Flow sorting of mitotic chromosomes in common wheat (Triticum aestivum L.)

The aim of this study was to develop an improved procedure for preparation of chromosome suspensions, and to evaluate the potential of flow cytometry for chromosome sorting in wheat. Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity (flow karyotypes) obtained after the analysis of DAPI-stained chromosomes were characterized and the chromosome content of all peaks on wheat flow karyotype was determined for the first time. Only chromosome 3B could be discriminated on flow karyotypes of wheat lines with standard karyotype. Remaining chromosomes formed three composite peaks and could be sorted only as groups. Chromosome 3B could be sorted at purity >95% as determined by microscopic evaluation of sorted fractions that were labeled using C-PRINS with primers for GAA microsatellites and for Afa repeats, respectively. Chromosome 5BL/7BL could be sorted in two wheat cultivars at similar purity, indicating a potential of various wheat stocks for sorting of other chromosome types. PCR with chromosome-specific primers confirmed the identity of sorted fractions and suitability of flow-sorted chromosomes for physical mapping and for construction of small-insert DNA libraries. Sorted chromosomes were also found suitable for the preparation of high-molecular-weight DNA. On the basis of these results, it seems realistic to propose construction of large-insert chromosome-specific DNA libraries in wheat. The availability of such libraries would greatly simplify the analysis of the complex wheat genome.     

Effects of an Agropyron chromosome on endosperm proteins in common wheat (Triticum aestivum L.)

An Agropyron chromosome having a gene conferring blue color on the aleurone layer of the kernel endosperm causes a 15% increase in total grain protein content when it is added to the common wheat (2n=42) complement. In contrast, there is no effect of this chromosome on total protein content if it replaced part of a wheat chromosome. Endosperm protein components of isolines having blue aleurone due to the Agropyron chromosome being added (2n=44) or translocated (2n=42) were compared to normal nonblue isoline counterparts. Gliadin proteins separated by aluminum lactate (pH 3.2) polyacrylamide gel electrophoresis (PAGE) in one or two dimensions showed greater staining intensity for the blue addition isolines (2n=44) than nonblue (2n=42) isolines. However, the 42-chromosome blue isoline did not show increased protein staining over the nonblue isoline, but at least five protein differences were detected between the lines. SDS-PAGE showed that blue and nonblue differences were expressed primarily in the gliadins, but also in the glutenin, globulin, and albumin proteins.This research was supported by a D. F. Jones Postdoctoral Fellowship to K. M. Soliman and by Western Regional Project W-132, Genotype-environment interactions related to end-product uses in small grains.     

Identification of three Wx proteins in wheat (Triticum aestivum L.)

Nullisomic analysis of waxy (Wx) protein of hexaploid wheat (Triticum aestivum L.) cv. “Chinese Spring” using two-dimensional polyacrylamide gel electrophoresis revealed that threeWx loci,Wx-A1, Wx-B1, andWx-D1, located on chromosome arms 7AS, 4AL, and 7DS, produce three distinct Wx subunit groups, subunit group-A (SGA), SGB, and SGD, respectively. SGA has a higher molecular weight and a more basic isoelectric point (pI) than the other two. SGB and SGD have the same molecular weight but a slightly different pI range. Owing to the detection of these three subunit groups, we were able to identify the expression of three waxy genes in wheat endosperm and to find two types of mutants among Japanese wheat cultivars, one lacking SGA and the others SGB. These results suggest the possibility of breeding a waxy wheat.     

The wheat (Triticum aestivum L.) leaf proteome

The wheat leaf proteome was mapped and partially characterized to function as a comparative template for future wheat research. In total, 404 proteins were visualized, and 277 of these were selected for analysis based on reproducibility and relative quantity. Using a combination of protein and expressed sequence tag database searching, 142 proteins were putatively identified with an identification success rate of 51%. The identified proteins were grouped according to their functional annotations with the majority (40%) being involved in energy production, primary, or secondary metabolism. Only 8% of the protein identifications lacked ascertainable functional annotation. The 51% ratio of successful identification and the 8% unclear functional annotation rate are major improvements over most previous plant proteomic studies. This clearly indicates the advancement of the plant protein and nucleic acid sequence and annotation data available in the databases, and shows the enhanced feasibility of future wheat leaf proteome research.     

Silicon absorption by wheat (Triticum aestivum L.)

Although silicon (Si) is a quantitatively major inorganic constituent of higher plants the element is not considered generally essential for them. Therefore it is not included in the formulation of any of the solution cultures widely used in plant physiological research. One consequence of this state of affairs is that the absorption and transport of Si have not been investigated nearly as much as those of the elements accorded 'essential' status. In this paper we report experiments showing that Si is rapidly absorbed by wheat (Triticum aestivum L.) plants from solution cultures initially containing Si at 0.5 mM, a concentration realistic in terms of the concentrations of the element in soil solutions. Nearly mature plants (headed out) 'preloaded' with Si absorbed it at virtually the same rate as did plants grown previously in solutions to which Si had not been added. The rate of Si absorption increased by more than an order of magnitude between the 2-leaf and the 7-8 leaf stage, with little change thereafter. This revised version was published online in June 2006 with corrections to the Cover Date.     

Identification of C-banded chromosomes in meiosis of common wheat,Triticum aestivum L.

Summary The C-banding pattern of nine meiotic chromosomes of common wheat (Triticum aestivum L.) as described. In F₁s of crosses between monosomics of Chinese Spring and two Spanish wheat cultivars, univalent chromosomes were used to aid the recognition and analysis of the C-banding pattern for the individual chromosomes. The identification of one chromosome involved in one translocation in Chinese Spring x Pané 247 has been made through heterochromatin bands observed in the chromosomes involved in multivalents.     

Linked sucrose synthase genes in group-7 chromosomes in hexaploid wheat (Triticum aestivum L.)

A cDNA library from developing wheat endosperm was screened for sucrose-synthase clones using a maize cDNA probe corresponding to the Sh1 locus under non-stringent conditions. Five positive clones were isolated and initially classified into two types on the basis of their relative ability to hybridize with the probe and of their partial restriction maps. Determination of the nucleotide sequences indicated homology between the two types of wheat clones, with type 1 showing higher homology to the maize Sh1 locus than to type-2 sequences. The inserts cloned in plasmids pST8 (type 1) and pST3 (type 2) were used as probes to determine the chromosomal locations of the two types of genes. DNAs from compensated nulli-tetrasomic and ditelosomic lines of wheat cultivar Chinese Spring were cleaved with EcoRI and analysed in Southern blots. DNA segments of the two types were thus identified in the short arms of chromosomes 7A, 7D, and, possibly, 7B. The two types of linked loci have been designated Ss1 and Ss2, respectively.     

Lead phytotoxicity on wheat (Triticum aestivum L.) seed germination and seedlings growth

Lead (Pb) is an environmental pollutant extremely toxic to plants and other living organisms including humans. To assess Pb phytotoxicity, experiments focusing on germination of wheat seeds were germinated in a solution containing Pb (NO(3))(2) (0.05; 0.1; 0.5; 1g/L) during 6 days. Lead accumulation in seedlings was positively correlated with the external concentrations, and negatively correlated with morphological parameters of plant growth. Lead increased lipid peroxidation, enhanced soluble protein concentrations and induced a significant accumulation of proline in roots. Esterase activity was enhanced in the presence of lead, whereas α-amylase activity was significantly inhibited. Antioxidant enzymes activities, such as, ascorbate peroxidase, peroxidase, superoxide dismutase, catalase and glutathione S-transferase were generally significantly increased in the presence of lead in a dose-dependent manner. The present results thus provide a model system to screen for natural compounds able to counteract the deleterious effects of lead.     

Molecular identification of powdery mildew resistance genes in common wheat (Triticum aestivum L.)

RFLP markers for the wheat powdery mildew resistance genes Pm1 and Pm2 were tagged by means of near-isogenic lines. The probe Whs178 is located 3 cM from the Pm1 gene. For the powdery mildew resistance gene Pm2, two markers were identified. The linkage between the Pm2 resistance locus and one of these two probes was estimated to be 3 cM with a F₂ population. Both markers can be used to detect the presence of the corresponding resistance gene in commercial cultivars. Bulked segregant analysis was applied to identify linkage disequillibrium between the resistance gene Pm18 and the abovementioned marker, which was linked to this locus at a distance of 4 cM. Furthermore, the RAPD marker OPH-11₁₉₀₀ (5-CTTCCGCAGT-3) was selected with pools created from a population segregating for the resistance of Trigo BR 34. The RAPD marker was mapped about 13 cM from this resistance locus.     

Genetic diversity of reaction of common wheat (Triticum aestivum L.) cultivars to light intensity

The effect of low light intensity (LI) on the period from sprouting to earing was studied in 12 cultivars of the spring common wheat under controlled conditions. Differences between cultivars with respect to their responses to LI (RLIs) were found both for those that were photoperiod-sensitive and those that were almost photoperiod-neutral. Specifically, a prolonged photoperiod and a low LI differently increased the period from sprouting to earling in different cultivars. Genetic analysis of the RLI demonstrated, for the first time, that the weak response was incompletely dominant in F1. The results of genetic analysis agree with the hypothesis that the cultivars Pitic 62 and Novosibirskaya 22 differ in alleles of two loci controlling the RLI in wheat.     

Hydrogen peroxide scavenging regulates germination ability during wheat (Triticum aestivum L.) seed maturation

Hydrogen peroxide (H₂O₂) promotes seed germination of cereal plants and ascorbic acid which acts as antioxidant suppresses the germination of wheat seeds, but the role of H₂O₂ scavenging on germination during seed maturation has not been demonstrated. We investigated relationship of germination, ascorbate, H₂O₂ scavenging enzymes and sensitivity to ascorbic acid (AsA) maturing seeds of two typical wheat (Triticum aestivum L.) cultivars, cvs. Shirogane-Komugi and Norin61. Shirogane-Komugi had marked high germination ability than Norin61 during seed maturation. Although the H₂O₂ content had no difference in the two culti-vars, sensitivity to AsA of Norin61 seeds was higher than that of Shirogane-Komugi seeds during seed maturation. The sensitivity to AsA closely correlated with germination characteristic in the two cultivars. Especially, at 28 days after pollination (DAP), sensitivity to AsA in Norin61 seeds was remarkably high. At that stage, no significant differences were observed in endogenous AsA level, ascorbate peroxidase (APX, EC 1.11.1.11) and dehydroascorbate reductase (DHAR, EC 1.8.5.1) activities in the two cultivars. However, catalase (CAT, EC 1.11.1.6) activity and CAT mRNA in Norin61 were remarkably higher than in Shirogane-Komugi. Sensitivity to AsA at 35 and 42 DAPs kept high levels in Norin61, and endogenous AsA and CAT activity in the seeds were significantly higher than in Shirogane-Komugi. These results revealed a direct correlation between germination and antioxidant sensitivity during the developmental stages of wheat seeds.Key words: ascorbic acid, germination, hydorogen peroxide, maturation, wheat seed     

Prevalence and control of wheat (Triticum aestivum L.) seed borne fungi in farmer-saved seeds

High incidence of diseases associated with the use of seeds saved from previous harvests as desire of maintaining local varieties with special attributes is of increased concern in wheat industry worldwide. Prevalent of seed-borne fungi in farmer-saved seeds and seed dressing fungicides to prevent infection from seeds to seedlings was studied in Northern Tanzania. One hundred and thirty five untreated farmer-saved seed lots were collected randomly from farmers. Alternaria alternata, Bipolaris sorokiniana, Drechslera tritici, Fusarium graminearum, Fusarium moniliforme, Aspergillus flavus, Cladosporium sphaerospermum, Epicoccum purpurascens, Pyricularia oryzae and Penicillium corylophilum were fungi isolated in farmer-saved seeds. Mean seed infection was 29% causing average grain yield loss of 1.2 mt/ha^?1. Seed dressing with Metalaxy plus (Methyl carboxaitide), Mancozeb (Manganase-zinc salt) and Baytan (Chlorophenoxy ethanol) increased seed germination by 14, 13 and 17%, respectively, and grain yield by 28, 20 and 18%, respectively. Farmer-saved seeds were heavily infected by fungi with low grain yield performance.     

The HPLC profile of proteins of thermoprotection-acquired wheat (Triticum aestivum L.) seedlings

A pre-treatment of 40 °Cprovided thermoprotection to wheat seedlings against 43 °C, which was otherwise a lethal temperature. Due to temperature pretreatment, the rate of protein synthesis at 45 °C increased in both plumules and radicles. The HPLC profile of plumule and radicle proteins of thermoprotection-acquired seedlings was different from the plumules and radicles of non-treated seedlings.     

Proteomic analysis on salicylic acid-induced salt tolerance in common wheat seedlings (Triticum aestivum L.)

The influence of salicylic acid (SA) on the salt tolerance mechanism in seedlings of common wheat (Triticum aestivum L.) was investigated using physiological measurements combined with global expression profiling (proteomics). In the present study, 0.5mM SA significantly reduced NaCl-induced growth inhibition in wheat seedlings, manifesting as increased fresh weights, dry weights, and photosynthetic pigments, but decreased lipid peroxidation. Two-week-old wheat seedlings treated with 0.5mM SA, 250mM NaCl and 250mM NaCl+0.5mM SA for 3days were used for the proteomic analyses. In total, 39 proteins differentially regulated by both salt and SA were revealed by 2D PAGE, and 38 proteins were identified by MALDI-TOF/TOF MS. The identified proteins were involved in various cellular responses and metabolic processes including signal transduction, stress defense, energy, metabolism, photosynthesis, and others of unknown function. All protein spots involved in signal transduction and the defense response were significantly upregulated by SA under salt stress, suggesting that these proteins could play a role in the SA-induced salt resistance in wheat seedlings.     

The effect of ovary-conditioned medium on microspore embryogenesis in common wheat (Triticum aestivum L.)

Microspores were isolated from wheat (Triticum aestivum L.) spikes of various genotypes following an effective pretreatment that induced microspore embryogenesis. The isolated microspores were cultured with or without live ovaries, and with or without medium pre-conditioned by ovaries for varying periods of time. Live ovaries alone increased androgenic embryoid yields up to 4.5-fold over the control for microspores isolated from responsive genotypes. While live ovary co-culture alone was not effective for microspores isolated from recalcitrant genotypes, the addition of medium preconditioned by ovaries to microspore cultures increased embryoid yield by more than 100-fold. Without ovary-conditioned medium, no embryoids could be obtained from some recalcitrant genotypes. Ovary-conditioned medium apparently functions to increase embryoid yields by providing essential substance(s) for elaboration of the embryogenic program already triggered during pretreatment.     

Assembling complex genotypes to resist Fusarium in wheat (Triticum aestivum L.)

Fusarium head blight of wheat is a major deterrent to wheat production world-wide. The genetics of FHB resistance in wheat are becoming clear and there is a good understanding of the genome location of FHB resistance QTL from different sources such as Sumai3, Wuhan, Nyubai and Frontana. All the components needed for assembling complex genotypes through large-scale molecular breeding experiments are now available. This experiment used high throughput microsatellite genotyping and half-seed analysis to process four independent crosses through a molecular breeding strategy to introduce multiple pest resistance genes into Canadian wheat. This included two backcrosses and selection for a total of six FHB resistance QTL, orange blossom wheat midge resistance (Sm1) and leaf rust resistance (Lr21). In addition, the fixation of the elite genetic background was monitored with 45–76 markers to accelerate restoration of the genetic background at each backcross. The strategy resulted in 87% fixation of the elite genetic background on average at the BC₂F₁ generation and successfully introduced all of the chromosome segments containing FHB, Sm1 and Lr21 resistance genes. The molecular breeding strategy was completed in 25 months, at an equal pace to conventional crossing and selection of spring wheat.     

A simplified AFLP method for fingerprinting of common wheat (Triticum aestivum L.) cultivars

The simplified AFLP method was developed and evaluated for identification and genetic diversity studies of wheat cultivars. Selective primers exploited in AFLP assay based on a single cutting enzyme PstI ((PstI)AFLP) generated total of 111 robust fragments, including 67 (60%) monomorphic and 12 (11%) cultivar-specific markers. Average similarity between 15 cultivars was 0.650, and varied from 0.293 ('Hope' vs. 'Aurora') to 0.865 ('Norman' vs. 'Hornet'). Mean similarities within groups of winter wheat cultivars with and without 1BL/1RS chromosome were 0.713 and 0.685, respectively. A higher variation was found in the group of spring wheats: 0.677. The obtained results confirm the usefulness of the proposed modification of the AFLP technique for diversity studies and identification of common wheat cultivars.