The GAMYB gene in rye: sequence, polymorphisms, map location, allele-specific markers, and relationship with ?-amylase activity.
ABSTRACT: BACKGROUND:Transcription factor (TF) GAMYB, belonging to MYB family (named after the gene of the avian myeloblastosis virus) is a master gibberellin (GA)-induced regulatory protein that is crucial for development and germination of cereal grain and involved in anther formation. It activates many genes including high-molecular-weight glutenin and ?-amylase gene families. This study presents the first attempt to characterize the rye gene encoding GAMYB in relation to its sequence, polymorphisms, and phenotypic effects. RESULTS:ScGAMYB was mapped on rye chromosome 3R using high-density Diversity Arrays Technology (DArT)/DArTseq-based maps developed in three mapping populations. The ScGAMYB sequences were identified in RNA-seq libraries of four rye inbred lines. The transcriptome used for the search contained almost 151,000 transcripts with a median contig length of 500?nt. The average amount of total base raw data was approximately 9?GB. Comparative analysis of the ScGAMYB sequence revealed its high level of homology to wheat and barley orthologues. Single nucleotide polymorphisms (SNPs) detected among rye inbred lines allowed the development of allele specific-PCR (AS-PCR) markers for ScGAMYB that might be used to detect this gene in wide genetic stocks of rye and triticale. Segregation of the ScGAMYB alleles showed significant relationship with ?-amylase activity (AMY). CONCLUSIONS:The research showed the strong similarity of rye GAMYB sequence to its orthologues in other Graminae and confirmed the position in the genome consistent with the collinearity rule of cereal genomes. Concurrently, the ScGAMYB coding sequence (cds) showed stronger variability (24 SNPs) compared to the analogous region of wheat (5 SNPs) and barley (7 SNPs). The moderate regulatory effect of ScGAMYB on AMY was confirmed, therefore, ScGAMYB was identified as a candidate gene for partial control of ?-amylase production in rye grain. The predicted structural protein change in the aa region 362-372, caused by a single SNP (C/G) at the 1100 position in ScGAMYB cds and single aa sequence change (S/C) at the 367 position, is the likely cause of the differences in the effectiveness of ScGAMYB regulatory function associated with AMY. The development of sequence-based, allele-specific (AS) PCR markers could be useful in research and application.
Project description:Agronomically important cereal crops wheat, barley, and rye of the Triticeace tribe under the genus Triticum were studied with special focus on their physical, proximal, and technological characteristics which are linked to their end product utilization. The physiochemical parameters showed variability among the three cereal grains. Lactic acid-solvent retention capacity (SRC) was found to be higher in wheat (95.86-111.92%) as compared to rye (53.78-67.97%) and barley (50.24-67.12%) cultivars, indicating higher gluten strength. Sucrose-SRC and sodium carbonate-SRC were higher in rye as compared to wheat and barley flours. The essential amino acid proportion in barley and rye cultivars was higher as compared to wheat cultivars. Barley and rye flours exhibited higher biological value (BV) owing to their higher lysine content. SDS-PAGE of wheat cultivars showed a high degree of polymorphism in the low molecular range of 27.03-45.24 kDa as compared to barley and rye cultivars. High molecular weight (HMW) proteins varied from 68.38 to 119.66 kDa (4-5 subunits) in wheat, 82.33 to 117.78 kDa (4 subunits) in rye, and 73.08 to 108.57 kDa (2-4 subunits) in barley. The comparative evaluation of barley and rye with wheat cultivars would help in the development of healthy food products.
Project description:Lunasin is a peptide from soybean seeds which has been demonstrated to have anticancer properties. It has also been reported in cereal seeds: wheat, rye, barley and Triticale. However, extensive searches of transcriptome and DNA sequence databases for wheat and other cereals have failed to identify sequences encoding either the lunasin peptide or a precursor protein. This raises the question of the origin of the lunasin reported in cereal grain.
Project description:Sequencing upstream of the Streptococcus mutans gene for a CcpA gene homolog, regM, revealed an open reading frame, named amy, with homology to genes encoding alpha-amylases. The deduced amino acid sequence showed a strong similarity (60% amino acid identity) to the intracellular alpha-amylase of Streptococcus bovis and, in common with this enzyme, lacked a signal sequence. Amylase activity was found only in S. mutans cell extracts, with no activity detected in culture supernatants. Inactivation of amy by insertion of an antibiotic resistance marker confirmed that S. mutans has a single alpha-amylase activity. The amylase activity was induced by maltose but not by starch, and no acid was produced from starch. S. mutans can, however, transport limit dextrins and maltooligosaccharides generated by salivary amylase, but inactivation of amy did not affect growth on these substrates or acid production. The amylase digested the glycogen-like intracellular polysaccharide (IPS) purified from S. mutans, but the amy mutant was able to digest and produce acid from IPS; thus, amylase does not appear to be essential for IPS breakdown. However, when grown on excess maltose, the amy mutant produced nearly threefold the amount of IPS produced by the parent strain. The role of Amy has not been established, but Amy appears to be important in the accumulation of IPS in S. mutans grown on maltose.
Project description:The transcription factor GAMYB is involved in gibberellin signalling in cereal aleurone cells and in plant developmental processes. Nucleotide diversity of HvGAMYB and TaGAMYB was investigated in 155 barley (Hordeum vulgare) and 42 wheat (Triticum aestivum) accessions, respectively. Polymorphisms defined 18 haplotypes in the barley collection and 1, 7 and 3 haplotypes for the A, B, and D genomes of wheat, respectively. We found that (1) Hv- and TaGAMYB genes have identical structures. (2) Both genes show a high level of nucleotide identity (>95%) in the coding sequences and the distribution of polymorphisms is similar in both collections. At the protein level the functional domain is identical in both species. (3) GAMYB genes map to a syntenic position on chromosome 3. GAMYB genes are different in both collections with respect to the Tajima D statistic and linkage disequilibrium (LD). A moderate level of LD was observed in the barley collection. In wheat, LD is absolute between polymorphic sites, mostly located in the first intron, while it decays within the gene. Differences in Tajima D values might be due to a lower selection pressure on HvGAMYB, compared to its wheat orthologue. Altogether our results provide evidence that there have been only few evolutionary changes in Hv- and TaGAMYB. This confirms the close relationship between these species and also highlights the functional importance of this transcription factor.
Project description:Abstract Stem rust, caused by Puccinia graminis, is a destructive group of diseases. The pathogen uses Berberis species as alternate hosts to complete its life cycle. B. vulgaris and the endemic species B. hispanica and B. garciae are present in Spain. The objective of this study was to investigate the functionality of the indigenous barberry as alternate hosts. Field surveys were conducted in 2018 and 2019 in Huesca, Teruel and Albacete provinces of Spain. Aecial samples on barberry were analysed via infection assays and DNA analysis. B. garciae was predominant in Huesca and Teruel provinces, often found in the field margins of cereal crops. Aecial infections on B. garciae were observed in May and uredinial infections on cereal crops in June. Scattered B. hispanica bushes were occasionally found near cereal crops in Albacete, where aecial infections on B. hispanica were observed in June when most cereal crops were mature. Infection assays using aeciospores resulted in stem rust infections on susceptible genotypes of wheat, barley, rye and oat, indicating the presence of the sexual cycle for P. graminis f. sp. tritici, f. sp. secalis and f. sp. avenae. Sequence analyses from aecial samples supported this finding as well as the presence of Puccinia brachypodii. This study provides the first evidence that indigenous Berberis species play an active role in the sexual cycle of P. graminis under natural conditions in Spain. Plants of indigenous Berberis spp. were frequently found growing near cereal crop fields in some areas of Spain and aeciospores from these barberries infected wheat, barley, rye and oats.
Project description:?-Amylase is a common enzyme for hydrolyzing starch. In the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), ?-amylase is found in both digestive fluid and hemolymph. Here, the complete genomic sequence of the Amy gene encoding ?-amylase from a local Thai silkworm, the Nanglai strain, was obtained. This gene was 7981 bp long with 9 exons. The full length Amy cDNA sequence was 1749 bp containing a 1503 bp open reading frame. The ORF encoded 500 amino acid residues. The deduced protein showed 81-54% identity to other insect ?-amylases and more than 50% identity to mammalian enzymes. Southern blot analysis revealed that in the Nanglai strain Amy is a single-copy gene. RT- PCR showed that Amy was transcribed only in the foregut. Transgenic B. mori also showed that the Amy promoter activates expression of the transgene only in the foregut.
Project description:Centromeres are essential for correct chromosome segregation during cell division and are determined by the presence of centromere-specific histone 3 (CENH3). Most of the diploid plant species, in which the structure and copy number of CENH3 genes have been determined, have this gene as a singleton; however, some cereal species in the tribe Triticeae have been found to have CENH3 in two variants. In this work, using the set of the wheat-rye addition lines we wanted to establish the chromosomal assignment of the CENH3 genes in the cultivated rye, Secale cereale (Linnaeus, 1753), in order to expand our knowledge about synteny conservation in the most important cereal species and about their chromosome evolution. To this end, we have also analyzed data in available genome sequencing databases. As a result, the ?CENH3 and ?CENH3 forms have been assigned to rye chromosomes 1R and 6R: specifically, the commonest variants ?CENH3v1 and ?CENH3v1 to chromosome 1R, and the rare variants, ?CENH3v2 and probably ?CENH3v2, to chromosome 6R. No other CENH3 variants have been found by analysis of the rye genome sequencing databases. Our chromosomal assignment of CENH3 in rye has been found to be the same as that in barley, suggesting that both main forms of CENH3 appeared in a Triticeae species before the barley and wheatrye lineages split.
Project description:Coeliac disease (CD) is an autoimmune disorder triggered by the ingestion of gluten that is associated with gastrointestinal issues, including diarrhea, abdominal pain, and malabsorption. Gluten is a general name for a class of cereal storage proteins of wheat, barley, and rye that are notably resistant to gastrointestinal digestion. After ingestion, immunogenic peptides are subsequently recognized by T cells in the gastrointestinal tract. The only treatment for CD is a life-long gluten-free diet. As such, it is critical to detect gluten in diverse food types, including those where one would not expect to find gluten. The utility of liquid chromatography-mass spectrometry (LC-MS) using cereal-specific peptide markers to detect gluten in heavily processed food types was assessed. A range of breakfast products, including breakfast cereals, breakfast bars, milk-based breakfast drinks, powdered drinks, and a savory spread, were tested. No gluten was detected by LC-MS in the food products labeled gluten-free, yet enzyme-linked immunosorbent assay (ELISA) measurement revealed inconsistencies in barley-containing products. In products containing wheat, rye, barley, and oats as labeled ingredients, gluten proteins were readily detected using discovery proteomics. Panels comprising ten cereal-specific peptide markers were analyzed by targeted proteomics, providing evidence that LC-MS could detect and differentiate gluten in complex matrices, including baked goods and milk-based products.
Project description:Rye, wheat and barley contain gluten, proteins that trigger immune-mediated inflammation of the small intestine in people with coeliac disease (CD). The only treatment for CD is a lifelong gluten-free diet. To be classified as gluten-free by the World Health Organisation the gluten content must be below 20 mg/kg, but Australia has a more rigorous standard of no detectable gluten and not made from wheat, barley, rye or oats. The purpose of this study was to devise an LC-MS/MS method to detect rye in food. An MS-based assay could overcome some of the limitations of current immunoassays, wherein antibodies often show cross-reactivity and lack specificity due to the diversity of gluten proteins in commercial food and the homology between rye and wheat gluten isoforms. Comprehensive proteomic analysis of 20 rye cultivars originating from 12 countries enabled the identification of a panel of candidate rye-specific peptide markers. The peptide markers were assessed in 16 cereal and pseudo-cereal grains, and in 10 breakfast cereals and 7 snacks foods. Spelt flour was contaminated with rye at a level of 2% and trace levels of rye were found in a breakfast cereal that based on its labelled ingredients should be gluten-free.
Project description:Rye (Secale cereale L.) is a cereal grass that is an important food crop in Central and Eastern Europe. In contrast to its close relatives wheat and barley, it was not a founder crop of Neolithic agriculture, but is considered a secondary domesticate that may have become a crop plant only after a transitory phase as a weed. As a minor crop of only local importance, genomic resources in rye are underdeveloped, and few population genetic studies using genomewide markers have been published to date. We collected genotyping-by-sequencing data for 603 individuals from 101 genebank accessions of domesticated rye and its wild progenitor S. cereale subsp. vavilovii and related species in the genus Secale. Variant detection in the context of a recently published draft sequence assembly of cultivated rye yielded 55,744 single nucleotide polymorphisms with present genotype calls in 90% of samples. Analysis of population structure recapitulated the taxonomy of the genus Secale. We found only weak genetic differentiation between wild and domesticated rye with likely gene flow between the two groups. Moreover, incomplete lineage sorting was frequent between Secale species because of either ongoing gene flow or recent speciation. Our study highlights the necessity of gauging the representativeness of ex situ germplasm collections for domestication studies and motivates a more in-depth analysis of the interplay between sequence divergence and reproductive isolation in the genus Secale.