Genome-wide identification of the plant homeodomain-finger family in rye and ScPHD5 functions in cold tolerance and flowering time.

KEY MESSAGE: 111 PHD genes were newly identified in rye genome and ScPHD5's role in regulating cold tolerance and flowering time was suggested. Plant homeodomain (PHD)-finger proteins regulate the physical properties of chromatin and control plant development and stress tolerance. Although rye (Secale cereale L.) is a major winter crop, PHD-finger proteins in rye have not been studied. Here, we identified 111 PHD genes in the rye genome that exhibited diverse gene and protein sequence structures. Phylogenetic tree analysis revealed that PHDs were genetically close in monocots and diverged from those in dicots. Duplication and synteny analyses demonstrated that ScPHDs have undergone several duplications during evolution and that high synteny is conserved among the Triticeae species. Tissue-specific and abiotic stress-responsive gene expression analyses indicated that ScPHDs were highly expressed in spikelets and developing seeds and were responsive to cold and drought stress. One of these genes, ScPHD5, was selected for further functional characterization. ScPHD5 was highly expressed in the spike tissues and was localized in the nuclei of rye protoplasts and tobacco leaves. ScPHD5-overexpressing Brachypodium was more tolerant to freezing stress than wild-type (WT), with increased CBF and COR gene expression. Additionally, these transgenic plants displayed an extremely early flowering phenotype that flowered more than two weeks earlier than the WT, and vernalization genes, rather than photoperiod genes, were increased in the WT. RNA-seq analysis revealed that diverse stress response genes, including HSPs, HSFs, LEAs, and MADS-box genes, were also upregulated in transgenic plants. Our study will help elucidate the roles of PHD genes in plant development and abiotic stress tolerance in rye.

CRISPR/CasΦ2-mediated gene editing in wheat and rye.

The compact CRISPR/CasΦ2 system provides a complementary genome engineering tool for efficient gene editing including cytosine and adenosine base editing in wheat and rye with high specificity, efficient use of the protospacer-adjacent motif TTN, and an alternative base-editing window.

Two functional CC-NBS-LRR proteins from rye chromosome 6RS confer differential age-related powdery mildew resistance to wheat.

Rye (Secale cereale), a valuable relative of wheat, contains abundant powdery mildew resistance (Pm) genes. Using physical mapping, transcriptome sequencing, barley stripe mosaic virus-induced gene silencing, ethyl methane sulfonate mutagenesis, and stable transformation, we isolated and validated two coiled-coil, nucleotide-binding site and leucine-rich repeat (CC-NBS-LRR) alleles, PmTR1 and PmTR3, located on rye chromosome 6RS from different triticale lines. PmTR1 confers age-related resistance starting from the three-leaf stage, whereas its allele, PmTR3, confers typical all-stage resistance, which may be associated with their differential gene expression patterns. Overexpression in Nicotiana benthamiana showed that the CC, CC-NBS, and CC-LRR fragments of PMTR1 induce cell death, whereas in PMTR3 the CC and full-length fragments perform this function. Luciferase complementation imaging and pull-down assays revealed distinct interaction activities between the CC and NBS fragments. Our study elucidates two novel rye-derived Pm genes and their derivative germplasm resources and provides novel insights into the mechanism of age-related resistance, which can aid the improvement of resistance against wheat powdery mildew.

Malate production, sugar metabolism, and redox homeostasis in the leaf growth zone of Rye (Secale cereale) increase stress tolerance to aluminum stress: A biochemical and genome-wide transcriptional study.

Global soil acidification is increasing, enlarging aluminum (Al) availability in soils, leading to reductions in plant growth. This study investigates the effect of Al stress on the leaf growth zones of Rye (Secale cereale, cv Beira). Kinematic analysis showed that the effect of Al on leaf growth rates was mainly due to a reduced cell production rate in the meristem. Transcriptomic analysis identified 2272 significantly (log2fold > |0.5| FDR < 0.05) differentially expressed genes (DEGs) for Al stress. There was a downregulation in several DEGs associated with photosynthetic processes and an upregulation in genes for heat/light response, and H2O2 production in all leaf zones. DEGs associated with heavy metals and malate transport were increased, particularly, in the meristem. To determine the putative function of these processes in Al tolerance, we performed biochemical analyses comparing the tolerant Beira with an Al sensitive variant RioDeva. Beira showed improved sugar metabolism and redox homeostasis, specifically in the meristem compared to RioDeva. Similarly, a significant increase in malate and citrate production, which are known to aid in Al detoxification in plants, was found in Beira. This suggests that Al tolerance in Rye is linked to its ability for Al exclusion from the leaf meristem.

Genome-wide identification and functional analysis of the TCP gene family in rye (Secale cereale L.).

TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) proteins are plant-specific transcription factors that play significant roles in plant growth, development, and stress response. Rye is a high-value crop with strong resistance to adverse environments. However, the functions of TCP proteins in rye are rarely reported. Based on a genome-wide analysis, the present study identified 26 TCP genes (ScTCPs) in rye. Mapping showed an uneven distribution of the ScTCP genes on the seven rye chromosomes and detected three pairs of tandem duplication genes. Phylogenetic analysis divided these genes into PCF (Proliferrating Cell Factors), CIN (CINCINNATA), and CYC (CYCLOIDEA)/TB1 (Teosinte Branched1) classes, which showed the highest homology between rye and wheat genes. Analysis of miRNA targeting sites indicated that five ScTCP genes were identified as potential targets of miRNA319. Promoter cis-acting elements analysis indicated that ScTCPs were regulated by light signals. Further analysis of the gene expression patterns and functional annotations suggested the role of a few ScTCPs in grain development and stress response. In addition, two TB1 homologous genes (ScTCP9 and ScTCP10) were identified in the ScTCP family. Synteny analysis showed that TB1 orthologous gene pairs existed before the ancestral divergence. Finally, the yeast two-hybrid assay and luciferase complementation imaging assay proved that ScTCP9, localized in the nucleus, interacts with ScFT (Flowering locus T), indicating their role in regulating flowering time. Taken together, this comprehensive study of ScTCPs provides important information for further research on gene function and crop improvement.

Subcellular compartmentalization of aluminum reduced its hazardous impact on rye photosynthesis.

Aluminum (Al) toxicity limits crops growth and production in acidic soils. Compared to roots, less is known about the toxic effects of Al in leaves. Al subcellular compartmentalization is also largely unknown. Using rye (Secale cereale L.) Beira (more tolerant) and RioDeva (more sensitive to Al) genotypes, we evaluated the patterns of Al accumulation in leaf cell organelles and the photosynthetic and metabolic changes to cope with Al toxicity. The tolerant genotype accumulated less Al in all organelles, except the vacuoles. This suggests that Al compartmentalization plays a role in Al tolerance of Beira genotype. PSII efficiency, stomatal conductance, pigment biosynthesis, and photosynthesis metabolism were less affected in the tolerant genotype. In the Calvin cycle, carboxylation was compromised by Al exposure in the tolerant genotype. Other Calvin cycle-related enzymes, phoshoglycerate kinase (PGK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), triose-phosphate isomerase (TPI), and fructose 1,6-bisphosphatase (FBPase) activities decreased in the sensitive line after 48 h of Al exposure. Consequentially, carbohydrate and organic acid metabolism were affected in a genotype-specific manner, where sugar levels increased only in the tolerant genotype. In conclusion, Al transport to the leaf and compartmentalization in the vacuoles tolerant genotype's leaf cells provide complementary mechanisms of Al tolerance, protecting the photosynthetic apparatus and thereby sustaining growth.

Use of the ß-Glucan-Producing Lactic Acid Bacteria Strains Levilactobacillus brevis and Pediococcus claussenii for Sourdough Fermentation-Chemical Characterization and Chemopreventive Potential of In Situ-Enriched Wheat and Rye Sourdoughs and Breads.

The aim of the present study was to examine ß-glucan production and the potential prebiotic and chemopreventive effects of wheat and rye sourdoughs and breads generated with wild-type and non-ß-glucan-forming isogenic mutant strains of Levilactobacillus brevis and Pediococcus claussenii. Sourdough and bread samples were subjected to in vitro digestion and fermentation. Fermentation supernatants (FS) and pellets (FP) were analyzed (pH values, short-chain fatty acids (SCFA), ammonia, bacterial taxa) and the effects of FS on LT97 colon adenoma cell growth, viability, caspase-2 and -3 activity, genotoxic and antigenotoxic effects and on gene and protein expression of p21, cyclin D2, catalase and superoxide dismutase 2 (SOD2) were examined. Concentrations of SCFA were increased and concentrations of ammonia were partly reduced in the FS. The relative abundance of Bifidobacteriaceae was increased in all FPs. Treatment with FS reduced the growth and viability of LT97 cells and significantly increased caspase-2 and -3 activities without exhibiting genotoxic or antigenotoxic effects. The p21 mRNA and protein levels were increased while that of cyclin D2 was reduced. Catalase and SOD2 mRNA and protein expression were marginally induced. The presented results indicate a comparable chemopreventive potential of wheat and rye sourdoughs and breads without an additional effect of the formed ß-glucan.

Proteolytic and Structural Changes in Rye and Triticale Roots under Aluminum Stress.

Proteolysis and structural adjustments are significant for defense against heavy metals. The purpose of this study was to evaluate whether the Al3+ stress alters protease activity and the anatomy of cereale roots. Azocaseinolytic and gelatinolytic measurements, transcript-level analysis of phytocystatins, and observations under microscopes were performed on the roots of Al3+-tolerant rye and tolerant and sensitive triticales exposed to Al3+. In rye and triticales, the azocaseinolytic activity was higher in treated roots. The gelatinolytic activity in the roots of rye was enhanced between 12 and 24 h in treated roots, and decreased at 48 h. The gelatinolytic activity in treated roots of tolerant triticale was the highest at 24 h and the lowest at 12 h, whereas in treated roots of sensitive triticale it was lowest at 12 h but was enhanced at 24 and 48 h. These changes were accompanied by increased transcript levels of phytocystatins in rye and triticale-treated roots. Light microscope analysis of rye roots revealed disintegration of rhizodermis in treated roots at 48 h and indicated the involvement of root border cells in rye defense against Al3+. The ultrastructural analysis showed vacuoles containing electron-dense precipitates. We postulate that proteolytic-antiproteolytic balance and structural acclimation reinforce the fine-tuning to Al3+.

Identification of rye B chromosome-associated peptides by mass spectrometry.

B chromosomes (Bs) are supernumerary dispensable components of the standard genome (A chromosomes, As) that have been found in many eukaryotes. So far, it is unkown whether the B-derived transcripts translate to proteins or if the host proteome is changed due to the presence of Bs. Comparative mass spectrometry was performed using the protein samples isolated from shoots of rye plants with and without Bs. We aimed to identify B-associated peptides and analyzed the effects of Bs on the total proteome. Our comparative proteome analysis demonstrates that the presence of rye Bs affects the total proteome including different biological function processes. We found 319 of 16 776 quantified features in at least three out of five +B plants but not in 0B plants; 31 of 319 features were identified as B-associated peptide features. According to our data mining, one B-specific protein fragment showed similarity to a glycine-rich RNA binding protein which differed from its A-paralogue by two amino acid insertions. Our result represents a milestone in B chromosome research, because this is the first report to demonstrate the existence of Bs changing the proteome of the host.

An efficient Oligo-FISH painting system for revealing chromosome rearrangements and polyploidization in Triticeae.

A chromosome-specific painting technique has been developed which combines the most recent approaches of the companion disciplines of molecular cytogenetics and genome research. We developed seven oligonucleotide (oligo) pools derivd from single-copy sequences on chromosomes 1 to 7 of barley (Hordeum vulgare L.) and corresponding collinear regions of wheat (Triticum aestivum L.). The seven groups of pooled oligos comprised between 10 986 and 12 496 45-bp monomers, and these then produced stable fluorescence in situ hybridization (FISH) signals on chromosomes of each linkage group of wheat and barley. The pooled oligo probes were applied to high-throughput karyotyping of the chromosomes of other Triticeae species in the genera Secale, Aegilops, Thinopyrum, and Dasypyrum, and the study also extended to some wheat-alien amphiploids and derived lines. We demonstrated that a complete set of whole-chromosome oligo painting probes facilitated the study of inter-species chromosome homologous relationships and visualized non-homologous chromosomal rearrangements in Triticeae species and some wheat-alien species derivatives. When combined with other non-denaturing FISH procedures using tandem-repeat oligos, the newly developed oligo painting techniques provide an efficient tool for the study of chromosome structure, organization, and evolution among any wild Triticeae species with non-sequenced genomes.

Application of Tris-HCl Allows the Specific Labeling of Regularly Prepared Chromosomes by CRISPR-FISH.

Visualizing the spatiotemporal organization of the genome will improve our understanding of how chromatin structure and function are intertwined. Here, we describe the further development of the RNA-guided endonuclease-in situ labeling (RGEN-ISL) method CRISPR-FISH. Using soybean and mouse chromosomes, we demonstrate that the treatment of conventionally fixed chromosomes (in ethanol or methanol:acetic acid) with 40 mM Tris-HCl (pH 9.0) for 30 minutes at 37°C prior to CRISPR-FISH allows the application of this method for the detection of high-copy sequences. Wheat, rye, maize, and Nicotiana benthamiana were used to confirm the applicability of the identified CRISPR-FISH conditions also in other species.

Identification and characterization of a new stripe rust resistance gene Yr83 on rye chromosome 6R in wheat.

KEY MESSAGE: A physical map of Secale cereale chromosome 6R was constructed using deletion mapping, and a new stripe rust resistance gene Yr83 was mapped to the deletion bin of FL 0.73-1.00 of 6RL. Rye (Secale cereale L., RR) possesses valuable genes for wheat improvement. In the current study, we report a resistance gene conferring stripe rust resistance effective from seedling to adult plant stages located on chromosome 6R. This chromosome was derived from triticale line T-701 and also carries highly effective resistance to the cereal cyst nematode species Heterodera avenae Woll. A wheat-rye 6R(6D) disomic substitution line exhibited high levels of seedling resistance to Australian pathotypes of the stripe rust (Puccinia striiformis f. sp. tritici; Pst) pathogen and showed an even greater resistance to the Chinese Pst pathotypes in the field. Ten chromosome 6R deletion lines and five wheat-rye 6R translocation lines were developed earlier in the attempt to transfer the nematode resistance gene to wheat and used herein to map the stripe rust resistance gene. These lines were subsequently characterized by sequential multicolor fluorescence in situ hybridization (mc-FISH), genomic in situ hybridization (GISH), mc-GISH, PCR-based landmark unique gene (PLUG), and chromosome 6R-specific length amplified fragment sequencing (SLAF-Seq) marker analyses to physically map the stripe rust resistance gene. The new stripe rust resistance locus was located in a chromosomal bin with fraction length (FL) 0.73-1.00 on 6RL and was named Yr83. A wheat-rye translocation line T6RL (#5) carrying the stripe rust resistance gene will be useful as a new germplasm in breeding for resistance.

Catcher of the Rye: Detection of Rye, a Gluten-Containing Grain, by LC-MS/MS.

Rye, wheat, and barley contain gluten, proteins that trigger immune-mediated inflammation of the small intestine in people with celiac disease (CD). The only treatment for CD is a lifelong gluten-free diet. To be classified as gluten-free by the World Health Organization 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 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 pseudocereal grains, and in 10 breakfast cereals and 7 snack foods. One of two spelt flours assessed was contaminated with rye at a level of 2%, and trace levels of rye were found in a breakfast cereal that should be gluten-free based on its labeled ingredients.

Qualitative Study on the Production of the Allelochemicals Benzoxazinones by Inducing Polyploidy in Gramineae with Colchicine.

The possibility of inducing polyploidy in grasses by treatment with colchicine and its effect on the production and root exudate content of 2,4-dihydroxy-7-methoxy-2 H-1,4-benzoxazin-3-one (DIMBOA) and 2,4-dihydroxy-2 H-benzoxazin-3-one (DIBOA) was studied in wheat, corn, and rye. Caryopses treated with colchicine at concentrations in the range of 0.1-10 mg/mL for 8 and 48 h and with inoculation of the growth medium are markedly affected in terms of both the distribution and concentration levels of allelochemicals in plants. A greater accumulation was observed in the root with respect to the stem, and this increased with an increasing concentration of colchicine and with treatment time. Analysis of the compounds released by root exudates showed that treatment with colchicine at a concentration higher than 1 mg/mL caused a significant increase in the concentrations of allelochemicals measured in the growth medium. It is proposed that treatment with colchicine of seedling caryopses mixoploids plant populations and that the overall effect is an increase in the levels of allelochemicals released. The ecological implications of this behavior are discussed along with the impact of plant-plant interactions (allelopathy).

Reorganization of wheat and rye genomes in octoploid triticale (× Triticosecale).

MAIN CONCLUSION: The analysis of early generations of triticale showed numerous rearrangements of the genome. Complexed transformation included loss of chromosomes, t-heterochromatin content changes and the emergence of retrotransposons in new locations. This study investigated certain aspects of genomic transformations in the early generations (F5 and F8) of the primary octoploid triticale derived from the cross of hexaploid wheat with the diploid rye. Most of the plants tested were hypoploid; among eliminated chromosomes were rye chromosomes 4R and 5R and variable number of wheat chromosomes. Wheat chromosomes were eliminated to a higher extent. The lower content of telomeric heterochromatin was also found in rye chromosomes in comparison with parental rye. Studying the location of selected retrotransposons from Ty1-copia and Ty3-gypsy families using fluorescence in situ hybridization revealed additional locations of these retrotransposons that were not present in chromosomes of parental species. ISSR, IRAP and REMAP analyses showed significant changes at the level of specific DNA nucleotide sequences. In most cases, the disappearance of certain types of bands was observed, less frequently new types of bands appeared, not present in parental species. This demonstrates the scale of genome rearrangement and, above all, the elimination of wheat and rye sequences, largely due to the reduction of chromosome number. With regard to the proportion of wheat to rye genome, the rye genome was more affected by the changes, thus this study was focused more on the rye genome. Observations suggest that genome reorganization is not finished in the F5 generation but is still ongoing in the F8 generation.

The changes in the reproductive barrier between hexaploid wheat (Triticum aestivum L.) and rye (Secale cereale L.): different states lead to different fates.

MAIN CONCLUSION: The changes in the reproductive barrier between hexaploid wheat ( Triticum aestivum L.) and rye ( Secale cereale L.) can be induced using in situ embryo rescue of abnormal embryos, yielding stable fertile amphidiploid plants. In intergeneric crosses between hexaploid wheat (Triticum aestivum L.) and rye (Secale cereale L.), postzygotic barriers may occur at different stages of hybrid development. One such mechanism is embryo lethality, which is genetically determined by the interaction and expression of two incompatible genes in wheat (Eml-A1) and rye (Eml-R1). Using in vitro culture methods as stressors, we overcame this hybrid lethality. Normal and abnormal embryos were observed to build embryogenic calli and produce regenerated plantlets in a similar manner. The high regenerative capacity of the abnormal embryos led us to conclude that the reproductive barrier in these intergeneric hybrids may have an epigenetic origin that can be easily overcome by culturing immature embryos via callus induction. After colchicine treatment during callus culture, amphidiploid plants were obtained. However, most of these plants did not produce seeds, due mainly to sterility of the pollen but also of the embryo sacs. These findings demonstrate that hybrid sterility affects both male and female gametophytes in plants obtained from abnormal embryos. The key roles of double fertilization and stress factors in the implementation of the apical meristem formation program in embryos from incompatible intergeneric crosses between hexaploid wheat and rye during in vitro culture are discussed. We also propose a hypothetical model for a wheat-rye lethality system involving differential expression of incompatible wheat Eml-A1 and rye Eml-R1b alleles in an identical genetic background.

Divergent Development of Hexaploid Triticale by a Wheat - Rye -Psathyrostachys huashanica Trigeneric Hybrid Method.

Hexaploid triticale is an important forage crop and a promising energy plant. Some forms were previously reported for developing the hexaploid triticale, such as crossing tetraploid wheat or hexaploid wheat with rye, crossing hexaploid triticale and/or hexaploid wheat with octoploid triticale, and spontaneously appearing in the selfed progenies of octoploid triticale. In the present study, we developed an effective method for production of diverse types of hexaploid triticale via wheat-rye-Psathyrostachys huashanica trigeneric hybrid. Genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH) karyotyping revealed that D genome chromosomes were completely eliminated and the whole A, B, and R genome chromosomes were retained in three lines. More interestingly, the composite genome of the line K14-489-2 consisted of complete A and B genomes and chromosomes 1D, 2R, 3R, 4R, 5R, 6R, and 7R, that of line K14-491-2 was 12 A-genome (1A-6A), 14 B-genome (1B-7B), 12 R-genome (1R-3R, 5R-7R), and chromosomes 1D and 3D, and that of the line K14-547-1 had 26A/B and 14R chromosomes, plus one pair of centric 6BL/2DS translocations. This finding implies that some of D genome chromosomes can be spontaneously and stably incorporated into the hexaploid triticale. Additionally, a variety of high-molecular-weight glutenin subunits (HMW-GS) compositions were detected in the six hexaploid triticale lines, respectively. Besides, compared with its recurrent triticale parent Zhongsi828, these lines showed high level of resistance to stripe rust (Puccinia striiformis f. sp. tritici, Pst) pathogens prevalent in China, including V26/Gui 22. These new hexaploid triticales not only enhanced diversification of triticale but also could be utilized as valuable germplasm for wheat improvement.

Cytogenetic Behavior of Trigeneric Hybrid Progeny Involving Wheat, Rye and Psathyrostachys huashanica.

Trigeneric hybrids are commonly used as bridges to transfer genes from some wild species to cultivated wheat and to measure the genomic interaction between donor species. We previously reported that trigeneric germplasms were produced by crossing wheat-Psathyrostachys huashanica amphiploids (PHW-SA, 2n = 8x = 56, AABBDDNsNs) with hexaploid triticale (Zhongsi 828, 2n = 6x = 42, AABBRR). In the present study, chromosome pairing behavior and the genome constitution of the F4 progenies of wheat-rye-P. huashanica trigeneric hybrids were studied. Cytological analysis showed that the chromosome number of F4 progenies ranged from 39 to 46, and 57.5% of them had 42 chromosomes. The mean meiotic configuration of F4 lines was 1.71 univalents, 20.26 bivalents, 0.04 trivalents, and 0.001 quadrivalents per pollen mother cell. Among the lines with 2n = 42, the average pairing configuration was 1.21 univalents, 16.22 ring bivalents, 4.16 rod bivalents, and 0.01 trivalents. This result indicated that these lines were cytologically stable. Other lines with 2n = 39, 40, 41, 43, 44, 45, and 46, bearing a high number of univalents or multivalents, showed abnormal meiotic behavior. Genomic in situ hybridization (GISH) revealed that all F4 lines had 11-14 rye chromosomes, but no P. huashanica chromosomes. The complete set of 14 rye chromosomes was found in 19 lines. At meiosis, GISH detected 1-6 univalents with hybridization signals of rye in 13 lines. Bivalents with fluorescence signals were identified in each line, ranging from 3 to 7. A quadrivalent with hybridization signals was observed in only 1 line, K13-714-8. Lagging chromosomes, chromosome bridges, micronuclei, and chromosome fragments hybridizing with the probe were not discovered in any of the lines. These results inferred that the behavior of rye chromosomes was normal during meiosis. In addition, 21 lines of 2n = 42 (91.3%) with 12 or 14 rye chromosomes, always contained 6 or 7 bivalents bearing fluorescence signals. This suggested that the rye chromosomes exhibiting complete pairing in these lines were cytologically stable during meiosis and may therefore be considered as new hexaploid triticales. Thus, these lines might be potential materials for further hexaploid triticale improvement.

Immunological Investigation for the Presence of Lunasin, a Chemopreventive Soybean Peptide, in the Seeds of Diverse Plants.

Lunasin, a 44 amino acid soybean bioactive peptide, exhibits anticancer and anti-inflammatory properties. All soybean varieties that have been examined contain lunasin. It has also been reported in a few other plant species including amaranth, black nightshade, wheat, barley, rye, and triticale. Interestingly, detailed searches of transcriptome and DNA sequence databases of cereals failed to identify lunasin-coding sequences, raising questions about the authenticity of lunasin in cereals. To clarify the presence or absence of lunasin in cereals and other plant species, an immunological investigation was conducted utilizing polyclonal antibodies raised against the first 20 amino acid N-terminal peptide (SKWQHQQDSCRKQLQGVNLT) and a 15 amino acid C-terminal peptide (CEKHIMEKIQGRGDD) of lunasin. Protein blot analyses revealed the presence of proteins from several plants that reacted against the lunasin N-terminal peptide antibodies. However, the same proteins failed to react against the lunasin C-terminal peptide antibodies. These results demonstrate that peptides identical to soybean lunasin are absent in seeds of diverse plants examined in this study.

Transmission of the Aegilops ovata chromosomes carrying gametocidal factors in hexaploid triticale (×Triticosecale Wittm.) hybrids.

The main aim of this work was to induce the chromosome rearrangements between Aegilops ovata (UUMM) and hexaploid triticale (AABBRR) by expression of the gametocidal factor located on the chromosome 4M. The Aegilops ovata × Secale cereale (UUMMRR) amphiploids and triticale 'Moreno' were used to produce hybrids by reciprocal crosses. Chromosome dynamics was observed in subsequent generations of hybrids during mitotic metaphase of root meristems and first metaphase of meiosis of pollen mother cells. Chromosomes were identified by genomic in situ hybridisation (GISH) and fluorescence in situ hybridisation (FISH) using pTa71, pTa791, pSc119.2 and pAs1 DNA probes. It has been shown that the origin of the genetic background had an influence on Aegilops chromosome transmission. Moreover, it has been reported that the preferential transmission of chromosome 4M appeared during both androgenesis and gynogenesis. It is also hypothesised that the expression of the triticale Gc gene suppressor had an influence on the semi-fertility of hybrids but did not inhibit the chromosome rearrangements. This paper also describes the double haploid production, which enabled to obtain plants with two identical copies of triticale chromosomes with translocations of Aegilops chromatin segments.