Molecular characterization of the celiac disease epitope domains in α-gliadin genes in Aegilops tauschii and hexaploid wheats (Triticum aestivum L.)

Nineteen novel full-ORF α-gliadin genes and 32 pseudogenes containing at least one stop codon were cloned and sequenced from three Aegilops tauschii accessions (T15, T43 and T26) and two bread wheat cultivars (Gaocheng 8901 and Zhongyou 9507). Analysis of three typical α-gliadin genes (Gli-At4, Gli-G1 and Gli-Z4) revealed some InDels and a considerable number of SNPs among them. Most of the pseudogenes were resulted from C to T change, leading to the generation of TAG or TAA in-frame stop codon. The putative proteins of both Gli-At3 and Gli-Z7 genes contained an extra cysteine residue in the unique domain II. Analysis of toxic epitodes among 19 deduced α-gliadins demonstrated that 14 of these contained 1–5 T cell stimulatory toxic epitopes while the other 5 did not contain any toxic epitopes. The glutamine residues in two specific ployglutamine domains ranged from 7 to 27, indicating a high variation in length. According to the numbers of 4 T cell stimulatory toxic epitopes and glutamine residues in the two ployglutamine domains among the 19 α-gliadin genes, 2 were assigned to chromosome 6A, 5 to chromosome 6B and 12 to chromosome 6D. These results were consistent with those from wheat cv. Chinese Spring nulli-tetrasomic and phylogenetic analysis. Secondary structure prediction showed that all α-gliadins had high content of β-strands and most of the α-helixes and β-strands were present in two unique domains. Phylogenetic analysis demonstrated that α-gliadin genes had a high homology with γ-gliadin, B-hordein, and LMW-GS genes and they diverged at approximate 39 MYA. Finally, the five α-gliadin genes were successfully expressed in E. coli, and their expression amount reached to the maximum after 4 h induced by IPTG, indicating that the α-gliadin genes can express in a high level under the control of T₇ promoter.     

Plant water uptake by hard red winter wheat (Triticum aestivum L.) genotypes at 2°C and low light intensity

Background

The moss Physcomitrella patens is an attractive model system for plant biology and functional genome analysis. It shares many biological features with higher plants but has the unique advantage of an efficient homologous recombination system for its nuclear DNA. This allows precise genetic manipulations and targeted knockouts to study gene function, an approach that due to the very low frequency of targeted recombination events is not routinely possible in any higher plant.

Results

As an important prerequisite for a large-scale gene/function correlation study in this plant, we are establishing a collection of Physcomitrella patens transformants with insertion mutations in most expressed genes. A low-redundancy moss cDNA library was mutagenised in E. coli using a derivative of the transposon Tn1000. The resulting gene-disruption library was then used to transform Physcomitrella. Homologous recombination of the mutagenised cDNA with genomic coding sequences is expected to target insertion events preferentially to expressed genes. An immediate phenotypic analysis of transformants is made possible by the predominance of the haploid gametophytic state in the life cycle of the moss. Among the first 16,203 transformants analysed so far, we observed 2636 plants ( = 16.2%) that differed from the wild-type in a variety of developmental, morphological and physiological characteristics.

Conclusions

The high proportion of phenotypic deviations and the wide range of abnormalities observed among the transformants suggests that mutagenesis by gene-disruption library transformation is a useful strategy to establish a highly diverse population of Physcomitrella patens mutants for functional genome analysis.     

Monosomic analysis of heading date and spikelet number in the common wheat (Triticum aestivum L.) multispikelet line ‘Noa’

Summary Genetic analysis of heading date and spikelet number was carried out in the common wheat (Triticum aestivum L.) multispikelet line Noa, by using the monosomic series of the regular line Mara. Noa's high number of spikelets was found to be controlled by a recessive major gene on chromosome 2D; a slight reduction in spikelet number was induced by another recessive gene on Noa's 7A chromosome. Noa's late heading date was found to be controlled by two recessive genes, located on chromosome 2D (a major effect) and 6B (a minor effect). The nature of the genes located on Noa's 2D chromosome and the relationship between spikelet number and heading date are discussed.     

Association between dwarfing genes ‘Rht1’ and ‘Rht2’ and resistance toSeptoria tritici Blotch in winter wheat (Triticum aestivum L. em Thell)

Summary Differences in levels of resistance toSeptoria tritici blotch were observed in plants with a specific height-reducing gene. When the gene Rht ₂ was present either as an isoline or in the progeny, a higher degree of resistance was found. The most susceptible plants were observed in populations carrying the Rht ₁ gene. Associations, as determined by phenotypic correlations, were detected betweenSeptoria tritici blotch and tall stature, late heading, and maturity. Plants having short stature, early heading, early maturity, and acceptable levels of resistance were identified in the F₂ population whenRht ₂ was present. Results of this study indicated that wheat breeders must select the appropriate dwarfing source that may confer resistance and grow large F₂ populations, in order to increase the probability of obtaining desired genotypes.     

Experimental and modelling studies of drought‐adaptive root architectural traits in wheat (Triticum aestivum L.)

Abstract

This paper presents an interdisciplinary approach to crop improvement that links physiology with plant breeding and simulation modelling to enhance the selection of high‐yielding, drought‐tolerant varieties. In a series of field experiments in Queensland, Australia, we found that the yield of CIMMYT wheat line SeriM82 ranged from 6% to 28% greater than the current cultivar Hartog. Physiological studies on the adaptive traits revealed that SeriM82 had a narrower root architecture and extracted more soil moisture, particularly deep in the profile. Results of a simulation analysis of these adaptive root traits with the cropping system model APSIM for a range of rain‐fed environments in southern Queensland indicated a mean relative yield benefit of 14.5% in water‐deficit seasons. Furthermore, each additional millimetre of water extracted during grain filling generated an extra 55 kg ha^?1 of grain yield. Further root studies of a large number of wheat genotypes revealed that wheat root architecture is closely linked to the angle of seminal roots at the seedling stage – a trait which is suitable for large‐scale and cost‐effective screening programmes. Overall, our results suggest that an interdisciplinary approach to crop improvement is likely to enhance the rate of yield improvement in rain‐fed crops.     

Identification of SNPs and development of allele-specific PCR markers for γ-gliadin alleles in Triticum aestivum

The coding regions of 28 entries of hexaploid wheat gamma-gliadin genes, gene fragments or pseudogenes in GenBank were used for nucleotide alignment. These sequences could be divided into nine subgroups based on nucleotide variation. The chromosomal locations of five of the seven unassigned subgroups were identified through subgroup-specific polymerase chain reactions (PCR) using Chinese Spring group-1 nulli-tetrasomic lines. Multiple single nucleotide polymorphisms (SNPs) and small insertions/deletions were identified in each subgroup. With further mining from wheat expressed sequence tag databases and targeted DNA sequencing, two SNPs were confirmed and one SNP was discovered for genes at the Gli-A1, Gli-B1 and Gli-D1 loci. A modified allele-specific PCR procedure for assaying SNPs was used to generate dominant DNA markers based on these three SNPs. For each of these three SNPs, two allele-specific primer sets were used to test Chinese Spring and 52 commercial Australian wheat varieties representing a range of low-molecular-weight (LMW) alleles. PCR results indicated that all were positive with one of the primer sets and negative with the other, with the exception of three varieties containing the 1BL/1RS chromosomal translocation that were negative for both. Furthermore, markers GliA1.1, GliB1.1 and GliD1.1 were found to be correlated with Glu-A3 a, b or c, Glu-B3 b, c, d or e and Glu-D3 a, b or e LMW glutenin alleles, respectively. Markers GliA1.2, GliB1.2 and GliD1.2 were found to be correlated with the Glu-A3 d or e, Glu-B3 a, g or h and Glu-D3 c alleles, respectively. These results indicated that the gamma-gliadin SNP markers could be used for detecting linked LMW glutenin subunit alleles that are important in determining the quality attributes of wheat products.     

Genetic control of heading date and spikelet number in common wheat (T. aestivum L.) line ‘Noa’

Summary The phenology and build-up of spikelet number under 10 h day-length were studied in five wheat lines: the multispikelet line Noa, the regular line Mara, the F₁ hybrid between them and monosomics 2D of Mara and of this hybrid (lacking the 2D chromosome of Mara). Noa had a longer spike development phase, a higher initial number of spikelet primordia and a slower rate of spikelet production than Mara. The F₁ hybrid was similar to Noa in its high initial number of spikelets and to Mara in its high rate of spikelet production. This hybrid had a shorter spikelet phase than both parents. Deletion of one dose of the Mara 2D chromosome from either Mara or the F₁ hybrid caused a reduction in the rate of spikelet production and an increase in the duration of the spikelet phase. These effects were due to the reduced dosage of the 2D chromosome. However, in the F₁ hybrid this deletion also caused an increase in the spike development phase — an indication that Noa carries on its 2D chromosome a recessive gene for late heading date which acts on the spike development phase. This gene of Noa is independent of the day-length sensitive gene ppd, and is different from Noas dominant gene for large initial number of spikelets.     

Culture-independent analysis of an endophytic core microbiome in two species of wheat: Triticum aestivum L. (cv. ‘Hondia’) and the first report of microbiota in Triticum spelta L. (cv. ‘Rokosz’)

The main goal of the study was to determine the structure of endophytic bacteria inhabiting different parts (endosperm, germ, roots, coleoptiles, and leaves) of two wheat species, Triticum aestivum L. (cv. ‘Hondia’) and Triticum spelta L. (cv. ‘Rokosz’), in order to provide new knowledge about the stability and/or changeability of the core microbiome in different plant organs. The endophytic core microbiome is associated with plants throughout their whole life cycle; however, plant organs can determine the actual endophytic community. Therefore, next generation sequencing with MiSeq Illumina technology was applied to identify the endophytic microbiome of T. aestivum and T. spelta. Bioinformatic analyses were performed with the use of the DADA2(1.8) package and R software (3.5.1).It was demonstrated that wheat, which is an important crop plant, was associated with beneficial endophytic bacteria inside the endosperms, germs, roots, leaves, and coleoptiles. Importantly, for the first time, biodiversity was recognized in the coleoptiles of the investigated wheat species. Flavobacterium, Pseudomonas and Janthinobacterium were shown to be common genera for both tested wheat cultivars. Among them, Pseudomonas was found to be the only endophytic genus accompanying both wheat species from the endosperm stage to the development of the leaf. Paenibacillus was recognized as a core genus for the ‘Hondia’ cv., whereas Pedobacter and Duganella constituted the core microbiome in the ‘Rokosz’ cv. In addition, the first insight into the unique and yet unrecognized endophytic microbiome of T. spelta is presented.     

Organelle genome stability in anther-derived doubled haploids of wheat (Triticum aestivum L., cv. ‘Moisson’)

Summary Chloroplast and mitochondrial compartments of a parental line of wheat (Triticum aestivum L., cv. Moisson) and its anther-derived doubled haploid lines have been analyzed and compared on the basis of their DNA restriction patterns. The results obtained show that no noticeable difference can be detected between doubled haploid lines and parental line at the level of ctDNA and mtDNA organization. It may be concluded that in vitro culture by itself does not systematically generate a cytoplasmic variation in germ cells.     

A ‘Chinese Spring’ wheat (Triticum aestivum L.) bacterial artificial chromosome library and its use in the isolation of SSR markers for targeted genome regions

A bacterial artificial chromosome (BAC) library was constructed from the bread wheat (Triticum aestivum L.) genotype ‘Chinese Spring’ (‘CS’). The library consists of 395,136 clones with an estimated average insert size of 157 kb. This library provides an estimated 3.4-fold genome coverage for this hexaploid species. The genome coverage was confirmed by RFLP analysis of single-copy RFLP clones. The CS BAC library was used to develop simple sequence repeat (SSR) markers for targeted genome regions using five sequence-tagged-site (STS) markers designed from the chromosome arm of 3BS. The SSR markers for the targeted genome region were successfully obtained. However, similar numbers of new SSR markers were also generated for the other two homoeologous group 3 chromosomes. This data suggests that BAC clones belonging to all three chromosomes of homoeologous group 3 were isolated using the five STS primers. The potential impacts of these results on marker isolation in wheat and on library screening in general are discussed.     

Purification and characterization of a hydroxamic acid glucoside β-glucosidase from wheat (Triticum aestivum L.) seedlings

A beta-glucosidase (EC 3.2.1.21) with a high affinity for cyclic hydroxamic acid beta-D-glucosides was purified from 48-h-old wheat (Triticum aestivum L.) seedlings. The activity occurred transiently at a high level during the non-autotrophic stage of growth, and the nature of the transient occurrence was correlated with that of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc). The glucosidase had maximum activity at an acidic pH (pH 5.5) and the purified enzyme showed a high affinity for DIMBOA-Glc, Vmax and Km being 4100 nkat/mg protein and 0.27 mM, respectively. It also hydrolyzed p-nitrophenol beta-glycosides, as well as flavone and isoflavone glucosides, but to a lesser extent. The results indicated that the primary natural substrate for the glucosidase is DIMBOA-Glc and that the enzyme is involved in defense against pathogens and herbivores in non-autotrophic wheat. The glucosidase was found to be present as oligomeric forms with a molecular mass of 260-300 kDa comprising 60- and 58-kDa monomers. The N-terminal 12-amino-acid sequences of the two monomers were identical (Gly-Thr-Pro-(Ser?)-Lys-Pro-Ala-Glu-Pro-Ile-Gly-Pro), and showed no similarity to those of other plant glucosidases. Polyacrylamide gel electrophoresis under nondenaturing condition indicated the existence of at least eight isozymes. Three cultivars of Triticum aestivum had the same zone of glucosidase activity on zymograms, but the activity zones of the Triticum species, T. aestivum L., T. spelta L. and T. turgidum L., had different mobilities.     

小麦(Triticum aestivum L.)野生资源的发掘、利用研究进展

小麦是世界主要的粮食作物,其生产面积居谷类作物之首。由于长期的定向选择,小麦遗传多样性不断降低,品种间杂交难以选育出抗性和综合性状优良的突破性品种。然而小麦近缘种如黑麦(Secale)、簇毛麦(Haynaldia)和偃麦草(Elytrigia)等包含很多优异基因,并且与小麦基因组间具有很好的互补性。可见远缘杂交在世界小麦育种实践中发挥了重要的作用。综述了小麦近缘野生资源的多样性、近缘野生种的利用方法和存在问题等的研究进展,讨论了小麦野生资源在小麦遗传改良的重要性和发展方向,以期为小麦优良品种培育及其遗传研究提供参考。     

Did the house mouse (Mus musculus L.) shape the evolutionary trajectory of wheat (Triticum aestivum L.)?

Wheat (Triticum aestivum L.) is one of the most successful domesticated plant species in the world. The majority of wheat carries mutations in the Puroindoline genes that result in a hard kernel phenotype. An evolutionary explanation, or selective advantage, for the spread and persistence of these hard kernel mutations has yet to be established. Here, we demonstrate that the house mouse (Mus musculus L.) exerts a pronounced feeding preference for soft over hard kernels. When allele frequencies ranged from 0.5 to 0.009, mouse predation increased the hard allele frequency as much as 10‐fold. Studies involving a single hard kernel mixed with ~1000 soft kernels failed to recover the mutant kernel. Nevertheless, the study clearly demonstrates that the house mouse could have played a role in the evolution of wheat, and therefore the cultural trajectory of humankind.     

Mapping of genes for male-fertility restoration in ’Pampa’ CMS winter rye (Secale cereale L.)

Hybrid rye breeding and seed production is based on the cytoplasmic male sterility (CMS)-inducing Pampa (P)-cytoplasm. For restoring male fertility in the hybrids, dominant, nuclear restorer genes are necessary. However, current pollinator lines are only partial restorers. Effective restorers were recently detected in the German inbred line L18 and in materials originating from the Argentinian rye cultivar Pico Gentario and an Iranian primitive rye accession called IRAN IX. F₂ populations were developed for each of these three restorer sources to map the responsible genes by means of RFLP (restriction fragment length polymorphism) markers. For this purpose, homo- and heterologous DNA probes were used leading to 101 polymorphic marker loci in total. For phenotypic evaluation, 100 to 134 randomly chosen plants from each of the populations were cloned and grown at two or three locations with two plants each. Segregation ratios of pollen fertility in the F₂ populations with L18 and IRAN IX were in accordance with a monogenic dominant inheritance. The segregation pattern for Pico Gentario indicated complementary gene action. Major dominant restorer genes were detected on chromosomes 1RS (L18) and 4RL (Pico Gentario, IRAN IX). The gene on 1RS explained 54% of the phenotypic variation and that on 4 RL 59% and 68% in the Pico Gentario and IRAN IX populations, respectively. Additionally, three minor genes from L18 were identified on chromosomes 3RL, 4RL and 5R. In the Pico Gentario population, a dominant modifier gene contributed by the female parent was found on chromosome 6R. This gene significantly enhanced the expression of the major restorer gene but on its own was not able to restore any degree of fertility. The map-distances between the major restorer loci and at least one flanking marker were small in all three F₂ populations (5–6 cM). In Pico Gentario an unfavorable linkage exists between the major restorer gene and a QTL for plant height. Since highly effective restorers are scarce in actual breeding populations, the major restorer genes detected on chromosomes 1 RS and 4RL should be introgressed into actual restorer lines. This is facilitated by using the closely linked molecular markers described. Received: 10 February 2000 / Accepted: 31 March 2000<@head-com-p1a.lf>Communicated by G. Wenzel     

Genetics of resistance to Puccinia graminis tritici in ‘Chris’ and ‘W3746’ wheats

Summary Chris wheat possessed genes Sr5, Sr7a, Sr8a, Sr9g and Sr12. W3746, derived from the cross Chris/Baart, possessed Sr7a and Sr12. The response conferred by Sr7a was influenced by the genetic background. Although Sr7a or Sr12 alone conferred no observable resistance upon adult plants, the adult resistances of Chris and W3746 to predominant pathotypes appeared to be associated with the interaction of Sr7a and Sr12, or genes at closely linked loci.     

Genetic analysis of tolerance to photo-oxidative stress induced by high light in winter wheat （Triticum aestivum L.）

High light induced photooxidation (HLIP) usually leads to leaf premature senescence and causes great yield loss in winter wheat. In order to explore the genetic control of wheat tolerance to HLIP stress, a quantitative trait loci (QTL) analysis was conducted on a set of doubled haploid population, derived from two winter wheat cultivars. Actual values of chlorophyll content (Chl), minimum fluorescence level (Fo), maximum fluorescence level (Fm), and the maximum quantum efficiency of photosystem Ⅱ (Fv/Fm) under both HLIP and non-stress conditions as well as the ratios of HLIP to non-stress were evaluated. HLIP considerably reduced Chl, Fm, and Fv/Fm, but increased Fo, compared with that under non-stress condition. A total of 27, 16, and 28 QTLs were associated with the investigated traits under HLIP and non-stress and the ratios of HLIP to non-stress, respectively. Most of the QTLs for the ratios of HLIP to non-stress collocated or nearly linked with those detected under HLIP condition. HLIP-induced QTLs were mapped on 15 chromosomes, involving in 1A, 1B, 1D,2A, 2B, 2D, 3A, 3B, 4A, 4D, 5B, 6A, 6B, 7A, and 7D while those expressed under non-stress condition involved in nine chromosomes, including 1B, 1D, 2A, 2B, 3B, 4A, 5A, 5B, and 7A. The expression patterns of QTLs under HLIP condition were different from that under non-stress condition except for six loci on five chromosomes. The phenotypic variance explained by individual QTL ranged from 5.0% to 19.7% under HLIP, 8.3% to 20.8% under non-stress, and 4.9% to 20.2% for the ratios of HLIP to non-stress, respectively. Some markers, for example,Xgwm192 and WMC331 on 4D regulating Chl, Fo, Fm, and Fv/Fm under HLIP condition, might be used in marker assistant selection.     

Identification and mapping QTL for high-temperature adult-plant resistance to stripe rust in winter wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) cultivar ‘Stephens’

High-temperature adult-plant (HTAP) resistance from the winter wheat (Triticum aestivum) cultivar 'Stephens' has protected wheat crops from stripe rust caused by Puccinia striiformis f. sp. tritici for 30 years. The objectives of this study were to identify quantitative trait loci (QTL) for HTAP resistance in Stephens through genetic linkage analysis and identify DNA markers linked to the QTL for use in marker-assisted breeding. Mapping populations consisted of 101 recombinant inbred lines (RILs) through single-seed descent from 'Stephens' (resistant) x 'Michigan Amber' (susceptible). F(5), F(6) and F(7) RILs were evaluated for stripe rust resistance at Pullman, WA in 1996, 1997 and 1998, respectively, whereas F(8) RILs were evaluated at Mt Vernon, WA, USA in 2005. The 101 F(8) RILs were evaluated with 250 resistance gene analog polymorphism (RGAP), 245 simple sequence repeat (SSR) and 1 sequence tagged site (STS) markers for genetic linkage map construction. Two QTL, which explained 48-61% of the total phenotypic variation of the HTAP resistance in Stephens, were identified. QYrst.wgp-6BS.1 was within a 3.9-cM region flanked by Xbarc101 and Xbarc136. QYrst.wgp-6BS.2 was mapped in a 17.5-cM region flanked by Xgwm132 and Xgdm113. Both two QTL were physically mapped to the short arm of chromosome 6B, but in different bins. Validation and polymorphism tests of the flanking markers in 43 wheat genotypes indicated that the molecular markers associated with these QTL should be useful in marker-assisted breeding programs to efficiently incorporate HTAP resistance into new wheat cultivars.     

Factors Affecting the Radiosensitivity of Hexaploid Wheat to γ-Irradiation: Radiosensitivity of Hexaploid Wheat (Triticum aestivum L.)

Understanding the radiosensitivity of plants, an important factor in crop mutation breeding programs, requires a thorough investigation of the factors that contribute to this trait. In this study, we used the highly radiosensitive wheat (Triticum aestivum L.) variety HY1 and J411, a γ-irradiation-insensitive control, which were screened from a natural population, to examine the factors affecting radiosensitivity, including free radical content and total antioxidant capacity, as well as the expression of TaKu70 and TaKu80 (DNA repair-related genes) as measured by real-time PCR. We also investigated the alternative splicing of this gene in the wild-type wheat ecotype by sequence analysis. Free radical contents and total antioxidant capacity significantly increased upon exposure of HY1 wheat to γ-irradiation in a dose-dependent manner. By contrast, in J411, the free radical contents exhibited a similar trend, but the total antioxidant capacity exhibited a downward trend upon increasing γ-irradiation. Additionally, we detected dose-dependent increases in TaKu70 and TaKu80 expression levels in γ-irradiated HY1, while in J411, TaKu70 expression levels increased, followed by a decline. We also detected alternative splicing of TaKu70 mRNA, namely, intron retention, in HY1 but not in J411. Our findings indicate that γ-irradiation induces oxidative stress and DNA damage in hexaploid wheat, resulting in growth retardation of seedlings, and they suggest that TaKu70 may play a causal role in radiosensitivity in HY1. Further studies are required to exploit these factors to improve radiosensitivity in other wheat varieties.