SPInDel Analysis of the Non-Coding Regions of cpDNA as a More Useful Tool for the Identification of Rye (Poaceae: Secale) Species.

Secale is a small but very diverse genus from the tribe Triticeae (family Poaceae), which includes annual, perennial, self-pollinating and open-pollinating, cultivated, weedy and wild species of various phenotypes. Despite its high economic importance, classification of this genus, comprising 3-8 species, is inconsistent. This has resulted in significantly reduced progress in the breeding of rye which could be enriched with functional traits derived from wild rye species. Our previous research has suggested the utility of non-coding sequences of chloroplast and mitochondrial DNA in studies on closely related species of the genus Secale. Here we applied the SPInDel (Species Identification by Insertions/Deletions) approach, which targets hypervariable genomic regions containing multiple insertions/deletions (indels) and exhibiting extensive length variability. We analysed a total of 140 and 210 non-coding sequences from cpDNA and mtDNA, respectively. The resulting data highlight regions which may represent useful molecular markers with respect to closely related species of the genus Secale, however, we found the chloroplast genome to be more informative. These molecular markers include non-coding regions of chloroplast DNA: atpB-rbcL and trnT-trnL and non-coding regions of mitochondrial DNA: nad1B-nad1C and rrn5/rrn18. Our results demonstrate the utility of the SPInDel concept for the characterisation of Secale species.

The distribution pattern of 5-methylcytosine in rye (Secale L.) chromosomes.

The rye (Secale L.) genome is large, and it contains many classes of repetitive sequences. Secale species differ in terms of genome size, heterochromatin content, and global methylation level; however, the organization of individual types of sequences in chromosomes is relatively similar. The content of the abundant subtelomeric heterochromatin fraction in rye do not correlate with the global level of cytosine methylation, hence immunofluorescence detection of 5-methylcytosine (5-mC) distribution in metaphase chromosomes was performed. The distribution patterns of 5-methylcytosine in the chromosomes of Secale species/subspecies were generally similar. 5-methylcytosine signals were dispersed along the entire length of the chromosome arms of all chromosomes, indicating high levels of methylation, especially at retrotransposon sequences. 5-mC signals were absent in the centromeric and telomeric regions, as well as in subtelomeric blocks of constitutive heterochromatin, in each of the taxa studied. Pericentromeric domains were methylated, however, there was a certain level of polymorphism in these areas, as was the case with the nucleolus organizer region. Sequence methylation within the region of the heterochromatin intercalary bands were also demonstrated to be heterogenous. Unexpectedly, there was a lack of methylation in rye subtelomeres, indicating that heterochromatin is a very diverse fraction of chromatin, and its epigenetic regulation or potential influence on adjacent regions can be more complex than has conventionally been thought. Like telomeres and centromeres, subtelomeric heterochromatin can has a specific role, and the absence of 5-mC is required to maintain the heterochromatin state.

Scale development and utilization of universal PCR-based and high-throughput KASP markers specific for chromosome arms of rye (Secale cereale L.).

BACKGROUND: Rye (Secale cereale L., 2n = 2x = 14, RR), a relative of common wheat, is a large gene resource pool for wheat improvement. Accurate and convenient identification of the rye chromatin in wheat background will facilitate the transfer and utilization of elite genes derived from rye in wheat breeding. RESULTS: In the present study, five rye cultivars including Imperial, German White, Jingzhouheimai, Baili and Guyuan were sequenced by specific-locus amplified fragment sequencing (SLAF-seq) to develop large-scale rye-specific markers. Based on SLAF-seq and bioinformatics analyses, a total of 404 universal PCR-based and a whole set of Kompetitive allele-specific PCR (KASP) markers specific for the 14 individual rye chromosome arms were developed and validated. Additionally, two KASP markers specific for 1RS and 2RL were successfully applied in the detection of 1RS translocations in a natural population and 2RL chromosome arms in wheat-rye derived progenies that conferred adult resistance to powdery mildew. CONCLUSION: The 404 PCR-based markers and 14 KASP markers specific for the 14 individual rye chromosome arms developed in this study can enrich the marker densities for gene mapping and accelerate the utilization of rye-derived genes in wheat improvement. Especially, the KASP markers achieved high-throughput and accurate detection of rye chromatin in wheat background, thus can be efficiently used in marker-assisted selection (MAS). Besides, the strategy of rye-specific PCR-based markers converting into KASP markers was high-efficient and low-cost, which will facilitate the tracing of alien genes, and can also be referred for other wheat relatives.

Isolation and characterization of a new MATE gene located in the same chromosome arm of the aluminium tolerance (Alt1) rye locus.

Aluminium (Al) toxicity is the major constraint for crop productivity in acid soils. Wild rye species (Secale spp.) exhibit high Al tolerance, being a good source of genes related to this trait. The Alt1 locus located on the 6RS chromosome arm is one of the four main loci controlling Al tolerance in rye and is known to harbour major genes but, so far, none have been found. Through synteny among the short arm of the rye chromosome 6R and the main grass species, we found a candidate MATE gene for the Atl1 locus, later named ScMATE3, which was isolated and characterized in different Secale species. The sequence comparisons revealed both intraspecific and interspecific variability, with high sequence conservation in the Secale genus. SNP with replacement substitution that changed the structure of the protein and can be involved in the Al tolerance trait were found in ScMATE3 gene. The predicted subcellular localization of ScMATE3 is the vacuolar membrane which, together with the phylogenetic relationships performed with other MATE genes of the Poaceae related to Al detoxification, suggest involvement of ScMATE3 in an internal tolerance mechanism. Moreover, expression studies of this gene in rye corroborate its contribution in some Al resistance mechanisms. The ScMATE3 gene is located on the 6RS chromosome arm between the same markers in which the Alt1 locus is involved in Al resistance mechanisms in rye, thus being a good candidate gene for this function.

Genetic diversity and relationship between cultivated, weedy and wild rye species as revealed by chloroplast and mitochondrial DNA non-coding regions analysis.

The genus Secale is small but very diverse. Despite the high economic importance, phylogenetic relationships of rye species have not been fully determined, and they are extremely important for the process of breeding of new cultivars that can be enriched with functional traits derived from wild rye species. The study analyzed the degree of relationship of 35 accessions of the genus Secale, representing 13 most often distinguished species and subspecies, originating from various seed collections in the world, based on the analysis of non-coding regions of the chloroplast (cpDNA) and mitochondrial genome (mtDNA), widely used in phylogenetic and population plant studies, because of a higher rate of evolution than the coding regions. There was no clear genetic structure between different species and subspecies, which may indicated the introgression between these taxa. The obtained data confirmed that S. vavilovii was very similar to S. cereale, which confirmed the assumption that they might share a common ancestor. The results also confirmed the divergence of S. sylvestre from other species and subspecies of rye. Areas that may be useful molecular markers in studies on closely related species of the genus Secale were also indicated.

Phylogeny and genetic structure in the genus Secale.

Secale L. is a small but important genus that includes cultivated rye. Although genetic diversity of cultivated rye is high, patterns of genetic diversity in the whole genus, and potential factors affecting the distribution of genetic diversity remain elusive. The population structure and distribution of genetic variation within Secale, and its correlation with taxonomic delimitation, cultivation status or spatial distribution in relation to geography and climate zones were analyzed in this study. A collection of 726 individual plants derived from 139 different accessions representing Secale cereale, S. vavilovii, S. strictum, and S. sylvestre were investigated using SSR analysis and sequence diversity analysis of a nuclear EST region. Our results indicated that perennial S. strictum subspecies are genetically divergent from annual forms of the genus. Existence of two distinct clusters within the annual taxa was observed, one corresponding to samples from Asia, and a second to those outside of Asia. No clear genetic structure was observed between different annual species/subspecies, indicating introgression between these taxa. The analysis of cultivated rye revealed that landrace populations from the Middle East have the highest genetic diversity, supporting the idea of the area being the center of origin for cultivated rye. Considering high adaptive potential of those populations, Middle Eastern landraces should be regarded as genetic resources reservoirs for new niches and future breeding programs.

Conserved molecular structure of the centromeric histone CENH3 in Secale and its phylogenetic relationships.

It has been repeatedly demonstrated that the centromere-specific histone H3 (CENH3), a key component of the centromere, shows considerable variability between species within taxa. We determined the molecular structure and phylogenetic relationships of CENH3 in 11 Secale species and subspecies that possess distinct pollination systems and are adapted to a wide range of abiotic and biotic stresses. The rye (Secale cereale) genome encodes two paralogous CENH3 genes, which differ in intron-exon structure and are transcribed into two main forms of the protein, αCENH3 and ßCENH3. These two forms differ in size and amino acid substitutions. In contrast to the reported differences in CENH3 structure between species within other taxa, the main forms of this protein in Secale species and subspecies have a nearly identical structure except some nonsynonymous substitutions. The CENH3 proteins are strictly controlled by genetic factors responsible for purifying selection. A comparison between Hordeum, Secale and Triticum species demonstrates that the structure of CENH3 in the subtribes Hordeinae and Triticinae evolved at different rates. The assumption that reticulate evolution served as a factor stabilizing the structure and evolutionary rate of CENH3 and that this factor was more powerful within Secale and Triticum than in Hordeum, is discussed.

In situ and in vitro ruminal starch degradation of grains from different rye, triticale and barley genotypes.

In recent years, advances in plant breeding were achieved, which potentially led to modified nutritional values of cereal grains. The present study was conducted in order to obtain a broad overview of ruminal digestion kinetics of rye, triticale and barley grains, and to highlight differences between the grain species. In total, 20 genotypes of each grain species were investigated using in situ and in vitro methods. Samples were ground (2 mm), weighed into polyester bags, and incubated in situ 1 to 48 h in three ruminally cannulated lactating dairy cows. The in vitro gas production of ground samples (1 mm) was measured according to the 'Hohenheim Gas Test', and cumulative gas production was recorded over different time spans for up to 72 h. There were significant differences (P<0.05) between the species for most parameters used to describe the in situ degradation of starch (ST) and dry matter (DM). The in situ degradation rate (c) and effective degradability (assuming a passage rate of 8%/h; ED8) of ST differed significantly between all grains and was highest for rye (rye: 116.5%/h and 96.2%; triticale: 85.1%/h and 95.0%; barley: 36.2%/h and 90.0% for c and ED8, respectively). With respect to DM degradation, the ranking of the species was similar, and predicted c values exhibited the highest variation within species. The in vitro gas production rate was significantly higher (P<0.05) for rye than for triticale and barley (rye: 12.5%/h; triticale: 11.5%/h; barley: 11.1%/h). A positive relationship between the potential gas production in vitro and the maximal degradable DM fraction in situ was found using all samples (r=0.84; P<0.001) as well as rye (P=0.002) and barley (P<0.001) alone, but not for triticale. Variation in ruminal in situ degradation parameters within the grain species resulted from the high c values, but was not reflected in the ED estimates. Therefore, the usage of mean values for the ED of DM and ST for each species appears reasonable. Estimated metabolisable energy concentrations (ME, MJ/kg DM) and the estimated digestibility of organic matter (dOM, %) were significantly lower (P<0.05) for barley than for rye and triticale. Rye and triticale dOM and ME values were not significantly different (P=0.386 and 0.485).

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.

Molecular diversity and genetic relationships in Secale.

The objective of this study was to quantify the molecular diversity and to determine the genetic relationships among Secale spp. and among cultivars of Secale cereale using RAPDs, ISSRs and sequence analysis of six exons of ScMATE1 gene. Thirteen ryes (cultivated and wild) were genotyped using 21 RAPD and 16 ISSR primers. A total of 435 markers (242 RAPDs and 193 ISSRs) were obtained, with 293 being polymorphic (146 RAPDs and 147 ISSRs). Two RAPD and nine ISSR primers generated more than 80% of polymorphism. The ISSR markers were more polymorphic and informative than RAPDs. Further, 69% of the ISSR primers selected achieved at least 70% of DNA polymorphism. The study of six exons of the ScMATE1 gene also demonstrated a high genetic variability that subsists in Secale genus. One difference observed in exon 1 sequences from S. vavilovii seems to be correlated with Al sensitivity in this species. The genetic relationships obtained using RAPDs, ISSRs and exons of ScMATE1 gene were similar. S. ancestrale, S. kuprijanovii and S. cereale were grouped in the same cluster and S. segetale was in another cluster. S. vavilovii showed evidences of not being clearly an isolate species and having great intraspecific differences.

Genome-wide characterization of genetic diversity and population structure in Secale.

BACKGROUND: Numerous rye accessions are stored in ex situ genebanks worldwide. Little is known about the extent of genetic diversity contained in any of them and its relation to contemporary varieties, since to date rye genetic diversity studies had a very limited scope, analyzing few loci and/ or few accessions. Development of high throughput genotyping methods for rye opened the possibility for genome wide characterizations of large accessions sets. In this study we used 1054 Diversity Array Technology (DArT) markers with defined chromosomal location to characterize genetic diversity and population structure in a collection of 379 rye accessions including wild species, landraces, cultivated materials, historical and contemporary rye varieties. RESULTS: Average genetic similarity (GS) coefficients and average polymorphic information content (PIC) values varied among chromosomes. Comparison of chromosome specific average GS within and between germplasm sub-groups indicated regions of chromosomes 1R and 4R as being targeted by selection in current breeding programs. Bayesian clustering, principal coordinate analysis and Neighbor Joining clustering demonstrated that source and improvement status contributed significantly to the structure observed in the analyzed set of Secale germplasm. We revealed a relatively limited diversity in improved rye accessions, both historical and contemporary, as well as lack of correlation between clustering of improved accessions and geographic origin, suggesting common genetic background of rye accessions from diverse geographic regions and extensive germplasm exchange. Moreover, contemporary varieties were distinct from the remaining accessions. CONCLUSIONS: Our results point to an influence of reproduction methods on the observed diversity patterns and indicate potential of ex situ collections for broadening the genetic diversity in rye breeding programs. Obtained data show that DArT markers provide a realistic picture of the genetic diversity and population structure present in the collection of 379 rye accessions and are an effective platform for rye germplasm characterization and association mapping studies.

Formation and expression of pseudogenes on the B chromosome of rye.

B chromosomes (Bs) are dispensable components of the genomes of numerous species. In contrast with the prevalent view that Bs do not harbor genes, our recent sequence analysis revealed that Bs of rye (Secale cereale) are rich in gene-derived sequences. We compared these gene-like fragments of the rye B with their ancestral A-located counterparts and confirmed an A chromosomal origin and the pseudogenization of B-located gene-like fragments. About 15% of the pseudogene-like fragments on Bs are transcribed in a tissue-type and genotype-specific manner. In addition, B-located sequences can cause in trans down- or upregulation of A chromosome-encoded genic fragments. Phenotypes and effects associated with the presence of Bs might be explained by the activity of B-located pseudogenes. We propose a model for the evolution of B-located pseudogenes.

Differentiation of triticale cultivars through FISH karyotyping of their rye chromosomes.

The aim of this work was to cytogenetically characterize triticale cultivars through fluorescence in situ hybridization (FISH) analysis of their rye chromosomes. In the present work, we studied six cultivars of triticale ('Cayú-UNRC', 'Cumé-UNRC', 'Genú-UNRC', 'Ñinca-UNRC', 'Quiñé-UNRC', and 'Tizné-UNRC'), released by the Universidad Nacional de Río Cuarto (UNRC), Córdoba, Argentina. The cultivars were obtained from the International Center for the Improvement of Maize and Wheat (CIMMYT) and improved for fresh forage, haymaking, and feed grain at UNRC. The distribution and organization of highly repetitive DNA sequences of Secale cereale (pSc74, pSc200, pSc250, and pSc119.2) using FISH analyses revealed a specific localization of the signals for several rye chromosomes, which allowed us to distinguish the cultivars. Cluster analysis showed a great cytogenetic similarity among the rye cultivars used to originate these hybrids. The knowledge of the variability among triticale cultivars is necessary to propose future crosses in breeding programs. This study will also be valuable to identify commercial seeds and to analyze the possible association between agronomic characters and the presence of certain rye chromosomes or specific regions in these chromosomes.

Alimentos integrais / Foods Integrals

Introdução: Alimento integral é aquele que não teve sua estrutura modificada no processo de industrialização e manteve as vitaminas, os minerais e as fibras. Objetivo: Verificar evidências do uso dos alimentos integrais na prevenção e tratamento de doenças. Métodos: Busca sistematizada da literatura, por meio de busca eletrônica de artigos que avaliem o uso de alimentos integrais na terapêutica médica. Resultados: Encontramos uma revisão sistemática com 10 estudos com pouca qualidade metodológica e de curta duração. Esse estudo evidenciou uma diminuição significativa do colesterol total e do LDL-colesterol (lipoproteína de baixa densidade), sem influência na glicemia, peso corpóreo, pressão arterial e triglicérides, no grupo suplementado com alimentos integrais. Encontramos um ensaio clínico randomizado com 18 meses de acompanhamento, no qual o grupo que ingeriu alimentos integrais teve perda de peso com diminuição significativa da circunferência abdominal, sem perda significativa de vitaminas, minerais e fibras. Conclusão: Pelos estudos apresentados, os efeitos redutores do colesterol e do LDL-colesterol estão demonstrados em estudos de curta duração. Há necessidade de mais estudos com boa qualidade metodológica para verificarmos em longo prazo todas as alterações metabólicas e consequentes benefícios circulatórios.

Polymorphism of the storage proteins in Portuguese rye (Secale cereale L.) populations.

Currently, due to the abandonment of traditional agricultural practices and the decline of rye production in Portugal, there is a need to assess the genetic diversity of rye in order to preserve its biodiversity. Furthermore, a greater knowledge of rye secalins is important for rye bread-making quality and other crop breeding purposes. The genetic variation and diversity of storage proteins were estimated for fourteen populations of rye (Secale cereale L.) sampled in northern Portugal. The work showed the high genetic diversity within the Portuguese rye gene-pool as an important source for plant breeding and emphasized the necessity of an integrated resources genetic program to allow a more efficient management and conservation of these resources. The rye populations were compared with 'Picasso' and 'Marder' varieties. Several alleles were identified by the single electrophoretic mobility patterns. We studied a set of 1600 rye seeds, including regional populations and varieties, having observed a total of 24, 5, 21 and 47 alleles for HMW, 75k γ-, 40k γ- and ω-secalins, respectively. The coefficient of similarity within populations is presented using cluster representation. The mean value of genetic variation indices (H) for rye storage proteins was very high in regional populations, ranging from 0.67 to 0.78, while in the varieties ranged from 0.57 to 0.58. Knowledge of the diversity of secalins will increase our understanding of the quality differences between rye varieties, especially considering the relative small number of rye cultivars grown around the world.

Diversity and evolution of four dispersed repetitive DNA sequences in the genus Secale.

We present the first characterization of 360 sequences in six species of the genus Secale of both cultivated and wild accessions. These include four distinct kinds of dispersed repetitive DNA sequences named pSc20H, pSc119.1, pSaO5(411), and pSaD15(940) belonging to the Revolver family. During the evolution of the genus Secale from wild to cultivated accessions, the pSaO5(411)-like sequences became shorter mainly because of the deletion of a trinucleotide tandem repeating unit, the pSc20H-like sequences displayed apparent homogenization in cultivated rye, and the second intron of Revolver became longer. In addition, the pSc20H-, pSc119.1-, and pSaO5(411)-like sequences cloned from wild rye and cultivated rye could be divided into two large clades. No single case of the four kinds of repetitive elements has been inherited by each Secale accession from a lone ancestor. It is reasonable to consider the vertical transmission of the four repetitive elements during the evolution of the genus Secale. The pSc20H- and pSaO5(411)-like sequences showed evolutionary elimination at specific chromosomal locations from wild species to cultivated species. These cases imply that different repetitive DNA sequences have played different roles in the chromosome development and genomic evolution of rye. The present study adds important information to the investigations dealing with characterization of dispersed repetitive elements in wild and cultivated rye.

Evolutionary trends of microsatellites during the speciation process and phylogenetic relationships within the genus Secale.

Eleven weedy or wild species or subspecies of the genus Secale L. were compared with a set of cultivated rye accessions, based on inter-simple sequence repeat (ISSR) markers to analyze their phylogenetic relationships. A total of 846 bands were amplified from reactions using 12 screening primers, including 79 loci with a mean of 10.1 alleles per locus. The number of amplified bands for each primer ranged from 12 to 134, with a mean of 70.5 amplified bands per primer. The presence and distribution of amplified bands in different accessions demonstrate that a rapid evolutionary trend of microsatellite repeats occurred during the speciation process from the perennial wild form to annual cultivated rye. In addition, variation, amplification, and deletion of microsatellites in genomes revealed phylogenetic relationships in the genus Secale. Analysis of the presence, number, and distribution of amplified bands in genomes, as well as the comparison with genetic similarity (GS) indices based on ISSR, indicate that Secale strictum subsp. africanum (Stapf) Hammer, Secale strictum anatolicum (Boiss.) Hammer, Secale sylvestre Host, and Secale strictum subsp. strictum (C. Presl) Hammer emerged in succession from a common ancestor of Secale following geographic separation and genetic differentiation. The annual weedy rye evolved from S. strictum subsp. strictum, which was domesticated as present-day cultivated rye. Data from ISSR analyses separated all investigated accessions of the genus Secale into three distinct groups. These results support the division of the genus Secale into three species: the annual wild species S. sylvestre; the perennial wild species S. strictum, including several differential subspecies forms such as strictum, africanum, and anatolicum; and S. cereale, including cultivated and weedy rye as subspecies forms.

Genetic variation in the extract viscosity of rye (Secale cereale L.) bread made from endosperm and wholemeal flour: impact of high-molecular-weight arabinoxylan, starch and protein.

BACKGROUND: To verify the viscous potential of rye bread made from population and hybrid cultivars, the overall extract viscosities (EVs) of endosperm and wholemeal breads (EBs and WMBs respectively) were assessed using extractants of different pH. Also, arabinoxylan-dependent EV (AX-EV) was determined after combined action of starch- and protein-degrading enzymes. RESULTS: The synergistic effect of a combination of α-amylase, amyloglucosidase and protease on EV was almost two times higher than the effects observed after single addition of each of them. However, water-extractable arabinoxylans (WE AXs) were the major contributors to EV. Unexpectedly, a decrease in the mean WE AX contents observed in the following order, WMB (31.7 g kg(-1) ) and EB (29.3 g kg(-1) ) of hybrid rye and WMB (28.8 g kg(-1) ) and EB (23.8 g kg(-1) ) of population rye, was accompanied by an increase in their AX-EVs (12, 21, 21 and 29 mPa s respectively). CONCLUSION: In some cases the structure of WE AXs is a more important determinant of AX-EV than their content in rye bread. Nevertheless, both factors influence AX-EV, which may reflect the viscosity of the human small intestine after rye bread consumption, related to some beneficial metabolic effects. Therefore AX-EV can be a preliminary parameter for selection towards high-viscosity bread.

Allelochemicals in rye (Secale cereale L.): cultivar and tissue differences in the production of benzoxazinoids and phenolic acids.

In the present study, a range of benzoxazinoid compounds and phenolic acids, all known to be allelochemicals of rye, were identified and quantified in 13 rye cultivars grown at three different localities. Plant samples were collected in the spring at the time when an autumn-sown rye cover crop would be incorporated into the soil. Significant variations in content among shoots and roots were seen for all of the secondary metabolites, with non-methoxy-substituted benzoxazinoids (BX) dominating the shoots, whereas comparable levels were found in the concentrations of BX and methoxy-substituted benzoxazinoids (MBX) in the roots. This distribution of compounds may indicate different biosynthetic pathways and/or different mechanisms of action of these compounds. Concentrations not only depended on plant part, but also on the geographical location--with differences in contents of up to a factor of 5. These differences can probably be attributed to differences in growing conditions. The variation among cultivars was similar to that among geographical localities, with differences within localities of up to a factor of 7 in the shoots and a factor of 14 in the roots. In roots, the contents of the four phenolic acids and the benzoxazinoid 6-methoxybenzoxazolin-2-one (MBOA) were correlated. In shoots, the contents of the two benzoic acids were correlated with each other, whereas the two cinnamic acids were correlated with MBOA and several other benzoxazinoids. The lack of correlation between MBOA and all other benzoxazinoids in the roots of rye might indicate that a hitherto unknown synthetic pathway exists for MBOA. The genes responsible for the synthesis of some of the benzoxazinoids have never been identified, and further gene expression studies are required to assess the observed correlation between the concentration of these compounds and other benzoxazinoids for which the responsible genes are known. The present study revealed a potential for breeding rye cultivars with a high content of biologically active secondary metabolites. However, growing conditions significantly influenced the level of these compounds.

Origin and genetic structure of feral rye in the western United States.

Feral rye (Secale cereale) is a serious, introduced weed of dry land agricultural regions of the western United States. It closely resembles cultivated cereal rye (Secale cereale cereale L.) with the exception of having a shattering seed head. Feral rye may have originated from hybridization of cultivated rye with mountain rye, Secale strictum, as past studies of northern Californian populations suggest, or directly from volunteer cultivated rye. We characterized the genetic structure of feral rye populations across a broad geographical range and reexamined evidence for hybrid origin versus direct evolution from domesticated cultivars. Eighteen feral populations were examined from three climatically distinct regions in the western United States. Seven cultivars, four mountain rye accessions, and one wild annual relative (Secale cereale ancestrale) were included in our analysis as possible progenitors of feral rye. Individual plants were scored for 14 allozyme and three microsatellite loci. Estimates of genetic diversity in feral populations were relatively high compared to those of the possible progenitors, suggesting that the weed had not undergone a genetic bottleneck. Weed populations had no geographical structure at either a broad or a local scale, suggesting idiosyncratic colonization and gene-flow histories at each site. Feral rye populations were no more closely related to mountain rye than cultivars were. They were, however, weakly clustered as a distinct lineage relative to cultivars. Our results do not support an interspecific hybrid origin for feral rye, but do suggest that the sampled populations of feral rye share a common ancestry that may explain its weedy nature.