Xiao-Ru Wang

Phylogenetic relationships of Eurasian pines (Pinus, Pinaceae) based on chloroplast rbcL, MATK, RPL20-RPS18 spacer, and TRNV intron sequences.

The sequence divergence of chloroplast rbcL, matK, trnV intron, and rpl20-rps18 spacer regions was analyzed among 32 Pinus species and representatives of six other genera in Pinaceae. The total aligned sequence length is 3570 bp. Of the four sequences examined, matK evolved much faster than rbcL in Pinus and in other Pinaceae genera. The two noncoding regions did not show more divergence than the two coding regions, especially within each Pinus subgenus. Phylogenetic analyses based on these four sequences gave consistent results and strongly supported the monophyly hypothesis for the genus Pinus and its two recognized subgenera. Pinus krempfii, the two-flat-needle pine endemic to Vietnam, was placed in subgen. Strobus and showed closer affinity to subsect. Gerardianae. The ancient character of sect. Parrya is further confirmed. However, monophyly of the sect. Parrya is not supported by our data. Among the Eurasian pines of subgen. Pinus, Mediterranean pines formed one clade and the Asian members of subsect. Sylvestres formed another. The Himalayan P. roxburghii showed considerable divergence from all the other hard pines from both regions. Pinus merkusii was distinctly separated from all the Asian members of subsect. Sylvestres. The implications of our results for Pinus classification are discussed.

Dysregulated Cannabinoid Signaling Disrupts Uterine Receptivity for Embryo Implantation

The mechanisms by which synchronized embryonic development to the blastocyst stage, preparation of the uterus for the receptive state, and reciprocal embryo-uterine interactions for implantation are coordinated are still unclear. We show in this study that preimplantation embryo development became asynchronous in mice that are deficient in brain-type (CB1) and/or spleen-type (CB2) cannabinoid receptor genes. Furthermore, whereas the levels of uterine anandamide (endocannabinoid) and blastocyst CB1 are coordinately down-regulated with the onset of uterine receptivity and blastocyst activation prior to implantation, these levels remained high in the nonreceptive uterus and in dormant blastocysts during delayed implantation and in pregnant, leukemia inhibitory factor (LIF)-deficient mice with implantation failure. These results suggest that a tight regulation of endocannabinoid signaling is important for synchronizing embryo development with uterine receptivity for implantation. Indeed this is consistent with our finding that while an experimentally induced, sustained level of an exogenously administered, natural cannabinoid inhibited implantation in wild-type mice, it failed to do so inCB1 −/− /CB2 −/−double mutant mice. The present study is clinically important because of the widely debated medicinal use of cannabinoids and their reported adverse effects on pregnancy.

Extensive Functional Diversification of the Populus Glutathione S-Transferase Supergene Family

Identifying how genes and their functions evolve after duplication is central to understanding gene family radiation. In this study, we systematically examined the functional diversification of the glutathione S-transferase (GST) gene family in Populus trichocarpa by integrating phylogeny, expression, substrate specificity, and enzyme kinetic data. GSTs are ubiquitous proteins in plants that play important roles in stress tolerance and detoxification metabolism. Genome annotation identified 81 GST genes in Populus that were divided into eight classes with distinct divergence in their evolutionary rate, gene structure, expression responses to abiotic stressors, and enzymatic properties of encoded proteins. In addition, when all the functional parameters were examined, clear divergence was observed within tandem clusters and between paralogous gene pairs, suggesting that subfunctionalization has taken place among duplicate genes. The two domains of GST proteins appear to have evolved under differential selective pressures. The C-terminal domain seems to have been subject to more relaxed functional constraints or divergent directional selection, which may have allowed rapid changes in substrate specificity, affinity, and activity, while maintaining the primary function of the enzyme. Our findings shed light on mechanisms that facilitate the retention of duplicate genes, which can result in a large gene family with a broad substrate spectrum and a wide range of reactivity toward different substrates.

Empirical assessment of allozyme and RAPD variation in Pinus sylvestris (L.) using haploid tissue analysis

We analysed 20 allozyme and 22 putative random amplified polymorphic DNA (RAPD) loci in two populations of Pinus sylvestris (L.) from northern Sweden. Genotypes for individual allozyme and RAPD loci were inferred from segregation patterns in haploid macrogametophytes. Therefore, it was possible to distinguish between homo- and heterozygotes carrying a RAPD fragment and to estimate directly the frequencies of RAPD fragments. The percentage of polymorphic loci and the expected and observed heterozygosity were lower for allozymes than for RAPDs. Average fixation indices for both types of markers were negative indicating a heterozygote excess over panmictic expectations. The apportionment of genetic variation within and among the investigated populations was similar for allozymes and RAPDs and showed that most of the variation resided within populations. RAPD genotypes inferred from haploid material were subsequently converted to diploid phenotypes and used to estimate indirectly the frequencies of RAPD fragments. Gene diversity measurements derived from indirectly estimated RAPD frequencies were distinctly lower than those based on directly estimated RAPD frequencies. This result was caused by the absence of the null homozygote at many loci which appeared as monomorphic in the diploid data set. Population differentiation coefficients based on the indirectly estimated RAPD frequencies were not concordant with those based on directly estimated RAPD frequencies. Our present results indicate that when complete genotype information can be obtained, RAPD analysis provides genetic information similar to that revealed by analysis of allozyme variation. On the other hand, our results are concordant with theoretical results suggesting that analysis of RAPD variation in diploid material can produce unreliable estimates of population-genetic parameters.

18S rRNA Gene Variation among Common Airborne Fungi, and Development of Specific Oligonucleotide Probes for the Detection of Fungal Isolates

ABSTRACT In this study, we sequenced 18S rRNA genes (rDNA) from 49 fungal strains representing 31 species from 15 genera. Most of these species are common airborne fungi and pathogens that may cause various public health concerns. Sequence analysis revealed distinct divergence between Zygomycota and Ascomycota. Within Ascomycota, several strongly supported clades were identified that facilitate the taxonomic placement of several little-studied fungi. Wallemia appeared as the group most diverged from all the other Ascomycota species. Based on the 18S rDNA sequence variation, 108 oligonucleotide probes were designed for each genus and species included in this study. After homology searches and DNA hybridization evaluations, 33 probes were verified as genus or species specific. The optimal hybridization temperatures to achieve the best specificity for these 33 probes were determined. These new probes can contribute to the molecular diagnostic research for environmental monitoring.

Detection and Quantification of Wallemia sebi in Aerosols by Real-Time PCR, Conventional PCR, and Cultivation

ABSTRACT Wallemia sebi is a deuteromycete fungus commonly found in agricultural environments in many parts of the world and is suspected to be a causative agent of farmers lung disease. The fungus grows slowly on commonly used culture media and is often obscured by the fast-growing fungi. Thus, its occurrence in different environments has often been underestimated. In this study, we developed two sets of PCR primers specific to W. sebi that can be applied in either conventional PCR or real-time PCR for rapid detection and quantification of the fungus in environmental samples. Both PCR systems proved to be highly specific and sensitive for W. sebi detection even in a high background of other fungal DNAs. These methods were employed to investigate the presence of W. sebi in the aerosols of a farm. The results revealed a high concentration of W. sebi spores, 107 m−3 by real-time PCR and 106 m−3 by cultivation, which indicates the prevalence of W. sebi in farms handling hay and grain and in cow barns. The methods developed in this study could serve as rapid, specific, and sensitive means of detecting W. sebi in aerosol and surface samples and could thus facilitate investigations of its distribution, ecology, clinical diagnosis, and exposure risk assessment.

Cytoplasmic composition in Pinus densata and population establishment of the diploid hybrid pine

Sequence and restriction site analyses of the paternally inherited chloroplast rbcL gene and maternally inherited mitochondrial nad1 fragments from the same set of populations and individuals were used to investigate cytoplasmic composition and population establishment of Pinus densata, a diploid pine that originated through hybridization between P. tabuliformis and P. yunnanensis. Two variable sites and three chlorotypes (TT, TC and GC) were detected on the rbcL gene of the three pines. P. densata harboured the three chlorotypes, two of which (TT, GC) were characteristic of the parental species, respectively. The third chlorotype (TC) was distributed extensively in seven of the 10 P. densata populations analysed, and might represent a mutation type or have been derived from an extinct parent. The distribution of chlorotypes, together with that of mitotypes, indicated that significant founder effect and backcross happened during the population establishment of the hybrid pine. P. tabuliformis and P. yunnanensis had acted as both mother and father donors, i.e. bi‐directional gene flow existed between the two parental species in the past. Population differentiation of P. densata is high, as detected from the cytoplasmic genomes: GST = 0.533 for cpDNA and GST = 0.905 for mtDNA. The differences in cytoplasmic composition among the hybrid populations suggest that the local populations have undergone different evolutionary histories.

Evaluation of PCR primers and PCR conditions for specific detection of common airborne fungi

We examined the selectivity of 53 sets of primers for environmental monitoring of indoor air quality. Thirty-six fungal strains, representing 26 species from 14 genera of commonly occurring fungi, and 16 different bacterial strains, representing both gram-negative and gram-positive species, were included in the experiment. We verified the specificity of 28 of the 53 sets of primers, which were classified as universal fungal, universal bacterial, group or species specific. The PCR conditions required for optimal specificity were also determined. These results can serve as a guide for the step-wise PCR-based detection and identification of airborne fungi commonly found in indoor environments.

Chloroplast DNA-based phylogeny of Asian Pinus species (Pinaceae)

AbstractThe genusPinus includes over 90 species with approximately 24 species native to Asia. We have analyzed the chloroplast (cp) DNA variation of 18Pinus species, including 15 Asian, two Eurasian, and one European species using seven restriction enzymes and ten non-overlapping probes and inferred their phylogenetic relationships. Results of phenetic and cladistic approaches to phylogeny reconstruction were largely in agreement, suggesting two major lineages within the genus and confirmed the ancient character of haploxylon and diploxylon subgenera. Species from sectionParrya appear to have diverged earliest from the hypothesized phylogenetic centre for the haploxylon pines, withP. bungeana andP. gerardiana forming two basal, monotypic lineages. The range of estimated pairwise nucleotide substitutions per site (