Mapping translocation breakpoints using a wheat microarray

We report mapping of translocation breakpoints using a microarray. We used complex RNA to compare normal hexaploid wheat (17 000 Mb genome) to a ditelosomic stock missing the short arm of chromosome 1B (1BS) and wheat-rye translocations that replace portions of 1BS with rye 1RS. Transcripts detected by a probe set can come from all three Triticeae genomes in ABD hexaploid wheat, and sequences of homoeologous genes on 1AS, 1BS and 1DS often differ from each other. Absence or replacement of 1BS therefore must sometimes result in patterns within a probe set that deviate from hexaploid wheat. We termed these ‘high variance probe sets’ (HVPs) and examined the extent to which HVPs associated with 1BS aneuploidy are related to rice genes on syntenic rice chromosome 5 short arm (5S). We observed an enrichment of such probe sets to 15–20% of all HVPs, while 1BS represents ∼2% of the total genome. In total 257 HVPs constitute wheat 1BS markers. Two wheat-rye translocations subdivided 1BS HVPs into three groups, allocating translocation breakpoints to narrow intervals defined by rice 5S coordinates. This approach could be extended to the entire wheat genome or any organism with suitable aneuploid or translocation stocks

Dynamics of Rye Chromosome 1R Regions with High or Low Crossover Frequency in Homology Search and Synapsis Development

In many organisms, homologous pairing and synapsis depend on the meiotic recombination machinery that repairs double-strand DNA breaks (DSBs) produced at the onset of meiosis. The culmination of recombination via crossover gives rise to chiasmata, which locate distally in many plant species such as rye, Secale cereale. Although, synapsis initiates close to the chromosome ends, a direct effect of regions with high crossover frequency on partner identification and synapsis initiation has not been demonstrated. Here, we analyze the dynamics of distal and proximal regions of a rye chromosome introgressed into wheat to define their role on meiotic homology search and synapsis. We have used lines with a pair of two-armed chromosome 1R of rye, or a pair of telocentrics of its long arm (1RL), which were homozygous for the standard 1RL structure, homozygous for an inversion of 1RL that changes chiasma location from distal to proximal, or heterozygous for the inversion. Physical mapping of recombination produced in the ditelocentric heterozygote (1RL/1RLinv) showed that 70% of crossovers in the arm were confined to a terminal segment representing 10% of the 1RL length. The dynamics of the arms 1RL and 1RLinv during zygotene demonstrates that crossover-rich regions are more active in recognizing the homologous partner and developing synapsis than crossover-poor regions. When the crossover-rich regions are positioned in the vicinity of chromosome ends, their association is facilitated by telomere clustering; when they are positioned centrally in one of the two-armed chromosomes and distally in the homolog, their association is probably derived from chromosome elongation. On the other hand, chromosome movements that disassemble the bouquet may facilitate chromosome pairing correction by dissolution of improper chromosome associations. Taken together, these data support that repair of DSBs via crossover is essential in both the search of the homologous partner and consolidation of homologous synapsis

Cytomolecular identification of individual wheat-wheat chromosome arm associations in wheat-rye hybrids

Chromosome pairing in the meiotic metaphase I of wheatrye hybrids has been characterized by sequential genomic and fluorescent in situ hybridization allowing not only the discrimination of wheat and rye chromosomes, but also the identification of the individual wheat and rye chromosome arms involved in the chromosome associations. The majority of associations (93.8%) were observed between the wheat chromosomes. The largest number of wheat-wheat chromosome associations (53%) was detected between the A and D genomes, while the frequency of B-D and A-B associations was significantly lower (32 and 8%, respectively). Among the A-D chromosome associations, pairing between the 3AL and 3DL arms was observed with the highest frequency, while the most frequent of all the chromosome associations (0.113/ cell) was found to be the 3DS-3BS. Differences in the pairing frequency of the individual chromosome arms of wheat-rye hybrids have been discussed in relation to the homoeologous relationships between the constituent genomes of hexaploid wheat

Integrated genetic map and genetic analysis of a region associated with root traits on the short arm of rye chromosome 1 in bread wheat

A rye–wheat centric chromosome translocation 1RS.1BL has been widely used in wheat breeding programs around the world. Increased yield of translocation lines was probably a consequence of increased root biomass. In an effort to map loci-controlling root characteristics, homoeologous recombinants of 1RS with 1BS were used to generate a consensus genetic map comprised of 20 phenotypic and molecular markers, with an average spacing of 2.5 cM. Physically, all recombination events were located in the distal 40% of the arms. A total of 68 recombinants was used and recombination breakpoints were aligned and ordered over map intervals with all the markers, integrated together in a genetic map. This approach enabled dissection of genetic components of quantitative traits, such as root traits, present on 1S. To validate our hypothesis, phenotyping of 45-day-old wheat roots was performed in five lines including three recombinants representative of the entire short arm along with bread wheat parents ‘Pavon 76’ and Pavon 1RS.1BL. Individual root characteristics were ranked and the genotypic rank sums were subjected to Quade analysis to compare the overall rooting ability of the genotypes. It appears that the terminal 15% of the rye 1RS arm carries gene(s) for greater rooting ability in wheat

Boron Nutrition of Tobacco BY-2 Cells. V. Oxidative Damage is the Major Cause of Cell Death Induced by Boron Deprivation

Boron (B) is an essential micronutrient for vascular plants. However, it remains unclear how B deficiency leads to various metabolic disorders and cell death. To understand this mechanism, we analyzed the physiological changes in suspension-cultured tobacco (Nicotiana tabacum) BY-2 cells upon B deprivation. When 3-day-old cells were transferred to B-free medium, cell death was detectable as early as 12 h after treatment. The B-deprived cells accumulated more reactive oxygen species and lipid peroxides than control cells, and showed a slight but significant decrease in the cellular ascorbate pool. Supplementing the media with lipophilic antioxidants effectively suppressed the death of B-deprived cells, suggesting that the oxidative damage is the immediate and major cause of cell death under B deficiency. Dead cells in B-free culture exhibited a characteristic morphology with a shrunken cytoplasm, which is often seen in cells undergoing programmed cell death (PCD). However, they did not display other hallmarks of PCD such as internucleosomal DNA fragmentation, decreased ascorbate peroxidase expression and protection from death by cycloheximide. These results suggest that the death of tobacco cells induced by B deprivation is not likely to be a typical PCD

Phenotypic, cytogenetic and spike fertility characterization of a population of male-sterile triticale

Triticale (X Triticosecale Wittmack) is a good cereal for production of flour and feed. A segregating population of triticale was developed from a male-sterile (MS) plant. To determine whether this new source of male sterility in triticale is appropriate for use in breeding programs the expression of the male sterility phenotype was characterized through spike fertility, meiotic behavior, and pollen. Controlled crosses between male-sterile plants and control varieties male-fertile (MF) of triticale were also conducted, and cytological analyses were performed in the F2 and backcross plants. Plants with male-sterile phenotypes displayed reduced spike fertility when compared to plants with male-fertile phenotypes. Compared to male-fertile plants, male-sterile plants exhibited a lower percentage of normal meiotic cells, a reduced meiotic index and reduced pollen viability. The F2 plants had improved pollen fertility when compared to the male-sterile population; however there were no corresponding improvements in the percentage of normal meiotic cells or in the meiotic index. A single generation of backcrosses resulted in an improved meiotic index and increased pollen viability. However, no changes in the percentage of normal meiotic cells were observed. Meiotic instability, which was shown to be inheritable, was the likely cause of male sterility. Therefore, the use of this population in triticale breeding was considered to be inappropriate because it could promote or contribute to the maintenance of meiotic instability, which is commonly observed in this species

Identification of the chromosome complement and the spontaneous 1R/1V translocations in allotetraploid Secale cereale × Dasypyrum villosum hybrids through cytogenetic approaches

Genome modifications that occur at the initial interspecific hybridization event are dynamic and can be consolidated during the process of stabilization in successive generations of allopolyploids. This study identifies the number and chromosomal location of ribosomal DNA (rDNA) sites between Secale cereale, Dasypyrum villosum, and their allotetraploid S. cereale × D. villosum hybrids. For the first time, we show the advantages of FISH to reveal chromosome rearrangements in the tetraploid Secale × Dasypyrum hybrids. Based on the specific hybridization patterns of ribosomal 5S, 35S DNA and rye species-specific pSc200 DNA probes, a set of genotypes with numerous Secale/Dasypyrum translocations of 1R/1V chromosomes were identified in successive generations of allotetraploid S. cereale × D. villosum hybrids. In addition we analyse rye chromosome pairs using FISH with chromosome-specific DNA sequences on S. cereale × D. villosum hybrids

AIP56: A Novel Bacterial Apoptogenic Toxin

Photobacterium damselae subsp. piscicida (Phdp) is a Gram-negative pathogen agent of an important fish septicemia. The key virulence factor of Phdp is the plasmid-encoded exotoxin AIP56, which is secreted by exponentially growing pathogenic strains. AIP56 has 520 amino acids including an N-terminal cleavable signal peptide of 23 amino acid residues, two cysteine residues and a zinc-binding region signature HEXXH that is typical of most zinc metallopeptidases. AIP56 induces in vitro and in vivo selective apoptosis of fish macrophages and neutrophils through a caspase-3 dependent mechanism that also involves caspase-8 and -9. In vivo, the AIP56-induced phagocyte apoptosis progresses to secondary necrosis with release of cytotoxic phagocyte molecules including neutrophil elastase. Fish injected with recombinant AIP56 die with a pathology similar to that seen in the natural infection

Caspase-3 Mediates the Pathogenic Effect of \u3cem\u3e Yersinia pestis \u3c/em\u3e YopM in Liver of C57BL/6 Mice and Contributes to YopM\u27s Function in Spleen

The virulence protein YopM of the plague bacterium Yersinia pestis has different dominant effects in liver and spleen. Previous studies focused on spleen, where YopM inhibits accumulation of inflammatory dendritic cells. In the present study we focused on liver, where PMN function may be directly undermined by YopM without changes in inflammatory cell numbers in the initial days of infection, and foci of inflammation are easily identified. Mice were infected with parent and ΔyopM-1 Y. pestis KIM5, and effects of YopM were assessed by immunohistochemistry and determinations of bacterial viable numbers in organs. The bacteria were found associated with myeloid cells in foci of inflammation and in liver sinusoids. A new in-vivo phenotype of YopM was revealed: death of inflammatory cells, evidenced by TUNEL staining beginning at d 1 of infection. Based on distributions of Ly6G+, F4/80+, and iNOS+ cells within foci, the cells that were killed could have included both PMNs and macrophages. By 2 d post-infection, YopM had no effect on distribution of these cells, but by 3 d cellular decomposition had outstripped acute inflammation in foci due to parent Y. pestis, while foci due to the Δ-1yopM strain still contained many inflammatory cells. The destruction depended on the presence of both PMNs in the mice and YopM in the bacteria. In mice that lacked the apoptosis mediator caspase-3 the infection dynamics were novel: the parent Y. pestis was limited in growth comparably to the ΔyopM-1 strain in liver, and in spleen a partial growth limitation for parent Y. pestis was seen. This result identified caspase-3 as a co-factor or effector in YopM\u27s action and supports the hypothesis that in liver YopM\u27s main pathogenic effect is mediated by caspase-3 to cause apoptosis of PMNs

A High Density Consensus Map of Rye (Secale cereale L.) Based on DArT Markers

L.) is an economically important crop, exhibiting unique features such as outstanding resistance to biotic and abiotic stresses and high nutrient use efficiency. This species presents a challenge to geneticists and breeders due to its large genome containing a high proportion of repetitive sequences, self incompatibility, severe inbreeding depression and tissue culture recalcitrance. The genomic resources currently available for rye are underdeveloped in comparison with other crops of similar economic importance. The aim of this study was to create a highly saturated, multilocus linkage map of rye via consensus mapping, based on Diversity Arrays Technology (DArT) markers.Recombinant inbred lines (RILs) from 5 populations (564 in total) were genotyped using DArT markers and subjected to linkage analysis using Join Map 4.0 and Multipoint Consensus 2.2 software. A consensus map was constructed using a total of 9703 segregating markers. The average chromosome map length ranged from 199.9 cM (2R) to 251.4 cM (4R) and the average map density was 1.1 cM. The integrated map comprised 4048 loci with the number of markers per chromosome ranging from 454 for 7R to 805 for 4R. In comparison with previously published studies on rye, this represents an eight-fold increase in the number of loci placed on a consensus map and a more than two-fold increase in the number of genetically mapped DArT markers.Through the careful choice of marker type, mapping populations and the use of software packages implementing powerful algorithms for map order optimization, we produced a valuable resource for rye and triticale genomics and breeding, which provides an excellent starting point for more in-depth studies on rye genome organization