Diploid Wheat Triticum Monococcum Research Articles

Intrinsic Allergenicity Potential of Salt-Soluble Protein Extracts from the Diploid, Tetraploid and Hexaploid Wheats: Validation Using an Adjuvant-Free Mouse Model

Wheat allergies are potentially life-threatening and, therefore, have become a major health concern at the global level. It is largely unknown at present whether genetic variation in allergenicity potential exists among hexaploid, tetraploid and diploid wheat species. Such information is critical in establishing a baseline allergenicity map to inform breeding efforts to identify hyper-, hypo- and non-allergenic varieties. We recently reported a novel mouse model of intrinsic allergenicity using the salt-soluble protein extract (SSPE) from durum, a tetraploid wheat (Triticum durum). Here, we validated the model for three other wheat species [hexaploid common wheat (Triticum aestivum), diploid einkorn wheat (Triticum monococcum), and the ancient diploid wheat progenitor, Aegilops tauschii], and then tested the hypothesis that the SSPEs from wheat species will exhibit differences in relative allergenicities. Balb/c mice were repeatedly exposed to SSPEs via the skin. Allergic sensitization potential was assessed by specific (s) IgE antibody responses. Oral anaphylaxis was quantified by the hypothermic shock response (HSR). The mucosal mast cell response (MMCR) was determined by measuring mast cell protease in the blood. While T. monococcum elicited the least, but significant, sensitization, others were comparable. Whereas Ae. taushcii elicited the least HSR, the other three elicited much higher HSRs. Similarly, while Ae. tauschii elicited the least MMCR, the other wheats elicited much higher MMCR as well. In conclusion, this pre-clinical comparative mapping strategy may be used to identify potentially hyper-, hypo- and non-allergenic wheat varieties via crossbreeding and genetic engineering methods.

Identification and characterization of Sr22b, a new allele of the wheat stem rust resistance gene Sr22 effective against the Ug99 race group.

SummaryWheat stem (or black) rust, caused by Puccinia graminis f. sp. tritici (Pgt), has been historically among the most devastating global fungal diseases of wheat. The recent occurrence and spread of new virulent races such as Ug99 have prompted global efforts to identify and isolate more effective stem rust resistance (Sr) genes. Here, we report the map‐based cloning of the Ug99‐effective SrTm5 gene from diploid wheat Triticum monococcum accession PI 306540 that encodes a typical coiled‐coil nucleotide‐binding leucine‐rich repeat protein. This gene, designated as Sr22b, is a new allele of Sr22 with a rare insertion of a large (13.8‐kb) retrotransposon into its second intron. Biolistic transformation of an ~112‐kb circular bacterial artificial chromosome plasmid carrying Sr22b into the susceptible wheat variety Fielder was sufficient to confer resistance to stem rust. In a survey of 168 wheat genotypes, Sr22b was present only in cultivated T. monococcum subsp. monococcum accessions but absent in all tested tetraploid and hexaploid wheat lines. We developed a diagnostic molecular marker for Sr22b and successfully introgressed a T. monococcum chromosome segment containing this gene into hexaploid wheat to accelerate its deployment and pyramiding with other Sr genes in wheat breeding programmes. Sr22b can be a valuable component of gene pyramids or transgenic cassettes combining different resistance genes to control this devastating disease.

Comparative Analysis of in vitro Digestibility and Immunogenicity of Gliadin Proteins From Durum and Einkorn Wheat.

Recent studies suggested that gliadin proteins from the ancient diploid einkorn wheat Triticum monococcum retained a reduced number of immunogenic peptides for celiac disease patients because of a high in vitro digestibility with respect to hexaploid common wheat. In this study, we compared the immunological properties of gliadins from two Triticum monococcum cultivars (Hammurabi and Norberto-ID331) with those of a Triticum durum cultivar (Adamello). Gliadins were digested by mimicking the in vitro gastrointestinal digestion process that includes the brush border membrane peptidases. Competitive ELISA, based on R5 monoclonal antibody, showed that gastrointestinal digestion reduced the immunogenicity of Triticum monococcum gliadins; conversely, the immunogenic potential of Triticum durum gliadins remained almost unchanged by the in vitro digestion. The immune stimulatory activity was also evaluated by detecting the IFN-γ production in gliadin-reactive T-cell lines obtained from the small intestinal mucosa of HLA-DQ2+ celiac disease patients. Interestingly, gastrointestinal digestion markedly reduced the capability of Triticum monococcum gliadins (p <0.05) of both cultivars to activate T cells, while it slightly affected the activity of Triticum durum. In conclusion, our results showed that Triticum durum was almost unaffected by the in vitro gastrointestinal digestion, while Triticum monococcum had a marked sensibility to digestion, thus determining a lower toxicity for celiac disease patients.

In Celiac Disease Patients the In Vivo Challenge with the Diploid Triticum monococcum Elicits a Reduced Immune Response Compared to Hexaploid Wheat.

Gluten from the diploid wheat Triticum monococcum (TM) has low content of immunostimulatory sequences and a high gastro-intestinal digestibility. Gluten-reactive T cells elicited by diploid and hexaploid (Triticum aestivum-TA) wheat in celiac disease (CD) patients upon a brief oral challenge are analyzed. Seventeen patients with CD (median age 13 years) consumed for 3 days sandwiches made with TM (cultivar Norberto-ID331, N=11), or TA (cultivar Sagittario, N=11) flours, corresponding to 12gr of gluten/die. Immunostimulatory properties are assessed in blood by measuring the IFN-γ-secreting T cells by EliSpot and the expression of inflammatory cytokines/receptors (IL-12A, IL-15, IL-18RAP, IFN-γ) by qPCR. TA mobilizes a remarkable number of gliadin-specific, IFN-γ-secreting T cells (p<0.05), while no significant cell mobilization is induced by TM (p=ns). Similar results are obtained in response to five immunogenic peptides from α-, ω-, and γ-gliadins, although with a large individual variability. An increased mRNA expression for IL-12A and IFN-γ is detected in the group eating TA compared to those consuming TM (p<0.05). Although T. monococcum is a cereal not suitable for the diet of celiacs, this diploid wheat elicits a reduced in vivo T-cell response compared to T. aestivum in celiac patients.

Wheat gene Sr60 encodes a protein with two putative kinase domains that confers resistance to stem rust.

Wheat stem rust, caused by Puccinia graminis Pers. f. sp. tritici (Pgt), is a devastating fungal disease threatening global wheat production. The present paper reports the identification of stem rust resistance gene Sr60, a race-specific gene from diploid wheat Triticum monococcum L. that encodes a protein with two putative kinase domains. This gene, designated asWHEAT TANDEM KINASE 2 (WTK2), confers intermediate levels of resistance to Pgt. WTK2 was identified by map-based cloning and validated by transformation of a c.10-kb genomic sequence including WTK2 into susceptible common wheat variety Fielder (Triticum aestivum L.). Transformation of Fielder with WTK2 was sufficient to confer Pgt resistance. Sr60 transcripts were transiently upregulated 1d post-inoculation with Pgt, but not in mock-inoculated plants. The upregulation of Sr60 was associated with stable upregulation of several pathogenesis-related genes. The Sr60-resistant haplotype found in T.monococcum was not found in polyploid wheat, suggesting an opportunity to introduce a novel resistance gene. Sr60 was successfully introgressed into hexaploid wheat, and wedeveloped a diagnostic molecular marker to accelerate its deployment and pyramiding with other resistance genes. The cloned Sr60 also can be a useful component of transgenic cassettes including other resistance genes with complementary resistance profiles.

Mapping and characterization of wheat stem rust\xa0resistance genes SrTm5 and Sr60 from Triticum monococcum

Key messageThe new stem rust resistance gene Sr60 was fine-mapped to the distal region of chromosome arm 5AmS, and the TTKSK-effective gene SrTm5 could be a new allele of Sr22.The emergence and spread of new virulent races of the wheat stem rust pathogen (Puccinia graminis f. sp. tritici; Pgt), including the Ug99 race group, is a serious threat to global wheat production. In this study, we mapped and characterized two stem rust resistance genes from diploid wheat Triticum monococcum accession PI 306540. We mapped SrTm5, a previously postulated gene effective to Ug99, on chromosome arm 7AmL, completely linked to Sr22. SrTm5 displayed a different race specificity compared to Sr22 indicating that they are distinct. Sequencing of the Sr22 homolog in PI 306540 revealed a novel haplotype. Characterization of the segregating populations with Pgt race QFCSC revealed an additional resistance gene on chromosome arm 5AmS that was assigned the official name Sr60. This gene was also effective against races QTHJC and SCCSC but not against TTKSK (a Ug99 group race). Using two large mapping populations (4046 gametes), we mapped Sr60 within a 0.44 cM interval flanked by sequenced-based markers GH724575 and CJ942731. These two markers delimit a 54.6-kb region in Brachypodium distachyon chromosome 4 and a 430-kb region in the Chinese Spring reference genome. Both regions include a leucine-rich repeat protein kinase (LRRK123.1) that represents a potential candidate gene. Three CC–NBS–LRR genes were found in the colinear Brachypodium region but not in the wheat genome. We are currently developing a Bacterial Artificial Chromosome library of PI 306540 to determine which of these candidate genes are present in the T. monococcum genome and to complete the cloning of Sr60.

Grain subproteome responses to nitrogen and sulfur supply in diploid wheat Triticum monococcum ssp. monococcum.

Wheat grain storage proteins (GSPs) make up most of the protein content of grain and determine flour end-use value. The synthesis and accumulation of GSPs depend highly on nitrogen (N) and sulfur (S) availability and it is important to understand the underlying control mechanisms. Here we studied how the einkorn (Triticum monococcum ssp. monococcum) grain proteome responds to different amounts of N and S supply during grain development. GSP composition at grain maturity was clearly impacted by nutrition treatments, due to early changes in the rate of GSP accumulation during grain filling. Large-scale analysis of the nuclear and albumin-globulin subproteomes during this key developmental phase revealed that the abundance of 203 proteins was significantly modified by the nutrition treatments. Our results showed that the grain proteome was highly affected by perturbation in the N:S balance. S supply strongly increased the rate of accumulation of S-rich α/β-gliadin and γ-gliadin, and the abundance of several other proteins involved in glutathione metabolism. Post-anthesis N supply resulted in the activation of amino acid metabolism at the expense of carbohydrate metabolism and the activation of transport processes including nucleocytoplasmic transit. Protein accumulation networks were analyzed. Several central actors in the response were identified whose variation in abundance was related to variation in the amounts of many other proteins and are thus potentially important for GSP accumulation. This detailed analysis of grain subproteomes provides information on how wheat GSP composition can possibly be controlled in low-level fertilization condition.

Mapping-by-sequencing in complex polyploid genomes using genic sequence capture: a case study to map yellow rust resistance in hexaploid wheat.

SummaryPreviously we extended the utility of mapping‐by‐sequencing by combining it with sequence capture and mapping sequence data to pseudo‐chromosomes that were organized using wheat–Brachypodium synteny. This, with a bespoke haplotyping algorithm, enabled us to map the flowering time locus in the diploid wheat Triticum monococcum L. identifying a set of deleted genes (Gardiner et al., 2014). Here, we develop this combination of gene enrichment and sliding window mapping‐by‐synteny analysis to map the Yr6 locus for yellow stripe rust resistance in hexaploid wheat. A 110 MB NimbleGen capture probe set was used to enrich and sequence a doubled haploid mapping population of hexaploid wheat derived from an Avalon and Cadenza cross. The Yr6 locus was identified by mapping to the POPSEQ chromosomal pseudomolecules using a bespoke pipeline and algorithm (Chapman et al., 2015). Furthermore the same locus was identified using newly developed pseudo‐chromosome sequences as a mapping reference that are based on the genic sequence used for sequence enrichment. The pseudo‐chromosomes allow us to demonstrate the application of mapping‐by‐sequencing to even poorly defined polyploidy genomes where chromosomes are incomplete and sub‐genome assemblies are collapsed. This analysis uniquely enabled us to: compare wheat genome annotations; identify the Yr6 locus – defining a smaller genic region than was previously possible; associate the interval with one wheat sub‐genome and increase the density of SNP markers associated. Finally, we built the pipeline in iPlant, making it a user‐friendly community resource for phenotype mapping.

Cloning and characterization of novel fast ω-gliadin genes in Triticum monococcum.

Fast ω-gliadin is the major allergen to wheat-dependent exercise-induced anaphylaxis (WDEIA). It is generally accepted that the proteins were expressed inGli-B1 loci located on the short arm of chromosome 1B in wheat. The knowledge of the orthologous genes in A and D genomes is still limited. In this study, the novel fastω-gliadin genes were cloned from genome A of diploid wheat Triticum monococcum. The deduced amino acid sequences of N-terminal domain showed that they referred to TRQ-type ω-gliadins. This type of ωgliadins have been identified in genome A by using mass spectroscopy and amino acid sequencing in previous studies (DuPont et al. 2004), but their coding sequences were lacking. Further analysis of IgE-binding epitopes suggested that the TRQ-typeω-gliadins have potential to trigger WDEIA. Wheat is one of the most important sources of food in the human diet. In the kernels of wheat, gliadins are the main components of the gluten. Traditionally, gliadins are further classified into α-gliadins, γ-gliadins and ω-gliadins on the basis of their mobility in electrophoresis at low pH (Wan et al. 2014). The ω-gliadins are minor components among wheat prolamins and they have not been studied well due to difficulties in cloning their genes caused by highly repetitive sequences (Anderson et al. 2009). In common wheat, most of the ω-gliadins were encoded by the Gli-A1, Gli-B1 and Gli-D1 on the short arm of chromosomes 1A, 1B and 1D, respectively (Pogna et al. 1990; Shewry et al. 1995; DuPont et al. 2000, 2004). Theω-gliadins have been categorized into ω1,ω2 andω5 types (DuPont et al. 2004). Theω5-gliadin, also referred as fastω-gliadin, are encoded byGli-B1 and the proteins can be distinguished by the amino acid sequences at the N-terminal domain, which are Ser-Arg-Leu (SRL) in ω5-gliadin, Ala-Arg-Gln (ARQ) or Lys-Glu-Leu (KEL)

Proteomic analysis shows that stress response proteins are significantly up-regulated in resistant diploid wheat (Triticum monococcum) in response to attack by the grain aphid (Sitobion avenae).

The grain aphid Sitobion avenae (F.) is a major pest of wheat, acting as a virus vector as well as causing direct plant damage. Commonly grown wheat varieties in the UK have only limited resistance to this pest. The present study was carried out to investigate the potential of a diploid wheat line (ACC20 PGR1755), reported as exhibiting resistance to S. avenae, to serve as a source of resistance genes. The diploid wheat line was confirmed as partially resistant, substantially reducing the fecundity, longevity and growth rate of the aphid. Proteomic analysis showed that approximately 200 protein spots were reproducibly detected in leaf extracts from both the resistant line and a comparable susceptible line (ACC5 PGR1735) using two-dimensional gel electrophoresis and image comparison software. Twenty-four spots were significantly up-regulated (>2-fold) in the resistant line after 24 h of aphid feeding (13 and 11 involved in local and systemic responses, respectively). Approximately 50 % of all differentially expressed protein spots were identified by a combination of database searching with MS and MS/MS data, revealing that the majority of proteins up-regulated by aphid infestation were involved in metabolic processes (including photosynthesis) and transcriptional regulation. However, in the resistant line only, several stress response proteins (including NBS–LRR-like proteins) and oxidative stress response proteins were identified as up-regulated in response to aphid feeding, as well as proteins involved in DNA synthesis/replication/repair. This study indicates that the resistant diploid line ACC20 PGR1755 may provide a valuable resource in breeding wheat for resistance to aphids.Electronic supplementary materialThe online version of this article (doi:10.1007/s11032-015-0220-x) contains supplementary material, which is available to authorized users.

Correction: De Novo Transcriptome Assembly and Analyses of Gene Expression during Photomorphogenesis in Diploid Wheat Triticum monococcum

Correction: De Novo Transcriptome Assembly and Analyses of Gene Expression during Photomorphogenesis in Diploid Wheat Triticum monococcum

De novo transcriptome assembly and analyses of gene expression during photomorphogenesis in diploid wheat Triticum monococcum.

Background Triticum monococcum (2n) is a close ancestor of T. urartu, the A-genome progenitor of cultivated hexaploid wheat, and is therefore a useful model for the study of components regulating photomorphogenesis in diploid wheat. In order to develop genetic and genomic resources for such a study, we constructed genome-wide transcriptomes of two Triticum monococcum subspecies, the wild winter wheat T. monococcum ssp. aegilopoides (accession G3116) and the domesticated spring wheat T. monococcum ssp. monococcum (accession DV92) by generating de novo assemblies of RNA-Seq data derived from both etiolated and green seedlings.Principal FindingsThe de novo transcriptome assemblies of DV92 and G3116 represent 120,911 and 117,969 transcripts, respectively. We successfully mapped ∼90% of these transcripts from each accession to barley and ∼95% of the transcripts to T. urartu genomes. However, only ∼77% transcripts mapped to the annotated barley genes and ∼85% transcripts mapped to the annotated T. urartu genes. Differential gene expression analyses revealed 22% more light up-regulated and 35% more light down-regulated transcripts in the G3116 transcriptome compared to DV92. The DV92 and G3116 mRNA sequence reads aligned against the reference barley genome led to the identification of ∼500,000 single nucleotide polymorphism (SNP) and ∼22,000 simple sequence repeat (SSR) sites.Conclusions De novo transcriptome assemblies of two accessions of the diploid wheat T. monococcum provide new empirical transcriptome references for improving Triticeae genome annotations, and insights into transcriptional programming during photomorphogenesis. The SNP and SSR sites identified in our analysis provide additional resources for the development of molecular markers.

A large-scale mutant panel in wheat developed using heavy-ion beam mutagenesis and its application to genetic research

Mutation analysis is a powerful tool for studying gene function. Heavy-ion beam mutagenesis is a comparatively new approach to inducing mutations in plants and is particularly efficient because of its high linear energy transfer (LET). High LET radiation induces a higher rate of DNA double-strand breaks than other mutagenic methods. Over the last 12years, we have constructed a large-scale mutant panel in diploid einkorn wheat (Triticum monococcum) using heavy-ion beam mutagenesis. Einkorn wheat seeds were exposed to a heavy-ion beam and then sown in the field. Selfed seeds from each spike of M1 plants were used to generate M2 lines. Every year, we obtained approximately 1000 M2 lines and eventually developed a mutant panel with 10,000 M2 lines in total. This mutant panel is being systematically screened for mutations affecting reproductive growth, and especially for flowering-time mutants. To date, we have identified several flowering-time mutants of great interest: non-flowering mutants (mvp: maintained vegetative phase), late-flowering mutants, and early-flowering mutants. These novel mutations will be of value for investigations of the genetic mechanism of flowering in wheat.

Characterization and gene mapping of a brittle culm mutant of diploid wheat (Triticum monococcum L.) with irregular xylem vessels development

Diploid wheat, Triticum monococcum L. (einkorn) is an ideal plant material for wheat functional genomics. Brittle culm mutant was identified by screening of the ethyl methane sulphonate-treated M 2 progenies of a T. monococcum accession pau14087 by banding the plant parts manually. The brittle culm mutant with drooping leaves, early flowering, reduced tiller numbers and susceptible to lodging had also exhibited brittleness in all plant parts than the wild-type parents. Comprehensive mechanical strength, histological, biochemical, scanning electron microscopy, and Fourier transform infrared spectroscopy analyses of brittle culm mutant supplemented and complemented the findings that the mutant had defective cellulose biosynthesis pathway and deposition of cell wall materials on secondary cell wall of mechanical tissues. Microscopic studies demonstrated that the decrease in cellulose contents resulted in the irregular cell wall organization in xylem vessels of leaf vascular bundles. To map the brc5 mutant, mapping populations were developed by crossing the brittle culm mutant with wild Triticum boeoticum acc. pau5088, having non-brittle characters. The brittle culm mutation was mapped between SSR markers, Xcfd39 and Xgwm126 on 5AmL chromosome of T. monococcum, with genetic distances of 2.6 and 4.8 cM, respectively. The brc5 mutant mapped on 5AmL, being distinct from a previously mapped brittle culm mutant in wheat, has been designated as brc5. The work on fine mapping and map-based cloning of brc5 gene regulating synthesis and deposition of cellulose on the secondary cell wall is in progress.

Characterization and gene mapping of a chlorophyll-deficient mutant clm1 of Triticum monococcum L.

Diploid wheat Triticum monococcum L. is a model plant for wheat functional genomics. Chlorophyll-deficient mutant (clm1) was identified during manual screening of the ethyl methanesulphonate (EMS)-treated M2 progenies of T. monococcum accession pau14087 in the field. The clm1 mutant, due to significantly decreased chlorophyll content compared with the wild-type (WT), exhibited pale yellow leaves which slowly recovered to green before flowering. The clm1 mutant showed early flowering, reduced number of tillers, trichome length and density, and different shape as compared with the WT. At the same time, clm1 mutant culm had more chlorophyll-containing parenchymatous tissues compared to WT, presumably to absorb more sunlight for photosynthesis. Genetic analysis indicated that the clm1 mutation was monogenic recessive. The clm1 mutant was mapped between Xgwm473 and Xwmc96 SSR markers, with genetic distances of 2.1 and 2.6 cM, respectively, on the 7AmL chromosome.

Identification of anAP2gene related to open flowering in diploid wheat(Triticum monococcum)

Cleistogamy involves the shedding of pollen within an enclosed flower. In barley, this trait is determined by the presence of a recessive allele at the gene Cly1, a member of the AP2 gene family. Here we show that the Cly1 ortholog in einkorn (diploid) wheat (Triticum monococcum) TmAP2 shares a similar structure and identical pattern of transcription as Cly1. The transcript abundance of TmAP2 was high in the spike around the time of anthesis, but low in the leaf, plumule and radicle. The TmAP2 transcript was cleaved at its miR172 target site. Flower gaping at anthesis in einkorn wheat is induced by the expansion of the lodicules.

Immunogenicity of monococcum wheat in celiac patients

Research is intense to find wheat of low or null toxicity for patients with celiac disease (CD). Among candidates, there are diploid wheat species. We compared the immunological properties of 2 lines of diploid monococcum wheat (Triticum monococcum ssp. monococcum), Monlis and ID331, with those of common wheat (Triticum aestivum). Interferon-γ production and the proliferation of intestinal gliadin-specific T cell lines and clones were measured as evidence of T cell activation by peptic and tryptic (PT) digests of gliadins from 2 monococcum lines. Furthermore, organ cultures of jejunal biopsies from 28 CD patients were set up to assess the effects of PT gliadin on innate and adaptive immune response by using immunohistochemistry. Monlis and ID331 induced interferon-γ production and proliferation in celiac mucosal T cells. In organ cultures, Monlis PT digest induced a significant increase of IL-15 epithelial expression and crypt enterocyte proliferation, whereas ID331 had no effect. Both monococcum lines caused intraepithelial T cell infiltration and lamina propria T cell activation. Our data show that the monococcum lines Monlis and ID331 activate the CD T cell response and suggest that these lines are toxic for celiac patients. However, ID331 is likely to be less effective in inducing CD because of its inability to activate the innate immune pathways.

Functional characterization of three cDNA libraries from the diploid wheatTriticum monoccocum(AmAm) with different growth habits

The diploid wheat Triticum monococcum (2n = 2x = 14, AmAm) constitutes an excellent and simplified model to study wheat genome expression since it has lower genome complexity compared to polyploid wheat species.The analysis of a collection of near 10,000 ESTs obtained from two libraries of T. monococcum (TM011XXX and TM043E1X) and one from T. monococcum ssp. aegilopoides (TM046E1X), lead to 2246, 1843 and 2405 unigenes, respectively. The unigenes from each library were individually analyzed through the Blast2Go interface revealing higher identity compared to EST databases. However, a low percentage of sequences showed significant similarity with Triticum databases, reflecting that several novel Triticum expressed genes have been identified in these libraries. The sequences annotation and classification under GO categories showed similar distribution for the three libraries, but differences were identified in some subcategories.When a virtual unique library was constructed including the sequences from the ...

Characterization and molecular mapping of EMS-induced brittle culm mutants of diploid wheat (Triticum monococcum L.)

Diploid wheat (Triticum monococcum L, AmAm) is an ideal material for induced mutations which can be easily characterized and transferred to polyploid wheats. The EMS-induced brittle culm mutants, brc1, brc2, and brc3 used in the present investigation, were isolated from T. monococcum. All the brittle mutants had brittle roots, leaves, leaf sheaths, culms, and spikes, and were also susceptible to lodging. The mutants had 47–57% reduced α-cellulose in the secondary cell walls than that of T. monococcum indicating that all of them had defective synthesis of cellulose. All the mutants were monogenic recessive. Bulk segregation analysis of the mutants, using Am genome anchored SSR markers in their F 2 populations with T. boeoticum, located the mutants, brc1, brc2, and brc3 on chromosome 6A, 3A, and 1A of T. monococcum, respectively. Molecular analysis of the putatively linked markers showed that brc1 mapped on chromosome 6AS between Xbarc37 and Xbarc113 markers, brc2 on chromosome 3AL between Xcfd62 and Xcfa2170 markers whereas brc3 mapped on chromosome 1AL between Xgwm135 and Xwmc470 markers. Isolation and mapping of three different brittle culm mutants in wheat for the first time shows that there might be many more genes in wheat which affect synthesis and deposition of cellulose.

Regulation of Freezing Tolerance and Flowering in Temperate Cereals: The VRN-1 Connection

In winter wheat (Triticum spp.) and barley (Hordeum vulgare) varieties, long exposures to nonfreezing cold temperatures accelerate flowering time (vernalization) and improve freezing tolerance (cold acclimation). However, when plants initiate their reproductive development, freezing tolerance decreases, suggesting a connection between the two processes. To better understand this connection, we used two diploid wheat (Triticum monococcum) mutants, maintained vegetative phase (mvp), that carry deletions encompassing VRN-1, the major vernalization gene in temperate cereals. Homozygous mvp/mvp plants never flower, whereas plants carrying at least one functional VRN-1 copy (Mvp/-) exhibit normal flowering and high transcript levels of VRN-1 under long days. The Mvp/- plants showed reduced freezing tolerance and reduced transcript levels of several cold-induced C-REPEAT BINDING FACTOR transcription factors and COLD REGULATED genes (COR) relative to the mvp/mvp plants. Diploid wheat accessions with mutations in the VRN-1 promoter, resulting in high transcript levels under both long and short days, showed a significant down-regulation of COR14b under long days but not under short days. Taken together, these studies suggest that VRN-1 is required for the initiation of the regulatory cascade that down-regulates the cold acclimation pathway but that additional genes regulated by long days are required for the down-regulation of the COR genes. In addition, our results show that allelic variation in VRN-1 is sufficient to determine differences in freezing tolerance, suggesting that quantitative trait loci for freezing tolerance previously mapped on this chromosome region are likely a pleiotropic effect of VRN-1 rather than the effect of a separate closely linked locus (FROST RESISTANCE-1), as proposed in early freezing tolerance studies.