Photosystems and antioxidative system of rye, wheat and triticale under Pb stress.

Lead (Pb2+) pollution in the soil sub-ecosystem has been a continuously growing problem due to economic development and ever-increasing anthropogenic activities across the world. In this study, the photosynthetic performance and antioxidant capacity of Triticeae cereals (rye, wheat and triticale) were compared to assess the activities of antioxidants, the degree of oxidative damage, photochemical efficiency and the levels of photosynthetic proteins under Pb stress (0.5 mM, 1 mM and 2 mM Pb (NO3)2). Compared with triticale, Pb treatments imposed severe oxidative damage in rye and wheat. In addition, the highest activity of major antioxidant enzymes (SOD, POD, CAT, and GPX) was also found to be elevated. Triticale accumulated the highest Pb contents in roots. The concentration of mineral ions (Mg, Ca, and K) was also high in its leaves, compared with rye and wheat. Consistently, triticale showed higher photosynthetic activity under Pb stress. Immunoblotting of proteins revealed that rye and wheat have significantly lower levels of D1 (photosystem II subunit A, PsbA) and D2 (photosystem II subunit D, PsbD) proteins, while no obvious decrease was noticed in triticale. The amount of light-harvesting complex II b6 (Lhcb6; CP24) and light-harvesting complex II b5 (Lhcb5; CP26) was significantly increased in rye and wheat. However, the increase in PsbS (photosystem II subunit S) protein only occurred in wheat and triticale exposed to Pb treatment. Taken together, these findings demonstrate that triticale shows higher antioxidant capacity and photosynthetic efficiency than wheat and rye under Pb stress, suggesting that triticale has high tolerance to Pb and could be used as a heavy metal-tolerant plant.

Genomic Scan of Male Fertility Restoration Genes in a 'Gülzow' Type Hybrid Breeding System of Rye (Secale cereale L.).

Efficient and stable restoration of male fertility (Rf) is a prerequisite for large-scale hybrid seed production but remains an inherent issue in the predominant fertility control system of rye (Secale cereale L.). The 'Gülzow' (G)-type cytoplasmic male sterility (CMS) system in hybrid rye breeding exhibits a superior Rf. While having received little scientific attention, one major G-type Rf gene has been identified on 4RL (Rfg1) and two minor genes on 3R (Rfg2) and 6R (Rfg3) chromosomes. Here, we report a comprehensive investigation of the genetics underlying restoration of male fertility in a large G-type CMS breeding system using recent advents in rye genomic resources. This includes: (I) genome-wide association studies (GWAS) on G-type germplasm; (II) GWAS on a biparental mapping population; and (III) an RNA sequence study to investigate the expression of genes residing in Rf-associated regions in G-type rye hybrids. Our findings provide compelling evidence of a novel major G-type non-PPR Rf gene on the 3RL chromosome belonging to the mitochondrial transcription termination factor gene family. We provisionally denote the identified novel Rf gene on 3RL RfNOS1. The discovery made in this study is distinct from known P- and C-type systems in rye as well as recognized CMS systems in barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.). We believe this study constitutes a stepping stone towards understanding the restoration of male fertility in the G-type CMS system and potential resources for addressing the inherent issues of the P-type system.

ß-1,3-Glucanases and chitinases participate in the stress-related defence mechanisms that are possibly connected with modulation of arabinogalactan proteins (AGP) required for the androgenesis initiation in rye (Secale cereale L.).

This work presents the biochemical, cytochemical and molecular studies on two groups of PR proteins, ß-1,3-glucanases and chitinases, and the arabinogalactan proteins (AGP) during the early stages of androgenesis induction in two breeding lines of rye (Secale cereale L.) with different androgenic potential. The process of androgenesis was initiated by tillers pre-treatments with low temperature, mannitol and/or reduced glutathione and resulted in microspores reprogramming and formation of androgenic structures what was associated with high activity of ß-1,3-glucanases and chitinases. Some isoforms of ß-1,3-glucanases, namely several acidic isoforms of about 26 kDa; appeared to be anther specific. Chitinases were well represented but were less variable. RT-qPCR revealed that the cold-responsive chitinase genes Chit1 and Chit2 were expressed at a lower level in the microspores and whole anthers while the cold-responsive Glu2 and Glu3 were not active. The stress pre-treatments modifications promoted the AGP accumulation. An apparent dominance of some AGP epitopes (LM2, JIM4 and JIM14) was detected in the androgenesis-responsive rye line. An abundant JIM13 epitopes in the vesicles and inner cell walls of the microspores and in the cell walls of the anther cell layers appeared to be the most specific for embryogenesis.

Characterization of an alkylresorcinol synthase that forms phenolics accumulating in the cuticular wax on various organs of rye (Secale cereale).

Alkylresorcinols are bioactive compounds produced in diverse plant species, with chemical structures combining an aliphatic hydrocarbon chain and an aromatic ring with characteristic hydroxyl substituents. Here, we aimed to isolate and characterize the enzyme that forms the alkylresorcinols accumulating in the cuticular wax on the surface of all above-ground organs of rye. Based on sequence homology with other type-III polyketide synthases, a candidate alkylresorcinol synthase was cloned. Yeast heterologous expression showed that the enzyme, ScARS, is highly specific for the formation of the aromatic resorcinol ring structure, through aldol condensation analogous to stilbene synthases. The enzyme accepts long-chain and very-long-chain acyl-CoA starter substrates, preferring saturated over unsaturated chains. It typically carries out three rounds of condensation with malonyl-CoA prior to cyclization, with only very minor activity for a fourth round of malonyl-CoA condensation and cyclization to 5-(2'-oxo)-alkylresorcinols or 5-(2'-hydroxy)-alkylresorcinols. Like other enzymes involved in cuticle formation, ScARS is localized to the endoplasmic reticulum. ScARS expression patterns were found correlated with alkylresorcinol accumulation during leaf development and across different rye organs. Overall, our results thus suggest that ScARS synthesizes the cuticular alkylresorcinols found on diverse rye organ surfaces.

Activity of Escherichia coli, Aspergillus niger, and Rye Phytase toward Partially Phosphorylated myo-Inositol Phosphates.

Kinetic parameters for the dephosphorylation of sodium phytate and a series of partially phosphorylated myo-inositol phosphates were determined at pH 3.0 and pH 5.0 for three phytase preparations (Aspergillus niger, Escherichia coli, rye). The enzymes showed lower affinity and turnover numbers at pH 3 compared to pH 5 toward all myo-inositol phosphates included in the study. The number and distribution of phosphate groups on the myo-inositol ring affected the kinetic parameters. Representatives of the individual phytate dephosphorylation pathways were identified as the best substrates of the phytases. Within the individual phytate dephosphorylation pathways, the pentakisphosphates were better substrates compared to the tetrakisphosphates or phytate itself. E. coli and rye phytase showed comparable activities at both pH values toward the tetrakis- and trisphosphate, whereas A. niger phytase exhibited a higher activity toward the tetrakisphosphate. A myo-inositol phosphate with alternate phosphate groups was shown to be not significantly dephosphorylated by the phytases.

Biosynthesis and chemical transformation of benzoxazinoids in rye during seed germination and the identification of a rye Bx6-like gene.

Benzoxazinoids are secondary metabolites with plant defense properties and possible health-promoting effects in humans. In this study, the transcriptional activity of ScBx genes (ScBx1-ScBx5; ScBx6-like), involved in benzoxazinoid biosynthesis, was analyzed during germination and early seedling development in rye. Our results showed that ScBx genes had highest levels of expression at 24-30 h after germination, followed by a decrease at later stages. For ScBx1-ScBx5 genes expression was higher in shoots compared with root tissues and vice versa for ScBx6-like gene transcripts. Moreover, methylated forms of benzoxazinoids accumulated in roots rather than in shoots during seedling development, in particular reaching high levels of HMBOA-glc in roots. Chemical profiles of benzoxazinoid accumulation in the developing seedling reflected the combined effects of de novo biosynthesis of the compounds as well as the turnover of compounds either pre-stored in the embryo or de novo biosynthesized. Bioinformatic analysis, together with the differential distribution of ScBx6-like transcripts in root and shoot tissues, suggested the presence of a ZmBx6 homolog encoding a 2-oxoglutarate dependent dehydrogenase in rye. The ScBx6-like cDNA was expressed in E. coli for functional characterization in vitro. LC-MS/MS analysis showed that the purified enzyme was responsible for the oxidation of DIBOA-glc into TRIBOA-glc, strongly suggesting the ScBX6-like enzyme in rye to be a functional ortholog of maize ZmBX6.

Rye B chromosomes encode a functional Argonaute-like protein with in vitro slicer activities similar to its A chromosome paralog.

B chromosomes (Bs) are supernumerary, dispensable parts of the nuclear genome, which appear in many different species of eukaryote. So far, Bs have been considered to be genetically inert elements without any functional genes. Our comparative transcriptome analysis and the detection of active RNA polymerase II (RNAPII) in the proximity of B chromatin demonstrate that the Bs of rye (Secale cereale) contribute to the transcriptome. In total, 1954 and 1218 B-derived transcripts with an open reading frame were expressed in generative and vegetative tissues, respectively. In addition to B-derived transposable element transcripts, a high percentage of short transcripts without detectable similarity to known proteins and gene fragments from A chromosomes (As) were found, suggesting an ongoing gene erosion process. In vitro analysis of the A- and B-encoded AGO4B protein variants demonstrated that both possess RNA slicer activity. These data demonstrate unambiguously the presence of a functional AGO4B gene on Bs and that these Bs carry both functional protein coding genes and pseudogene copies. Thus, B-encoded genes may provide an additional level of gene control and complexity in combination with their related A-located genes. Hence, physiological effects, associated with the presence of Bs, may partly be explained by the activity of B-located (pseudo)genes.

Characterization and expression analyses of the H⁺-pyrophosphatase gene in rye.

The H⁺-pyrophosphatase (H⁺-PPase) gene plays an important role in maintaining intracellular proton gradients. Here, we characterized the full-length complementary DNA (cDNA) and DNA of the H⁺-PPase gene ScHP1 in rye (Secale cereale L. 'Qinling'). We determined the subcellular localization of this gene and predicted the corresponding protein structure. We analysed the evolutionary relationship between ScHP1 and H⁺-PPase genes in other species, and did real-time quantitative polymerase chain reaction to explore the expression patterns of ScHP1 in rye plants subjected to N, P and K deprivation and to cold, high-salt and drought stresses. ScHP1 cDNA included a 2289 bp open reading frame (ORF) encoding 762 amino acid residues with 14 transmembrane domains. The genomic ScHP1 DNA was 4354 bp and contained eight exons and seven introns. ScHP1 was highly homologous with other members of the H⁺-PPase gene family. When the full-length ORF was inserted into the expression vector pA7-YFP, the fluorescent microscopy revealed that ScHP1-YFP fusion protein was located in the plasma membrane. Rye plants that were subjected to N deprivation, cold and high-salt stresses, ScHP1 expression was higher in the leaves than roots. Conversely, plants subjected to P and K deprivation and drought stress, ScHP1 expression was higher in the roots than leaves. Under all the investigated stress conditions, expression of ScHP1 was lower in the stem than in the leaves and roots. Our results imply that ScHP1 functions under abiotic stress response.

Soluble and cell wall-bound phenolic acids and ferulic acid dehydrodimers in rye flour and five bread model systems: insight into mechanisms of improved availability.

BACKGROUND: The bread-making process influences bread components, including phenolics that significantly contribute to its antioxidant properties. Five bread model systems made from different rye cultivars were investigated to compare their impact on concentration of ethanol-soluble (free and ester-bound) and insoluble phenolics. RESULTS: Breads produced by a straight dough method without acid addition (A) and three-stage sourdough method with 12 h native starter preparation (C) exhibited the highest, genotype-dependent concentrations of free phenolic acids. Dough acidification by direct acid addition (method B) or by gradual production during prolonged starter fermentation (24 and 48 h, for methods D and E) considerably decreased their level. However, breads B were enriched in soluble ester-bound fraction. Both direct methods, despite substantial differences in dough pH, caused a similar increase in the amount of insoluble ester-bound fraction. The contents of phenolic fractions in rye bread were positively related to activity level of feruloyl esterase and negatively to those of arabinoxylan-hydrolysing enzymes in wholemeal flour. CONCLUSION: The solubility of rye bread phenolics may be enhanced by application of a suitable bread-making procedure with respect to rye cultivar, as the mechanisms of this process are also governed by a response of an individual genotype with specific biochemical profile.

On the consequences of aluminium stress in rye: repression of two mitochondrial malate dehydrogenase mRNAs.

Plants have developed several external and internal aluminium (Al) tolerance mechanisms. The external mechanism best characterised is the exudation of organic acids induced by Al. Rye (Secale cereale L.), one of the most Al-tolerant cereal crops, secretes both citrate and malate from its roots in response to Al. However, the role of malate dehydrogenase (MDH) genes in Al-induced stress has not been studied in rye. We have isolated the ScMDH1 and ScMDH2 genes, encoding two different mitochondrial MDH isozymes, in three Al-tolerant rye cultivars (Ailés, Imperial and Petkus) and one sensitive inbred rye line (Riodeva). These genes, which have seven exons and six introns, were located on the 1R (ScMDH1) and 3RL (ScMDH2) chromosomes. Exon 1 of ScMDH1 and exon 7 of ScMDH2 were the most variable among the different ryes. The hypothetical proteins encoded by these genes were classified as putative mitochondrial MDH isoforms. The phylogenetic relationships obtained using both cDNA and protein sequences indicated that the ScMDH1 and ScMDH2 proteins are orthologous to mitochondrial MDH1 and MDH2 proteins of different Poaceae species. The expression studies of the ScMDH1 and ScMDH2 genes indicate that it is more intense in roots than in leaves. Moreover, the amount of their corresponding mRNAs in roots from plants treated and not treated with Al was higher in the tolerant cultivar Petkus than in the sensitive inbred line Riodeva. In addition, ScMDH1 and ScMDH2 mRNA levels decreased in response to Al stress (repressive behaviour) in the roots of both the tolerant Petkus and the sensitive line Riodeva.

Application of the Taguchi method in poultry science: estimation of the in vitro optimum intrinsic phytase activity of rye, wheat and barley.

1. In poultry investigations, the main interest is often to study the effects of many factors simultaneously. Two or three level factorial designs are the most commonly used for this type of investigation. However, it is often too costly to perform when number of factors increase. So a fractional factorial design, which is a subset or a fraction of a full factorial design, is an alternative. The Taguchi method has been proposed for simplifying and standardising fractional factorial designs. 2. An experiment was conducted to evaluate the applicability of the Taguchi method to optimise in vitro intrinsic phytase activity (IPA) of rye, wheat and barley under different culture conditions. 3. In order to have a solid base for judging the suitability of the Taguchi method, the results of the Taguchi method were compared with those of an experiment that was conducted as a 3(4) full factorial arrangement with three feed ingredients (rye, wheat and barley), three temperatures (20°C, 38°C and 55°C), three pH values (3.0, 5.5 and 8.0) and three incubation times (30, 60 and 120 min), with two replicates per treatment. 4. After data collection, a Taguchi L 9 (3(4)) orthogonal array was used to estimate the effects of different factors on the IPA, based on a subset of only 9 instead of 81 treatments. The data were analysed with both Taguchi and full factorial methods and the main effects and the optimal combinations of these 4 factors were obtained for each method. 5. The results indicated that according to both the full factorial experimental design and the Taguchi method, the optimal culture conditions were obtained with the following combination: rye, pH = 3, temperature = 20 °C and time of incubation = 30 min. The comparison between the Taguchi and full factorial results showed that the Taguchi method is a sufficient and resource saving alternative to the full factorial design in poultry science.

Complete subsite mapping of a "loopful" GH19 chitinase from rye seeds based on its crystal structure.

Crystallographic analysis of a mutated form of "loopful" GH19 chitinase from rye seeds a double mutant RSC-c, in which Glu67 and Trp72 are mutated to glutamine and alanine, respectively, (RSC-c-E67Q/W72A) in complex with chitin tetrasaccharide (GlcNAc)4 revealed that the entire substrate-binding cleft was completely occupied with the sugar residues of two (GlcNAc)4 molecules. One (GlcNAc)4 molecule bound to subsites -4 to -1, while the other bound to subsites +1 to +4. Comparisons of the main chain conformation between liganded RSC-c-E67Q/W72A and unliganded wild type RSC-c suggested domain motion essential for catalysis. This is the first report on the complete subsite mapping of GH19 chitinase.

Rye oxidative stress under long term Al exposure.

Aluminium (Al) toxicity decreases plant growth. Secale cereale L. is among the most Al-tolerant crop species. In order to study the response to Al-long term exposure, two rye genotypes with different Al sensitivity ('D. Zlote' and 'Riodeva') were exposed to 1.11 and 1.85mM Al and the antioxidant responses were followed for 2 and 3 weeks in roots and leaves. Al toxicity signals, such as a severe decrease in root growth, occurred sooner in 'Riodeva.' The antioxidant response was dependent on the genotype, the organ, Al concentration and the exposure period. Al-exposed roots of 'D. Zlote' showed earlier enhancements of APX, SOD and G-POX activities than those of 'Riodeva.' 'D. Zlote' roots showed stimulation of the AsA-GSH cycle after the second week (when root growth inhibition was less severe), while later (when severe root growth inhibition was observed), oxidation of AsA and GSH pools was observed. In leaves of both genotypes, CAT, SOD and G-POX activities increased with Al exposure. In these leaves, the effect of AsA-GSH was time dependent, with maximum oxidation at the second week, followed by recovery. We confirmed that the oxidation state of AsA and GSH pools is involved in the detoxification of Al-induced oxidative stress. Moreover, our data demonstrate that the production of ROS does not correlate with the Al-induced root growth decrease. Finally, the differences observed over time indicate that long term exposure may provide additional information on rye sensitivity to Al, and contribute to a better understanding of this species' mechanisms of Al tolerance.

Crystal structure and chitin oligosaccharide-binding mode of a 'loopful' family GH19 chitinase from rye, Secale cereale, seeds.

The substrate-binding mode of a 26-kDa GH19 chitinase from rye, Secale cereale, seeds (RSC-c) was investigated by crystallography, site-directed mutagenesis and NMR spectroscopy. The crystal structure of RSC-c in a complex with an N-acetylglucosamine tetramer, (GlcNAc)(4) , was successfully solved, and revealed the binding mode of the tetramer to be an aglycon-binding site, subsites +1, +2, +3, and +4. These are the first crystallographic data showing the oligosaccharide-binding mode of a family GH19 chitinase. From HPLC analysis of the enzymatic reaction products, mutation of Trp72 to alanine was found to affect the product distribution obtained from the substrate, p-nitrophenyl penta-N-acetyl-ß-chitopentaoside. Mutational experiments confirmed the crystallographic finding that the Trp72 side chain interacts with the +4 moiety of the bound substrate. To further confirm the crystallographic data, binding experiments were also conducted in solution using NMR spectroscopy. Several signals in the (1) H-(15) N HSQC spectrum of the stable isotope-labeled RSC-c were affected upon addition of (GlcNAc)(4) . Signal assignments revealed that most signals responsive to the addition of (GlcNAc)(4) are derived from amino acids located at the surface of the aglycon-binding site. The binding mode deduced from NMR binding experiments in solution was consistent with that from the crystal structure.

Detergent-resistant plasma membrane proteome in oat and rye: similarities and dissimilarities between two monocotyledonous plants.

The plasma membrane (PM) is involved in important cellular processes that determine the growth, development, differentiation, and environmental signal responses of plant cells. Some of these dynamic reactions occur in specific domains in the PM. In this study, we performed comparable nano-LC-MS/MS-based large-scale proteomic analysis of detergent-resistant membrane (DRM) fractions prepared from the PM of oat and rye. A number of proteins showed differential accumulation between the PM and DRM, and some proteins were only found in the DRM. Numerous proteins were identified as DRM proteins in oat (219 proteins) and rye (213 proteins), of which about half were identified only in the DRM. The DRM proteins were largely common to those found in dicotyledonous plants (Arabidopsis and tobacco), which suggests common functions associated with the DRM in plants. Combination of semiquantitative proteomic analysis and prediction of post-translational protein modification sites revealed differences in several proteins associated with the DRM in oat and rye. It is concluded that protein distribution in the DRM is unique from that in the PM, partly because of the physicochemical properties of the proteins, and the unique distribution of these proteins may define the functions of the specific domains in the PM in various physiological processes in plant cells.

Dispersed benzoxazinone gene cluster: molecular characterization and chromosomal localization of glucosyltransferase and glucosidase genes in wheat and rye.

Benzoxazinones (Bxs) are major defensive secondary metabolites in wheat (Triticum aestivum), rye (Secale cereale), and maize (Zea mays). Here, we identified full sets of homeologous and paralogous genes encoding Bx glucosyltransferase (GT) and Bx-glucoside glucosidase (Glu) in hexaploid wheat (2n = 6x = 42; AABBDD). Four GT loci (TaGTa-TaGTd) were mapped on chromosomes 7A, 7B (two loci), and 7D, whereas four glu1 loci (Taglu1a-Taglu1d) were on chromosomes 2A, 2B (two loci), and 2D. Transcript levels differed greatly among the four loci; B-genome loci of both TaGT and Taglu1 genes were preferentially transcribed. Catalytic properties of the enzyme encoded by each homeolog/paralog also differed despite high levels of identity among amino acid sequences. The predominant contribution of the B genome to GT and Glu reactions was revealed, as observed previously for the five Bx biosynthetic genes, TaBx1 to TaBx5, which are separately located on homeologous groups 4 and 5 chromosomes. In rye, where the ScBx1 to ScBx5 genes are dispersed to chromosomes 7R and 5R, ScGT and Scglu were located separately on chromosomes 4R and 2R, respectively. The dispersal of Bx-pathway loci to four distinct chromosomes in hexaploid wheat and rye suggests that the clustering of Bx-pathway genes, as found in maize, is not essential for coordinated transcription. On the other hand, barley (Hordeum vulgare) was found to lack the orthologous GT and glu loci like the Bx1 to Bx5 loci despite its close phylogenetic relationship with wheat and rye. These results contribute to our understanding of the evolutionary processes that the Bx-pathway loci have undergone in grasses.

Active-site architecture of benzoxazinone-glucoside ß-D-glucosidases in Triticeae.

The ß-D-glucosidases from wheat (Triticum aestivum) and rye (Secale cereale) hydrolyze benzoxazinone-glucose conjugates. Although wheat and rye glucosidases have high sequence identity, they have different substrate preferences; the wheat enzyme favors DIMBOA-Glc (2-O-ß-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one) over DIBOA-Glc (7-demethoxy-DIMBOA-Glc), whereas the rye enzyme preference is the opposite. To investigate the mechanism of substrate binding, we analyzed crystal structures of an inactive mutant of the wheat glucosidase complexed with the natural substrate DIMBOA-Glc, wheat and rye glucosidases complexed with an aglycone DIMBOA, and wheat and rye glucosidases complexed with an inhibitor 2-fluoro-2-deoxy-ß-D-glucose. The binding position of substrate in the active site was determined but interaction between the substrate and Ser-464 or Leu-465 was not observed, although amino acid residues at these two positions are the only structural distinctions between wheat and rye glucosidase catalytic pockets. Variation at these two positions alters the width of the pocket entrance, which may relate to observed differences in substrate specificity. The side chain of Glu-462 that forms hydrogen bonds with the glucose moiety of DIMBOA-Glc moved deeper into the pocket upon substrate binding, and mutation of this residue dramatically decreased enzyme activity.

Proteolysis and bioconversion of cereal proteins to glutamate and γ-Aminobutyrate (GABA) in Rye malt sourdoughs.

This study aimed to achieve the conversion of cereal proteins to the alternative end products glutamate or γ-aminobutyrate (GABA). Rye malt, fungal proteases, and lactobacilli were employed to convert wheat gluten or barley proteins. Glutamate and GABA formations were strain-dependent. Lactobacillus reuteri TMW1.106 and Lactobacillus rossiae 34J accumulated glutamate; L. reuteri LTH5448 and LTH5795 accumulated GABA. Glutamate and GABA accumulation by L. reuteri TMW1.106 and LTH5448 increased throughout fermentation time over 96 h, respectively. Peptides rather than amino acids were the main products of proteolysis in all doughs, and barley proteins were more resistant to degradation by rye malt proteases than wheat gluten. However, addition of fungal protease resulted in comparable degradation of both substrates. Glutamate and GABA accumulated to concentrations up to 63 and 90 mmol kg(-1) DM, respectively. Glutamate levels obtained through bioconversion of cereal proteins enable the use of hydrolyzed cereal protein as condiment.

Genomic architecture of alpha-amylase activity in mature rye grain relative to that of preharvest sprouting.

Bi-directional selective genotyping (BSG) carried out on two opposite groups of F(9)(541 × Ot1-3) recombinant inbred lines (RILs) with extremely low and extremely high alpha-amylase activities in mature (dry) grain of rye, followed by molecular mapping, revealed a complex system of selection-responsive loci. Three classes of loci controlling alpha-amylase activity were discerned, including four major AAD loci on chromosomes 3R (three loci) and 6RL (one locus) responding to both directions of the disruptive selection, 20 AAR loci on chromosomes 2RL (three loci), 3R (three loci), 4RS (two loci), 5RL (three loci), 6R (two loci) and 7R (seven loci) responding to selection for low alpha-amylase activity and 17 AAE loci on chromosomes 1RL (seven loci), 2RS (two loci), 3R (two loci), 5R (two loci) and 6RL (four loci) affected by selection for high alpha-amylase activity. The majority of the discerned AA loci also showed responsiveness to selection for preharvest sprouting (PHS). Two AAD loci on chromosome arm 3RL coincided with PHSD loci. The AAD locus on chromosome arm 3RS was independent from PHS, whereas that on chromosome 6RL belonged to the PHSR class. AAR-PHSR loci were found on chromosomes 4RS (one locus) and 5R (two loci) and AAE-PHSE loci were identified on chromosomes 1RL (one locus) and 5RL (one locus). Some PHSD loci represented the AAE (chromosomes 1RL, 3RS and 3RL) or AAR classes (chromosome 5RL). AAR and AAE loci not related to PHS were found on chromosomes 1RL, 2R, 3RS, 4R, 6RL and 7RL. On the other hand, several PHS loci (1RL, 3RS, 5RL, 6RS and 7RS) had no effect on alpha-amylase activity. Allele originating from the parental line 541 mapped in six AA loci on chromosomes 2R (two loci), 5R (three loci) and 7R (one locus) exerted opposite effects on PHS and alpha-amylase activity. Differences between the AA and PHS systems of loci may explain the weak correlation between these two traits observed among recombinant inbred lines. Strategies for the breeding of sprouting-resistant varieties with low alpha-amylase and high PHS resistance are discussed.

Mapping of sequence-specific markers and loci controlling preharvest sprouting and alpha-amylase activity in rye (Secale cereale L.) on the genetic map of an F2 (S120×S76) population.

Location of the loci that control preharvest sprouting and alpha-amylase activity in rye was studied based on intercross S120×S76, consisting of 110 genotypes of F2 and F3 progenies. The genetic map currently consists of 141 loci distributed in 11 linkage groups, covering a distance of 506.4 cM, and was enriched during this study with 24 sequence-specific markers (7 SCARs, 7 SSRs, and 10 STSs). The extended map was applied for composite interval mapping of the loci controlling preharvest sprouting and α-amylase activity, revealing 3 significant QTLs for preharvest sprouting, located on chromosomes 3R, 5R and 6R (in 1999), and one QTL for α-amylase activity found on chromosome 2R (in 2000).