Construction and Characterization of the Microclone Library from Chromosome 1R in Rye

Two and five 1R chromosomes were microdissected from the metaphase spreads of rye ( Secale cereale L. ) root-rip cells with the aids of glass needles. The dissected chromosomes were amplified in vitro by the Sau3A linker adaptor mediated PCR technique, by which 0.3 to 2.5 kb smear DNA fragments were obtained. After hybridized with DIG labeled probes, it was confirmed that the PCR products of the microdissected chromosomes were homologous with the rye genomic DNA, and derived from the 1R chromosome as well. Then, the second round PCR products from five chromosomes of 1R were microcloned to construct the plasmid library, including 220 000 clones. 172 randomly selected clones were evaluated ranged in size from 300 to 1 800 bp. Furthermore, the genomic dot hybridization results indicated that the library contained nearly 42% medium/high repetitive sequences and 58% low/single copy sequences, and its redundancy was very low. In this research, many aspects of the 1R chromosome microclone library exceeded or approached those of the previous reports in the literatures. Those are potential for construction of a high density genetic map of chromosome IR, from which some important genes can be tagged and isolated.     

Microdissection and Recovery of 1R Chromosome in Rye ( Secale cereale )

The technique of mieromanipulaion has been used to establish a system of single chromosome mierodissection. According to the standard karyotype of rye ( Secale cereale L. ), 1R chromosome carrying disease-resistant gene has been identified, microdissected and transferred into Ep-pendorf tube using mieromanipulator. The results showed that IR chromosome in cells pretreated with α-bromonaphthalene could be identified quickly and the efficiency of microdissection was greatly improved when the technique of wall degradation and hypotonic treatment was applied.     

Chloroplast Development in Rye Coleoptiles

In the parenchyma cells of 1-d-old dark-grown rye coleoptiles (Secale cereale) proplastids occurred which sometimes contained starch grains. During coleoptile growth in darkness starch-filled amyloplasts are formed from the preexisting proplastids. No prolamellar bodies were observed in the stroma of the plastids of the etiolated coleoptile. After irradiation of 3-d-old etiolated coleoptiles with continuous white light three different types of plastids occurred. In the epidermal cells proplastids were observed. The parenchyma cells below the stomata of the outer epidermis (above the two vascular bundles) contained mature, spindle-shaped chloroplasts with a well-developed thylakoid system. In the parenchyma cells that surround the vascular bundles amyloplasts with some thylakoid membranes (chloroamyloplasts) occurred. The mesophyll cells of the primary leaves of dark-grown seedlings contained etioplasts with large prolamellar bodies. In the primary leaves of irradiated plants chloroplasts similar to those of the parenchyma cells of the coleoptile were observed. Our results show that the rye coleoptile, which grows underground as a heterotrophic organ, is capable of developing mature chloroplasts upon reaching the light above the soil surface. The significance of this expression of photosynthetic capacity for the carbon economy of the developing seedling is discussed.     

Studies on Microdissection and Microcloning of the Rye Chromosome 1R

Two 1R chromosomes of Secale cereale L. were isolated from one metaphase cell by means of chromosome micro-isolation, and the chromosomal DNA was amplified adopting the cohesive adapters single primer polymerase chain reaction (CASP-PCR) technique. The CASP-PCR products were labeled as probes. The results of Southern blot hybridization confirmed that the CASP- PCR products derived from the chromosome IR were homologous with the genomic DNA of S. cereale. The clones of PCR products were obtained with high efficiency. Over 10 000 recombinant clones were obtained from one-tenth of the ligation mixture which was transferred into the competent E. coli DH5a. The size of the inserted fragments of clones ranged from 250 bp to 500 bp. This research has established the foundation for further selection of chromosome 1R markers.     

Pigment Accumulation and Photosynthesis in Developing Rye Coleoptiles

Rye seedlings (Secale cereale L.) were grown in darkness or irradiated with white light (WL) of medium intensity (100 μmol quanta . m^?2 . s^?1). In the coleoptile and primary leaf of irradiated plants synthesis of chlorophylls a and b with average ratios of 2.4 and 2.7, respectively, were measured. The chlorophyll content of the fully green primary leaf was about 10-fold larger than that of the pale green coleoptile. In darkness a large increase in carotenoid content occurred in the primary leaf, but in the coleoptile the level of carotenoids remained very low. In both organs a WL-induced increase in carotenoid synthesis was observed. The level of anthocyanin was likewise enhanced by WL. In the coleoptile and primary leaf of irradiated seedlings light-dependent oxygen evolution was measured. The specific activity of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase was enhanced after WL treatment. The results indicate that the mature rye coleoptile is a photosynthetically active plant organ.     

黑麦基因组特异DNA片段的分离与SCAR标记的建立

以2个栽培黑麦、2个野生黑麦和4个普通小麦为材料,从200条10碱基RAPD随机引物中筛选出1条引物H11。H11在小麦中有1条低拷贝扩增,而在黑麦中却有极高拷贝的扩增。对H11在黑麦中的高拷贝片段进行克隆、测序,得其全长679 bp,记作OPH11679。根据OPH11679设计特异PCR引物H11-F和H11-R,对小麦族其它物种和含黑麦染色质的物种进行验证,结果发现仅含黑麦染色质的物种能扩增出长为643 bp的片段(命名为pScH643),这表明该片段为黑麦所特有。用H11-F和H11-R对1套中国春-Imperial黑麦附加系等进行扩增,结果显示pScH643片段分布在黑麦整套染色体上,这一结果在小麦-黑麦异源材料的分析中得到进一步验证。即表明pScH643片段可作为SCAR标记用于含黑麦染色质材料的检测。     

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.     

Water Potential Modulates Extensibility of Rye Coleoptile Cell Walls

Extensibility of walls of frozen/thawed rye (Secale cereale) coleoptile segments as a function of the water potential of the incubation solution (Ψ₀) was analyzed employing the creep test method. Negative Ψ₀ exerts an inhibiting effect on extension of isolated walls. The lower the Ψ₀ of polyethylene glycol 6000 (PEG), the less the walls of frozen/thawed segments extended under load. This inhibiting effect of Ψ₀ on wall creep was reversible and independent of the preincubation temperature of the segments. An increase in Ψ₀ resulted in increased extension rate within 2–4 min, whereas a decrease in Ψ₀ resulted in gradually decreasing extension rate after 8–12 min. This finding implies that wall extension changes during growth induced by changes of Ψ₀ in vivo are not only due to changes of turgor pressure but also due to a direct influence by negative Ψ₀ on physical wall properties. The results are discussed with respect to the regulation of extension growth during conditions of water stress.     

Putative MADS-Box Retropseudogenes in Rye (Secale L.)

The fragments of MADS-box genes belonging to the agamous and agamous-like structural classes were isolated by direct amplification of genomic DNA from annual rye (Secale cereale L.) and perennial rye (Secale montanum Guss.). The characterized fragments (deposited in the Genbank as the accession nos. AF332885–AF332887 and AF346894) comprise the complete sequences of exons 1 to 5 and lack corresponding introns. Their nearest homologs are the maize genes zag1 and zag5 (the Genbank accession nos. L18924 and L46398). One more agamous-like fragment isolated from annual rye (AF362364) is similar to theTaMADS12 wheat gene (AB007505). The fragment comprises exons 3–5 and contains a 105-bp insert between the exons 3 and 4; this insert does not resemble any MADS-box introns presently known. We assume that all these fragments of MADS-box genes are retropseudogenes.     

Resistance to Ergot in Self-incompatible Germplasm Resources of Winter Rye

The poor progress in breeding for resistance to ergot ( Claviceps purpurea [Fr.] Tul.) in rye ( Secale cereale L.) is attributed to the lack of appreciable genotypic variation for this trait. The present study was, therefore, undertaken to evaluate 52 indigenous and exotic genetic resources and 13 open-pollinated cultivars for ergot resistance. These 65 self-incompatible populations were evaluated in four environments with each entry plot surrounded by wheat plots on four sides. Inoculation with a pathogen mixture was done thrice during flowering and harvesting was done early by hand. Resistance traits were ergot incidence and severity. Logit-transformed data were subjected to analyses of variance. The lowest disease severity in any environment was 0.18%, and that was still higher than the official threshold of 0.1% for feed. In the pooled analysis across environments, significant genotypic differences as well as genotype × environment interaction were present. The correlation between ergot severity and pollen shedding was not significant indicating that genotypic differences were not affected by pollen shedding. Broad-sense heritability ( h ² ) estimates were moderate (0.54–0.65). 'Halo', an old German cultivar, and the genetic resources 'Schmidt-Roggen' and 'Dukat', had low ergot severity (Halo having the lowest severity) as well as low disease incidence. The results document a significant, but moderate genotypic variance for ergot resistance among self-incompatible rye that can be exploited to breed ergot-resistant rye.     

Recombination Landscape Divergence Between Populations is Marked by Larger Low-Recombining Regions in Domesticated Rye

The genomic landscape of recombination plays an essential role in evolution. Patterns of recombination are highly variable along chromosomes, between sexes, individuals, populations, and species. In many eukaryotes, recombination rates are elevated in sub-telomeric regions and drastically reduced near centromeres, resulting in large low-recombining (LR) regions. The processes of recombination are influenced by genetic factors, such as different alleles of genes involved in meiosis and chromatin structure, as well as external environmental stimuli like temperature and overall stress. In this work, we focused on the genomic landscapes of recombination in a collection of 916 rye (Secale cereale) individuals. By analyzing population structure among individuals of different domestication status and geographic origin, we detected high levels of admixture, reflecting the reproductive biology of a self-incompatible, wind-pollinating grass species. We then analyzed patterns of recombination in overlapping subpopulations, which revealed substantial variation in the physical size of LR regions, with a tendency for larger LR regions in domesticated subpopulations. Genome-wide association scans (GWAS) for LR region size revealed a major quantitative-trait-locus (QTL) at which, among 18 annotated genes, an ortholog of histone H4 acetyltransferase ESA1 was located. Rye individuals belonging to domesticated subpopulations showed increased synaptonemal complex length, but no difference in crossover frequency, indicating that only the recombination landscape is different. Furthermore, the genomic region harboring rye ScESA1 showed moderate patterns of selection in domesticated subpopulations, suggesting that larger LR regions were indirectly selected during domestication to achieve more homogeneous populations for agricultural use.     

Fluorescence Banding in Several Species of Plant

Chromosomal fluorescence banding in Haynaldia villosa (2n=14),Zea mays (2n=20) and Secale cereale (2n=14) have been studied. Stained with Hoe-chst 33258, all three species show chromosomal banding clearly and brightly, but withquinacrine. 2HCl only the staining of H. villosa chromosomes are satisfactory. While thequinacrine fluorescence banding of maize and Secale is not clearer and quench morequickly. The positions of the Ho-banding are basically identical with the Q-banding. A comparison has been made between the fluorescence banding and the C-banding in great detail. The results show that the two barding patterns are about the same, but there are also some differences ,between them. We have also discussed the reason of the similarities and the differences between Ho-banding, Q-banding and C-banding in chromosomes of same species.     

Physiological Evidence for the Accumulation of Restrained Wall Loosening Potential on the Growth-Inhibited Side of Graviresponding Rye Coleoptiles1

Abstract: Subsequent to inversion, horizontally pre-gravistim-ulated coleoptiles exhibit an increased gravicurvature capacity as compared to vertically pre-gravistimulated and subsequently horizontally gravistimulated coleoptiles. This indicates that gravistimulated inhibition of growth of the upper organ flank during gravicurvature is mediated wo the retention of wall loos ening potential. After inversion, this potential contributes to en hanced cell extension on the then physiologically lower side.     

An ecological approach to study the physical and chemical effects of rye cover crop residues on Amaranthus retroflexus, Echinochloa crus-galli and maize

Weeds can be suppressed in the field by cover crop residues, extracts of which have been demonstrated to exert chemical inhibition of crop and weed germination and early growth in bioassays. In this study, two complementary bioassays were developed with soil and mulch material originating from a long‐term maize–cover crop experiment to determine the relative physical and chemical effect of rye cover crop residues on weed and maize germination and early growth. This was compared with the effect exerted by residue material from the natural vegetation that developed in the crop stubble during the winter before maize sowing. Germination percentage and early growth of maize and two maize weeds, Amaranthus retroflexus and Echinochloa crus‐galli, were assessed in a seed incubator in tilled (green manured) and nontilled (surface mulched) soil, with and without N fertilisation, at various dates after cover crop destruction. Responses were compared to those of the same species in a standard soil without mulch or with an inert poplar mulch. A second bioassay was set up in a glasshouse to determine the effect of different quantities of fresh residue material and additional N fertilisation on emergence speed and percentage and on plant vigour during the first 22 days after cover crop destruction. These results were compared with no‐mulch controls and poplar mulch controls. Results of these trials were compared with weed density and biomass that developed in the maize crop sown after cover crop destruction. Soil and mulch chemical and biological properties were determined for material collected in the field at different times after cover crop destruction. Chemical properties of the mulch differed only occasionally between the treatments, but variation in cover crop biomass production led to significantly different soil chemical properties. Although soil total phenolic acid content did not always correlate to weed and maize germination and early growth inhibition, soil microbial activity did. In suboptimal conditions, as is often the case in the field, plant residue material exerted both a physical and a chemical effect on maize and weed emergence and early growth. Nitrogen fertilisation and application timing can give the maize crop a competitive advantage with respect to the weeds, but the final response and the practical consequences depended largely on the weed species involved.     

Fusarium Head Blight Reactions and Accumulation of Deoxynivalenol (DON) and Some of its Derivatives in Kernels of Wheat, Triticale and Rye

Fifty-three commercially grown cultivars and germplasm lines of winter triticale (n = 18), wheat (n = 13), and rye (n = 5) and spring triticale (n = 8), wheat (n = 7) and rye (n = 2) were inoculated at mid anthesis with a spore suspension consisting of a mixture of Fusarium culmorum, Fusarium avenaceum and Fusarium graminearum isolates of known toxinogenic activity. Reactions to Fusarium head blight were measured as disease severity, reductions of kernel number/head, kernel weight/head and 1000 kernel weight, number of Fusarium-damaged kernels and kernel content of deoxynivalenol (DON) and its acetyl-derivatives 3-AcDON, 15-AcDON, and moniliformin. None of the cereal genotypes was completely resistant to Fusarium head blight. Wheat suffered from the largest kernel weight reductions, and accumulated the largest amounts of deoxynivalenol (up to 39.5 mg/kg) and 3AcDON (up to 6.0 mg/kg) in kernels. Deoxynivalenol was not detected in grain samples of winter rye cv. Dańkowskie Z?ote, and spring rye cv. Ludowe. 15-AcDON was only detected in genotypes of triticale, and 3AcDON only in a few genotypes of winter wheat and rye. Moniliformin was detected at low concentrations (up to 0.092 mg/kg) in kernels of some genotypes selected for the mycotoxin analysis. A moderately strong Pearson correlation was found between head blight severity parameters and the accumulation of deoxynivalenol and its derivatives in grain of the cereal genotypes studied. Fusarium head blight severity parameters were correlated with the percentage of Fusarium-damaged kernels and reductions of yield components. However, some head blight-susceptible genotypes realized their potential yields, but accumulated high levels of mycotoxins in kernels. Both Fusarium head blight resistant and susceptible genotypes of the three cereal species accumulated deoxynivalenol in kernels. This finding suggests that the system regulating deoxynivalenol accumulation may be independent of Fusarium head blight reaction.     

Variation of B Chromosome Associated with Tissue Culture in Wheat-rye Cross

In vitro variation of B chromosomes was studied by examining the callus cells derived from the immature embryos from a cross of Chinese Spring wheat ( Triticum aestivum L.) and Fin 7416 rye ( Secale cereale L.) carrying two B chromosomes. In 40-d-old callus cells, the numbers of B chromosomes ranged from one to four in 65.6% of the cells observed. The distribution of B chromosome numbers was associated with the ploidy levels of the normal chromosomes (A chromosomes). The frequency of the cells with high numbers of B chromosomes (i.e., three or four B chromosomes) in the amphiploid cells with 56 A chromosomes was greater than those in the haploid cells with 28 A chromosomes. Although structural changes in the rye A chromosomes were observed, cytological observation and genomic in situ hybridization demonstrated that the rye B chromosomes were conserved in morphological appearance following tissue culture.     

Interconversions of Chlorophylls a and b Synthesized from Exogenous Chlorophyllides a and b in Etiolated and Post-Etiolated Rye Seedlings

A comparative study of reciprocal conversions of chlorophylls a and b (Chl aand Chl b) in etiolated and post-etiolated rye seedlings (Secale cereale L.) was performed. The production of these pigments was initiated by infiltration of exogenous chlorophyllides a and b (Chlide a and b). It was shown that Chlide b, when infiltrated into etiolated rye seedlings, was esterified, producing Chl b. A major portion of Chl b (more than 80%) was transformed into Chl aduring long-term seedling dark exposure. The high rate of Chl b conversion into Chl a in the pool of pigments of exogenous origin was also observed during the lag-phase when there was no chlorophyll formation from endogenous precursors. The infiltration of Chlide a resulted in Chl a formation. The efficiency of its conversion into Chl b was low (about 1%) in the etiolated seedlings but increased during their greening. In the post-etiolated seedlings infiltrated with Chlide b, which were preliminary illuminated for 6–12 h, the Chl /Chl a ratio was almost similar in the pools of pigments synthesized from both exogenous and endogenous precursors. The rates of direct and reverse reactions responsible for the interconversion of Chl aand Chl b depended on the stage of the formation of the photosynthetic apparatus during greening of etiolated seedlings, when the particular structural components are formed in a definite sequence.     

Aging and Loss of Germination in Rye Seeds Is Accompanied by a Decreased Fragmentation of Nuclear DNA at Loop Domain Boundaries

To investigate the processes that occur in the embryo cell nuclei in the course of natural and accelerated aging of rye seeds, nuclear DNA structural organization into chromatin loop domains was studied. The loss of germination was shown to be accompanied by a decreased excision of chromatin loop domains. The study of chromatin accessibility to DNase I did not reveal any considerable changes in chromatin architecture that would explain the decreased DNA fragmentation at matrix attachment regions. A soluble nuclear protein of ca. 31 kD was found to manifest nuclease activity, which declined with the loss of germination. The study of DNA fragmentation in histone-depleted nuclei (nucleoids) disclosed a nuclease activity resistant to 2 M NaCl extraction and sensitive to the specific inhibitors of DNA topoisomerase II; the latter activity also declined with aging. The authors conclude that the changes in DNA fragmentation patterns in aging seeds were primarily caused by a decreased activity of the enzymes accounting for the excision of chromatin loop domains.     

Variation of B Chromosome Associated with Tissue Culture in Wheat-rye Cross

In vitro variation of B chromosomes was studied by examining the callus cells derived from the immature embryos from a cross of Chinese Spring wheat （Triticum aestivum L.） and Fin 7416 rye （Secale cereale L.） carrying two B chromosomes. In 40-d-old callus cells, the numbers of B chromosomes ranged from one to four in 65.6% of the cells observed. The distribution of B chromosome numbers was associated with the ploidy levels of the normal chromosomes （A chromosomes）. The frequency of the cells with high numbers of B chromosomes （i.e., three or four B chromosomes） in the amphiploid cells with 56 A chromosomes was greater than those in the haploid cells with 28 A chromosomes. Although structural changes in the rye A chromosomes were observed, cytological observation and genomic in situ hybridization demonstrated that the rye B chromosomes were conserved in morphological appearance following tissue culture.     

Cold pretreatment to enhance green plant regeneration from rye anther culture

Cold pretreatment of detached tillers of rye, Secale cereale, was tested under two light regimes. The ryes included two spring and two winter cultivars. Significant increases in green plant regeneration were recorded in each experiment when cold pretreatments of two to four weeks were applied. Dim light during the stress period improved green plant regeneration for two of the four cultivars tested. The highest regeneration rate, 30.6 green plants per 100 plated anthers, was reached following three weeks at +4 °C under dim light, for spring rye Jo02. Starvation stress applied to plated anthers in mannitol medium suppressed anther response. This revised version was published online in June 2006 with corrections to the Cover Date.