Phenotypic effects of Am genomes in nascent synthetic hexaploids derived from interspecific crosses between durum and wild einkorn wheat.

Allopolyploid speciation is a major evolutionary process in wheat (Triticum spp.) and the related Aegilops species. The generation of synthetic polyploids by interspecific crosses artificially reproduces the allopolyploidization of wheat and its relatives. These synthetic polyploids allow breeders to introduce agriculturally important traits into durum and common wheat cultivars. This study aimed to evaluate the genetic and phenotypic diversity in wild einkorn Triticum monococcum ssp. aegilopoides (Link) Thell., to generate a set of synthetic hexaploid lines containing the various Am genomes from wild einkorn, and to reveal their trait characteristics. We examined the genetic diversity of 43 wild einkorn accessions using simple sequence repeat markers covering all the chromosomes and revealed two genetically divergent lineages, L1 and L2. The genetic divergence between these lineages was linked to their phenotypic divergence and their habitats. L1 accessions were characterized by early flowering, fewer spikelets, and large spikelets compared to L2 accessions. These trait differences could have resulted from adaptation to their different habitats. We then developed 42 synthetic hexaploids containing the AABBAmAm genome through interspecific crosses between T. turgidum cv. Langdon (AABB genome) as the female parent and the wild einkorn accessions (AmAm genome) as the male parents. Two of the 42 AABBAmAm synthetic hexaploids exhibited hybrid dwarfness. The phenotypic divergence between L1 and L2 accessions of wild einkorn, especially for days to flowering and spikelet-related traits, significantly reflected phenotypic differences in the synthetic hexaploids. The differences in plant height and internodes between the lineages were more distinct in the hexaploid backgrounds. Furthermore, the AABBAmAm synthetic hexaploids had longer spikelets and grains, long awns, high plant heights, soft grains, and late flowering, which are distinct from other synthetic hexaploid wheat lines such as AABBDD. Utilization of various Am genomes of wild einkorn resulted in wide phenotypic diversity in the AABBAmAm synthetic hexaploids and provides promising new breeding materials for wheat.

Do ancient wheats contain less gluten than modern bread wheat, in favour of better health?

Popular media messaging has led to increased public perception that gluten-containing foods are bad for health. In parallel, 'ancient grains' have been promoted with claims that they contain less gluten. There appears to be no clear definition of 'ancient grains' but the term usually includes einkorn, emmer, spelt and Khorasan wheat. Gluten is present in all wheat grains and all can induce coeliac disease (CD) in genetically susceptible individuals. Analyses of 'ancient' and 'modern' wheats show that the protein content of modern bread wheat (Triticum aestivum) has decreased over time while the starch content increased. In addition, it was shown that, compared to bread wheat, ancient wheats contain more protein and gluten and greater contents of many CD-active epitopes. Consequently, no single wheat type can be recommended as better for reducing the risks of or mitigating the severity of CD. An estimated 10% of the population of Western countries suffers from gastrointestinal symptoms that lack a clear organic cause and is often referred to as irritable bowel syndrome (IBS). Many of these patients consider themselves gluten sensitive, but in most cases this is not confirmed when tested in a medical setting. Instead, it may be caused by gas formation due to fermentation of fructans present in wheat or, in some patients, effects of non-gluten proteins. A significant overlap of symptoms with those of CD, IBS and inflammatory bowel disease makes a medical diagnosis a priority. This critical narrative review examines the suggestion that 'ancient' wheat types are preferred for health and better tolerance.

Phenotypic effects of the U-genome variation in nascent synthetic hexaploids derived from interspecific crosses between durum wheat and its diploid relative Aegilops umbellulata.

Aegilops umbellulata is a wild diploid wheat species with the UU genome that is an important genetic resource for wheat breeding. To exploit new synthetic allohexaploid lines available as bridges for wheat breeding, a total of 26 synthetic hexaploid lines were generated through crossing between the durum wheat cultivar Langdon and 26 accessions of Ae. umbellulata. In nascent synthetic hexaploids with the AABBUU genome, the presence of the set of seven U-genome chromosomes was confirmed with U-genome chromosome-specific markers developed based on RNA-seq-derived data from Ae. umbellulata. The AABBUU synthetic hexaploids showed large variations in flowering- and morphology-related traits, and these large variations transmitted well from the parental Ae. umbellulata accessions. However, the variation ranges in most traits examined were reduced under the AABBUU hexaploid background compared with under the diploid parents. The AABBUU and AABBDD synthetic hexaploids were clearly discriminated by several morphological traits, and an increase of plant height and in the number of spikes and a decrease of spike length were commonly observed in the AABBUU synthetics. Thus, interspecific differences in several morphological traits between Ae. umbellulata and A. tauschii largely affected the basic plant architecture of the synthetic hexaploids. In conclusion, the AABBUU synthetic hexaploid lines produced in the present study are useful resources for the introgression of desirable genes from Ae. umbellulata to common wheat.

Introgression of chromosomal segments conferring early heading date from wheat diploid progenitor, Aegilops tauschii Coss., into Japanese elite wheat cultivars.

The breeding of agriculturally useful genes from wild crop relatives must take into account recent and future climate change. In Japan, the development of early heading wheat cultivars without the use of any major gene controlling the heading date is desired to avoid overlap of the harvesting time before the rainy season. Here, we backcrossed two early heading lines of a synthetic hexaploid wheat, derived from a crossing between durum wheat and the wild wheat progenitor Aegilops tauschii, with four Japanese elite cultivars to develop early heading lines of bread wheat. In total, nine early heading lines that showed a heading date two to eight days earlier than their parental cultivars in field conditions were selected and established from the selfed progenies of the two- or three-times backcrossed populations. The whole appearance and spike shape of the selected early heading lines looked like their parental wheat cultivars. The mature grains of the selected lines had the parental cultivars' characteristics, although the grains exhibited longer and narrower shapes. RNA sequencing-based genotyping was performed to detect single nucleotide polymorphisms between the selected lines and their parental wheat cultivars, which revealed the chromosomal regions transmitted from the parental synthetic wheat to the selected lines. The introgression regions could shorten wheat heading date, and their chromosomal positions were dependent on the backcrossed wheat cultivars. Therefore, early heading synthetic hexaploid wheat is useful for fine-tuning of the heading date through introgression of Ae. tauschii chromosomal regions.

Molecular characterization of gliadins of Chinese Spring wheat in relation to celiac disease elicitors.

The wheat seed storage proteins gliadin and glutenin are encoded by multigenes. Gliadins are further classified into α-, γ-, δ- and ω-gliadins. Genes encoding α-gliadins belong to a large multigene family, whose members are located on the homoeologous group 6 chromosomes at the Gli-2 loci. Genes encoding other gliadins are located on the homoeologous group 1 chromosomes at the Gli-1 loci. Two-dimensional polyacrylamide gel electrophoresis (2-DE) was used to characterize and profile the gliadins. The gliadins in aneuploid Chinese Spring wheat lines were then compared in this study. Gliadin proteins separated into 70 spots after 2-DE and a total of 10, 10 and 16 spots were encoded on chromosomes 6A, 6B and 6D, respectively, which suggested that they were α-gliadins. Similarly, six, three and seven spots were encoded on chromosomes 1A, 1B and 1D, respectively, which indicated that they were γ-gliadins. Spots that could not be assigned to chromosomes were N-terminally sequenced and were all determined to be α-gliadins or γ-gliadins. The 2-DE profiles showed that specific α-gliadin spots assigned to chromosome 6D were lost in tetrasomic chromosome 2A lines. Furthermore, western blotting against the Glia-α9 peptide, an epitope for celiac disease (CD), suggested that α-gliadins harboring the CD epitope on chromosome 6D were absent in the tetrasomic chromosome 2A lines. Systematic analysis of α-gliadins using 2-DE, quantitative RT-PCR and genomic PCR revealed that tetrasomic 2A lines carry deletion of a chromosome segment at the Gli-D2 locus. This structural alteration at the Gli-D2 locus may provide a genetic resource in breeding programs for the reduction of CD immunotoxicity.

Genetic mapping reveals a dominant awn-inhibiting gene related to differentiation of the variety anathera in the wild diploid wheat Aegilops tauschii.

Aegilops tauschii, a wild wheat relative, is the D-genome donor of common wheat. Subspecies and varieties of Ae. tauschii are traditionally classified based on differences in their inflorescence architecture. However, the genetic information for their diversification has been quite limited in the wild wheat relatives. The variety anathera has no awn on the lemma, but the genetic basis for this diagnostic character is unknown. Wide variations in awn length traits at the top and middle spikes were found in the Ae. tauschii core collection, and the awn length at the middle spike was significantly smaller in the eastward-dispersed sublineage than in those in other sublineages. To clarify loci controlling the awnless phenotype of var. anathera, we measured awn length of an intervariety F2 mapping population, and found that the F2 individuals could be divided into two groups mainly based on the awn length at the middle of spike, namely short and long awn groups, significantly fitting a 3:1 segregation ratio, which indicated that a single locus controls the awnless phenotype. The awnless locus, Anathera (Antr), was assigned to the distal region of the short arm of chromosome 5D. Quantitative trait locus analysis using the awn length data of each F2 individual showed that only one major locus was at the same chromosomal position as Antr. These results suggest that a single dominant allele determines the awnless diagnostic character in the variety anathera. The Antr dominant allele is a novel gene inhibiting awn elongation in wheat and its relatives.

Dough properties and bread-making quality-related characteristics of Yumechikara near-isogenic wheat lines carrying different Glu-B3 alleles.

We investigated the relationships of three allelic variations in Glu-B3 (ab, g, and h) with dough properties and bread-making quality-related characteristics using near-isogenic lines (NILs) of 'Yumechikara' that commonly carry Glu-A1a, Glu-B1b, Glu-D1d, Glu-A3f, Glu-B3ab and Glu-D3a. Measurement of peak time (PT) in a 2-g mixograph indicated that Glu-B3g was the most effective for a strong dough property, followed by Glu-B3ab, with Glu-B3h being the least effective. The results of measurement of mixing time during bread-making were similar to those for PTs, i.e., the lines carrying Glu-B3g showed the longest mixing time, followed by those of Glu-B3ab, and those of Glu-B3h showed the shortest mixing time. Since two parameters of bread-making quality, loaf volume (LV) and specific loaf volume (SLV), were affected by flour protein contents in all groups of the Glu-B3 genotype, we compared the effects of the three Glu-B3 alleles on those parameters using analysis of covariance (ANCOVA) to remove the effect of protein content. The results indicated that the Glu-B3h group showed the largest SLV, followed by the Glu-B3ab group, and the Glu-B3g group showed the smallest SLV. These results suggest that the introduction of Glu-B3h into 'Yumechikara' makes it possible to breed varieties with good bread-making quality-related characteristics.

Identification and distribution of Puroindoline b-2 variant gene homologs in Hordeum.

The barley hordoindoline genes (Hina and Hinb) are homologous to the wheat puroindoline genes (Pina and Pinb). These genes are involved in grain hardness, which is an important quality for barley processing. We identified novel variants of Hina and Hinb in 10 wild Hordeum species (H. bogdanii, H. brachyantherum, H. bulbosum, H. chilense, H. comosum, H. marinum, H. murinum, H. patagonicum, H. pusillum, and H. roshevitzii) covering all Hordeum genomes and preliminarily named them Hinc. These nucleotide sequences were highly similar to those of Puroindoline b-2 variant genes (Pinb-2v) and were located on chromosome 7I in H. chilense. The Hinc genes in H. bogdanii, H. bulbosum, H. patagonicum, and H. roshevitzii were pseudogenes possessing in-frame stop codons. We also found a partial Hinc sequence in H. murinum. This gene was not found in cultivated barley and H. vulgare subsp. spontaneum. The phylogenetic tree of Gsp-1, Hin, and Pin genes demonstrates that Hinc and Pinb-2v genes formed one cluster. Therefore, we considered that Hinc and Pinb-2v genes shared a common ancestral gene and were homologous to each other. We also studied the evolutional process of Gsp-1, Hin, and Pin genes. Our results suggested that Gsp-1 might be the most closely related to a putative ancestral gene on Ha locus.

Spatial and temporal progress of programmed cell death in the developing starchy endosperm of rice.

Programmed cell death (PCD) is the genetically regulated disassembly of cells, and occurs in the endosperm of cereals during seed maturation. Since PCD determines the lifetime of cells, it can affect endosperm growth and, therefore, cereal yield. However, the features and mechanisms of PCD in the developing starchy endosperm in the Poaceae remain unclear. In the present study, we investigated the characteristics of PCD in developing starchy endosperm of rice (Oryza sativa L.) by fluorescence microscopy, focusing on the spatial and temporal progress of PCD-associated responses. Cell death commenced in the central region of starchy endosperm, and then spread to the peripheral region. PCD-associated responses, such as mitochondrial membrane permeabilization and activation of the protease that cleaves the amino acid sequence VEID, showed similar spatial patterns to that of cell death, but preceded cell death. Degradation of nuclear DNA could not be detected in developing starchy endosperm by the TUNEL assay. These results indicated that PCD in developing starchy endosperm of rice proceeds via a highly organized pattern. In addition, these results suggested that PCD in developing starchy endosperm of rice is characterized by the involvement of mitochondrial signaling and the activity of a caspase-like protease that cleaves the VEID sequence.

Evidence from principal component analysis for improvement of grain shape- and spikelet morphology-related traits after hexaploid wheat speciation.

Grain shape and size are involved in the main components of the domestication syndrome in cereals. Wheat grain shape has been dramatically altered at each stage of the domestication of tetraploid wheat and through common wheat speciation. To elucidate the evolutionary change of wheat grain shape, principal component (PC) analysis of grain shape-related traits was first conducted using wild and cultivated tetraploid, synthetic hexaploid, and common wheat accessions. The synthetic hexaploid wheat lines were previously produced through interspecific crosses between two common wheat progenitors, tetraploid wheat and Aegilops tauschii, and produced grains similar to those of cultivated tetraploid wheat. To identify genetic loci related to the difference in grain shape between common wheat and the synthetic wheat, the 15 traits related to grain and spikelet shape were measured in 108 F(2) individuals between Norin 61 and a synthetic wheat line, and the first three PC values for the 15 traits, PC1, PC2 and PC3, were mapped as quantitative traits in the F(2) population. In total, six QTLs, found on chromosomes 1A, 5A, 1D, 2D and 7D, showed significant LOD scores. Among them, a QTL for PC2, located on the 2DS chromosomal region near the Ppd-D1 locus, mainly contributed to the phenotypic difference in grain shape. Tg-D1, controlling tenacious glume phenotype, was located at a similar region to the 2DS QTL, which suggested that the Tg-D1 locus pleiotropically affects not only glume toughness but also spikelet and grain shape in hexaploid wheat. Therefore, it was predicted that wheat grains were rapidly improved toward a shorter and rounder phenotype accompanied with free-threshing wheat formation.

Evaluation of fresh pasta-making properties of extra-strong common wheat (Triticum aestivum L.).

The relationship between characterictics of flour of common wheat varieties and fresh pasta-making qualitites was examined, and the fresh pasta-making properties of extra-strong varieties that have extra-strong dough were evaluated. There was a positive correlation between mixing time (PT) and hardness of boiled pasta, indicating that the hardness of boiled pasta was affected by dough properties. Boiled pasta made from extra-strong varieties, Yumechikara, Hokkai 262 and Hokkai 259, was harder than that from other varieties and commercial flour. There was a negative correlation between flour protein content and brightness of boiled pasta. The colors of boiled pasta made from Yumechikara and Hokkai 262 grown under the condition of standard manuring culture were superior to those of boiled pasta made from other varieties. Discoloration of boiled pasta made from Yumechikara grown under the condition of heavy manuring culture was caused by increase of flour protein content. On the other hand, discoloration of boiled pasta made from Hokkai 262 grown under the condition of heavy manuring culture was less than that of boiled pasta made from Yumechikara. These results indicate that pasta made from extra-strong wheat varieties has good hardness and that Hokkai 262 has extraordinary fresh pasta-making properties.

Distribution of Hordoindoline genes in the genus Hordeum.

Hordoindoline (Hin) genes, which are known to comprise Hina, Hinb-1, and Hinb-2, are associated with grain hardness in barley. However, the interspecific variation in the Hin genes in the genus Hordeum has not been studied in detail. We examined the variation in Hin genes and used it to infer the phylogenetic relationships between the genes found in two H. vulgare subspecies (cultivated barley and H. vulgare subsp. spontaneum) and 10 wild relatives (H. bogdanii, H. brachyantherum, H. bulbosum, H. chilense, H. comosum, H. marinum, H. murinum, H. patagonicum, H. pusillum, and H. roshevitzii). The Hina and Hinb genes of these species were amplified by PCR. We found two Hinb genes in three wild species (H. bogdanii, H. brachyantherum, and H. roshevitzii) and preliminarily named them Hinb-A and Hinb-B. Cluster analysis showed that the 17 Hinb genes present in Hordeum formed two distinct clusters (named A and B). Seven Hinb genes were included in Cluster-A, and 10 Hinb genes were included in Cluster-B. All Hinb-A genes were included in Cluster-A, while all of the Hinb-B genes were included in Cluster-B. In contrast, the Hinb-1 and Hinb-2 genes in H. vulgare were included in Cluster-B. These results suggest that the Hinb genes duplicated during the early stages of diversification in the genus Hordeum. On the other hand, the Hinb-1 and Hinb-2 genes in H. vulgare seem to have been generated by a duplication of the Hinb gene after the split of the lineages leading to H. vulgare and H. bulbosum.

Chromosome 5H of Hordeum species involved in reduction in grain hardness in wheat genetic background.

Grain hardness is an important factor affecting end-use quality in wheat. Mutations of the puroindoline genes, which are located on chromosome 5DS, control a majority of grain texture variations. Hordoindoline genes, which are the puroindoline gene homologs in barley, are located on chromosome 5HS and are also responsible for grain texture variation. In this study, we used three types of wheat-barley species (Hordeum vulgare, H. vulgare ssp. spontaneum, and H. chilense) chromosome addition lines and studied the effect of chromosome 5H of these species on wheat grain characteristics. The 5H chromosome addition lines showed significantly lower grain hardness and higher grain weight than the corresponding wheat parents. The effect of enhancing grain softness was largest in the wheat-H. chilense line regardless of having an increase in grain weight similar to those in the wheat-H. vulgare and wheat-H. spontaneum lines. Our results indicated that chromosome 5H of the Hordeum species plays a role in enhancing grain softness and increasing grain weight in the wheat genetic background, and the extent of effect on grain hardness depends on the type of Hordeum species. Protein analysis of hordoindolines indicated that profiles of 2D-electrophoresis of hordoindolines were different among Hordeum species and hordoindolines in the addition lines appeared to be most abundant in wheat-H. chilense line. The differences in enhancing grain softness among the Hordeum species might be attributed to the quantity of hordoindolines expressed in the 5H chromosome addition lines. These results suggested that the barley hordoindolines located on chromosome 5HS play a role in reducing grain hardness in the wheat genetic background.

Impact of ω-5 gliadin on wheat-dependent exercise-induced anaphylaxis in mice.

The effects of ω-5 gliadin on wheat-dependent exercise-induced anaphylaxis (WDEIA) were investigated by using a mouse model. The gliadin fraction was prepared as a 70% ethanol-soluble solution, and ω-5 gliadin was purified by chromatography. Purified ω-5 gliadin was run on SDS-PAGE gel to reveal three bands with a molecular mass of 53-60 kDa and had the characteristic N-terminal sequence of ω-5 gliadin. The mice were sensitized to the gliadin fraction, and the anaphylactic response was assessed by measuring the body temperature and voluntary physical activity. An oral administration of ω-5 gliadin evoked a significant drop in both the body temperature and voluntary physical activity, similar to the effects of the whole gliadin fraction. ELISA and immunoblotting analyses revealed that the IgE expression from sensitized mice reacted most strongly to ω-5 gliadin. Taken together, these results indicate ω-5 gliadin to be a major allergen responsible for stimulating WDEIA in mice, with the characteristic potential for stimulating IgE production.

Comparison of low molecular weight glutenin subunits identified by SDS-PAGE, 2-DE, MALDI-TOF-MS and PCR in common wheat.

BACKGROUND: Low-molecular-weight glutenin subunits (LMW-GS) play a crucial role in determining end-use quality of common wheat by influencing the viscoelastic properties of dough. Four different methods - sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2-DE, IEF x SDS-PAGE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and polymerase chain reaction (PCR), were used to characterize the LMW-GS composition in 103 cultivars from 12 countries. RESULTS: At the Glu-A3 locus, all seven alleles could be reliably identified by 2-DE and PCR. However, the alleles Glu-A3e and Glu-A3d could not be routinely distinguished from Glu-A3f and Glu-A3g, respectively, based on SDS-PAGE, and the allele Glu-A3a could not be differentiated from Glu-A3c by MALDI-TOF-MS. At the Glu-B3 locus, alleles Glu-B3a, Glu-B3b, Glu-B3c, Glu-B3g, Glu-B3h and Glu-B3j could be clearly identified by all four methods, whereas Glu-B3ab, Glu-B3ac, Glu-B3ad could only be identified by the 2-DE method. At the Glu-D3 locus, allelic identification was problematic for the electrophoresis based methods and PCR. MALDI-TOF-MS has the potential to reliably identify the Glu-D3 alleles. CONCLUSIONS: PCR is the simplest, most accurate, lowest cost, and therefore recommended method for identification of Glu-A3 and Glu-B3 alleles in breeding programs. A combination of methods was required to identify certain alleles, and would be especially useful when characterizing new alleles. A standard set of 30 cultivars for use in future studies was chosen to represent all LMW-GS allelic variants in the collection. Among them, Chinese Spring, Opata 85, Seri 82 and Pavon 76 were recommended as a core set for use in SDS-PAGE gels. Glu-D3c and Glu-D3e are the same allele. Two new alleles, namely, Glu-D3m in cultivar Darius, and Glu-D3n in Fengmai 27, were identified by 2-DE. Utilization of the suggested standard cultivar set, seed of which is available from the CIMMYT and INRA Clermont-Ferrand germplasm collections, should also promote information sharing in the identification of individual LMW-GS and thus provide useful information for quality improvement in common wheat.

A barley Hordoindoline mutation resulted in an increase in grain hardness.

Barley seed proteins, Hordoindolines, are homologues of wheat Puroindolines, which are associated with grain hardness. Barley Hordoindoline genes are known to comprise Hina and Hinb, and Hinb consists of two Hinb genes, Hinb-1 and Hinb-2. Two types of allele were found for Hina, Hinb-1 and Hinb-2 genes, respectively, among Japanese two- and six-rowed barley lines. One of the alleles of Hinb-2 (Hinb-2b) had a frame-shift mutation resulting in an in-frame stop codon. For two-rowed barley lines, grain hardness was significantly higher among lines with the Hinb-2b than those with the wild type Hinb-2 gene (Hinb-2a). Protein spots corresponding to HINa, HINb-1, and HINb-2 were identified by 2D-gel electrophoresis among barley lines with Hinb-2a. Among the lines with Hinb-2b, HINa and HINb-1 were expressed at similar levels as those in the wild type, but HINb-2 was not detected. A DNA (cleaved amplified polymorphic sequence) marker was developed to distinguish between the Hinb-2a and Hinb-2b gene sequences. Analysis of grain hardness among F(2) lines derived from a cross between a line with Hinb-2a (Shikoku hadaka 115) and a line with the Hinb-2b (Shikoku hadaka 84) showed significantly higher grain hardness in the mutant lines. From these results, the Hinb-2b frame-shift (null) mutation might play a critical role in barley grain hardness. The DNA marker will be useful in barley breeding to select lines having harder grain texture.