The <Emphasis Type="Italic">GAL10</Emphasis> Gene is Located 40 kbp Away from the <Emphasis Type="Italic">GAL7</Emphasis>-<Emphasis Type="Italic">GAL1</Emphasis> Region in the Yeast <Emphasis Type="Italic">Kazachstania naganishii</Emphasis>

Of the genes involved in galactose metabolism, GAL7, GAL10, and GAL1 are tightly linked in this order on chromosome II in Saccharomyces cerevisiae. While several species of the order Saccharomycetales have similar gene organization, Kazachstania naganishii is unique, in which GAL7 and GAL1 are close to each other whereas GAL10 is substantially apart from them on chromosome XI. In this study, we inserted the recognition sequence of I-SceI homing-endonuclease into GAL10 and also into the intervening segment of GAL7-GAL1. By cleaving chromosome DNA of the gene-manipulated strain with I-SceI, we obtained evidence that chromosome XI (610 kbp) was replaced with three fragments (305, 265, and 40 kbp). Using appropriate probes, we further found that GAL10 was about 40 kbp apart from the GAL7-GAL1 cluster and that orientation of GAL10 was reversed comparing to the S. cerevisiae counter part. We, therefore, contend that comparison of the organization of the GAL cluster among Saccharomycetales is of importance to elucidate evolution of chromosomes and that the experimental scheme developed in this study is useful for this line of investigation.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

<Emphasis Type="Italic">Carex</Emphasis> × <Emphasis Type="Italic">involuta</Emphasis> and <Emphasis Type="Italic">Carex juncella</Emphasis> in the flora of Slovakia

The paper brings information on an isolated occurrence and morphological characters of Carex × involuta and C. juncella populations in the Vel’ká Fatra Mts. Their presence has been known neither from the territory of Slovakia nor from the whole Western Carpathians till now.     

Brood cell size of <Emphasis Type="Italic">Apis</Emphasis> <Emphasis Type="Italic">mellifera</Emphasis> modifies the reproductive behavior of <Emphasis Type="Italic">Varroa</Emphasis> <Emphasis Type="Italic">destructor</Emphasis>

We undertook a field study to determine whether comb cell size affects the reproductive behavior of Varroa destructor under natural conditions. We examined the effect of brood cell width on the reproductive behavior of V. destructor in honey bee colonies, under natural conditions. Drone and worker brood combs were sampled from 11 colonies of Apis mellifera. A Pearson correlation test and a Tukey test were used to determine whether mite reproduction rate varied with brood cell width. Generalized additive model analysis showed that infestation rate increased positively and linearly with the width of worker and drone cells. The reproduction rate for viable mother mites was 0.96 viable female descendants per original invading female. No significant correlation was observed between brood cell width and number of offspring of V. destructor. Infertile mother mites were more frequent in narrower brood cells.     

High-throughput genotyping-by-sequencing facilitates molecular tagging of a novel rust resistance gene, <Emphasis Type="Italic">R</Emphasis><Subscript><Emphasis Type="Italic">15</Emphasis></Subscript>, in sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.)

Key message

A novel rust resistance gene, R ₁₅ , derived from the cultivated sunflower HA-R8 was assigned to linkage group 8 of the sunflower genome using a genotyping-by-sequencing approach. SNP markers closely linked to R ₁₅ were identified, facilitating marker-assisted selection of resistance genes.

Abstract

The rust virulence gene is co-evolving with the resistance gene in sunflower, leading to the emergence of new physiologic pathotypes. This presents a continuous threat to the sunflower crop necessitating the development of resistant sunflower hybrids providing a more efficient, durable, and environmentally friendly host plant resistance. The inbred line HA-R8 carries a gene conferring resistance to all known races of the rust pathogen in North America and can be used as a broad-spectrum resistance resource. Based on phenotypic assessments of 140 F₂ individuals derived from a cross of HA 89 with HA-R8, rust resistance in the population was found to be conferred by a single dominant gene (R ₁₅ ) originating from HA-R8. Genotypic analysis with the currently available SSR markers failed to find any association between rust resistance and any markers. Therefore, we used genotyping-by-sequencing (GBS) analysis to achieve better genomic coverage. The GBS data showed that R ₁₅ was located at the top end of linkage group (LG) 8. Saturation with 71 previously mapped SNP markers selected within this region further showed that it was located in a resistance gene cluster on LG8, and mapped to a 1.0-cM region between three co-segregating SNP makers SFW01920, SFW00128, and SFW05824 as well as the NSA_008457 SNP marker. These closely linked markers will facilitate marker-assisted selection and breeding in sunflower.

     

Gibberellic acid promotes in vitro regeneration and shoot multiplication in <Emphasis Type="Italic">Lotus</Emphasis><Emphasis Type="Italic">corniculatus</Emphasis> L.

Shoots of Lotus corniculatus L., previously transformed with Agrobacterium tumefaciens LBA4404/pTOK233, were grown in gibberellic acid (GA₃)-containing media in an attempt to improve their growth and multiplication. Nodal stem segments of four poorly multiplying clones, a clone with very good multiplication, and two non-transformed clones were incubated in media containing GA₃ for 3 weeks, and then returned to a hormone-free medium for a further 3–5 subculture periods. Gibberellic acid increased the number of axillary buds from one in controls to 2–13 shoots during the first 3 weeks of subculture in GA₃-containing media. The multiplied buds, which apparently derived from additional axillaries, continued to multiply in hormone-free medium, producing bunches of more than 50 shoots after 9 weeks. In addition, many nodal segments also produced callus tissues at the basal end, or along the internode below the node, in which abundant shoot meristems formed de novo. Histological examination confirmed their origin via organogenesis. About 25% of regenerated shoots rooted spontaneously. The application of auxins significantly improved rooting in more than 75% of all clones. Well-developed plantlets were produced upon the transfer of rooted shoots to pots. No differences in responses to hormones were observed between LBA4404/pTOK233-transformed clones and non-transformed clones, indicating that the reaction to GA₃ was not the consequence of transformation events. The results point to a possible involvement of GA₃ in lateral branching. They might also be recommended as a procedure suitable for increasing the production of transformed plants in L. corniculatus.     

<Emphasis Type="Italic">Prunus serotina</Emphasis> unleashed: invader dominance after 70 years of forest development

Propagule pressure and disturbance have both been found to facilitate invasion. Therefore, knowledge on the history of introduction and disturbance is vital for understanding an invasion process, and research should focus on areas in which the invasive species has not been deliberately introduced or managed to study unconfounded colonization patterns. Comparing the outcome of such spontaneous colonization processes for different ecosystems might provide a useful framework for setting management priorities for invasive species that enter new, uninvaded areas. We focused on the 70-year spontaneous spread of the invasive tree species Prunus serotina in a pine forest in the Netherlands. To reconstruct the invasion pattern, we combined historical maps, tree ring analysis, spatially explicit tree inventory data, seed density data, and regeneration data for both native and non-native species. Prunus serotina was the only species that showed successful regeneration: the species was present throughout the forest in the tree, shrub, and herb layer. Native species were not able to outgrow the seedling stage. Our data demonstrate that P. serotina is a gap-dependent species with high seed production that builds up a seedling bank. We also compared the results of this study with a similar study on P. serotina colonization in a deciduous forest in Belgium, where P. serotina invasion was not successful. The sharp contrast between the outcomes of the two invasion processes shows the importance of studying an invasive species and the recipient ecosystem jointly and made us raise the hypothesis that herbivore pressure may facilitate P. serotina invasion.     

Inheritance of the<Emphasis Type="Italic"> Md-ACS1</Emphasis> gene and its relationship to fruit softening in apple (<Emphasis Type="Italic">Malus</Emphasis> ×<Emphasis Type="Italic"> domestica</Emphasis> Borkh.)

The 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene is a member of the ACS gene family that is involved in apple (Malus × domestica Borkh.) fruit ripening. Presence of an allele (Md-ACS1-2) of this gene is associated with low internal ethylene concentration in some apple cultivars. In this study, inheritance of Md-ACS1 was determined for 50 apple cultivars/advanced selections and 101 F₁ seedlings from five populations. Following this, the softening pattern of apples stored at 20°C for up to 40 days was examined using 35 fruiting cultivars/selections of defined Md-ACS1 status. Md-ACS1 is inherited in a Mendelian fashion and was found to be linked to fruit softening. Maturity season of genotypes also significantly affected fruit softening. Late-season genotypes in the Md-ACS1-2/2 class had the slowest rate of softening, while early-season Md-ACS1-1/1 genotypes had the most rapid softening rate. The implications of these results are discussed in relation to parental selection and breeding for storage ability in apple.Communicated by H. Nybom     

Blooms of the invasive ctenophore, <Emphasis Type="Italic">Mnemiopsis</Emphasis> <Emphasis Type="Italic">leidyi</Emphasis>, span the Mediterranean Sea in 2009

Blooms of the invasive ctenophore, Mnemiopsis leidyi, occurred in 2009 along the Mediterranean Sea coasts of Spain and Israel. This voracious zooplanktivore spread throughout the Black Sea basin after its introduction in the early 1980s, throughout northern European coastal waters, and now occurs throughout the Mediterranean Sea. M. leidyi occurred throughout the summer along the entire Catalan Spanish and Israeli coasts in 2009. Those locations had high temperatures (18–26°C) and salinities (37–38) during the blooms. The patterns of abundance of large jellyfish along the Catalan coast were unusual in 2009, with low numbers during July, August, and September when ctenophores were abundant. Small populations of those potential predators and food competitors of M. leidyi could have contributed to the ctenophore bloom. The identity of the ctenophores from Spain and Israel was confirmed as M. leidyi by molecular analysis based on DNA sequencing of the nuclear internal transcribed spacer (ITS) regions. This is the first molecular confirmation of M. leidyi in the Mediterranean Sea. Most ctenophores had an ITS genotype previously found in M. leidyi from other invaded regions (the Black, Azov, and Mediterranean seas), as well as native regions in the United States, suggesting common ancestry. Based on the circulation patterns of Mediterranean surface waters and shipping activities, we conclude that the spread of M. leidyi in the Mediterranean probably resulted from re-introductions by ballast water transport and subsequent distribution by currents. We also conclude that the near-simultaneous blooms in opposite ends of both the Mediterranean basins indicate that M. leidyi is resident around the Mediterranean. We discuss environmental conditions, food, and predators of M. leidyi in both regions that would influence the future effects of this voracious consumer on the pelagic food web of the Mediterranean Sea.     

<Emphasis Type="Italic">Astyanax</Emphasis> <Emphasis Type="Italic">bockmanni</Emphasis> Vari and Castro, 2007: an ambiguous karyotype in the <Emphasis Type="Italic">Astyanax</Emphasis> genus

Despite the widespread distribution of Astyanax bockmanni in streams from Upper Paraná River system in central, southeastern, and southern Brazil, just recently, it has been identified as a distinct Astyanax species. Cytogenetic studies were performed in two populations of this species, revealing conservative features. A. bockmanni shows 2n = 50 chromosomes, a karyotypic formula composed of 10 M + 12SM + 12ST + 16A and multiple Ag-NORs. Eight positive signals in subtelocentric/acrocentric chromosomes were identified by fluorescent in situ hybridization (FISH) with 18S rDNA probes. After FISH with 5S rDNA probes, four sites were detected, comprising the interstitial region of a metacentric pair and the terminal region on long arms of another metracentric pair. Little amounts of constitutive heterochromatin were observed, mainly distributed at distal region in two chromosomal pairs. Additionally, heterochromatin was also located close to the centromeres in some chromosomes. No positive signals were detected in the chromosomes of A. bockmanni by FISH with the As-51 satellite DNA probe. The studied species combines a set of characteristics previously identified in two different Astyanax groups. The chromosomal evolution in the genus Astyanax is discussed.     

Heterologous expression of a gene for thermostable xylanase from <Emphasis Type="Italic">Chaetomium</Emphasis> <Emphasis Type="Italic">thermophilum</Emphasis> in <Emphasis Type="Italic">Pichia</Emphasis> <Emphasis Type="Italic">pastoris</Emphasis> GS115

We studied heterologous expression of xylanase 11A gene of Chaetomium thermophilum in Pichia pastoris and characterized the thermostable nature of the purified gene product. For this purpose, the xylanase 11A gene of C. thermophilum was cloned in P. pastoris GS115 under the control of AOX1 promoter. The maximum extracellular activity of recombinant xylanase (xyn698: gene with intron) was 15.6 U ml⁻¹ while that of recombinant without intron (xyn669) was 1.26 U ml⁻¹ after 96 h growth. The gene product was purified apparently to homogeneity level. The optimum temperature of pure recombinant xylanase activity was 70°C and the enzyme retained its 40.57% activity after incubation at 80°C for 10 min. It exhibited quite lower demand of activation energy, enthalpy, Gibbs free energy, entropy, and xylan binding energy during substrate hydrolysis than that required by that of the donor, thus indicating its thermostable nature. pH-dependent catalysis showed that it was quite stable in a pH range of 5.5–8.5. This revealed that gene was successfully processed in P. pastoris and remained heat stable and may qualify for its potential use in paper and pulp and animal feed applications.     

Carbohydrate and Ethane Release with <Emphasis Type="Italic">Erwinia carotovora</Emphasis> Subspecies <Emphasis Type="Italic">betavasculorum</Emphasis><Emphasis Type="Italic">–</Emphasis>Induced Necrosis

Erwinia carotovora subspecies betavasculorum, also known as E. betavasculorum and Pectobacterium betavasculorum, is a soil bacterium that has the capacity to cause root rot necrosis of sugarbeets. The qualitatively different pathogenicity exhibited by the virulent E. carotovora strain and two avirulent strains, a Citrobacter sp. and an Enterobacter cloacae, was examined using digital analysis of photographic evidence of necrosis as well as for carbohydrate, ethane, and ethylene release compared with uninoculated potato tuber slices. Visual scoring of necrosis was superior to digital analysis of photographs. The release of carbohydrates and ethane from potato tuber slices inoculated with the soft rot necrosis-causing Erwinia was significantly greater than that of potato tuber slices that had not been inoculated or that had been inoculated with the nonpathogenic E. cloacae and Citrobacter sp. strains. Interestingly, ethylene production from potato slices left uninoculated or inoculated with the nonpathogenic Citrobacter strain was 5- to 10-fold higher than with potato slices inoculated with the pathogenic Erwinia strain. These findings suggest that (1) carbohydrate release might be a useful measure of the degree of pathogenesis, or relative virulence; and that (2) bacterial suppression of ethylene formation may be a critical step in root rot disease formation.     

Regeneration of transformed verbena (<Emphasis Type="Italic">Verbena </Emphasis>× <Emphasis Type="Italic">hybrida</Emphasis>) by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Verbena (Verbena x hybrida), an important floricultural species, was successfully regenerated from stem segments on Murashige and Skoog's basal medium supplemented with thidiazuron and indole-3-acetic acid. A transformation system was developed using cvs. Temari Scarlet, Temari Sakura, Tapien Rose and TP-P2. Agrobacterium tumefaciens strain Agl0 harboring the sGFP gene was infected into stem segments. Transformation efficiency was improved by evaluating and manipulating the age of the plant material, the concentration of kanamycin in the medium during selection, and the length of the culture period in the dark. After 2-3 months of culture on the selection medium, GFP-positive shoots were obtained in all four of the cultivars tested. These shoots were successfully acclimated and set flowers within 2-3 months in a greenhouse. GFP was expressed in all of the organs including the floral parts. Stable genomic transformation was confirmed by Southern blot analysis. No morphological differences were observed between the transformed plants and their host plants.     

Involvement of Carbohydrates in Response to Preconditioning Flooding in Two Clones of <Emphasis Type="Italic">Populus deltoides</Emphasis> Marsh. × <Emphasis Type="Italic">P.</Emphasis> <Emphasis Type="Italic">nigra</Emphasis> L.

Poplar, a fast-growing species widely used outside its original area of distribution, was evaluated in the present study to verify its tolerance and hardening to waterlogging in anticipation of its implementation in rehabilitation practices for marginal lands subjected to flooding. Three water regimes were applied to the plants, grown in pots, with two clones (I-488 and D-64) with different sensitivities to flooding. These plants included a lot consisting of control plants (C), which were irrigated with good drainage, a non-preconditioned lot (NPr), and a third lot that was preconditioned to flooding (Pr). Furthermore, flooding was imposed on NPr and Pr plants by submerging pots up to 5 cm above the collar for 60 days followed by a 40-day recovery period. At the end of these two periods, shoot dry mass, foliar concentrations of certain ions, soluble sugars, starch and proline, and the relative electrolyte leakage were evaluated. The preconditioning treatment conferred different degrees of hardness on the treated plants compared with NPr plants. Our results revealed that high carbohydrate availability (soluble sugars and starch) is suggested to participate in sustaining membrane integrity which may affect the recuperative potential of Pr plants, notably those of clone I-488, from flooding damage.