, race T. The pathotoxin inhibits oxidation of α ketoglutarate and malate and stimulates NADH oxidation. The time taken for the pathotoxin to induce these changes is a measure of the sensitivity of the mitochondria to the pathotoxin. A range of nine different pairs of genotypes, each pair differing principally in the presence of nuclear male fertility restorer alleles has been compared in their sensitivity to pathotoxin. In every case the line carrying the restorer alleles is more resistant to the pathotoxin. The restored genotypes can be quantitatively arranged into groups which correspond to the four different sources of the restorer genes in these lines. It is suggested that the restorer genes cause changes in mitochondria, which modify the functional aberration introduced by the cytoplasmically-inherited mutation causing sterility.

The ribosomal RNA gene contents of wheat aneuploid lines, differing in the numbers of 5 A, 5B, 5D, 6A, 6B and 6D chromosomes, have been estimated by ribosomal RNA/DNA hybridisation. These and other estimates on intervarietal substitution lines, provide some evidence for ribosomal RNA genes being localised on chromosomes 5D and 6B. Chromosome 6B of the variety Chinese Spring possesses approximately 5500 rRNA genes, 60 per cent of the total ribosomal RNA gene complement of the variety. In the variety Hold-fast, chromosome 6B possess approximately 2000 ribosomal RNA genes. This value comes from examining interspecies substitution lines in which chromosomes 6A, 6B and 6D are replaced by rye chromosomes. The approximate number of ribosomal RNA genes on the four nucleolar organiser chromosomes of the variety Chinese Spring are presented.

The DNAs of wheat and rye plants with rye B chromosomes have been compared with wheat, rye and oats DNAs by DNA/DNA hybridisation. The presence of DNA from B chromosomes made no significant difference to the proportion of repeated sequence DNA. The repeated sequence fractions of these cereal DNAs were quantitatively divided into eight different groups on the basis of the amount of DNA/DNA hybridisation occurring between the different DNAs. Rye A and B chromosomes contained similar proportions of three of the groups. These results, together with estimates of the thermal stabilities of all the renatured DNA duplexes suggest that rye B chromosome DNA is very similar to rye A chromosome DNA in the proportion and heterogeneity of its repeated sequences.

Labelled RNA, transcribed in vitro from wheat ribosomal DNA cloned in a bacterial plasmid, has been hybridised to metaphase chromosomes of five diploid wheats. Autoradiography of the chromosomes has provided unequivocal evidence that these genotypes possess two pairs of nucleolus organizer chromosomes. The diploid wheat accessions used possess widely differing numbers of ribosomal RNA genes.

Maize plants carrying Texas (T) cytoplasm are male-sterile and sensitive to Drechslera maydis race T toxin, whereas plants carrying Normal (N) cytoplasm are male-fertile and resistant to the toxin. Some plants regenerated from T cytoplasm tissue cultures exhibit a N cytoplasm-like phenotype with respect to malefertility and toxin-resistance. Analysis of the high molecular weight mitochondrial DNA (mtDNA) of such regenerants has shown that the plants do not contain N cytoplasm mtDNA. However, their mtDNAs do show sequence differences from each other and from the mtDNA of plants possessing T cytoplasm. No single alteration detected correlates with the change to malefertility or toxin resistance. Sequence alterations were also evident in high molecular weight mtDNA isolated from a plant regenerated from N cytoplasm callus. No changes in low molecular weight mtDNA molecules were observed in regenerants from N or T cytoplasm callus.

The amounts of a 1.9 kb mitochondrial plasmid relative to sequences in another mitochondrial DNA replicon and also to nuclear ribosomal DNA sequences have been compared in maize leaves and anthers. Similar comparisons have been made between plants with the same nuclear genotype but containing normal, S, or T cytoplasms. The ratio of 1.9 kb plasmid to nuclear rDNA is lower in plants with normal cytoplasm than in plants with S or T cytoplasm. It also differs between leaves and anthers. Furthermore, the relative concentration of the mitochondrial DNA sequences belonging to different replicons differs between leaves and anthers. It is concluded that components of different mitochondrial replicons are not maintained in fixed ratios during development and that the concentration of the 1.9 kb plasmid is regulated, in part, by cytoplasmically-inherited determinants. The 1.9 kb plasmid is absent from lines with the Vg cytoplasm, but related sequences are found in the maize nuclear genome.

In hexaploid wheat and diploid rye, different varieties have different numbers of ribosomal RNA genes as indicated by rRNA/DNA hybridisation. Wheat has four different chromosomes which carry nucleolar organisers. Analyses of DNA isolated from substitution lines in which each of these nucleolar organiser chromosomes of several varieties has been substituted one at a time into a common genetic background, have indicated that none of the four organiser chromosomes possess an invariant number of ribosomal RNA genes. The ribosomal RNA gene complement of the varieties investigated can be approximately accounted for by the sum of the ribosomal RNA genes on each of the four nucleolar organiser chromosomes.

A quantitative analysis of malate dehydrogenase isozymes has been carried out in a hexaploid wheat Triticum aestivum variety Holdfast, a diploid rye Secale cereale variety King II, a series of seven addition lines each having the Holdfast wheat chromosome complement, and also a different homologous pair of King II rye chromosomes. In young shoots of three of these addition lines grown in a defined salts medium lacking sucrose, at least one isozyme activity was elevated. This did not occur in shoots grown in a medium containing 0.5% sucrose or in the Triticale possessing the full wheat and rye chromosomal complements grown in the absence of exogenous sucrose. On the basis of cellular localization and substrate inhibition studies, the particular isozyme activities enhanced by the rye chromosomes were indistinguishable from isozyme activities in Holdfast wheat and dissimilar to all malate dehydrogenase isozyme activities observed in King II rye. These results suggest that three different rye chromosomes produce gene products which can interact with the wheat malate dehydrogenase regulatory system.

Reassociation kinetics of sheared hexaploid wheat DNA in 0.18 M Na+ at 60 °C show the presence of three major classes of nucleotide sequences; (1) a very rapidly reannealing fracton that comprises about 4 to 10% of the genome and reanneals virtually instantly. This fraction contains sequences which appear to be randomly distributed through at least 40% of the genome; they may be palindromic sequences; (2) a heterogeneous intermediate reannealing fraction comprising about 70–80% of the genome which consists of families with apparent repetition frequencies ranging from about 100–100000; and (3) a slow reannealing fraction. These fractions have been isolated and studied separately. Seventy percent of the slow reannealing fraction (12 to 20% of the genome) was found to contain sequences present in approximately six copies per hexaploid genome. The single copy sequences in the three constituent diploid genomes of hexaploid wheat appear to show near complete homology to one another.