Recombination changes at the boundaries of fully and partially sex-linked regions between closely related Silene species pairs.

The establishment of a region of suppressed recombination is a critical change during sex chromosome evolution, leading to such properties as Y (and W) chromosome genetic degeneration, accumulation of repetitive sequences and heteromorphism. Although chromosome inversions can cause large regions to have suppressed recombination, and inversions are sometimes involved in sex chromosome evolution, gradual expansion of the non-recombining region could potentially sometimes occur. We here test whether closer linkage has recently evolved between the sex-determining region and several genes that are partially sex-linked in Silene latifolia, using Silene dioica, a closely related dioecious plants whose XY sex chromosome system is inherited from a common ancestor. The S. latifolia pseudoautosomal region (PAR) includes several genes extremely closely linked to the fully Y-linked region. These genes were added to an ancestral PAR of the sex chromosome pair in two distinct events probably involving translocations of autosomal genome regions causing multiple genes to become partially sex-linked. Close linkage with the PAR boundary must have evolved since these additions, because some genes added in both events now show almost complete sex linkage in S. latifolia. We compared diversity patterns of five such S. latifolia PAR boundary genes with their orthologues in S. dioica, including all three regions of the PAR (one gene that was in the ancestral PAR and two from each of the added regions). The results suggest recent recombination suppression in S. latifolia, since its split from S. dioica.

Frequent, geographically structured heteroplasmy in the mitochondria of a flowering plant, ribwort plantain (Plantago lanceolata).

Recent research has convincingly documented cases of mitochondrial heteroplasmy in a small set of wild and cultivated plant species. Heteroplasmy is suspected to be common in flowering plants and investigations of additional taxa may help understand the mechanisms generating heteroplasmy as well as its effects on plant phenotypes. The role of mitochondrial heteroplasmy is of particular interest in plants as cytoplasmic male sterility is controlled by mitochondrial genotypes, sometimes leading to co-occurring female and hermaphroditic individuals (gynodioecy). Paternal leakage may be important in the evolution of mating systems in such populations. We conducted a genetic survey of the gynodioecious plant Plantago lanceolata, in which heteroplasmy has not previously been reported, and estimated the frequencies of mitochondrial genotypes and heteroplasmy. Sanger sequence genotyping of 179 individuals from 15 European populations for two polymorphic mitochondrial loci, atp6 and rps12, identified 15 heteroplasmic individuals. These were distributed among 6 of the 10 populations that had polymorphisms in the target loci and represented 8% of all sampled individuals and 15% of the individuals in those 6 populations. The incidence was highest in Northern England and Scotland. Our results are consistent with geographic differences in the incidence of paternal leakage and/or the rates of nuclear restoration of male fertility.

RAD mapping reveals an evolving, polymorphic and fuzzy boundary of a plant pseudoautosomal region.

How loss of genetic exchanges (recombination) evolves between sex chromosomes is a long-standing question. Suppressed recombination may evolve when a sexually antagonistic (SA) polymorphism occurs in a partially sex-linked 'pseudoautosomal' region (or 'PAR'), maintaining allele frequency differences between the two sexes, and creating selection for closer linkage with the fully sex-linked region of the Y chromosome in XY systems, or the W in ZW sex chromosome systems. Most evidence consistent with the SA polymorphism hypothesis is currently indirect, and more studies of the genetics and population genetics of PAR genes are clearly needed. The sex chromosomes of the plant Silene latifolia are suitable for such studies, as they evolved recently and the loss of recombination could still be ongoing. Here, we used RAD sequencing to genetically map sequences in this plant, which has a large genome (c. 3 gigabases) and no available whole-genome sequence. We mapped 83 genes on the sex chromosomes, and comparative mapping in the related species S. vulgaris supports previous evidence for additions to an ancestral PAR and identified at least 12 PAR genes. We describe evidence that recombination rates have been reduced in meiosis of both sexes, and differences in recombination between S. latifolia families suggest ongoing recombination suppression. Large allele frequency differences between the sexes were found at several loci closely linked to the PAR boundary, and genes in different regions of the PAR showed striking sequence diversity patterns that help illuminate the evolution of the PAR.

Identifying new sex-linked genes through BAC sequencing in the dioecious plant Silene latifolia.

BACKGROUND: Silene latifolia represents one of the best-studied plant sex chromosome systems. A new approach using RNA-seq data has recently identified hundreds of new sex-linked genes in this species. However, this approach is expected to miss genes that are either not expressed or are expressed at low levels in the tissue(s) used for RNA-seq. Therefore other independent approaches are needed to discover such sex-linked genes. RESULTS: Here we used 10 well-characterized S. latifolia sex-linked genes and their homologs in Silene vulgaris, a species without sex chromosomes, to screen BAC libraries of both species. We isolated and sequenced 4 Mb of BAC clones of S. latifolia X and Y and S. vulgaris genomic regions, which yielded 59 new sex-linked genes (with S. vulgaris homologs for some of them). We assembled sequences that we believe represent the tip of the Xq arm. These sequences are clearly not pseudoautosomal, so we infer that the S. latifolia X has a single pseudoautosomal region (PAR) on the Xp arm. The estimated mean gene density in X BACs is 2.2 times lower than that in S. vulgaris BACs, agreeing with the genome size difference between these species. Gene density was estimated to be extremely low in the Y BAC clones. We compared our BAC-located genes with the sex-linked genes identified in previous RNA-seq studies, and found that about half of them (those with low expression in flower buds) were not identified as sex-linked in previous RNA-seq studies. We compiled a set of ~70 validated X/Y genes and X-hemizygous genes (without Y copies) from the literature, and used these genes to show that X-hemizygous genes have a higher probability of being undetected by the RNA-seq approach, compared with X/Y genes; we used this to estimate that about 30% of our BAC-located genes must be X-hemizygous. The estimate is similar when we use BAC-located genes that have S. vulgaris homologs, which excludes genes that were gained by the X chromosome. CONCLUSIONS: Our BAC sequencing identified 59 new sex-linked genes, and our analysis of these BAC-located genes, in combination with RNA-seq data suggests that gene losses from the S. latifolia Y chromosome could be as high as 30 %, higher than previous estimates of 10-20%.

A new plant sex-linked gene with high sequence diversity and possible introgression of the X copy.

We describe patterns of DNA sequence diversity in a newly identified sex-linked gene, SlX9/SlY9, in Silene latifolia (Caryophyllaceae). The copies on both sex chromosomes seem to be functional, and each maps close to the respective X- and Y-linked copy of another sex-linked gene pair, SlCypX/SlCypY. The Y-linked copy has low diversity, similar to what has been found for several other Y-linked genes in S. latifolia, and consistent with the theoretical expectations of hitch-hiking processes occurring on a non-recombining chromosome. However, SlX9 has higher diversity than other genes on the S. latifolia X chromosome. We evaluate the hypothesis of introgression from the closely related species S. dioica as an explanation for the high sequence diversity observed.

Active miniature transposons from a plant genome and its nonrecombining Y chromosome.

Mechanisms involved in eroding fitness of evolving Y chromosomes have been the focus of much theoretical and empirical work. Evolving Y chromosomes are expected to accumulate transposable elements (TEs), but it is not known whether such accumulation contributes to their genetic degeneration. Among TEs, miniature inverted-repeat transposable elements are nonautonomous DNA transposons, often inserted in introns and untranslated regions of genes. Thus, if they invade Y-linked genes and selection against their insertion is ineffective, they could contribute to genetic degeneration of evolving Y chromosomes. Here, we examine the population dynamics of active MITEs in the young Y chromosomes of the plant Silene latifolia and compare their distribution with those in recombining genomic regions. To isolate active MITEs, we developed a straightforward approach on the basis of the assumption that recent transposon insertions or excisions create singleton or low-frequency size polymorphisms that can be detected in alleles from natural populations. Transposon display was then used to infer the distribution of MITE insertion frequencies. The overall frequency spectrum showed an excess of singleton and low-frequency insertions, which suggests that these elements are readily removed from recombining chromosomes. In contrast, insertions on the Y chromosomes were present at high frequencies. Their potential contribution to Y degeneration is discussed.

Defining regions and rearrangements of the Silene latifolia Y chromosome.

We combine data from published marker genotyping of three sets of S. latifolia Y chromosome deletion mutants with changed sex phenotypes and add genotypes for several new genic markers to refine the deletion map of the Y chromosome and compare it with the X chromosome genetic map. We conclude that the Y chromosome of this species has been derived through multiple rearrangements of the ancestral gene arrangement and that none of the rearrangements so far detected was involved in stopping X-Y recombination. Different Y genotypes may also differ in their gene content and possibly arrangements, suggesting that mapping the Y-linked sex-determining genes will be difficult, even if many further genic markers are obtained. Even in determining the map of Y chromosome markers to discover all the rearrangements, physical mapping by FISH or other experiments will be essential. Future deletion mapping work should ensure that markers are studied in the parents of deletion mutants and should probably include additional deletions that were not ascertained by causing mutant sex phenotypes.

Prospective randomized study of steroids in the prevention of ischaemic injury during hepatic resection with pedicle clamping.

BACKGROUND: The major drawback of hepatic pedicle clamping is ischaemia-reperfusion injury with impairment of liver function. Perioperative steroid administration has been advocated to reduce liver damage. The aim of this prospective, randomized study was to determine whether steroid administration can reduce liver injury and improve short-term outcome. METHODS: Fifty-three patients undergoing liver resection were randomized to a steroid group (group 1) or to a control group (group 2); patients in group 1 received methylprednisolone 30 mg/kg 30 min before liver resection whereas those in group 2 did not. Serum levels of interleukin (IL) 6, total bilirubin, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and prothrombin time (PT) were measured. Length of stay, and type and number of complications were recorded. RESULTS: Serum IL-6 levels were significantly lower in the steroid group than in the control group 24 h after surgery. Steroid administration significantly modified AST, ALT and PT levels only in patients with chronic liver disease. Overall and lung-related morbidity were not significantly different between the two groups. CONCLUSIONS: Steroid administration suppresses serum IL-6 levels, but has no effect on short-term outcome.

Impact of biocontrol Pseudomonas fluorescens CHA0 and a genetically modified derivative on the diversity of culturable fungi in the cucumber rhizosphere.

Little is known about the effects of Pseudomonas biocontrol inoculants on nontarget rhizosphere fungi. This issue was addressed using the biocontrol agent Pseudomonas fluorescens CHA0-Rif, which produces the antimicrobial polyketides 2,4-diacetylphloroglucinol (Phl) and pyoluteorin (Plt) and protects cucumber from several fungal pathogens, including Pythium spp., as well as the genetically modified derivative CHA0-Rif(pME3424). Strain CHA0-Rif(pME3424) overproduces Phl and Plt and displays improved biocontrol efficacy compared with CHA0-Rif. Cucumber was grown repeatedly in the same soil, which was left uninoculated, was inoculated with CHA0-Rif or CHA0-Rif(pME3424), or was treated with the fungicide metalaxyl (Ridomil). Treatments were applied to soil at the start of each 32-day-long cucumber growth cycle, and their effects on the diversity of the rhizosphere populations of culturable fungi were assessed at the end of the first and fifth cycles. Over 11,000 colonies were studied and assigned to 105 fungal species (plus several sterile morphotypes). The most frequently isolated fungal species (mainly belonging to the genera Paecilomyces, Phialocephala, Fusarium, Gliocladium, Penicillium, Mortierella, Verticillium, Trichoderma, Staphylotrichum, Coniothyrium, Cylindrocarpon, Myrothecium, and Monocillium) were common in the four treatments, and no fungal species was totally suppressed or found exclusively following one particular treatment. However, in each of the two growth cycles studied, significant differences were found between treatments (e.g., between the control and the other treatments and/or between the two inoculation treatments) using discriminant analysis. Despite these differences in the composition and/or relative abundance of species in the fungal community, treatments had no effect on species diversity indices, and species abundance distributions fit the truncated lognormal function in most cases. In addition, the impact of treatments at the 32-day mark of either growth cycle was smaller than the effect of growing cucumber repeatedly in the same soil.

Ericoid mycorrhizal fungi are common root associates of a Mediterranean ectomycorrhizal plant (Quercus ilex).

Mycorrhiza samples of neighbouring Quercus ilex and Erica arborea plants collected in a postcutting habitat were processed to see whether plants differing in mycorrhizal status harbour the same root endophytes. Three experiments were performed in parallel: (i) isolation, identification and molecular characterization of fungi from surface-sterilized roots of both plant species; (ii) re-inoculation of fungal isolates on axenic E. arborea and Q. ilex seedlings; (iii) direct inoculation of field-collected Q. ilex ectomycorrhizas onto E. arborea seedlings. About 70 and 150 fungal isolates were obtained from roots of Q. ilex and E. arborea, respectively. Among them, Oidiodendron species and five cultural morphotypes of sterile isolates formed typical ericoid mycorrhizas on E. arborea in vitro. Fungi with such mycorrhizal ability were derived from both host plants. Isolates belonging to one of these morphotypes (sd9) also exhibited an unusual pattern of colonization, with an additional extracellular hyphal net. Ericoid mycorrhizas were also readily obtained by direct inoculation of E. arborea seedlings with Q. ilex ectomycorrhizal tips. Polymerase chain-restriction fragment length polymorphism and random amplified polymorphic DNA analyses of the shared sterile morphotypes demonstrate, in the case of sd9, the occurrence of the same genet on the two host plants. These results indicate that ericoid mycorrhizal fungi associate with ectomycorrhizal roots, and the ecological significance of this finding is discussed.