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High-resolution physical mapping of the secalin-1 locus of rye on extended DNA fibers.

High-resolution mapping of secalin-1 (Sec-1) locus has been performed by fluorescence in situ hybridization to extended DNA fibers of rye (Secale cereale, 2n = 14), employing DNA probes of lambda phage clones containing the omega-secalin gene. The fluorescent signals to rye extended DNA fibers revealed continuous strings of 45 microm, corresponding to the size of 147 kb DNA. To determine the copy number of Sec-1 locus on DNA fibers, a 1.2-kb fragment including the entire coding region of the omega-secalin gene and a 1.0-kb fragment of the promoter region were amplified by PCR as probes for another fiber FISH. The physical position of these sequences was visualized as alternating fluorescent spots by multicolor in situ hybridization. Alternating signals of two DNA probes reflected the tandem repeated organization of the Sec-1 locus having 15 copies of the gene. The present findings based on fiber FISH analysis support the contention that the omega-secalin genes are arranged in a head-to-tail fashion separated by 8 kb of spacer sequences with a total length of 145 kb.

Base Sequence↗

Strategies for the study of meiosis in rye.

We describe how we are furthering our understanding of meiosis in rye (Secale cereale L.) using a combination of cytogenetic and molecular biological approaches. Fluorescent in situ hybridisation, electron microscopy of synaptonemal complexes, sequencing of meiosis-specific genes, and the immunolocalisation of recombinogenic proteins are being combined to build up phenotypic "identikits" of wild type, asynaptic mutants sy1 and sy9, and desynaptic mutant sy10. From this information, we review the status of our current understanding of the genetic control of meiosis in rye, and consider strategies for determining more precisely the interrelationships between meiosis-specific genes and their products.

DNA, Plant↗

Effects of phytoestrogen extracts isolated from rye, green and yellow pea seeds on hormone production and proliferation of trophoblast tumor cells Jeg3.

BACKGROUND: Phytoestrogens are a diverse group of non-steroidal plant compounds. Because they have chemical structures similar to estrogens they are able to bind on estrogen receptors in humans. OBJECTIVES: In this study, we tested the effects of crude phytoestrogen extracts from rye (Secale cereale), green pea (Pisum sativum) and yellow pea seeds (Pisum sativum cv.) on cell proliferation and the production of progesterone in trophoblast tumor cells of the cell line Jeg3. METHODS: Isoflavone extracts from green and yellow pea seeds and lignan extracts from rye seeds were obtained, using different extraction methods. Isolated extracts were incubated in different concentrations with trophoblast tumor cells. Untreated cells were used as controls. At designated times, aliquots were removed and tested for estradiol and progesterone production. In addition, we tested the effects of the phytoestrogen extracts on cell proliferation. RESULTS: Cell proliferation is significantly inhibited by potential phytoestrogens isolated from rye, green and yellow pea seeds in trophoblast tumor cells of the cell line Jeg3. We found a correlation between the effects of proliferation and production of estradiol in isoflavone extracts from green and yellow pea seeds in Jeg3 cells. In addition, higher concentrations of isoflavones isolated from green pea seeds and lignans from rye showed also a inhibition of progesterone production whereas higher concentrations of rye lignans elevated estradiol production in Jeg3 cells. CONCLUSION: A useful indicator test system for potential phytoestrogens could be established. Based on the obtained results it is proposed that green and yellow pea seeds contain measurable concentrations of isoflavones and rye seeds contain lignans which can be isolated and used for special human diet programs.

Cell Line, Tumor↗

Different patterns of rDNA organization at interphase in nuclei of wheat and rye.

The physical location of the rDNA repeating units (25 S, 18 S and 5.8 S rRNA genes and the intergenic spacer sequences) was investigated in rye (Secale cereale L.) and wheat (Triticum aestivum L.) root tip meristematic cells by in situ hybridization using light and electron microscopy. The rDNA sequences are organized differently in the two related and intercrossable species. In rye (2n = 14, one pair of chromosomes with nucleolar organizing regions, NORs), two condensed blocks of rDNA-containing chromatin occurred in each interphase nucleus. The blocks were associated with the periphery of nucleoli and a single-labelled, decondensed rDNA fibre extended into the nucleolus from the block. We term this expression pattern terminal decondensation. In wheat (2n = 6x = 42, five pairs of chromosomes with NORs), inactive condensed labelled chromatin was found unassociated with nucleoli. Active NORs had some condensed rDNA associated with the nucleolar periphery, but, in contrast to rye, condensed rDNA was also found within the nucleolus. The condensed labelled rDNA in wheat nucleoli was visible as fluorescent foci in the light microscope and labelled condensed chromatin in the electron microscope. Its absence in rye shows that condensed rDNA need not be present in active plant nucleoli. Diffuse labelled sites of rDNA, likely to represent actively transcribed rDNA, were found in both rye and wheat. Active rDNA loci in wheat have many expressed segments separated by unexpressed, condensed, rDNA-fragmented decondensation-while each locus in rye has a single, unexpressed perinucleolar condensed block of rRNA genes. Thus the positions of actively transcribed genes within the tandem arrays of rDNA at each locus are fundamentally different in the two cereals. The NOR chromosome appeared to extend through the nucleolus, and active rDNA sequences did not loop out from chromatin into the nucleolus as is frequently described in nucleolar models.

Cell Nucleolus↗

Detection of the Sec-1 locus of rye by a PCR-based method.

The structural genes for the omega-secalins of rye (Secale cereale) are located in the Sec-1 locus on the short arm of rye chromosome 1R. We applied PCR (polymerase chain reaction) to detect the Sec-1 locus in a wheat genomic background. A primer set we designed based on a published sequence of a omega-secalin gene amplified not only the omega-secalin sequence, but also a putative omega-gliadin sequence. We determined partial sequences of both PCR-amplified fragments and designed different primers for the specific amplification of the omega-secalin sequence. One of the new primer sets amplified DNA fragments only in rye and wheat lines carrying chromosome 1R or telosome 1RS; no amplification occurred in either euploid wheats or 1RS deletion lines. This PCR-based method would provide efficient screening for the Sec-1 locus in progeny of wheat lines carrying chromosome 1R.

Base Sequence↗

Bale compression and hydrogen phosphide fumigation to control cereal leaf beetle (Coleoptera: Chrysomelidae) in exported rye straw.

Control of larvae and adults of cereal leaf beetle, Oulema melanopus (L.), by bale compression and hydrogen phosphide fumigation was studied in rye straw, Secale cereale L., in Aurora, OR. Natural mortality of larvae after transport was 4.0 +/- 1.0% (mean +/- SEM). Compression (105 kg/cm2) of larvae in standard bales (122 cm long) of rye straw resulted in 100% mortality. Compression of adults in standard bales plus storage of the compressed bales (56 cm long) for 1 d in a freight container resulted in 100% mortality. A KD50 of 102 ppm hydrogen phosphide for 1 h was estimated from the probit regression line developed from dose-response data at 21 degrees C in basic laboratory tests. The LD50s and LD99s were 163 and 6,910 ppm for 2-h exposures and were 18 and 42 ppm for 6-h exposures at 21 degrees C, respectively. A tested close of 400 ppm for 24 h at 21 degrees C resulted in 100% mortality of the adults. Larvae (n = 10,560) and adults (n = 18,602) did not survive exposure to bale compression followed by hydrogen phosphide fumigation (60 g/28.3 m3) for 3 d in rye straw loaded in freight containers in large-scale tests. Copper plate corrosion values indicating the severity of exposure to hydrogen phosphide were 13 and 12, and mean temperatures of five locations in the freight container were 25 and 26 degrees C in large-scale tests with the larvae and adults, respectively. Hydrogen phosphide concentrations were > or = 400 ppm throughout the 3-d fumigation for larvae and during the first day of fumigation for adults. We propose that cereal leaf beetle can be controlled by a single treatment of bale compression followed by a 1-d storage period or by a fumigation in which 400 ppm hydrogen phosphide is maintained for 1 d at 21 degrees C or above. We confirmed that a multiple quarantine treatment of bale compression) followed by a 3-d fumigation will control cereal leafbeetle in exported rye straw.

Animals↗

Incorporation of plant carbon into the soil animal food web of an arable system.

We used stable isotopes to examine the incorporation of plant carbon into the belowground food web of an agricultural system. Plots were established and planted with maize (Zea mays) in a rye field (Secale cereale) near Göttingen (northern Germany) in May 1999. In October 1999, April 2000, and October 2000, meso- and macrofauna and maize and rye litter were collected in each plot and analyzed for 13C and 15N content. 15N signatures suggested that the soil animal species analyzed span three trophic levels with the trophic position of species varying little in time. The species investigated formed a continuum from primary to secondary decomposers to predators. On average, predator species differed from primary and secondary decomposers by 3.9 sigma15N suggesting that they fed on a mixed diet of both decomposer groups. The combined analysis of 13C and 15N signatures allowed us to identify links between prey and consumer species. In October 1999, shortly after maize residues had been incorporated into the plots, maize-born carbon was present in each of the animal species investigated, including top predators. The incorporation of maize carbon into the belowground food web increased during the following 12 months but the concentration of maize-born carbon never exceeded 50% in any of the species. Furthermore, the ranks of the incorporation of maize-born carbon of the species changed little. The results suggest that the belowground food web relies heavily on carbon originating from plant residues from before the recent two growing seasons. In most species the amount of maize-born carbon increased continuously; however, in some species it decreased during winter, suggesting that these species switched to a diet based more on C3 plants during winter, or predominantly metabolized carbon incorporated during the last growing season. The study documents that the combined analysis of 13C and 15N signatures in soil invertebrate species, after replacement of C3 by C4 plants, is a powerful tool to better understand the structure of the belowground food web and the flux of carbon through it.

Agriculture↗

Use of vegetative furrows to mitigate copper loads and soil loss in runoff from polyethylene (plastic) mulch vegetable production systems.

The transport of runoff with high copper concentrations and sediment loads into adjacent surface waters can have adverse effects on nontarget organisms as a result of increased turbidity and degraded water quality. Runoff from vegetable production utilizing polyethylene mulch can contain up to 35% of applied copper, a widely used fungicide/bactericide that has adverse effects on aquatic organisms. Copper is primarily transported in runoff with suspended particulates; therefore, implementation of management practices that minimize soil erosion will reduce copper loads. Replacing bare-soil furrows with furrows planted in rye (Secale cereale) significantly improved the sustainability of vegetable production with polyethylene mulch and reduced the potential environmental impact of this management practice. Vegetative furrows decreased runoff volume by >40% and soil erosion by >80%. Copper loads with runoff were reduced by 72% in 2001, primarily as a result of reduced soil erosion since more than 88% of the total copper loads were transported in runoff with suspended soil particulates. Tomato yields in both years were similar between the polyethylene mulch plots containing either bare-soil or vegetative furrows. Replacing bare-soil furrows with vegetative furrows greatly reduces the effects of sediments and agrochemicals on sensitive ecosystems while maintaining crop yields.

Agriculture↗

Nitrogen removal and nitrate leaching for two perennial, sod-based forage systems receiving dairy effluent.

In northern Florida, year-round forage systems are used in dairy effluent sprayfields to reduce nitrate leaching. Our purpose was to quantify forage N removal and monitor nitrate N (NO3(-)-N) concentration below the rooting zone for two perennial, sod-based, triple-cropping systems over four 12-mo cycles (1996-2000). The soil is an excessively drained Kershaw sand (thermic, uncoated Typic Quartzip-samment). Effluent N rates were 500, 690, and 910 kg ha(-1) per cycle. Differences in N removal between a corn (Zea mays L.)-bermudagrass (Cynodon spp.)-rye (Secale cereale L.) system (CBR) and corn-perennial peanut (Arachis glabrata Benth.)-rye system (CPR) were primarily related to the performance of the perennial forages. Nitrogen removal of corn (125-170 kg ha(-1)) and rye (62-90 kg ha(-1)) was relatively stable between systems and among cycles. The greatest N removal was measured for CBR in the first cycle (408 kg ha(-1)), with the bermudagrass removing an average of 191 kg N ha(-1). In later cycles, N removal for bermudagrass declined because dry matter (DM) yield declined. Yield and N removal of perennial peanut increased over the four cycles. Nitrate N concentrations below the rooting zone were lower for CBR than CPR in the first two cycles, but differences were inconsistent in the latter two. The CBR system maintained low NO3(-)-N leaching in the first cycle when the bermudagrass was the most productive; however, it was not a sustainable system for long-term prevention of NO3(-)-N leaching due to declining bermudagrass yield in subsequent cycles. For CPR, effluent N rates > or = 500 kg ha(-1) yr(-1) have the potential to negatively affect ground water quality.

Biodegradation, Environmental↗

Cover cropping to reduce nitrate loss through subsurface drainage in the northern U.S. corn belt.

Despite the use of best management practices for nitrogen (N) application rate and timing, significant losses of nitrate nitrogen (NO3(-)-N) in drainage discharge continue to occur from row crop cropping systems. Our objective was to determine whether a autumn-seeded winter rye (Secale cereale L.) cover crop following corn (Zea mays L.) would reduce NO3(-)-N losses through subsurface tile drainage in a corn-soybean [Glycine mar (L.) Merr.] cropping system in the northern Corn Belt (USA) in a moderately well-drained soil. Both phases of the corn-soybean rotation, with and without the winter rye cover crop following corn, were established in 1998 in a Normania clay loam (fine-loamy, mixed, mesic Aquic Haplustoll) soil at Lamberton, MN. Cover cropping did not affect subsequent soybean yield, but reduced drainage discharge, flow-weighted mean nitrate concentration (FWMNC), and NO3(-)-N loss relative to winter fallow, although the magnitude of the effect varied considerably with annual precipitation. Three-year average drainage discharge was lower with a winter rye cover crop than without (p = 0.06). Over three years, subsurface tile-drainage discharge was reduced 11% and NO3(-)-N loss was reduced 13% for a corn-soybean cropping system with a rye cover crop following corn than with no rye cover crop. We estimate that establishment of a winter rye cover crop after corn will be successful in one of four years in southwestern Minnesota. Cover cropping with rye has the potential to be an effective management tool for reducing NO3(-)-N loss from subsurface drainage discharge despite challenges to establishment and spring growth in the north-central USA.

Agriculture↗

Repeated compost application effects on phosphorus runoff in the Virginia Piedmont.

Increasing amounts of animal and municipal wastes are being composted before land application to improve handling and spreading characteristics, and to reduce odor and disease incidence. Repeated applications of composted biosolids and manure to cropland may increase the risk for P enrichment of agricultural runoff. We conducted field research in 2003 and 2004 on a Fauquier silty clay loam (Ultic Hapludalfs) to compare the effects of annual (since 1999) applications of composted and uncomposted organic residuals on P runoff characteristics. Biosolids compost (BSC), poultry litter-yard waste compost (PLC), and uncomposted poultry litter (PL) were applied based on estimated plant-available N. A commercial fertilizer treatment (CF) and an unamended control treatment (CTL) were also included. Corn (Zea mays L.) and a cereal rye (Secale cereal L.) cover crop were planted each year. We applied simulated rainfall in fall 2004 and analyzed runoff for dissolved reactive P (DRP), total dissolved P (TDP), total P (TP), total organic C (TOC), and total suspended solids (TSS). End of season soil samples were analyzed for Mehlich-3 P (M3P), EPA 3050 P (3050P), water soluble P (WSP), degree of P saturation (DPS), soil C, and bulk density. Compost treatments significantly increased soil C, decreased bulk density, and increased M3P, 3050P, WSP, and DPS. The concentration of DRP, TDP, and TP in runoff was highest in compost treatments, but the mass of DRP and TDP was not different among treatments because infiltration was higher and runoff lower in compost-amended soil. Improved soil physical properties associated with poultry litter-yard waste compost application decreased loss of TP and TSS.

Agriculture↗

Recognition of two types of positive staining chromosomal material by manipulation of critical steps in the N-banding technique.

The nucleolar regions on chromosomes 1B and 6B of Triticum aestivum L. cv Chinese Spring wheat can reliably be observed after careful control of the Giemsa N-banding technique. Identification of rye (Secale cereale) chromosomes using N-banding is demonstrated and compared to a simple C-banding method. The N-banding in rye chromosomes and the nucleolar sites on 1B and 6B of wheat differ from the normal N-banding sites of wheat chromosomes. Further, the banding of these nucleolar regions and of the rye chromosomes does not reappear in preparations that have been retreated with hot acid buffer. These differences provide evidence for at least two types of chromatin that stain darkly (positively) using N-banding. The critical procedures in the N-banding technique and the use of alternatives to 1 M NaH2PO4 buffer are discussed along with the possible basis of N-band formation.

Cell Nucleolus↗

Effect of maturity on degradation kinetics of sod-seeded cereal grain forage grown in northern Arkansas.

Wheat (Triticum aestivum L.), oat (Avena sativa L.), and rye (Secale cereale L.) were overseeded into a dormant bermudagrass (Cynodon dactylon (L.) Pers.) sod and harvested at 3-wk intervals throughout March, April, May, and early June. Plant growth stage was documented for each forage on each harvest date, and harvested forages were evaluated for forage quality characteristics. Degradation kinetics of DM and NDF for these forages were evaluated by the in situ method. Fractional degradation rates for DM and NDF in all three species were relatively rapid for vegetative forage (> or =0.086 h(-1)) but declined rapidly by the heading stage of development and stabilized thereafter. Forage quality declined and forages were more resistant to ruminal degradation as plants entered the reproductive stages of growth. Based on these findings, growth stage is an effective predictor of most characteristics of in situ DM and NDF disappearance. The relationships between these degradation parameters and growth stage were typically explained with quadratic or cubic models. Clearly, forage quality characteristics of overseeded rye deteriorated more rapidly with phenological development and growth stage than quality characteristics of overseeded wheat and oat grown in the same environment. For rye, this problem is further complicated by its accelerated phenological development. These factors combine to permit a very narrow harvest window in early spring, relative to the other cereal grains evaluated. Acceptable forage quality may persist for an extended period in wheat and oat; this suggests that producers wishing to utilize these forages may lengthen the harvest window by planting more than one species, either as a mixture or preferably in independent stands.

Animal Feed↗

[Genetic determination of stem-rust resistance in rye].

The harmful effect of stem rust on the crops of short-stem diploid winter rye was studied. If stem rust affected the plants by 70-100%, this decreased the mass of 1000 grains by about 35.8%. The genes that control the stemrust resistance of rye might originate from the following cultivars and forms: Ilmen, Orlovskii Gibrid, Kharkovskaya 55, Kharkovskaya 60, Kustovka, Kombaininyai, Kazanskaya, Krupnozernaya, Novozybkovskaya 4, Alfa, Derzhavinskaya 29, Chulpan, and Rossul, as well as wild populations of the perennial rye Secale montanum. This study was first to demonstrate that the resistance of the Kharkovskaya 55 and Rossul rye cultivars to the population of stem rust was controlled by a single dominant gene, which was designated Sr1.

Fungi↗

Components of the Arabidopsis C-repeat/dehydration-responsive element binding factor cold-response pathway are conserved in Brassica napus and other plant species.

Many plants increase in freezing tolerance in response to low, nonfreezing temperatures, a phenomenon known as cold acclimation. Cold acclimation in Arabidopsis involves rapid cold-induced expression of the C-repeat/dehydration-responsive element binding factor (CBF) transcriptional activators followed by expression of CBF-targeted genes that increase freezing tolerance. Here, we present evidence for a CBF cold-response pathway in Brassica napus. We show that B. napus encodes CBF-like genes and that transcripts for these genes accumulate rapidly in response to low temperature followed closely by expression of the cold-regulated Bn115 gene, an ortholog of the Arabidopsis CBF-targeted COR15a gene. Moreover, we show that constitutive overexpression of the Arabidopsis CBF genes in transgenic B. napus plants induces expression of orthologs of Arabidopsis CBF-targeted genes and increases the freezing tolerance of both nonacclimated and cold-acclimated plants. Transcripts encoding CBF-like proteins were also found to accumulate rapidly in response to low temperature in wheat (Triticum aestivum L. cv Norstar) and rye (Secale cereale L. cv Puma), which cold acclimate, as well as in tomato (Lycopersicon esculentum var. Bonny Best, Castle Mart, Micro-Tom, and D Huang), a freezing-sensitive plant that does not cold acclimate. An alignment of the CBF proteins from Arabidopsis, B. napus, wheat, rye, and tomato revealed the presence of conserved amino acid sequences, PKK/RPAGRxKFxETRHP and DSAWR, that bracket the AP2/EREBP DNA binding domains of the proteins and distinguish them from other members of the AP2/EREBP protein family. We conclude that components of the CBF cold-response pathway are highly conserved in flowering plants and not limited to those that cold acclimate.

Acclimatization↗

Microdissection and chromosome painting of plant B chromosomes.

Plant chromosome microdissection techniques together with different isolation and amplification methods of microisolated DNA are described. Such isolated DNA was used to 'chromosome paint' B chromosomes of the dicot Brachycome dichromosomatica and the monocot Secale cereale. It is demonstrated that the specific painting of the described chromosomes was possible because of enrichment for chromosome-specific repetitive sequences, rather than the chromosome specific low- and single-copy sequences which are responsible for the painting of mammalian chromosomes. The feasibility of 'chromosome painting' of standard chromosomes in plant species with relatively small or large genomes is discussed.

Asteraceae↗

Combinatorial labelling of DNA probes enables multicolour fluorescence in situ hybridisation in plants.

This paper demonstrates a simple but effective use of combinatorial probes to label plant chromosomes by multicolour fluorescence in situ hybridisation (FISH). Three different DNA probes were labelled with only two different fluorophores, hybridised to somatic metaphase chromosomes of Secale cereale and Triticum aestivum, simultaneously visualised, and unequivocally distinguished in a single FISH experiment. Combinatorial labelling can augment karyotypical investigations, physical mapping of chromosomes and other analyses in plants based upon FISH.

Chromosomes↗

Morphological, cytological and BSA-based testing on limited segregation population AFLPs.

Cytoplasmic male sterility (cms) in rye (Secale cereale L.), especially cytoplasma PAMPA, is used commercially in hybrid breeding programmes. The development of molecular markers that are tightly linked to the numerous genes coding for pollen fertility is expected to have great impact in the field. Morphological and cytological analyses of plants from a three-way cross C394: [(S67P/94 x S38/94) x CHD296] indicated the presence of at least several genes acting at different stages of pollen grain development, and proved the concurrence of both approaches in plant classification. The AFLP technique combined with the Bulk Segregant Analysis (BSA) were applied to identify DNA fragments linked to the genes of interest. All the 256 possible primer pair combinations based on the MseI and EcoRI restriction sites generated distinct band patterns allowing the identification of 31143 DNA fragments, visualised using the isotopic method. On average, any given primer combination generated 122 fragments. Among 1111 and 431 potential genetic markers respectively identified in the restorer form and the maternal lines, 775 and 295 were present in the F2 population. These numbers were then reduced to 109 and 51. The identified DNA fragments were tested on a limited segregating population, C394-F2, in order to eliminate false signals and to select markers for a future marker-assisted selection programme. Twenty-five markers were selected. Four of these markers were not identified via the BSA approach, indicating that if a highly polymorphic component is used for a cross, or a polygenic trait is studied, then the use of a limited population may be required.

Breeding↗