A large DNA repeat of the dispersion pattern common to wheat and rye genomes

A Hind III-generated fragment of wheat embryo nuclear DNA has been cloned and sequenced. The cloned fragment corresponds to a 1241 bp long, moderately repeated (60 000 copies/genome) segment of the genomic DNA. The repeat is AT-rich (67%), contains an open reading frame for 151 amino acids and several nucleotide blocks resembling the consensus domain of autonomously replicating sequences. Southern blot hybridization analyses indicate that the repeat is scattered through the wheat genome. A sequence homologous to this repeat occurs also in rye embryo nuclear DNA where it shows the same dispersion pattern as that observed for the wheat repeat.     

A molecular linkage map of rye

A genetic map of six chromosomes of rye, (all of the rye chromosomes except for 2R), was constructed using 77 RFLP and 12 RAPD markers. The map was developed using an F₂ population of 54 plants from a cross between two inbred lines. A rye genomic library was constructed as a source of clones for RFLP mapping. Comparisons were made between the rye map and other rye and wheat maps by including additional probes previously mapped in those species. These comparisons allowed (1) chromosome arm orientation to the linkage groups to be given, (2) the corroboration of several evolutionary translocations between rye chromosomes and homoeologous chromosomes of wheat; (3) an increase in the number of available markers for target regions of rye that show colinearity with wheat. Inconsistencies in the location of markers between the wheat and rye maps were mostly detected by multi-copy probes.     

Dosage response of rye genes in a wheat background

Summary A series of hexaploid wheat lines containing zero, two or four doses of rye chromosome arm 1RS was used to investigate the response to changes in dosage by the rye genes when in a wheat background. The quantity of protein produced by the secalin protein genes contained on 1RS was directly related to the number of copies of 1RS present in the line. No response could be detected by representative wheat proteins suggesting that the increase in secalin protein observed was due to an increase in mRNA produced when four copies of the secalin gene was present. These results suggest that increasing the dosage of alien genes introgressed into wheat may be a useful tool for enhancing their expression.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 suitable.Contribution from the Missouri Agricultural Experiment Station. Journal Series No. 11,413     

A new type of cytoplasmic male sterility in rye (Secale cereale L.): analysis of mitochondrial DNA

Summary Mitochondrial (mt) DNA of a new type of rye cytoplasm (Gülzow, G) that induces cytoplasmic male sterility (CMS) was analyzed and compared with rye mtDNAs of different origins MtDNA of the G type was easily distinguishable from mtDNA of another CMS source, Pampa (P) type, and from mtDNA of fertile lines with respect to restriction fragment patterns and hybridization with mitochondrial genes. The results of the molecular analyses indicate a close, but not identical relationship between the mtDNA of the G type cytoplasm and that of cv Pluto.     

Identification of a repeat sequence of rye DNA in wheat and related species

Polymerase chain reaction (PCR) and enhanced chemiluminescence (ECL) were used to determine the distribution of the rye-specific sequence contained in the pSc119.1 probe among wheat and related species. A specific pair of primers targeting this rye-specific sequence was used. A 745-bp fragment, the predicted size of pSc119.1, was present inSecale cereale, Triticum aestivum, XTriticosecale, Hordeum vulgare, H. bogdanii, andH. parodii. PCR results were verified by hybridizing the rye-specific probe pSc119.1 to dotblots of DNA from the different species used. Strong hybridization signals detected by ECL were consistent with the PCR results. The results demonstrate the effectiveness of PCR and dot-blot ECL in screening plants for defined DNA sequences, and indicate that pSc119.1 has counterparts with strong homology inT. aestivum, H. vulgare, H. bogdanii, andH. parodii.     

The effect of B-chromosomes of rye on the chromosome association in F1 hybrids Triticum aestivum x Secale cereale in the absence of chromosomes 5B or 5D

Summary T. aestivum var. Chinese Spring (monosomic 5B and 5D, respectively) was crossed with S. cereale (with and without B-chromosomes). The resulting nullisomic 5B hybrids exhibited a high degree of chromosome association both at 20°C and 10°C. The presence of B-chromosomes reduced association slightly whether 5B was present or not.In nullisomic 5D hybrids B-chromosomes of rye raise chromosome association at 20°C when compared to hybrids with 5D, with as well as without, B's. At 10°C, due to the absence of the Ltp gene on 5D, chromosome association in nullisomic 5D hybrids is low, and no effects of rye B-chromosomes is detectable.The hypothesis that B-chromosomes of rye carry (an) asynaptic gene(s) decreasing effective pairing, and (an) independent post-synaptic gene(s) increasing chiasma frequency on effective pairing sites, is presented.The work was supported by a fellowship of the Gulbenkian Foundation and partly carried out while the author was at the Department of Genetics, Agricultural University, Wageningen, the Netherlands     

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.     

A linkage map of rye

A linkage map of rye (Secale cereale L.) is presented which comprises 60 loci including RFLPs, RAPDs, isozyme, morphological and physiological markers. The genetics and linkage relationships of these markers were investigated in several inbred lines of rye. For the RFLP mapping a genomic library of PstI-digested DNA was constructed from which 50 size-selected clones were analysed. The portion of single-copy and multi-copy DNA and the frequency of polymorphic DNA was determined. The markers are unequally distributed over the seven chromosomes of rye. Many of them exhibit a distorted segregation. The main region of deviating segregation ratios could be localized near the self-incompatibility loci.     

Structural heterogeneity in the R173 family of rye-specific repetitive DNA sequences

The rye-specific R173 family of repeated DNA sequences consists of ca. 15 000 individual copies per diploid rye (Secale cereale) genome and is distributed over all 7 rye chromosomes in a dispersed manner. Individual R173 elements vary in size between 3 and 6 kb, are generally not arranged as tandem repeats and are flanked by both multi-copy and single-copy sequences. DNA sequence analysis of three R173 elements (R173-1, R173-2 and R173-3) demonstrated a high degree of homology in conserved domains. The structure of R173-1 was quite different from the other two elements: long direct repeats, which represent a rye-specific repetitive sequence, were found at the ends and a 600 bp long domain was replaced by an unrelated sequence of approximately equal size. R173-2 and R173-3 were extremely similar to each other with the exception of a terminal truncation of R173-2. No open reading frames for proteins >20 kDa were present and a database search failed to detect significant homologies to published protein sequences. Despite the transposon like genomic organisation of the R173 family, individual elements lacked sequence features frequently associated with transposons and retrotransposons. In contrast, two of the regions flanking R173 elements showed strong DNA homologies to a 850 bp long region of a proposed wheat retrotransposon and to a 300 bp long region downstream of the wheatGlu-D1 gene.     

The pattern of amyloplast DNA accumulation during wheat endosperm development

The accumulation of amyloplast DNA during endosperm development was studied in two cultivars of spring wheat, Triticum aestivum L. Chinese Spring (CS) and Spica, small and relatively larger-grained cultivars, respectively. Endosperms were isolated between 9 and 45 days post anthesis (dpa) and the amyloplast DNA content of endosperm nucleic-acid extracts was measured by quantitative hybridisation with a homologous chloroplast-DNA probe. The endosperm cells of CS and Spica accumulated amyloplast DNA during development in a similar way. In both cultivars there was a large increase in the amount of plastid DNA (ptDNA) per endosperm between 9 and about 15 dpa, after which there was no further increase. Because nuclear DNA continued to accumulate until 24 dpa, the percentage contribution of amyloplast DNA to total DNA fluctuated in both cultivars during development, reaching maxima at 12 dpa of about 1.00% and 0.85%, and dropping to apparently constant levels of 0.60% and 0.52% in CS and Spica, respectively, by 24 dpa. In both cultivars, the average number of ptDNA copies per amyloplast was calculated to increase from about 10 copies at 9 dpa to about 50 copies in the mature amyloplasts at 31 dpa. However, the heavier endosperms of Spica contain more cells than those of CS and the varieties therefore differed in the amount of ptDNA that accumulated per endosperm: Spica endosperms accumulated 110 ng of ptDNA by 15 dpa, compared with only 85 ng in CS. The apparent accumulation of ptDNA copies in wheat amyloplasts during endosperm development contrasts with the decline in chloroplast-DNA copies in wheat chloroplasts during leaf development.Abbreviations CS Chinese Spring - ctDNA chloroplast DNA - dpa days post anthesis - kbp 10³ base pairs - nDNA nuclear DNA - ptDNA plastid DNA - mtDNA mitochondrial DNA     

Mapping the nucleolus organizer region,seed protein loci and isozyme loci on chromosome 1R in rye

Summary The nucleolus organizer region located on the short arm of chromosome 1R of rye consists of a large cluster of genes that code for ribosomal RNA (designated the Nor-R1 locus). The genes in the cluster are separated by spacer regions which can vary in length in different rye lines. Differences in the spacer regions were scored in two families of F₂ progeny. Segregation also occurred, in one or both of the families, at two seed protein loci and at two isozyme loci also located on chromosome 1R. The seed protein loci were identified as the Sec 1 locus controlling -secalins located on the short arm of chromosome 1R and the Sec 3 locus controlling high-molecular-weight secalins located on the long arm of 1R. The two isozyme loci were the Gpi-R1 locus controlling glucose-phosphate isomerase isozymes and the Pgd 2 locus controlling phosphogluconate dehydrogenase isozymes. The data indicated linkage between all five loci and map distances were calculated. The results indicate a gene order: Pgd 2 ... Sec 3 ... [centromere] ... Nor-R1 ... Gpi-R1 ... Sec 1. Evidence was obtained that rye possesses a minor 5S RNA locus (chromosome location unknown) in addition to the major 5S RNA locus previously shown to be located on the short arm of chromosome 1R.     

A BamHI family of highly repeated DNA sequences of Nicotiana tabacum

Summary HRS60.1, a monomer unit (184 bp) of a highly repeated nuclear DNA sequence of Nicotiana tabacum, has been cloned and sequenced. Following BamHI digestion of tobacco DNA, Southern hybridization with HRS60.1 revealed a ladder of hybridization bands corresponding to multiples of the basic monomer unit. If the tobacco DNA was digested with restriction endonucleases which have no target site in HRS60.1, the larger part of DNA homologous to HRS60.1 remained as uncleaved relic DNA. These results suggest a tandem arrangement of this DNA repeat unit. Four other clones of tobacco nuclear DNA cross-hybridized with HRS60.1, thus forming a HRS60-family. Sequencing their inserts has shown their strong mutual homology. HRS60-family comprised about 2% of the nuclear genome of N. tabacum. Computer comparisons with other tandem plant-repeated DNA sequences could not detect any other homologous sequence.     

A consensus map of rye integrating mapping data from five mapping populations

A consensus map of rye (Secale cereale L.) was constructed using JoinMap 2.0 based on mapping data from five different mapping populations, including ‘UC90’ × ‘E-line’, ‘P87’ × ‘P105’, ‘I_0.1-line’ × ‘I_0.1-line’, ‘E-line’ × ‘R-line’, and ‘Ds2’ × ‘RxL10’. The integration of the five mapping populations resulted in a 779-cM map containing 501 markers with the number of markers per chromosome ranging from 57 on 1R to 86 on 4R. The linkage sizes ranged from 71.5 cM on 2R to 148.7 cM on 4R. A comparison of the individual maps to the consensus map revealed that the linear locus order was generally in good agreement between the various populations, but the 4R orientations were not consistent among the five individual maps. The 4R short arm and long arm assignments were switched between the two population maps involving the ‘E-line’ parent and the other three individual maps. Map comparisons also indicated that marker order variations exist among the five individual maps. However, the chromosome 5R showed very little marker order variation among the five maps. The consensus map not only integrated the linkage data from different maps, but also greatly increased the map resolution, thus, facilitating molecular breeding activities involving rye and triticale.     

A minichromosome-like structure in wheat embryos

A crude nuclear fraction of resting wheat embryos was used as the source of putative plant minichromosomes: unique DNA sequences the size of genes and flanked by telomere-type repeats. Preliminary separation of low-molecular-weight DNA species from chromosomal DNA (Hirt's method), velocity sedimentation, and isopycnic centrifugation were followed by PCR amplification of minichromosome-like sequences. The most abundant PCR product was cloned and sequenced. In addition to telomeric repeats (defined by a PCR primer), which were the expected sequences, the linear DNA molecule (637 pb) contained an ARS-like element, RAP1-binding site, and two relatively long ORFs. The whole sequence seems to represent a naturally occurring plant minichromosome.     

Influence of wheat and rye parents on agronomic characters in primary hexaploid and octoploid triticale

Summary Thirty-five hexaploid and twenty octoploid primary triticales (xTriticosecale Wittmack) derived from homozygous wheat and rye inbred lines were used (1) to investigate the parental wheat, rye, and interaction effects and (2) to estimate quantitative genetic parameters for agronomic traits. The winter triticales were tested in four environments in a three-replicate split-plot design with drilled 1 m² plots. Superior performance of hexaploid triticales as compared to the octoploids was revealed. Substantial genetic variation and high heritability estimates were found for nearly all of the characters investigated. Estimates of wheat, rye, and wheat×rye interaction variance components disclosed parental main effects to be the most important source of genetic variation in primary triticales. The rye parent was dominant for all characters affecting fertility, and the wheat parent was more important for vegetative development. Character correlations were very similar for triticales of both ploidy levels. The lack of association between grain yield and tillering and the positive correlation between kernels per spike and thousand kernel weight indicated physiological disorders specific for primary triticales.     

An extrachromosomal fragment of telomeric DNA in wheat

A procedure developed orginally for selective extraction of viral (extrachromosomal) DNA from virus-infected mammalian cells was applied to cell nuclei isolated from uninfected wheat embryos. The resulting nuclear extrachromosomal DNA (exDNA) was enriched for telomere-type sequences by isopycnic centrifugation and inserted into the Sma I site of pUC119. A cloned DNA fragment (241 bp) was found to consist primarily of tandemly repeated heptamere units of the same sequence (5-CCCTAAA-3) that is known to predominate in telomeric DNA of Arabidopsis thaliana. Hybridization experiments indicate that extrachromosomal telomeric repeats are abundant in resting embryos and disappear rapidly during germination.     

A highly repeated DNA sequence in Arabidopsis thaliana

Summary Three members of a family of highly repeated DNA sequences from Arabidopsis thaliana have been cloned and characterized. The repeat unit has an average length of 180 bp and is tandemly repeated in arrays longer than 50 kb. This family represents more than one percent of the Arabidopsis genome. Sequence comparisons with tandemly repeated DNA sequences from other Cruciferae species show several regions of homology and a similar length of the repeat unit. Homologies are also found to highly repeated sequences from other plant species. When the sequence CCGG occurs in the repeated DNA, the inner cytosine is generally methylated.     

Nucleotide sequence of a highly repeated DNA sequence and its chromosomal localization in Allium fistulosum

A highly repeated DNA sequence with a repeating unit of approximately 380bp was found in EcoRV digests of the total genomic DNA of Allium fistulosum. Three independent clones containing this unit were isolated, and their repeating units sequenced. These units showed more than 94% sequence homology, and the copy number was estimated to be about 2.8×10⁶ per haploid genome. In situ hybridization, with the repeating unit as a probe, and C-banding analyses indicated that the repeated DNA sequence of A. fistulosum is closely associated with the major C-heterochromatin in the terminal regions of all 16 chromosomes at mitotic metaphase. The characters of the repeating unit are similar to those of the A. cepa unit, which is taxonomically closely related to A. fistulosum.