The peroxidase isozymes of the wheat kernel: tissue and substrate specificity and their chromosomal location

Summary Peroxidase isozymes were studied in the Triticum aestivum L. kernel and in nullisomic-tetrasomic and ditelocentric combinations of Chinese Spring wheat. Analyses were carried out on different parts of dry kernels (embryo plus scutellum and endosperm) using polyacrylamide and starch gel electrophoresis, different electrophoretic buffer systems and various staining methods. The peroxidase isozymes showed a low substrate-specificity and a high tissue-specificity. The embryo plus scutellum and the endosperm always presented different peroxidase patterns. Endosperm peroxidases were associated with chromosome arms 7DS, 4BL and 7AS; whereas the embryo plus scutellum isozymes were related to chromosome arms 3AL, 3BL and 3DS. The different results obtained using various electrophoretic techniques are due to the buffer system used. All staining procedures employed revealed the same peroxidase isozymes.     

Identification and chromosomal location of four subfamilies of the rubisco small subunit genes in common wheat

Summary Three different 3 noncoding sequences of wheat rubisco small subunit (SSU) genes (RbcS) were used as probes to identify the gene members of different RbcS subfamilies in the common wheat cultivar Chinese Spring (CS). All genes of the wheat RbcS multigene family were previously assigned to the long arm of homoeologous group 5 and to the short arm of homoeologous group 2 chromosomes of cv CS. Extracted DNA from various aneuploids of these homoeologous groups was digested with four restriction enzymes and hybridized with three different 3 noncoding sequences of wheat SSU clones. All RbcS genes located on the long arm of homoeologous group 5 chromosomes were found to comprise a single subfamily, while those located on the short arm of group 2 comprised three subfamilies. Each of the ancestral diploid genomes A, B, and D has at least one representative gene in each subfamily, suggesting that the divergence into subfamilies preceded the differentiation into species. This divergence of the RbcS genes, which is presumably accompanied by a similar divergence in the 5 region, may lead to differential expression of various subfamilies in different tissues and in different developmental stages, in response to different environmental conditions. Moreover, members of one subfamily that belong to different genomes may have diverged also in the coding sequence and, consequently, code for distinguishable SSU. It is assumed that such utilization of the RbcS multigene family increases the adaptability and phenotypic plasticity of common wheat over its diploid progenitors.     

The chromosomal location of peroxidase isozymes of the wheat kernel

Summary The analysis of the individual parts of the Triticum aestivum L. kernel yields a total of 11 peroxidase isozymes: m, n, a, c, d₁, d, d₂, e, f, g and h (in order from faster to slower migration). Isozymes a, c and d are found in the endosperm (Ed) and seed coats (C), while m, n, d₁, d₂, e, f, g and h are peculiar to the embryo and scutellum (E + S). The use of the nullitetrasomic and ditellosomic series of Chinese Spring wheat allows peroxidase isozymes to be associated with specific chromosome arms. Isozymes a, c and d (Ed) are associated with chromosome arms 7D^S, 4B^L and 7A^S; whereas isozymes m, d₂, e and f are associated with chromosome arms 3D^S, 3B^L, 3D^L and 3D^L, respecitvely. Thus, the E + S isozymes are associated with homoeology group 3 and the Ed isozymes with homoeology groups 7 (a and d isozymes) or 4 (c isozymes).     

Abundance, variability and chromosomal location of microsatellites in wheat

The potential of microsatellite sequences as genetic markers in hexaploid wheat (Triticum aestivum) was investigated with respect to their abundance, variability, chromosomal location and usefulness in related species. By screening a lambda phage library, the total number of (GA)_n blocks was estimated to be 3.6 x 10⁴ and the number of (GT)_n blocks to be 2.3 x 10⁴ per haploid wheat genome. This results in an average distance of approximately 270 kb between these two microsatellite types combined. Based on sequence analysis data from 70 isolated microsatellites, it was found that wheat microsatellites are relatively long containing up to 40 dinucleotide repeats. Of the tested primer pairs, 36% resulted in fragments with a size corresponding to the expected length of the sequenced microsatellite clone. The variability of 15 microsatellite markers was investigated on 18 wheat accessions. Significantly, more variation was detected with the microsatellite markers than with RFLP markers with, on average, 4.6 different alleles per microsatellite. The 15 PCR-amplified microsatellites were further localized on chromosome arms using cytogenetic stocks of Chinese Spring. Finally, the primers for the 15 wheat microsatellites were used for PCR amplification with rye (Secale cereale) and barley accessions (Hordeum vulgare, H. spontaneum). Amplified fragments were observed for ten primer pairs with barley DNA and for nine primer pairs with rye DNA as template. A microsatellite was found by dot blot analysis in the PCR products of barley and rye DNA for only one primer pair.     

The chromosomal location of factors determining the presence of phenolic compounds in wheat (Triticum aestivum L.)

Summary Using thin-layer chromatography and nulli-tetrasomic and ditellosomic series of Triticum aestivum L. cv. Chinese Spring, it has been possible to relate the phenolic compounds found in adult plant leaves and 12 day-old seedling leaves with the chromosomes or chromosome arms 1 B, 2 BL, 3 BL, 5 A, 6 AL, 7 B and 7 DS.     

Chromosomal location of genes controlling heat shock proteins in hexaploid wheat

Summary The low molecular weight heat shock protein (HSP) profiles of the hexaploid wheat cultivar Chinese Spring and its ditelosomic series were characterized by isoelectric focusing polyacrylamide gel electrophoresis of denatured in vivo radiolabeled proteins. Comparisons of the ditelosomics (DTs) to the euploid Chinese Spring enabled the assignment of genes controlling 9 of the 13 targeted HSPs to seven chromosome arms. There did not appear to be a genome-specific action in the regulation of expression of these HSPs. There did appear to be a higher frequency of controlling genes within homoeologous DT lines 3, 4 and 7. Significant variation in protein quantity was evident among the DT lines for some HSPs, while other HSPs were remarkably stable in their expression across all DTs examined. The results are useful in identifying specific DT lines for the investigation of HSP functions in hexaploid wheat.     

Chromosomal location of a gene suppressing powdery mildew resistance genes Pm8 and Pm17 in common wheat (Triticum aestivum L. em. Thell.)

The chromosomal location of a suppressor for the powdery mildew resistance genes Pm8 and Pm17 was determined by a monosomic set of the wheat cultivar Caribo. This cultivar carries a suppressor gene inhibiting the expression of Pm8 in cv Disponent and of Pm17 in line Helami-105. In disease resistance assessments, monosomic F₁ hybrids (2n=41) of Caribo x Disponent and Caribo x Helami-105 lacking chromosome 7D were resistant, whereas monosomic F₁ hybrids involving the other 20 chromosomes, as well as disomic F₁ hybrids (2n=42) of all cross combinations, were susceptible revealing that the suppressor gene for Pm8 and Pm17 is localized on chromosome 7D. It is suggested that genotypes without the suppressor gene be used for the exploitation of genes Pm8 and Pm17 in enhancing powdery mildew resistance in common wheat.     

Sequence variation, differential expression and chromosomal location of rice chitinase genes

Rice chitinases are encoded by a small multigene family. To clarify the overall organization of rice chitinase genes, we have isolated and characterized the genes Cht-1, Cht-2 and Cht-3. Although all the three genes encode class I chitinase, the nucleotide sequences of the coding regions of Cht-1 and Cht-3 are very similar (90%), while that of Cht-2 is clearly more divergent (78%). Only Cht-2 has a 130 by intron and encodes a C-terminal peptide sequence similar to that known to function as a vacuolar targeting signal. In 5 flanking regions of Cht-1 and Cht-3, but not of Cht-2, conserved sequences (GGCCGGCYGCCCYAG) were found. Related sequences were found also in the 5 flanking regions of another chitinase gene and a -glucanase gene which has also been reported to be stress-induced in rice. RNA blot hybridization analysis demonstrated that the stress-induced expression patterns of the Cht-1 and Cht-3 genes are similar, but quite different from that of Cht-2. However, all three genes are active in unstressed roots. By restriction fragment length polymorphism (RFLP) linkage analysis, Cht-1 and Cht-3 were mapped onto chromosome 6 and shown to be closely linked (0.8 cM). Cht-2 was mapped onto chromosome 5. All these features suggest that the expression patterns of rice class I chitinase genes may be correlated with their levels of sequence divergence and their chromosomal location.     

Chromosome substitutions of Triticum timopheevii in common wheat and some observations on the evolution of polyploid wheat species

Whether the two tetraploid wheat species, the well known Triticum turgidum L. (macaroni wheat, AABB genomes) and the obscure T. timopheevii Zhuk. (A^tA^tGG), have monophyletic or diphyletic origin from the same or different diploid species presents an interesting evolutionary problem. Moreover, T. timopheevii and its wild form T. araraticum are an important genetic resource for macaroni and bread-wheat improvement. To study these objectives, the substitution and genetic compensation abilities of individual T. timopheevii chromosomes for missing chromosomes of T. aestivum Chinese Spring (AABBDD) were analyzed. Chinese Spring aneuploids (nullisomic-tetrasomics) were crossed with a T. timopheevii x Aegilops tauschii amphiploid to isolate T. timopheevii chromosomes in a monosomic condition. The F₁ hybrids were backcrossed one to four times to Chinese Spring aneuploids without selection for the T. timopheevii chromosome of interest. While spontaneous substitutions involving all A^t- and G-genome chromosomes were identified, the targeted T. timopheevii chromosome was not always recovered. Lines with spontaneous substitutions from T. timopheevii were chosen for further backcrossing. Six T. timopheevii chromosome substitutions were isolated: 6A^t (6A), 2G (2B), 3G (3B), 4G (4B), 5G (5B) and 6G (6B). The substitution lines had normal morphology and fertility. The 6A^t of T. timopheevii was involved in a translocation with chromosome 1G, resulting in the transfer of the group-1 gliadin locus to 6A^t. Chromosome 2G substituted for 2B at a frequency higher than expected and may carry putative homoeoalleles of gametocidal genes present on group-2 chromosomes of several alien species. Our data indicate a common origin for tetraploid wheat species, but from separate hybridization events because of the presence of a different spectrum of intergenomic translocations.     

Legumin of Vicia faba major: accumulation in developing cotyledons,purification, mRNA characterization and chromosomal location of coding genes

Summary Experiments were carried out on Vicia faba major involving (1) determination of the pattern of legumin accumulation during seed development, (2) protein purification from mature cotyledons, (3) the characterization of legumin mRNA, and (4) the chromosomal localization of the genes coding for legumins. In developing cotyledons the synthesis of legumin begins 28 days after petal desiccation (DAPD), and 4 days after initiation of vicilin synthesis. The two subunits (_A and _A) of legumin A appear 2 days earlier than those (_B and _B) of legumin B. While the accumulation of vicilin peaks on the 30th DAPD, that of legumin continues during further seed development, and the synthesis of legumin mRNA peaks on the 37th DAPD. Northern blot hybridizations using two DNA plasmids containing cDNA inserts with sequence homology to the A- and B-type legumin genes, respectively, indicated that legumin mRNAs extracted from cotyledons 36 DAPD band below the 18S RNA band. In addition, a faint band below that of the 25S RNA band can be observed in legumin mRNAs extracted from cotyledons at an earlier developmental stage (30 DAPD). By means of polyacrylamide gel electrophoresis in the presence or absence of SDS and 2-mercaptoethanol, two fractions could be eluted after zonal isoelectric precipitation of the globulins from mature seeds: one fraction contains mainly vicilin, the other, legumin. In situ hybridization showed that legumin genes are arranged in two clusters: the genes coding for legumin A are located in the longer arm of the one between the two shortest subtelocentric chromosome pairs whose centromere is in a less terminal position; those coding for legumin B are located in the non-satellited arm of the longer submetacentric pair.     

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     

Variation of starch granule proteins and chromosome mapping of their coding genes in common wheat

Starch granule proteins (SGPs) of common wheat (Triticum aestivum L.) were analyzed by two electrophoretic techniques: sodium dodecyl sulphate polyacrylamide-gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2D-PAGE). These analyses identified three kinds of SGPs which were tentatively designated SGP-1, SGP-2 and SGP-3. SDS-PAGE resolved the products of three homoeologous genes for SGP-1 into three protein fractions, SGP-A1, -B1 and -D1. While SDS-PAGE resolved SGP-3 into one fraction, 2D-PAGE separated it into three protein fractions encoded by homoeologous genes Sgp-A3, B3 and -D3. SGP-2 was detected as one protein by SDS-PAGE and was present as one protein on 2D-PAGE. Aneuploid (nullisomic-tetrasomic and ditelosomic) analyses in the cultivar Chinese Spring showed that the genes for two SGPs (SGP-1 and -3) were located on the short arms of group-7 chromosomes. The results obtained from deletion lines for chromosome arms 7AS, 7BS and 7DS suggested that the gene order along the arms is centromere-Sgp-1-Sgp-3-Wx. An electrophoretic survey of wheat germ plasm identified a few cultivars lacking one of the proteins SGP-A1, -B1, -D1, SGP-A3 and -B3. The null alleles Sgp-A1b, Sgp-B1b and Sgp-D1b will be useful for the production of a variant wheat lacking SGP-1.     

Transfer of Ph I genes promoting homoeologous pairing from Triticum speltoides to common wheat

Diploid-like chromosome pairing in polyploid wheat is controlled by several Ph (pairing homoeologous) genes with major and minor effects. Homoeologous pairing occurs in either the absence of these genes or their inhibition by genes from other species (Ph ^I genes). We transferred Ph ^I genes from Triticum speltoides (syn Aegilops speltoides) to T. aestivum, and on the basis of further analysis it appears that two duplicate and independent Ph ^I genes were transferred. Since Ph ^I genes are epistatic to the Ph genes of wheat, homoeologous pairing between the wheat and alien chromosomes occurs in the F₁ hybrids. Using the Ph ^I gene stock, we could demonstrate homoeologous pairing between the wheat and Haynaldia villosa chromosomes. Since homoeologous pairing occurs in F₁ hybrids and no cytogenetic manipulation is needed, the Ph ^I gene stock may be a versatile tool for effecting rapid and efficient alien genetic transfers to wheat.Contribution no. 93-435-J from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan, KS 66506-5502, USA     

The effect of polyamines on protein kinase activities of wheat (Triticum aestivum) L. anthers

Protein kinase activities were extracted from anther tissue of wheat (Triticum aestivum L. cv. Zaragoza) and characterized with regard to the effects of polyamines, second messengers and plant growth hormones. The protein kinases were inhibited by polyamines and cyclic nucleotides, but were stimulated by the addition of auxins, gibberellic acid and kinetin. The dominant polyamine-sensitive kinase activity was partially purified and characterized. The optimal pH of the reaction was 7.5 to 8.0 and casein was the preferred exogenous substrate. Polyamines were inhibiting in the decreasing order of putrecine > spermidine > spermine. The results are discussed against the context of the anther culture technique.     

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.     

Development of a chromosomal arm map for wheat based on RFLP markers

Summary A chromosomal arm map has been developed for common wheat (Triticum aestivum L. em. Thell.) using aneuploid stocks to locate more than 800 restriction fragments corresponding to 210 low-copy DNA clones from barley cDNA, oat cDNA, and wheat genomic libraries. The number of restriction fragments per chromosome arm correlates moderately well with relative DNA content and length of somatic chromosomes. The chromosomal arm locations of loci detected with 6 different clones support an earlier hypothesis for the occurrence of a two-step translocation (4AL to 5AL, 5AL to 7BS, and 7BS to 4AL) in the ancestral wheat genomes. In addition, 1 clone revealed the presence of a 5AL segment translocated to 4AL. Anomalies in aneuploid stocks were also observed and can be explained by intrahomoeologous recombination and polymorphisms among the stocks. We view the development of this chromosomal arm map as a complement to, rather than as a substitute for, a conventional RFLP linkage map in wheat.Paper No. 802 of the Cornell Plant Breeding Series     

Improvement of freezing tolerance in tobacco plants expressing a cold-responsive and chloroplast-targeting protein WCOR15 of wheat

Cold acclimation, an adaptive process for developing freezing tolerance in over-wintering plants, is associated with increased expression levels of a series of cold-responsive (Cor)/late embryogenesis abundant (Lea) genes. To investigate the function of Wcor15, a member of the wheat Cor/Lea gene family, for improvement of freezing tolerance, two types of transgenic tobacco lines expressing Wcor15-containing chimeric genes were produced and characterized. Immunoblot and gene expression analyses of a transgenic tobacco line expressing the Wcor15-GFP fusion gene under control of the CaMV35S promoter showed transport and abundant accumulation of the WCOR15 protein in the stromal compartment of the chloroplasts. The 5' upstream region of Wcor15 induced expression of the GFP reporter gene under low-temperature conditions in the transgenic tobacco. Both transgenic lines expressing the Wcor15-GFP fusion gene showed a similar and significantly improved level of freezing tolerance compared with the wild-type tobacco plants. Our results demonstrate that the induced expression of the wheat Wcor15 gene positively contributes to the development of freezing tolerance in the heterologous tobacco plants.     

Differential effects of Wx-A1, -B1 and -D1 protein deficiencies on apparent amylose content and starch pasting properties in common wheat

Waxy (Wx) protein is a granule-bound starch synthase (GBSS) responsible for amylose production in cereal endosperm. Eight isolines of wheat (Triticum aestivum L.) having different combinations of presence and absence of three Wx proteins, Wx-A1, -B1, and -D1, were produced in order to elucidate the effect of Wx protein deficiencies on the apparent amylose content and starch-pasting properties. An improved SDS gel electrophoresis showed that ’Bai Huo’ (a parental wheat) carried a variant Wx-B1 protein from an allele, Wx-B1e. Thus, wheat lines of types 1, 2, 4, and 6 examined in this study contained a variant Wx-B1 allele and not the standard allele, Wx-B1a. The results from 3 years of experiments using 176 lines derived from two cross-combinations showed that apparent amylose content increased the least in type 8 (waxy) having no Wx proteins and, in ascending order, increased in type 5 (only the Wx-A1 protein is present) <type 7 (Wx-D1) <type 6 (Wx-B1) <type 3 (Wx-A1 and -D1) <type 4 (Wx-A1 and -B1) <type 2 (Wx-B1 and -D1) <type 1 (three Wx proteins). However, Tukey’ s studentized range test did not detect significant differences in some cases. Densitometric analysis suggested that the amylose content was related to the amount of the Wx protein in the eight types. Parameters in the Rapid Visco-Analyzer test and swelling power were correlated to amylose content. Consequently, amylose content and pasting properties of starch were determined to be influenced the most by the lack of the Wx-B1 protein, followed by a lack of Wx-D1, and leastly by the Wx-A1 deficiency, which indicated the presence of differential effects of the three null alleles for the Wx protein. Received: 1 February 1999 / Accepted: 10 April 1999