Mitochondrial DNA copy number is maintained during spermatogenesis and in the development of male larvae to sustain the doubly uniparental inheritance of mitochondrial DNA system in the blue mussel Mytilus galloprovincialis

Doubly uniparental inheritance (DUI) of mitochondrial (mt) DNA has been reported in the blue mussel Mytilus galloprovincialis. In DUI, males inherit both paternal (M type) and maternal (F type) mtDNA. Here we investigated changes in M type mtDNA copy numbers and mitochondrial mass in testicular cells by real‐time polymerase chain reaction and flow cytometry. The ratios of M type mtDNA copy numbers to nuclear DNA content were not different between haploid (1n), diploid (2n) and tetraploid (4n) spermatogenic cells. The mitochondrial mass decreased gradually during spermatogenesis. These results suggest that mtDNA and mitochondrial mass are maintained during spermatogenesis. We then traced M type mtDNA in larvae after fertilization. M type mtDNA was maintained up to 24 h after fertilization in the male‐biased crosses, but decreased significantly in female‐biased crosses (predicted by Mito Tracker staining pattern). These results are strikingly different from those reported for mammals and fish, where it is well known that the mitochondria and mtDNA are reduced during spermatogenesis and that sperm mitochondria and mtDNA are eliminated soon after fertilization. Thus, the M type mtDNA copy number is maintained during spermatogenesis and in the development of male larvae to sustain the DUI system in the blue mussel.     

土霉素胁迫下拟南芥基因组DNA甲基化的MSAP分析

以拟南芥 (Arabidopsis thaliana)为材料,研究不同土霉素浓度下拟南芥幼苗生长发育及基因组DNA的甲基化水平和变化模式。结果表明,3、5、7\,9 μmol/L土霉素胁迫对拟南芥幼苗的根长和株高有显著抑制作用;但对拟南芥幼苗的侧根数量有显著促进作用。甲基化敏感扩增多态性 (methylation-sensitive amplification polymorphism, MSAP)分析表明,经3、5、7\,9 μmol/L土霉素处理后基因组DNA甲基化比率分别为17.91%、12.50%、11.81%和14.62%,均低于对照 (18.18%)。结果表明,拟南芥经土霉素胁迫后存在基于 DNA甲基化水平和模式改变的表观遗传变异,5-甲基胞嘧啶百分含量的变化无统一趋势或规律。与对照相比,3、5、7\,9 μmol/L土霉素胁迫下拟南芥幼苗基因组DNA的甲基化和去甲基化分别为13.29%、9.22%、8.03%、12.59%和2.80%、4.26％、5.11%、4.90％。由此推测,DNA甲基化可能是植物适应土霉素胁迫机制的机制之一。     

Septal pore complex morphology in the Agaricomycotina (Basidiomycota) with emphasis on the Cantharellales and Hymenochaetales

The ultrastructure of septa and septum-associated septal pore caps are important taxonomic markers in the Agaricomycotina (Basidiomycota, Fungi). The septal pore caps covering the typical basidiomycetous dolipore septum are divided into three main phenotypically recognized morphotypes: vesicular-tubular (including the vesicular, sacculate, tubular, ampulliform, and globular morphotypes), imperforate, and perforate. Until recently, the septal pore cap-type reflected the higher-order relationships within the Agaricomycotina. However, the new classification of Fungi resulted in many changes including revision of existing and addition of new orders. Therefore, the septal pore cap ultrastructure of more than 325 species as reported in literature was related to this new classification. In addition, the septal pore cap ultrastructures of Rickenella fibula and Cantharellus formosus were examined by transmission electron microscopy. Both fungi have dolipore septa associated with perforate septal pore caps. These results combined with data from the literature show that the septal pore cap-type within orders of the Agaricomycotina is generally monomorphic, except for the Cantharellales and Hymenochaetales.It appears from the fungal phylogeny combined with the septal pore cap ultrastructure that the vesicular-tubular and the imperforate type both may have arisen from endoplasmic reticulum. Thereafter, the imperforate type eventually gave rise to the perforate septal pore cap-type.     

陕西省林麝mtDNA D-loop区序列结构和种群遗传多样性

林麝(Moschus berezovskii)曾广泛分布于中国,由于盗猎和栖息地缩小,秦岭地区野生种群数量迅速下降,圈养繁殖种群已成立了几十年,但大多数圈养种群的遗传背景不清,种群规模增长非常缓慢。为了给这一物种的保护和管理提供有用的信息,调查了陕西省林麝1个圈养种群3个野生种群线粒体DNA(mt DNA)D-Loop 632 bp片段的遗传多样性和种群结构。在69个个体中其碱基组成为A+T的平均含量63.2%高于G+C含量36.8%,共检测到变异位点171个(约占总位点数的27.05%)。核苷酸多样性(Pi)为0.04424,平均核苷酸差异数(K)为19.908。69个个体分属32个单倍型,单倍型间的平均遗传距离(P)为0.070。32个单倍型构建的NJ系统树聚为3个分支,4个林麝群体中的单倍型是随机分布的。4个群体的平均遗传距离为0.043(标准误SE为0.005),凤县养殖场群体与留坝和陇县群体的亲缘关系较远。单倍型间的平均遗传距离为0.043,可见其遗传分化尚未达到种群分化的水平。结果表明,陕西省林麝群体mt DNA D-loop区序列存在着较丰富的变异和遗传多样性,凤县野生群体和凤县养殖场群体的核苷酸多样性和单倍型多样较高,养殖场种群没有出现近亲繁殖及遗传多样性下降的情况。凤县野生群体和凤县养殖场群体两者遗传分化较小,存在着较高的基因流水平。     

Genetic Polymorphism of Microsatellite DNA in Two Populations of Northern Sheatfish (Silurus soldatovi)

In this article, population variations and genetic structures of two populations of northern sheatfish (Silurus soldatovi) were analyzed using 24 microsatellite loci enriched from southern catfish (S. meriaionalis Chen) by magnetic beads. Gene frequency (P), observed heterozygosity (Ho), expected heterozygosity (He), polymorphism information contents (PIC), and number of effective alleles (Ne) were determined. One population was wild, ripe individuals collected from Heilongjiang River (HNS); the other was cultured fry collected from Songhuajiang River (SNS). The Hardy-Weinberg equilibrium (HWE) was tested by the genetic departure index (d). The coefficient of gene differentiation G_ST and Φ_ST by AMOVA (Analysis of Molecular Variety) was imputed using Arlequin software in this study. In addition, a phylogenetic tree was constructed by UPGMA method based on the pairwise Nei's standard distances using PHYLIP. A total of 1 357 fragments with sizes ranging between 102 bp and 385 bp were acquired by PCR amplifications. The average number of alleles of the two populations was 8.875. Results indicated that these microsatellite loci were highly polymorphic and could be used as genetic markers. The mean values of the parameters P, Ho, He, PIC, and Ne were 0.165, 0.435, 0.758, 0.742, and 5.019 for HNS and 0.147, 0.299, 0.847, 0.764, and 5.944 for SNS, respectively. Although there were differences, there were no significant differentiations except for the locus HLJcf37. These populations to a certain extent deviated from HWE, such as excessive and deficient heterozygote numbers. The value of G_ST was 0.078 and above 98% of the variation were differences among individuals within the population, so the variation between populations was insignificant. Cluster analysis also showed that the relationships among individuals were very close. In conclusion, the microsatellite markers that were developed through this study are useful for genetic analysis and the genetic culture that was proposed in this study has no significant impact on S. soldatovi.     

Lavandula angustifolia essential oil as a novel and promising natural candidate for tick (Rhipicephalus (Boophilus) annulatus) control

Lavandula angustifolia is a well known herbal medicine with a variety of useful properties, including its acaricidal effect. This experiment was carried out to study the bioacaricidal activity of L. angustifolia essential oil (EO) against engorged Rhipicephalus (Boophilus) annulatus (Acari; Ixodidae) females. For this purpose six serial concentrations (0.5, 1.0, 2.0, 4.0, 6.0 and 8.0% w/v) of L. angustifolia EO were used. There was considerable mortality in concentrations more than 4.0% although there were no differences between 6.0 and 8.0% in all measured criteria. The mortality rate 24 h after inoculation was 73.26 and 100% in groups treated with 4.0 and 8.0% EO, respectively. Lavender EO also reduced tick egg weight in a concentration-dependent manner. The amount of eggs produced varied from 0.12 g (at 0.5% EO) to 0.00 g (at 8.0% EO) but did not differ statistically from the control. L. angustifolia EO caused 100% failure in egg laying at 6.0 and 8.0% whereas this value in the control group was zero. A positive correlation between L. angustifolia EO concentration and tick control, assessed by relative mortality rate and egg-laying weight, was observed by the EO LC/EC₅₀, which, when calculated using the Probit test, was 2.76-fold higher than the control. Lavender is a promising acaricidal against R. (B.) annulatusin vitro.     

Temporal expression and steroidal regulation of piRNA pathway genes (mael, piwi, vasa) during Silurana (Xenopus) tropicalis embryogenesis and early larval development

It has been extensively documented that exposure of amphibians and teleost fish to exogenous steroid hormones like estrogen, androgen, xenoestrogen or steroid biosynthesis inhibitors can impair their gonadal development or induce sex reversal against genotypic sex. However, the molecular pathways underlying sexual development and the effects of sex steroids or other exogenous hormones in these aquatic vertebrates remain elusive. Recently, a germ plasm-associated piRNA (piwi-interacting RNA) pathway has been shown to be a determinant in the development of animal gonadal germline cells. In the current study, we examined whether this piRNA pathway is involved in the regulation of sex steroid hormones in gonadal development. We firstly established developmental expression patterns of three key piRNA pathway genes (mael, piwi and vasa), during Silurana (Xenopus) tropicalis embryogenesis and early larval development. All three genes exhibit high expression at early developmental stages and have significantly decreased expression thereafter, indicating a very active involvement of piRNA pathway at the beginning of embryogenesis. We further examined gene expression changes of those genes in frog larvae exposed to two sex steroid biosynthesis inhibitors, fadrozole and finasteride, both of which are known to result in male-biased or female-biased phenotypes, respectively. We found that fadrozole and finasteride exposures increased the expression of piRNA pathway genes such as mael and vasa at the larval stage when the expression of piRNA pathway genes is programmed to be very low. Therefore, our results indicate that the piRNA pathway is likely a common pathway by which different sex steroid hormones regulate gonadal sex differentiation.     

RPB2 sequences reveal a close phylogenetic relationship between tetraploid Hordelymus and diploid Hordeum species in Triticeae (Poaceae)

The origin of Hordelymus genome has been debated for years, and no consensus conclusion was reached. In this study, we sequenced and analyzed the RPB2 (RNA polymerase subunit II) gene from Hordelymus europaeus (L.) Harz, and its potential diploid ancestor species those were suggested in previous studies. The focus of this study was to examine the phylogenetic relationship of Hordelymus genomes with its potential donor Hordeum, Psathyrostachys, and Taeniatherum species. Two distinguishable copies of sequences were obtained from H. europaeus. The obvious difference between the two copies of sequences is a 24 bp indel (insertion/deletion). Phylogenetic analysis showed a strong affinity between Hordeum genome and Hordelymus with 85% bootstrap support. These results suggested that one genome in tetraploid H. europaeus closely related to the genome in Hordeum species. Another genome in H. europaeus is sister to the genomes in Triticeae species examined here, which corresponds well with the recently published EF-G data. No obvious relationship was found between Hordelymus and either Ta genome donor, Taeniatherum caput-medusae or Ns genome donor, Psathyrostachys juncea. Our data does not support the presence of Ta and Ns genome in H. europaeus, and further confirms that H. europaeus is allopolyploid.     

Systematics and evolution of the genus Torrubiella (Hypocreales, Ascomycota)

Torrubiella is a genus of arthropod-pathogenic fungi that primarily attacks spiders and scale insects. Based on the morphology of the perithecia, asci, and ascospores, it is classified in Clavicipitaceae s. lat. (Hypocreales), and is considered a close relative of Cordyceps s. 1., which was recently reclassified into three families (Clavicipitaceae s. str., Cordycipitaceae, Ophiocordycipitaceae) and four genera (Cordyceps s. str, Elaphocordyceps, Metacordyceps, and Ophiocordyceps). Torrubiella is distinguished morphologically from Cordyceps s. lat. mainly by the production of superficial perithecia and the absence of a well-developed stipitate stroma. To test and refine evolutionary hypotheses regarding the placement of Torrubiella and its relationship to Cordyceps s. lat., a multi-gene phylogeny was constructed by conducting ML and Bayesian analyses. The monophyly of Torrubiella was rejected by these analyses with species of the genus present in Clavicipitaceae, Cordycipitaceae, and Ophiocordycipitaceae, and often intermixed among species of Cordyceps s. lat. The morphological characters traditionally used to define the genus are, therefore, not phylogenetically informative, with the stipitate stromata being gained and/or lost several times among clavicipitaceous fungi. Two new genera (Conoideocrella, Orbiocrella) are proposed to accommodate two separate lineages of torrubielloid fungi in the Clavicipitaceae s. str. In addition, one species is reclassified in Cordyceps s. str. and three are reclassified in Ophiocordyceps. The phylogenetic importance of anamorphic genera, host affiliation, and stipitate stromata is discussed.     

Relationships between Bambusa species (Poaceae,Bambusoideae) revealed by random amplified polymorphic DNA

RAPD (random amplified polymorphic DNA) molecular markers were used to investigate relationships between a sample of Bambusa species from South Eastern China that have been placed in Bambusa or in several segregate genera, Dendrocalamopsis, Leleba, Lingnania, Neosinocalamus and Sinocalamus by different authors. On the resultant neighbor-joining tree, a thorny core Bambusa cluster was distinguished, as was a Lingnania group, and a cluster of Dendrocalamus species with more capitate inflorescences. However, Leleba was found to be a polyphyletic group in the present study.     

Mycobiota associated with the ambrosia beetle Scolytodes unipunctatus (Coleoptera: Curculionidae, Scolytinae)

Ambrosia beetles (Coleoptera: Scolytinae) are associated with strictly entomochoric and mutualistic fungi. We studied the mycobiota associated with Scolytodes unipunctatus, ambrosia beetles that infest Cecropia trees in Central America. Isolates were characterized using morphology and rDNA sequences (ITS region, LSU, and SSU rDNA). Four species are described here: Raffaelea scolytodis sp. nov. (Ophiostomatales), Gondwanamyces scolytodis sp. nov., Custingophora cecropiae sp. nov., and Graphium sp. (Microascales). The genus Custingophora is emended to include Knoxdaviesia anamorphs of Gondwanamyces based on uniformity of DNA sequences and phenotype.     

8-Oxoguanine DNA glycosylase 1 (ogg1) maintains the function of cardiac progenitor cells during heart formation in zebrafish

Genomic damage may devastate the potential of progenitor cells and consequently impair early organogenesis. We found that ogg1, a key enzyme initiating the base-excision repair, was enriched in the embryonic heart in zebrafish. So far, little is known about DNA repair in cardiogenesis. Here, we addressed the critical role of ogg1 in cardiogenesis for the first time. ogg1 mainly expressed in the anterior lateral plate mesoderm (ALPM), the primary heart tube, and subsequently the embryonic myocardium by in situ hybridisation. Loss of ogg1 resulted in severe cardiac morphogenesis and functional abnormalities, including the short heart length, arrhythmia, decreased cardiomyocytes and nkx2.5⁺ cardiac progenitor cells. Moreover, the increased apoptosis and repressed proliferation of progenitor cells caused by ogg1 deficiency might contribute to the heart phenotype. The microarray analysis showed that the expression of genes involved in embryonic heart tube morphogenesis and heart structure were significantly changed due to the lack of ogg1. Among those, foxh1 is an important partner of ogg1 in the cardiac development in response to DNA damage. Our work demonstrates the requirement of ogg1 in cardiac progenitors and heart development in zebrafish. These findings may be helpful for understanding the aetiology of congenital cardiac deficits.     

Real-time PCR detection of Holophagae (Acidobacteria) and Verrucomicrobia subdivision 1 groups in bulk and leek (Allium porrum) rhizosphere soils

In the light of the poor culturability of Acidobacteria and Verrucomicrobia species, group-specific real-time (qPCR) systems were developed based on the 16S rRNA gene sequences from culturable representatives of both groups. The number of DNA targets from three different groups, i.e. Holophagae (Acidobacteria group 8) and Luteolibacter/Prosthecobacter and unclassified Verrucomicrobiaceae subdivision 1, was determined in DNA extracts from different leek (Allium porrum) rhizosphere soil compartments and from bulk soil with the aim to determine the distribution of the three bacterial groups in the plant-soil ecosystem. The specificity of the designed primers was evaluated in three steps. First, in silico tests were performed which demonstrated that all designed primers 100% matched with database sequences of their respective groups, whereas lower matches with other non-target bacterial groups were found. Second, PCR amplification with the different primer sets was performed on genomic DNA extracts from target and from non-target bacteria. This test demonstrated specificity of the designed primers for the target groups, as single amplicons of expected sizes were found only for the target bacteria. Third, the qPCR systems were tested for specific amplifications from soil DNA extracts and 48 amplicons from each primer system were sequenced. All sequences were > 97% similar to database sequences of the respective target groups. Estimated cell numbers based on Holophagae-, Luteolibacter/Prosthecobacter- and unclassified Verrucomicrobiaceae subdivision 1-specific qPCRs from leek rhizosphere compartments and bulk soils demonstrated higher preference for one or both rhizosphere compartments above bulk soil for all three bacterial groups.     

Inheritance of mitochondrial DNA in lentil (Lens culinaris Medik.)

Restriction fragment analysis was used to examine the inheritance of lentil mitochondrial DNA (mtDNA) in F₁ and F₅ progeny from intrasubspecific (Lens culinaris ssp. culinaris) crosses and in F₁ progeny from intersubspecific (Lens culinaris ssp. orientalis x L. culinaris ssp. culinaris) crosses. Southern blots of digested parental and progeny DNA were hybridized to heterologous maize mtDNA probes specific to coxI and atp6 genes. Two restriction fragment polymorphisms separated L.c. ssp. culinaris Laird and Eston from L.c. ssp. culinaris ILL5588, and one restriction fragment polymorphism distinguished L.c. ssp. culinaris Laird and Eston from L.c. ssp. orientalis LO4. Twelve of 13 f₁ progeny and all F₅ progeny from the intrasubspecific crosses, and all F₁ progeny from intersubspecific crosses had only maternal mtDNA restriction fragments. One f₁ plant from an Eston x ILL5588 cross inherited mtDNA fragments from both parents. Nuclear DNA inheritance was biparental in all F₁ progeny.NRCC No. 38451     

Flow cytometric determination of zoospore DNA content and population DNA distribution in cultured Pfiesteria spp. (Pyrrhophyta)

The relative cellular DNA content from 23 different clonal cultures of Pfiesteria spp. zoospores was determined using a DNA fluorochrome and flow cytometry. Significant differences between Pfiesteria piscicida and P. shumwayae were detected, both in mean zoospore DNA content and population cell cycle DNA distribution. Intraspecific differences in DNA content were found between clonal zoospore cultures established from different geographical regions. Long-term cultures (years) of P. piscicida were available for testing, and a negative correlation was observed between zoospore DNA content and time in culture. Zoospore cell cycle-related DNA distributions were also markedly different between the two species in these clonal cultures. In most cultures tested, P. piscicida zoospores exhibited bimodal DNA flow histograms with G1-S-G2+M distributions, typical of eukaryotic asynchronously cycling cells. In contrast, cultures of P. shumwayae zoospores exhibited one DNA peak distribution, indicative of synchronized cells. The data are consistent with the hypothesis that P. shumwayae zoospores are interphasic cells, and mitosis in zoospore cultures of this species predominantly occurs as benthic or adherent non-motile division cysts. Light microscopy observations of the nuclear condition of electrostatically sorted zoospores of each Pfiesteria species also support this hypothesis. If highly conserved, this disparity in modes of vegetative reproduction would ramify the population dynamics of the two Pfiesteria species.