Current applications of wheat and wheat–alien precise genetic stocks

A comprehensive collection of wheat aneuploids, whole chromosome substitutions (both intervarietal and interspecific) and wheat–alien addition lines, along with various introgression and near-isogenic lines, has been created over a period of years, primarily to provide the means of localizing the genes underpinning traits and to introduce novel genes into the bread wheat genome. For a time, interest in this class of genetic material was on the wane, but more recently it has revived in the context, for example, of localizing DNA-based markers, designing chromosome-specific bacterial artificial chromosome libraries, and establishing functional differences between alleles and homoeoalleles. Here, a brief review is provided of recent applications of precise genetic stocks in the field of molecular genetics, functional genetics and genomics of the Triticeae species.     

Culturable and metagenomic approaches of wheat bran and wheat straw phyllosphere’s highlight new lignocellulolytic microorganisms

The phyllosphere, defined as the aerial parts of plants, is one of the most prevalent microbial habitats on earth. The microorganisms present on the phyllosphere can have several interactions with the plant. The phyllosphere represents then a unique niche where microorganisms have evolved through time in that stressful environment and may have acquired the ability to degrade lignocellulosic plant cell walls in order to survive to oligotrophic conditions. The dynamic lignocellulolytic potential of two phyllospheric microbial consortia (wheat straw and wheat bran) has been studied. The microbial diversity rapidly changed between the native phyllospheres and the final degrading microbial consortia after 48 h of culture. Indeed, the initial microbial consortia was dominated by the Ralstonia (35·8%) and Micrococcus (75·2%) genera for the wheat bran and wheat straw whereas they were dominated by Candidatus phytoplasma (59%) and Acinetobacter (31·8%) in the final degrading microbial consortia respectively. Culturable experiments leading to the isolation of several new lignocellulolytic isolates (belonging to Moraxella and Atlantibacter genera) and metagenomic reconstruction of the microbial consortia highlighted the existence of an unpredicted microbial diversity involved in lignocellulose fractionation but also the existence of new pathways in known genera (presence of CE2 for Acinetobacter, several AAs for Pseudomonas and several GHs for Bacillus in different metagenomes-assembled genomes). The phyllosphere from agricultural co-products represents then a new niche as a lignocellulolytic degrading ecosystem.     

Taxonomical classification and origin of Kamut® wheat

Bioagriculture and healthy lifestyle are trends of the twenty-first century. Bioagriculture involves the breeding of crops without using modern synthetic substances. Kamut brand wheat is one of the popular biocereals grown mainly in the USA and Europe. This cereal has the status of ancient wheat, not only because it has been grown since the era of the ancient Egyptian civilization, but also for its properties favorable for modern breeding programs and modern food marketing. In spite of Kamut’s^® interesting history and stable place in the market, it is not a common subject of genetic studies. It is also interesting that it has not been successfully taxonomically classified yet. There are a few studies which classify this tetraploid wheat as Triticum polonicum L., T. turanicum Jakubz., T. turgidum L. and T. durum Desf. These studies are based on cytological and comparative methods. We chose molecular (transposable element resistance gene analog polymorphism, diversity arrays technology, sequencing of genes SBEIIa, and ψLpx-A1_like) and statistical methods to classify Kamut^® wheat. According to our experiments we suggest that Kamut brand wheat originated as a natural hybrid between Triticum dicoccon conv. dicoccon and T. polonicum and is not original ancient Egyptian wheat. We suggest that Etruscan wheat has the same parents as Kamut^®.     

Isolation and characterization of wheat ω-gliadin genes

The DNA sequences of two full-length wheat ω-gliadin prolamin genes (ωF20b and ωG3) containing significant 5′ and 3′ flanking DNA sequences are reported. The ωF20b DNA sequence contains an open reading frame encoding a 30,460-Dalton protein, whereas the ωG3 sequence would encode a putative 39,210-Dalton protein except for a stop codon at amino-acid residue position 165. These two ω-gliadin genes are closely related and are of the ARQ-/ARE-variant type as categorized by the derived N-terminal amino-acid sequences and amino-acid compositions. The ω-gliadins were believed be related to the ω-secalins of rye and the C-hordeins of barley, and analyses of these complete ω-gliadin sequences confirm this close relationship. Although the ω-type sequences from all three species are closely related, in this analysis the rye and barley ω-type sequences are the most similar in a pairwise comparison. A comparison of ω-gliadin flanking sequences with respect to that of their orthologs and with respect to wheat gliadin genes suggests the conservation of flanking DNA necessary for gene function. Sequence data for members of all major wheat prolamin families are now available. Received: 24 August 2000 / Accepted: 15 December 2000     

High-density genetic map of durum wheat × wild emmer wheat based on SSR and DArT markers

A genetic linkage map of tetraploid wheat was constructed based on a cross between durum wheat [Triticum turgidum ssp. durum (Desf.) MacKey] cultivar Langdon and wild emmer wheat [T. turgidum ssp. dicoccoides (K?rn.) Thell.] accession G18-16. One hundred and fifty-two single-seed descent derived F(6) recombinant inbred lines (RILs) were analyzed with a total of 690 loci, including 197 microsatellite and 493 DArT markers. Linkage analysis defined 14 linkage groups. Most markers were mapped to the B-genome (60%), with an average of 57 markers per chromosome and the remaining 40% mapped to the A-genome, with an average of 39 markers per chromosome. To construct a stabilized (skeleton) map, markers interfering with map stability were removed. The skeleton map consisted of 307 markers with a total length of 2,317 cM and average distance of 7.5 cM between adjacent markers. The length of individual chromosomes ranged between 112 cM for chromosome 4B to 217 cM for chromosome 3B. A fraction (30.1%) of the markers deviated significantly from the expected Mendelian ratios; clusters of loci showing distorted segregation were found on chromosomes 1A, 1BL, 2BS, 3B, and 4B. DArT markers showed high proportion of clustering, which may be indicative of gene-rich regions. Three hundred and fifty-two new DArT markers were mapped for the first time on the current map. This map provides a useful groundwork for further genetic analyses of important quantitative traits, positional cloning, and marker-assisted selection, as well as for genome comparative genomics and genome organization studies in wheat and other cereals.     

Expression and secretion of wheat α-amylase in Kluyveromyces lactis

The plasmid pCR1 has been constructed to express a wheat -amylase enzyme in Kluyveromyces lactis strains. The contruct is based on the vector pCXJ-kan1, which has been derived from pDK1, a native plasmid of K. lactis var. drosophilarum containing the essential regions for plasmid replication and stability. Contruct pCR1 produces an -amylase by DNA isolated from a wheat cDNA clone and is controlled by a Saccharomyces cerevisia PGK promoter. Correspondence to: C. Russell     

Comparison of wheat albumin inhibitors of α-amylase and trypsin

Wheat albumins were extracted from whole wheat flour with 150 mM sodium chloride solution and precipitated between 0·4 and 1·8 M ammonium sulphate. The albumin precipitate was separated by gel filtration on Sephadex G100 into five peaks. Three peaks (II, III, and IV), whose MWs were 60 000, 24 000 and 12 500 daltons respectively, were active toward several insect α-amylases, whereas only peak III inhibited human saliva and pancreatic α-amylases. Peaks III and IV also inhibited trypsin. In each active peak, we found several α-amylase inhibitors slightly different in their electrophoretic mobilities in a Tris—glycine buffer system (pH 8·5), whereas only one major trypsin inhibitor was present in peaks III and IV. In contrast to α-amylase inhibitors that were all anodic, trypsin inhibitors migrated to the cathode under our experimental conditions. From a quantitative standpoint, wheat albumins that inhibit trypsin are negligible, whereas about 2/3 of the total albumin inhibits amylases from different origins. All inhibitor components of peak III were active toward both insect and mammalian α-amylases. Moreover, they reversibly dissociated in the presence of 6 M guanidine hydrochloride giving two similar subunits.     

Location of β-amylase sequences in wheat and its relatives

Summary A -amylase cDNA clone isolated from barley has been used to locate -amylase encoding sequences on wheat, rye, and Aegilops umbellulata chromosomes by hybridisation to restriction endonuclease digested DNA obtained from wheat aneuploid and wheat-alien addition lines. Structural genes were identified on homoeologous group 4 and 5 chromosomes, confirming the results of isozyme studies. In addition, a further set of structural genes was found on homoeologous group 2 chromosomes. It is proposed that there are two homoeoallelic series, -Amy-1 on group 4 or 5 chromosomes, and -Amy-2 on group 2 chromosomes. Evidence is presented that each locus contains one or two -amylase structural genes, and it is suggested that the large number of isozymes seen upon IEF are due to post-translational modifications.     

The wheat ω-gliadin genes: structure and EST analysis

A survey and analysis is made of all available ω-gliadin DNA sequences including ω-gliadin genes within a large genomic clone, previously reported gene sequences, and ESTs identified from the large wheat EST collection. A contiguous portion of the Gli-B3 locus is shown to contain two apparently active ω-gliadin genes, two pseudogenes, and four fragments of the 3′ portion of ω-gliadin sequences. Comparison of ω-gliadin sequences allows a phylogenetic picture of their relationships and genomes of origin. Results show three groupings of ω-gliadin active gene sequences assigned to each of the three hexaploid wheat genomes, and a fourth group thus far consisting of pseudogenes assigned to the A-genome. Analysis of ω-gliadin ESTs allows reconstruction of two full-length model sequences encoding the AREL- and ARQL-type proteins from the Gli-A3 and Gli-D3 loci, respectively. There is no DNA evidence of multiple active genes from these two loci. In contrast, ESTs allow identification of at least three to four distinct active genes at the Gli-B3 locus of some cultivars. Additional results include more information on the position of cysteines in some ω-gliadin genes and discussion of problems in studying the ω-gliadin gene family. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization and solvent engineering of wheat β-amylase for enhancing its activity and stability

The kinetic and thermodynamic parameters of wheat β-amylase (WBA) were characterized and various additives were evaluated for enhancing its activity and thermostability. WBA activity was examined by neocuproine method using soluble starch as substrate. The Michaelis constant (K(m)) and molecular activity (k(cat)) were determined to be 1.0±0.1% (w/v) and 94±3s(-1), respectively, at pH 5.4 and at 25°C. The optimum reaction temperature (T(opt)) for WBA activity was 55°C and the temperature (T(50)) at which it loses half of the activity after 30-min incubation was 50±1°C. Modifications of the solvent with 182mM glycine and 0.18% (w/v) gelatin have increased the T(50) by 5°C. Glycerol, ethylene glycol, dimethylformamide (DMF) and dimethyl sulfoxide have also slightly enhanced the thermostability plausibly through weakening the water structure and decreasing the water shell around the WBA protein. Ethanol and DMF activated WBA by up to 24% at 25°C probably by inducing favorable conformation for the active site or changing the substrate structure by weakening the hydrogen bonding. Its half-life in the inactivation at 55°C was improved from 23 to 48min by 182mM glycine. The thermodynamic parameters indicate that WBA is thermo-labile and sufficient stabilization was achieved through solvent modification with additives and that the heat inactivation of WBA is entropic-driven. It is suggested that WBA could be applied more widely in starch-saccharification industries with employing suitable additives.     

Anthocyanin biosynthesis genes location and expression in wheat–rye hybrids

Studies into gene expression in a foreign background contribute toward understanding of how genes derived from different species or genera manages to co-exist in a common nucleus, on the one hand, and help to estimate possible effectiveness of wide hybridization for cultivated plant improvement, on the other hand. The aim of this study was to investigate conservation of wheat and rye expression networks, using the anthocyanin biosynthesis pathway (ABP) genes as a model system. We isolated and analyzed ABP genes encoding enzymes acting at different steps of the pathway: chalcone-flavanone isomerase (CHI), flavanone 3-hydroxylase (F3H), anthocyanidin synthase (ANS), and anthocyanidin-3-glucoside rhamnosyltransferase (3RT). The rye ABP genes locations we determined (Chi on chromosome 5RL, F3h on 2RL, Ans on 6RL, 3Rt on 5RL, the regulatory Rc—red coleoptile—gene on 4RL) were in agreement with the rearrangements established between rye and wheat chromosomes. Expression of the ABP structural genes was studied in wheat–rye chromosome addition and substitution lines. F3h activation by the Rc gene was found to be critical for the red coleoptile trait formation. It was shown that the rye regulatory Rc gene can activate the wheat target gene F3h and vice versa wheat Rc induces expression of rye F3h. However, lower level of expression of rye F3h in comparison with that of the two wheat orthologues in the wheat–rye chromosome substitution line 2R(2D) was observed. Thus, although work of the wheat and rye ABP gene systems following the formation of wheat–rye hybrids is finely coordinated, some divergence exists between rye and wheat ABP genes, affecting level of gene expression.     

Anther culture and chromosome reduction in wheat × Thinopyrum wide crosses

Callus cultures of Ambrosia tenuifolia were established from sub-apical leaves. The explants were grown on basal media MS (Murashige and Skoog) supplemented with 10 μM kinetin, 1 μM 2-4 dichlorophenoxyacetic, ascorbic acid and cysteine and cultivated either in light or darkness. To determine the effect of each individual salt component on the growing rate of the callus and the psilostachyinolides and altamisine production in the culture, the concentrations of each nutrient was tested at different levels, ranging from 50% above to 50% below the standard medium. Interestingly, increased concentration of boron in the medium, resulted in a four fold increase in the production of sesquiterpene lactones by the callus. However, positive production of psilostachynolides and altamisine was only detected when the basal concentrations of the others components of the basal media were used. In addition, production of altamisine was highly sensitive to the variations of nutrients. No statiscally effect on terpenoide production by the callus was detected by varying light exposure. These results indicates that conditions for the optimal production of these terpenoids can be enhanced by modifying nutrients of the MS basal media. This revised version was published online in June 2006 with corrections to the Cover Date.     

HpaII library indicates ‘methylation-free islands’ in wheat and barley

Summary A library of wheat genomic DNA HpaII tiny fragments (HTF), sized below 500 bp, has been constructed. Of the clones in the library 80% belong to the single/low-copy category, while 12% of the clones are nuclear repetitive sequences and 8% originate from the chloroplast and mitochondrial DNA. This result shows a substantial enrichment in the single/low-copy sequences of the wheat genome, which contains at least 80% repetitive sequences. Twenty-nine random single/lowcopy clones were analysed further for wheat chromosome location, cross-hybridisation to barley DNA and their association with rare-cutting, C-methylation-sensitive restriction sites. The results show that the HTF clones are associated more frequently than expected with NotI, MluI, NruI and PstI sites in wheat and barley genomic DNA. The 12% repetitive fraction of the clones contain both moderately and highly repetitive sequences, but no tandemly repeated sequences. The level of enrichment for single/low-copy sequences indicates that libraries of this type are a valuable source of probes for RFLP mapping. In addition, the close association of the HTF clones with rare-cutting restriction enzyme sites ensures that HTF clones will have a useful role in the construction of long-range physical maps in wheat.     

Isozyme variation and RFLPs at the β-amylase loci in wheat

Summary Forty-one hexaploid wheat genotypes have been examined for RFLPs detected by a -amylase probe using three restriction enzymes, and for mature grain -amylase isozyme polymorphism following IEF. The two homoeoallelic series assayed for RFLPs differed: little variation was found at group 2 chromosome homoeoloci, while the group 4/5 chromosome homoeoloci displayed considerable variation. Varieties that displayed a RFLP with one RE almost always did likewise with the other two REs, suggesting that most of the polymorphisms observed were due to large DNA rearrangements. Comparison of the variation in grain -amylase isozymes with the RFLP results indicated strong associations between particular RFLP and isozyme alleles.     

High-frequency generation and characterization of intergeneric hybrids and haploids from new wheat–barley crosses

Key message

Hybrid plants and a high frequency of maternal haploids were obtained using an efficient wheat–barley hybridization system (with new genotype combinations) and confirmed by several cytological and molecular tools.

Abstract

An efficient hybridization system between wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) is presented on the basis of three new genotype combinations. A particularly high, 14 % frequency of plant regeneration per florets was achieved in the wheat–barley genotype combination of ‘Sichuan’ × ‘Morex’. The genome composition in 42 of the 95 plants regenerated by embryo rescue was determined using ploidy analysis, genomic in situ hybridization and the application of chromosome arm-specific molecular markers (SSR and STS). A high overall frequency (76 %) of maternal (wheat) haploids was observed in all the tests for all three cross combinations. A major implication of this observation is that this new hybridization system represents a useful tool to study the mechanism of uniparental chromosome elimination in cereals.     

Comparative analysis of the meiotic effects of wheat ph1b and ph2b mutations in wheat×rye hybrids

 Wheat-wheat and wheat-rye homoeologous pairing at metaphase I and wheat-rye recombination at anaphase I were examined by genomic in situ hybridization (GISH) in wild-type (Ph1Ph2) and mutant ph1b and ph2b wheat×rye hybrids. The metaphase-I analysis revealed that the relative contribution of wheat-rye chromosome associations in ph2b wheat×rye was similar to that of the wild-type hybrid genotype but differed from the effect of the ph1b mutation. The greater pairing promotion effect of the ph1b mutation appears to be relatively more on distant homoeologous partner metaphase-I associations, whereas the lower promoting effect of ph2b is evenly distributed among all types of homoeologous associations. This finding reveals that distinct mechanisms are involved in the control of wheat homoeologous pairing by the two Ph genes. The frequency of wheat-rye recombination calculated from anaphase-I analysis was lower than expected from the metaphase-I data. A greater discrepancy was found in ph2b than in ph1b wheat×rye hybrids, which may suggest a more distal chiasma localization in the former hybrid genotype. Received: 20 June 1997 / Accepted: 9 December 1997     

Phenotypic and genotypic intra-diversity among Anatolian durum wheat “Kunduru” landraces

Kunduru is an important Anatolian landrace having peculiar traits that are appreciated by farmers and breeders. 33 accessions known as Kunduru collected by ICARDA from six geographical provinces of Turkey, were used to study the phenotypic and genotypic intra-diversity. Kunduru landraces exhibited high intra-diversity for most of the studied morphological traits. GPC (12.10–14.90%), vitreousness (75–100%), TKW (31.80–56.70 g), YP (4.70–8.00 ppm), b*-value (14.30–19.50), ash content (1.60–2.0%) and gluten strength (14–60 ml) showed marked variations. Gliadin and glutenin banding patterns showed high polymorphism. 65 alleles were detected with 14 SSR markers, giving a mean of 6.77 alleles per locus. The average PIC value was 0.44 and ranged from 0.11 to 0.70. The average genetic distance between pairs of landraces was 0.47 and ranged between 0.11 and 0.72. This study showed that Kunduru landraces maintains high allelic variation. PCoA indicated that eco-geographical variables have a significant effect on SSR diversity as well as morphological traits. Many of the landraces studied are in danger of disappearing from the local farmers' fields; this study demonstrates the importance of maintaining and conserving this precious genetic resources.     

NH4 + and NO3 − requirement for wheat somatic embryogenesis

The influence of three nitrogen salts: NH₄NO₃, KNO₃ and NH₄Cl on wheat in vitro cultures was investigated. Both NO ₃ ⁻ and NH ₄ ⁺ ions were indispensable for proliferation of embryogenic calli and development of wheat somatic embryos. It is possible to obtain wheat somatic embryos when the medium is enriched with NH₄NO₃ only as a source of inorganic nitrogen. The results of the statistical analysis showed that the level of NH₄NO₃ and KNO₃ in the medium had a great influence on the efficiency of somatic embryogenesis. We observed tendency that calli on media containing 50 mM NH₄NO₃ and 0 to 20 mM KNO₃ turned out to be more embryogenic than on control MS medium. High concentrations of KNO₃- 100 mM inhibited somatic embryogenesis, while 100 mM NH₄NO₃ did not. The level of total N did not have significant influence on wheat somatic embryogenesis. Ratio NO ₃ ⁻ :NH ₄ ⁺ also turned out to be not substantial. We observed that mutual connection of concentration levels between NH₄NO₃ and KNO₃ and between NH₄Cl and KNO₃ was more important. The efficiency of somatic embriogenesis obtained in the experiment with NH₄Cl and KNO₃ was significantly lower than in experiment with NH₄NO₃ and KNO₃.