Flow cytometry and cytogenetic tools in eucalypts: genome size variation × karyotype stability

The eucalypts comprise a group of woody plants used in commercial forest plantations owing to their high growth rates, adaptability to various ecological conditions and multiple applications. Despite the enormous amount of molecular data available for eucalypts, a basic understanding of the nature of its genome still requires information regarding the DNA amount in the genus. In this work, we estimated the genome size and base composition of 25 eucalypt species. With a comparative karyotype approach, we aimed to identify possible chromosomal alterations correlated with the genome size variation. Classical cytogenetic and genomic in situ hybridization experiments were conducted for this purpose. The studied species showed genome size ranging from 2C = 0.91 (Corymbia intermedia) to 2C = 1.37 pg (Eucalyptus paniculata) and AT/CG ratios varying from AT = 61.3 (Eucalyptus urophylla) to AT = 62.85% (C. intermedia). Comparative karyotype analysis revealed no remarkable differences in chromosome number (2n = 22) or morphology among eucalypt species despite considerable differences in nuclear DNA content. The genome in situ hybridization method did not distinguish non-homologous chromosomal regions of Eucalyptus baileyana and Corymbia citriodora, despite the difference of 0.45 pg between their genome sizes. The results found in the present work corroborate the consideration of small and dispersed DNA changes as the main cause of genome size variation in eucalypts.     

Expression profiles of <Emphasis Type="Italic">amh</Emphasis> and <Emphasis Type="Italic">foxl2</Emphasis> in <Emphasis Type="Italic">Schizothorax kozlovi</Emphasis>, and their response to temperature during the early developmental stage

To elucidate the role of amh and foxl2 in sex differentiation of the teleost fish Schizothorax kozlovi, the full-length cDNAs were cloned from the mature testis and ovary by rapid amplification of cDNA ends (RACE), and their relative mRNA expression levels were determined by quantitative real-time polymerase chain reaction among tissues and temperature groups. The complete amh and foxl2 cDNAs of S. kozlovi were 2060 bp and 1750 bp, which encoded 568 and 306 amino acids, respectively. The amh were expressed only in gonads, while foxl2 was expressed in the gills, brain and gonads, both exhibiting relatively high tissue specificity. The amh exhibited sex-specific expression pattern in the gonads. No sex differences in the foxl2 expression were observed in the brain and gonads, but significant sex differences were found in the gills. No significant differences were found in the foxl2 expression, from the larval to the juvenile stage, and also between different temperature groups. However, significant differences were found in the expression levels of amh from the larval (12–63 days posthatching (dph)) to the juvenile stage (190 dph), and also among the \(18{^{\circ }}\hbox {C}\) and \(10{^{\circ }}\hbox {C}\) groups at 31 dph. This result suggested that amh plays an important role in male sex differentiation of S. kozlovi during the early developmental stage, but no similar effect was observed in foxl2.     

Is the interspecific variation of body size of land snails correlated with rainfall in Israel and Palestine?

The hypothesis that body size of land snail species increases with aridity in Israel and Palestine because large snails lose relatively less water due to their lower surface to volume ratio has been investigated. Data on rainfall amplitudes of 84 land snail species in Israel and Palestine and on their body sizes were used to test for interspecific correlations between body size and rainfall. Four methods, means of body sizes in rainfall categories, the midpoint method, the across-species method, and a phylogenetically controlled analysis (CAIC) showed that there is no significant correlation between body size of land snail species and their rainfall amplitude in Israel and Palestine. The lack of an interspecific correlation between body size and rainfall amplitude may be the result of conflicting selective forces on body size.     

Streaked horned lark <Emphasis Type="Italic">Eremophila alpestris strigata</Emphasis> has distinct mitochondrial DNA

The Streaked Horned Lark (STHL; Eremophila alpestris strigata) is a federal candidate for listing under the Endangered Species Act. We evaluated the conservation status and level of genetic diversity of the STHL using the complete mitochondrial ND2 gene. We sampled 32 STHLs from the southern Puget Sound region, the Pacific coast, and Whites Island in the Columbia River of Washington, and additional 68 horned larks from Alaska, alpine and eastern Washington, Oregon, California, and Asia (outgroups). Our Maximum Likelihood analysis of 32 haplotypes identified three geographically concordant clades in Pacific coast states: Pacific Northwest (alpine and eastern Washington, Alaska), Pacific Coast (western Washington, California), and Great Basin (eastern Oregon). Each of the three clades was supported by bootstrap values ≥86%. The distance among them varied from 0.72 to 0.79% nucleotide divergence excluding intraclade variation. The relationship among the clades was not resolved. AMOVA also showed significant structuring of haplotypes among the three clades. Differences among clades accounted for 75.7% of sequence variation, differences among localities within clades accounted for 12.1%, and differences among individuals within localities accounted for the remaining 12.2%. Although STHL populations were closely related to the Californian sample, they appeared unique and isolated. All pairwise F _st values involving the STHL samples were significant (except between themselves). STHLs appear to have remarkably low genetic diversity; all 32 STHLs shared the same haplotype. Even with small sample sizes, all other localities had multiple haplotypes. Because the STHL appears to be unique and isolated, and to have little genetic diversity our data suggest it should be a conservation priority.     

Variation in alphoid DNA size and trisomy 21: a possible cause of nondisjunction

The cause of nondisjunction of chromosome 21 remains largely unknown. In the present report, we investigate the hypothesis that variation in alphoid DNA size has a role in trisomy formation. Pulsed-field gel electrophoresis was used to examine the chromosome 21 alphoid DNA array lengths in 23 families (all of Northern European ancestry) with an affected child with trisomy 21 in whom the parental and meiotic origin of nondisjunction had been determined as maternal meiosis I, and in 38 controls. Initially, the combined alphoid size of both chromosome 21 homologues was assessed. This indicated an association between small combined alphoid size and maternal meiosis I nondisjunction. Moreover, in a subset of the families under study (n=12), it was possible to study the alpha21-I size of individual chromosome 21 homologues (simple alphoid size); this provided further evidence that the risk for nondisjunction is related to the size of the alphoid array of one of the two chromosome 21 homologues being small.     

Group size and composition of black-and-gold howler monkeys (<Emphasis Type="Italic">Alouatta caraya</Emphasis>) on the Upper Paraná River,Southern Brazil

In social mammals, group size, sex and age-class composition are important parameters that are required to understand population dynamics and determine conservation strategies. These parameters are known only poorly for the black-and-gold howler monkey (Alouatta caraya). Here, we studied groups of A. caraya on islands and adjacent banks of the Upper Paraná River of southern Brazil, to examine variability in group size and composition. This location is important for this species because of the high density of howlers. Group size was large, varying from 6 to 18 individuals (average = 11.5, standard deviation = 3.3, n = 13). Groups were multi-male, and adult females outnumbered adult males. On average, groups had the following ratios: 1 adult male: 0.5 subadult male: 1.9 adult females: 0.9 juveniles: 0.5 infants. The ratio of 0.2 infant: 1 adult female is less than that in other species, but typical of other studies of the black-and-gold howlers. Here, we discuss environmental and social pressures that may impose structure on large groups of howlers in terms of strategies for living in high densities. We also compare these data with those of the area of sympatry shared between A. caraya and A. clamitans, and suggest that competition may occur between the two species and that reduced fitness may be a consequence of mixed groups. Contribution number 1,746 of the Departamento de Zoologia of Universidade Federal do Paraná (UFPR).     

Anthraquinones from in vitro root culture of <Emphasis Type="Italic">Morinda royoc</Emphasis> L.

Morinda royoc L. (Rubiaceae) root cultures were established for the production of anthraquinones (AQs). Three independent experiments were performed to evaluate the effects of different levels of indole-3-acetic acid (0–22.8 μM), culture duration (15–75 days) and subculture number (0–4). The following indicators were recorded: root fresh weight per Erlenmeyer and intracellular and extracellular AQ production. The experiments performed in this study allowed an increase of intracellular AQ content up to a maximum of 4.5 mg g⁻¹ of fresh mass, after 30 days of culture in a medium 5.7 μM of IAA. In addition, isolation and identification of seven AQs from M. royoc L. roots is described, one of them being reported for the first time for this species. The structures of isolated compounds were determined from ¹H-NMR data. To the best of our knowledge, this is the first report on AQ production from root culture of this plant.     

<Emphasis Type="Bold">The Dangers of Direct-to-Consumer Genetic Testing for Alzheimer’s Disease</Emphasis>

The overarching issue with this case study is poor regulation and quality control over direct-to-consumer genetic testing, delivered in the absence of any medical oversight.     

<Emphasis Type="Italic">Apis mellifera</Emphasis> and <Emphasis Type="Italic">Osmia cornuta</Emphasis> as carriers for the secondary spread of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> on apple flowers

The efficiency of two pollinators, Apis mellifera L. (Hymenoptera: Apidae) and the mason bee Osmia cornuta (Latreille) (Hymenoptera: Megachilidae), as carriers of biocontrol agents (BCA) from flower to flower (secondary colonisation) was investigated on apple cv ‘Golden Delicious’. The BCA tested was Bacillus subtilis, strain BD170 (Biopro®) developed for the control of the ‘fire blight’ caused by Erwinia amylovora (Burril) Winslow et al. The two insect species were studied as secondary BCA carriers on apple plants in pots under net screened tunnels. Their behaviour and capacity to deposit the BCA in the most receptive flower parts were compared both by washing, diluting and plating the flower organs on a recovery medium and by means of PCR analyses based on a molecular marker. O. cornuta showed better performances with respect to A. mellifera. For the field trials, pollinators were introduced in four apple orchards. During apple’s flowering, the BD170 (100 g hl^?l) was sprayed once in two fields, and twice in the others. The pollinators’ efficacy in carrying the BCA from sprayed flowers to the stigmas of newly opened ones at different times after the spray treatment was evaluated. The detection of the BCA was performed by PCR analysis. The percentages of positive PCR flower samples were higher in the internal treated areas of the fields with respect to the external untreated ones, but the high colonisation level found in the latter and in the flowers opened in both areas several days after the treatment(s) demonstrated that pollinators can play an important role as secondary carriers.     

Cryopreservation of hairy root cultures of <Emphasis Type="Italic">Maesa lanceolata</Emphasis> and <Emphasis Type="Italic">Medicago truncatula</Emphasis>

To study the production of secondary metabolites of Maesa lanceolata and Medicago truncatula, hairy root cultures of both plant species were established. Because maintenance of large numbers of cultures is laborious and costly, we developed a cryopreservation protocol and stored different isolated lines over time. Using encapsulation-dehydration, high survival rates were observed for both Maesa and Medicago hairy roots. Root tips were isolated and encapsulated in calcium-alginate beads, containing 0.1 M sucrose. The encapsulated hairy roots were precultured for 3 days using basal medium containing high sucrose concentrations. Medicago root tip growth during the preculturing time lead to unwanted outgrowth which could be tempered by addition of plant growth inhibitors. After preculturing, the beads were dehydrated in the air flow of a laminar flow until 35–40% of the initial bead weight was reached. Dehydrated beads were plunged into liquid nitrogen and after different storage times thawed in a water bath at 40°C. The survival rates were 90% for Maesa and 53% for Medicago, which are sufficient to allow implementation in large storage experimental set-ups.     

Geographic trend in sexual size dimorphism and body size of Osteopilus septentrionalis (Cuban treefrog): implications for invasion of the southeastern United States

We collected Osteopilus septentrionalis from Central Florida for analysis of sexual size dimorphism and compared our data to published information from populations across the native and introduced range of the species. We found significant sexual size dimorphism (females larger), with the degree of dimorphism decreasing with increasing degrees north latitude, a trend largely driven by a pronounced decrease in female mean snout to vent length. Potential explanations for this trend include reduced growth rates, increased time to maturity, and reduced life expectancy, all of which may be tied to climatic variation. A trend of decreasing female mean size toward the northernmost extent of the current introduced range of this species may be indicative of diminished impacts and invasive success. Geographical variation in morphology and its associated implications should be evaluated when considering the potential impacts of invasive species.     

Association of methylenetetrahydrofolate reductase gene 677C > T polymorphism and Down syndrome

The association between Down syndrome (DS) and maternal polymorphisms in genes encoding folic acid metabolizing enzymes remains a controversial issue. A meta-analysis was performed to evaluate the association of maternal MTHFR 677C > T polymorphism and the risk of having a child with DS. Case–control studies were screened from major literature databases. Twenty articles from 13 countries worldwide, with a total of 2,101 DS and 2,702 control mothers, attended the inclusion criteria. We found a 50 % increase for the association of maternal homozygous TT genotype and DS in both fixed (OR = 1.51; 95 % CI 1.22–1.87) and random effects models (OR 1.54; 95 % 1.15–2.05). Similarly, a significant pooled OR was found for the heterozygote CT, with an OR 1.26; 95 % CI 1.10–1.43 (fixed effects model) and OR 1.28; 95 % 1.08–1.51 (random effects model). As ultra-violet B solar radiation highly depends on latitude, and can promote, in less pigmented skin, intravascular folate photolysis, we stratified the analysis by latitude region, defining as Tropical (between 23.5^° S and 23.5^° N), Sub-Tropical (between 23.5^° and 40^° N and S), and Northern (≥40^o N). Significant association was only found for Sub-Tropical area, both using fixed and random effect models. In conclusion, MTHFR 677C > T polymorphism is a moderate risk factor for DS for some populations, and populations located in Sub-Tropical region seem to be at greater risk. Latitude, ethnicity, skin pigmentation, and red blood cell folate are important variables to be considered in future studies.     

Gene editing and mutagenesis reveal inter-cultivar differences and additivity in the contribution of <Emphasis Type="Italic">TaGW2</Emphasis> homoeologues to grain size and weight in wheat

Key message

CRISPR-Cas9-based genome editing and EMS mutagenesis revealed inter-cultivar differences and additivity in the contribution of TaGW2 homoeologues to grain size and weight in wheat.

Abstract

The TaGW2 gene homoeologues have been reported to be negative regulators of grain size (GS) and thousand grain weight (TGW) in wheat. However, the contribution of each homoeologue to trait variation among different wheat cultivars is not well documented. We used the CRISPR-Cas9 system and TILLING to mutagenize each homoeologous gene copy in cultivars Bobwhite and Paragon, respectively. Plants carrying single-copy nonsense mutations in different genomes showed different levels of GS/TGW increase, with TGW increasing by an average of 5.5% (edited lines) and 5.3% (TILLING mutants). In any combination, the double homoeologue mutants showed higher phenotypic effects than the respective single-genome mutants. The double mutants had on average 12.1% (edited) and 10.5% (TILLING) higher TGW with respect to wild-type lines. The highest increase in GS and TGW was shown for triple mutants of both cultivars, with increases in 16.3% (edited) and 20.7% (TILLING) in TGW. The additive effects of the TaGW2 homoeologues were also demonstrated by the negative correlation between the functional gene copy number and GS/TGW in Bobwhite mutants and an F₂ population. The highest single-genome increases in GS and TGW in Paragon and Bobwhite were obtained by mutations in the B and D genomes, respectively. These inter-cultivar differences in the phenotypic effects between the TaGW2 gene homoeologues coincide with inter-cultivar differences in the homoeologue expression levels. These results indicate that GS/TGW variation in wheat can be modulated by the dosage of homoeologous genes with inter-cultivar differences in the magnitude of the individual homoeologue effects.

     

Does size really matter? Species–area relationships in human settlements

Aim To describe species–area relationships in human settlements and compare them with those from a non‐urban habitat. Location West‐central Mexico. Methods We surveyed breeding birds in 13 human settlements and five shrubland patches. We estimated bird species richness using an abundance‐based coverage estimator with equal sample sizes to eliminate biases related to sampling effort differences. To assess species–area relationships, we performed log–log linear regressions between the size of the studied patches and their estimated bird richness. We also used a logarithmic approach to determine how the species–area relationship asymptoted and made use of the Michaelis–Menten model to identify the size at which the studied patches reached their maximum species richness. We also investigated (1) possible relationships among the estimated bird richness and other variables known to affect urban‐dwelling birds (built cover, plant species richness, tree cover or human population density) and (2) changes in bird community composition related to the size of the studied human settlements. Results Species–area relationships exhibited different patterns among the studied habitats. The log–log regression slope was steeper in human settlements, while the intercept was higher in shrublands. The maximum number of species was more than twofold higher in shrublands. Human settlement patch size was the only variable significantly related to bird richness. Our community composition results show that two main bird groups are related to human settlement size, and that as the size of human settlements increases, bird community similarity in relation to the largest city increases. Main conclusions Human settlements act as ecological islands, with pronounced species–area relationships. Our results suggest that an important threshold for bird species richness and community composition is reached in human settlements > 10.2 km². This threshold is unlikely to be generalizable among bio‐regions, and thus should be quantified and considered when studying, managing and/or planning urban systems.     

Body size variation in Audouin's Gull Larus audouinii; a density-dependent effect?

A significant decrease in the body size of Audouin's Gulls Larus audouinii breeding at the Chafarinas Islands is reported. The decrease in linear measurements in the current breeding population ranged from 2.5% to 5.6% in males and from 0.61% to 4.4% in females. This was detected when assessing the reliability of a sex-discriminating function derived for the same colony 13 years earlier. When applied to the current population, this function failed to predict the sex of a large proportion of males (44%). The relative decrease in mean size was significantly greater in males than in females for culmen, nalospi and tarsus lengths, while for bill depth at culmen, wing length and body mass the relative decrease was similar in both sexes. Since the extent of differences depended on sex, these differences cannot be attributed to a systematic between-observer bias. Mean body size reduction might be either the result of a greater proportion of small breeding birds in the current population, because of increased availability of nesting sites (competition relaxation hypothesis), or an outcome of environmental factors affecting growth parameters (environmental constraint hypothesis). According to the first hypothesis, the changes observed would be associated with higher variability values. Conversely, if the second is true, the degree of variability should be similar. Since there are no significant differences in the degree of variability shown in the two data sets, our results support the second hypothesis. The environmental constraint acting via growth parameters is probably related to the increase in the number of Audouin's Gull breeding pairs while food availability was depleted. Our data suggest that changes in the duration of the growth period, rather than in the growth rates themselves, are involved in the body size differences found.     

Serological identification and molecular characterization of B (A) 02 subtype in patients and blood donors from Eastern China

This study was designed to identify the rare?type?ABO?blood?groups, B(A) 02, from Eastern China. Three samples with discordant serological results during routine blood type identification and four samples from one sample’ family were selected. All of them were detected by serological method. The exon 6 and 7 of the ABO genes were amplified by PCR and sequenced. They were typed as AsubB by serology and as BO by genotype. In AsubB samples, nt 700C>G mutation was detected in B gene, which was previously defined as B(A)02 alleles. In these seven samples, six showed B(A)02/O01 and one showed B(A)02/O02.B(A)02 allele was found to be more common in this study than B(A)04 which is considered to be more frequent than B(A)02. The careful identification of rare blood types is important for the safety of clinical blood transfusion.     

Real-time fluid dynamics investigation and physiological response for erythromycin fermentation scale-up from 50 L to 132 m<Superscript>3</Superscript> fermenter

The physiological response of erythromycin fermentation scale-up from 50 L to 132 m³ scale was investigated. A relatively high oxygen uptake rate (OUR) in early phase of fermentation was beneficial for erythromycin biosynthesis. Correspondingly, the maximal consistency coefficient (K) reflecting non-Newtonian fluid characteristics in 50 L and 132 m³ fermenter also appeared in same phase. Fluid dynamics in different scale bioreactor was further investigated by real-time computational fluid dynamics modeling. The results of simulation showed that the impeller combination in 50 L fermenter could provide more modest flow field environment compared with that in 132 m³ fermenter. The decrease of oxygen transfer rate (OTR) in 132 m³ fermenter was the main cause for impairing cell physiological metabolism and erythromycin biosynthesis. These results were helpful for understanding the relationship between hydrodynamic environment and physiological response of cells in bioreactor during the scale-up of fermentation process.     

Deep Dermatophytosis Caused by Zoophilic Strain of <Emphasis Type="Italic">Trichophyton interdigitale</Emphasis> with Successful Treatment of Itraconazole

We report a 66-year-old female patient with deep dermatophytosis caused by zoophilic strain of Trichophyton interdigitale, a rare granulomatous presentation of Trichophyton species infection in patients with underlying systemic diseases, and she was successfully cured by itraconazole. Since the identification of Trichophyton mentagrophytes complex had been misused for years, a brief discussion of molecular diagnosis and taxonomy of T. mentagrophytes complex is given. The pathogenesis and comparison with cases reported in the literature are also discussed.     

An efficient in vitro propagation protocol for <Emphasis Type="Italic">Pinguicula lusitanica</Emphasis>, a rare insectivorous plant

In this study, an efficient protocol was developed for in vitro propagation of Pinguicula lusitanica L., a rare insectivorous plant with pharmacological value and limited reproductive capacity. The effects of two concentrations (0.1 and 0.5 mg l⁻¹) of a range of plant growth regulators, including cytokinins (BA, KIN, and ZEA) and auxins (IAA, IBA, and NAA), and three concentrations of MS medium macronutrients (total, 1/2 and 1/4MS) on proliferation and rooting, were investigated. P. lusitanica shoots showed abundant proliferation and rooting capacity, both of which were significantly influenced by MS medium strength. The use of 1/2MS supplemented with 0.5 mg l⁻¹ BA or KIN ensure a 29-fold rate of proliferation. Best rooting frequency and higher root number and length were attained in 1/4MS medium containing 0.2 mg l⁻¹ IAA. Sixty percent of the plantlets were successfully acclimatized to ex vitro conditions, exhibiting normal development. These plantlets can be used to replenish declining populations in the wild and/or for the extraction of bioactive compounds, reducing pressure on wild stocks.