EST‐SSR markers from Fragaria vesca L. cv. Yellow Wonder

Fourteen microsatellite primer pairs were developed from a cDNA library of heat‐treated seedlings of Fragaria vesca cv. yellow wonder. Transferability to 13 species of Fragaria ranged from 71% in diploid species F. gracilis Losinsk., F. iinumae Makino, F. nilgerrensis Schltdl. ex J. Gay and F. nipponica Makino, to 100% in octoploid domestic strawberry and its progenitors. Polymorphism was high in polyploid Fragaria species. However, polymorphism and heterozygosity of eight EST‐SSRs (expressed sequence tag–simple sequence repeats) was low in 14 F. vesca genotypes.     

Elevation of Francisella philomiragia subsp. noatunensisMikalsen et al. (2007) to Francisella noatunensis comb. nov. [syn. Francisella piscicida Ottem et al. (2008) syn. nov.] and characterization of Francisella noatunensis subsp. orientalis subsp. nov., two important fish pathogens

Aims: This study was conducted to clarify the taxonomic status of Francisella sp. strain Ehime‐1, a fish pathogen, in relation to the fish pathogens F. piscicida and F. philomiragia subsp. noatunensis and to F. philomiragia subsp. philomiragia. Methods and Results: Francisella sp. Ehime‐1 was compared to F. piscicida, F. philomiragia subsp. noatunensis and several F. philomiragia subsp. philomiragia isolates through sequencing of the 16S rRNA‐gene and several house‐keeping genes and determination of biochemical and phenotypic properties. Results show that F. piscicida is indistinguishable from F. philomiragia subsp. noatunensis by sequence and phenotypic traits. Francisella sp. Ehime‐1 and F. philomiragia subsp. noatunensis are clearly separated from F. philomiragia. Francisella sp. Ehime‐1 is biochemically, phenotypically and genetically different from F. philomiragia subsp. noatunensis (=F. piscicida), but DNA–DNA hybridization does not clearly support establishment as a separate species (level of relatedness 64% and 73·4%, mean 68·7%). Conclusions: We propose to elevate F. philomiragia subsp. noatunensis to species rank as F. noatunensis comb. nov., while F. piscicida is considered a heterotypic synonym of F. noatunensis comb. nov. Evidence suggests that Francisella sp. Ehime‐1 represents a novel subspecies of F. noatunensis, for which the name F. noatunensis subsp. orientalis subsp. nov. is proposed (=DSM21254^T, = LMG24544^T). Significance and Impact of the Study: This study contributes to the taxonomy and characteristics of fish‐pathogenic Francisella spp.     

Adaptive significance of amylase polymorphism in Drosophila . XII. density‐ and frequency‐dependent selection at the Amy locus in Drosophila subobscura reared on media with different carbohydrate composition

Egg‐to‐adult viability is studied in the progeny of the flies of different genotypes according to S and F alleles of Amy locus of Drsophila subobscura . This component of fitness is observed in the single and mixed cultures with various frequencies of three genotypes (S/S, F/F and S/F) under conditions of low (LD) and high densities (HD) on three types of media with different carbohydrate composition. In such multifactorial experimental conditions, density‐ and frequency‐dependent selection on certain Amy genotypes was observed. Genotype frequencies and carbohydrate composition have significant effect on the viability of Amy genotypes. The significant intergenotypic differences exist, mostly at HD conditions. The heterozygous genotype S/F has generally lower viability which decreases with its increased frequencies, on all media at LD or HD. The results suggest a high level of complexity and interaction between these two types of balanced selection.     

Pathogenic analyses of fungus strains isolated from medicinal Fritillaria przewalskii Maxim. bulb rot

Bulb rot causes a great loss of rare perennial medicinal Fritillaria przewalskii Maxim., which is exacerbated with growth year and seriously constrains the plant productivity, but the pathogens responsible for the disease were still unknown. In this paper, we attempted to explore the potential pathogens leading to bulb rotting and their occurrence patterns in artificial domesticated F. przewalskii. Fungus strains were isolated from the bulb rot spot of the 3‐ to 5‐year‐old diseased F. przewalskii plants by tissue separation, further assayed for pathogenicity according to Koch's law and finally identified by both morphological and molecular methods. Seven pathogenic strains (F1‐F7) were detected in 5‐year‐old rot bulbs, six (F1‐F6) in 4‐year‐old rot bulbs and four (F1, F2, F5, F6) in 3‐year‐old rot bulbs. All of the strains were able to infect bulbs by stabbing and some exhibited varying levels of aggressiveness. Relative to the non‐stabbing controls, the bulbs stab‐inoculated with F5, F2, F7, F4, F1, F6, and F3 showed 76.65%, 75.15%, 71.44%, 40.37%, 39.09%, 36.87%, and 34.93% rot after 8 days, respectively. Phylogenetic analysis revealed that these seven strains were clustered into Bionectria ochroleuca (F1, F3, F4), Fusarium oxysporum (F2, F7), Fusarium tricinctum (F5), and Clonostachys rosea (F6). The two species of Fusarium had the strongest pathogenicity, followed by Bionectria ochroleuca and Clonostachys rosea. Although leading to low bulb rot incidence by stab inoculation, F1 showed the highest isolation rate (48.9%) among all strains. Thus, the edible and medicinal bulbs of F. przewalskii are susceptible to synergetic contamination by these seven pathogens at some point after their third year of growth, which has contributed to the species endangered status, with the two strains of Fusarium being the predominant pathogens. To our knowledge, this is the first report on the seven strains of four fungal species causing F. przewalskii bulb rot in China.     

A Phytoalexin-Like Flavonol Involved in the Carnation (Dianthus caryophyllus)-Fusarium oxysporum f. sp. dianthi Pathosystem

The fungitoxic flavonol triglycoside, kaempferide 3‐O‐[2^G‐β‐d ‐glucopyranosyl]‐β‐rutinoside, is a constituent of the carnation cultivar ‘Novada’, known as one of the most resistant cultivar to Fusarium oxysporum f. sp. dianthi, causative agent of Fusarium wilt. Due to its constitutive presence within the carnation tissues, this antifungal flavonol should be considered as a phytoanticipin; its biosynthesis, however, is stimulated by the inoculation with F. oxysporum f. sp. dianthi, just as is the rule for a typical phytoalexin. The results seem to indicate that in carnation the concentration of some preformed antifungal flavonoids may be significantly increased by a fungal presence: owing to their fungitoxic properties, these molecules could cooperate, together with the unconstitutive and postinfectional anthranilic acid‐derivative phytoalexins, to the plant defensive response against Fusarium attacks.     

Aldehyde oxidase carrying an unusual subunit structure from Pseudomonas sp. MX‐058

Pseudomonas sp. MX‐058 produces aldehyde oxidase catalysing glyoxal to glyoxylic acid. Two aldehyde oxidases (F10 and F13) were purified to homogeneity from Pseudomonas sp. MX‐058. F10 and F13 had subunit structures, a heterotetramer and heteropentamer respectively. The N‐terminal amino acid sequences of all subunits were highly homologous to amino acid sequences of the putative oxidoreductases of Pseudomonas strains. All of these homologous oxidoreductases have a heterotrimer structure consisting of 85‐88 (α), 37‐39 (β) and 18‐23 (γ) kDa subunits. However, the α‐subunits of F10 and F13 might have decomposed into two [80 (α¹) and 9 kDa (α²)] and three [58 (α^1′), 22 (α^1″) and 9 (α²) kDa] subunits, respectively, while the β‐ and γ‐subunits remained intact. Both F10 and F13 show high activity toward several aliphatic and aromatic aldehydes. The aldehyde oxidases of Pseudomonas sp. MX‐058 has unique protein structures, α¹α²βγ for F10 and α^1′α^1″α²βγ for F13, a heterotetramer and heteropentamer respectively. The enzymes exhibit significantly low activity toward glyoxylic acid compared with glyoxal, which is an advantageous property for glyoxylic acid production from glyoxal.     

Investigation of penetratin peptides. Part 2. In vitro uptake of penetratin and two of its derivatives

As endocytic uptake of the Antennapedia homeodomain‐derived penetratin peptide (RQIKIWFQNRRMKWKK) is finally being revealed, some of the early views about penetratin need to be reconsidered. Endocytic uptake seems to contradict the indispensability of tryptophans and also the minimum length of 16 amino acid residues for efficient internalization. To revise the membrane translocation of penetratin, two penetratin analogs were designed and synthesized: a peptide in which tryptophans were replaced by phenylalanines (Phe^{6, 14}‐penetratin, RQIKI F FQNRRMK F KK) and a shortened analog (dodeca‐penetratin, RQIKIWF‐R‐KWKK) made up of only 12 residues. The peptides were fluorescently labeled and applied to live, unfixed cells from various lines. Cellular uptake was analysed by confocal microscopy and flow cytometry. Low temperature or ATP‐depletion blocked the intracellular entry of all three penetratin peptides. A decrease in membrane fluidity or cholesterol depletion with methyl‐β‐cyclodextrin greatly inhibited peptide uptake, showing the involvement of cholesterol‐rich lipid rafts in internalization. Exogenous heparan sulfate also diminished the internalization of penetratin and its derivatives, reflecting the paramount importance of electrostatic interactions with polyanionic cell‐surface proteoglycans. The beneficial presence of tryptophans is supported by observations on the decreased cellular uptake of Phe^{6, 14}‐penetratin. The maintained translocational efficiency of dodeca‐penetratin demonstrates that a thorough understanding of penetratin internalization can yield new penetratin analogs with unaltered translocational abilities. This study provides evidence on the energy‐dependent and lipid raft‐mediated endocytic uptake of penetratin and highlights the necessity of revealing those pathways that cationic cell‐penetrating peptides employ to enter live cells. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.     

The Ficus erecta genome aids Ceratocystis canker resistance breeding in common fig (F. carica)

Ficus erecta, a wild relative of the common fig (F. carica), is a donor of Ceratocystis canker resistance in fig breeding programmes. Interspecific hybridization followed by recurrent backcrossing is an effective method to transfer the resistance trait from wild to cultivated fig. However, this process is time consuming and labour intensive for trees, especially for gynodioecious plants such as fig. In this study, genome resources were developed for F. erecta to facilitate fig breeding programmes. The genome sequence of F. erecta was determined using single‐molecule real‐time sequencing technology. The resultant assembly spanned 331.6 Mb with 538 contigs and an N50 length of 1.9 Mb, from which 51 806 high‐confidence genes were predicted. Pseudomolecule sequences corresponding to the chromosomes of F. erecta were established with a genetic map based on single nucleotide polymorphisms from double‐digest restriction‐site‐associated DNA sequencing. Subsequent linkage analysis and whole‐genome resequencing identified a candidate gene for the Ceratocystis canker resistance trait. Genome‐wide genotyping analysis enabled the selection of female lines that possessed resistance and effective elimination of the donor genome from the progeny. The genome resources provided in this study will accelerate and enhance disease‐resistance breeding programmes in fig.     

Bothriospermum longistylum sp. nov. (Boraginaceae) from north China

Based on field investigations as well as morphological and molecular systematic studies we found a new species of Bothriospermum which can be easily distinguished from all other known congeneric species by its unlobed faucal appendages and very long styles. We conducted molecular phylogenetic analyses based on a combined dataset from nrITS, plastid rps16, trnL‐trnF and trnG‐trnS regions. The results indicated that the new species indeed belongs in Bothriospermum and that it is well separated from other congeneric clusters, but that the genus Bothriospermum is paraphyletic with Nihon and Thyrocarpus clusetered within it. As a result, Bothriospermum longistylum sp. nov. is described and illustrated here. A revised key to species of Bothriospermum and a distributional map are also presented.     

Craniological differentiation between European wildcats (Felis silvestris silvestris), African wildcats (F. s. lybica) and Asian wildcats (F. s. ornata): implications for their evolution and conservation

Intraspecific diversification of the wildcat (Felis silvestris), including the European wildcat (F. s. silvestris), the Asian wildcat (F. s. ornata) and the African wildcat (F. s. lybica), was examined based on 39 cranial morphology variables. The samples of free‐ranging cats originated from Britain, Europe, Central Asia and southern Africa, consisting of both nominal wildcat specimens (referred to henceforth as ‘wildcats’) and nominal non‐wildcat specimens (‘non‐wildcats’) based on museum labels. The skull morphology of ‘wildcats’ from Britain and Europe is clearly different from that of ‘wildcats’ of Central Asia and southern Africa. The latter are characterized especially by their proportionately larger cheek teeth. On the basis of principal component, discriminant function and canonical variate analyses, the skull morphology of British ‘non‐wildcats’ is less distinct than is that of British ‘wildcats’ from the skull morphologies of ‘wildcats’ of Central Asia and southern Africa. On the other hand, the skull morphology of southern African ‘non‐wildcats’ is as distinct from those of ‘wildcats’ of Britain and Europe as is that of southern African ‘wildcats’. We suggest that the evolution of the modern wildcat probably consisted of at least three different distribution expansions punctuated by two differentiation events: the exodus from Europe during the late Pleistocene, coinciding with the emergence of the steppe wildcat lineage (phenotype of Asian–African wildcat), followed by its rapid range expansion in the Old World. The second differentiation event was the emergence of the domestic cat followed by its subsequent colonization of the entire world with human assistance. Considering the recent evolutionary history of, and morphological divergence in, the wildcat, preventing hybridization between the European wildcat and the domestic cat is a high conservation priority. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 83 , 47–63.     

Apple S‐RNase interacts with an actin‐binding protein,MdMVG, to reduce pollen tube growth by inhibiting its actin‐severing activity at the early stage of self‐pollination induction

In S‐RNase‐mediated self‐incompatibility, S‐RNase secreted from the style destroys the actin cytoskeleton of the self‐pollen tubes, eventually halting their growth, but the mechanism of this process remains unclear. In vitro biochemical assays revealed that S‐RNase does not bind or sever filamentous actin (F‐actin). In apple (Malus domestica), we identified an actin‐binding protein containing myosin, villin and GRAM (MdMVG), that physically interacts with S‐RNase and directly binds and severs F‐actin. Immunofluorescence assays and total internal reflection fluorescence microscopy indicated that S‐RNase inhibits the F‐actin‐severing activity of MdMVG in vitro. In vivo, the addition of S‐RNase to self‐pollen tubes increased the fluorescence intensity of actin microfilaments and reduced the severing frequency of microfilaments and the rate of pollen tube growth in self‐pollination induction in the presence of MdMVG overexpression. By generating 25 single‐, double‐ and triple‐point mutations in the amino acid motif E‐E‐K‐E‐K of MdMVG via mutagenesis and testing the resulting mutants with immunofluorescence, we identified a triple‐point mutant, MdMVG^{(E167A/E171A/K185A)}, that no longer has F‐actin‐severing activity or interacts with any of the four S‐haplotype S‐RNases, indicating that all three amino acids (E167, E171 and K185) are essential for the severing activity of MdMVG and its interaction with S‐RNases. We conclude that apple S‐RNase interacts with MdMVG to reduce self‐pollen tube growth by inhibiting its F‐actin‐severing activity.     

A photo‐responsive F‐box protein FOF2 regulates floral initiation by promoting FLC expression in Arabidopsis

Floral initiation is regulated by various genetic pathways in response to light, temperature, hormones and developmental status; however, the molecular mechanisms underlying the interactions between different genetic pathways are not fully understood. Here, we show that the photoresponsive gene FOF2 (F‐box of flowering 2) negatively regulates flowering. FOF2 encodes a putative F‐box protein that interacts specifically with ASK14, and its overexpression results in later flowering under both long‐day and short‐day photoperiods. Conversely, transgenic plants expressing the F‐box domain deletion mutant of FOF2 (FOF2ΔF), or double loss of function mutant of FOF2 and FOL1 (FOF2‐LIKE 1) present early flowering phenotypes. The late flowering phenotype of the FOF2 overexpression lines is suppressed by the flc‐3 loss‐of‐function mutation. Furthermore, FOF2 mRNA expression is regulated by autonomous pathway gene FCA, and the repressive effect of FOF2 in flowering can be overcome by vernalization. Interestingly, FOF2 expression is regulated by light. The protein level of FOF2 accumulates in response to light, whereas it is degraded under dark conditions via the 26S proteasome pathway. Our findings suggest a possible mechanistic link between light conditions and the autonomous floral promotion pathway in Arabidopsis.     

Surviving climate changes: high genetic diversity and transoceanic gene flow in two arctic–alpine lichens,Flavocetraria cucullata and F. nivalis (Parmeliaceae,Ascomycota)

Aim We examined genetic structure and long‐distance gene flow in two lichenized ascomycetes, Flavocetraria cucullata and Flavocetraria nivalis, which are widespread in arctic and alpine tundra. Location Circumpolar North. Methods DNA sequences were obtained for 90 specimens (49 for F. cucullata and 41 for F. nivalis) collected from various locations in Europe, Asia and North America. Sequences of the nuclear internal transcribed spacer (ITS) + 5.8S ribosomal subunit gene region were generated for 89 samples, and supplemented by beta‐tubulin (BTUB) and translation elongation factor 1‐alpha gene (EF1) sequences for a subset of F. cucullata specimens. Phylogenetic, nonparametric permutation methods and coalescent analyses were used to assess population divergence and to estimate the extent and direction of migration among continents. Results Both F. cucullata and F. nivalis were monophyletic, supporting their morphology‐based delimitation, and had high and moderately high intraspecific genetic diversity, respectively. Clades within each species contained specimens from both North America and Eurasia. We found only weak genetic differentiation among North American and Eurasian populations, and evidence for moderate to high transoceanic gene flow. Main conclusions Our results suggest that both F. cucullata and F. nivalis have been able to migrate over large distances in response to climatic fluctuations. The high genetic diversity observed in the Arctic indicates long‐term survival at high latitudes, whereas the estimated migration rates and weak geographic population structure suggest a continuing long‐distance gene flow between continents that has prevented pronounced genetic differentiation. The mode of long‐distance dispersal is unknown, but wind dispersal of conidia and/or ascospores is probably important in the open arctic landscapes. The high genetic diversity and efficient long‐distance dispersal capability of F. cucullata and F. nivalis suggest that these species, and perhaps other arctic lichens as well, will be able to track their potential niche in the changing Arctic.     

Molecular identification of species in Prunus sect. Persica (Rosaceae), with emphasis on evaluation of candidate barcodes for plants

Abstract Species of Prunus L. sect. Persica are not only important fruit trees, but also popular ornamental and medicinal plants. Correct identification of seedlings, barks, or fruit kernels is sometimes required, but no reliable morphological characters are available. Nowadays, the technique of DNA barcoding has the potential to meet such requirements. In this study, we evaluated the suitability of 11 DNA loci (atpB‐rbcL, trnH‐psbA, trnL‐F, trnS‐G, atpF‐H, rbcL, matK, rpoB, rpoC1, nad1, and internal transcribed spacer [ITS]) as candidate DNA barcodes for peaches, using samples from 38 populations, covering all the species in sect. Persica. On the whole, the primers worked well in this group and sequencing difficulties were met only in the case of ITS locus. Five loci (rbcL, matK, rpoB, rpoC, and nad1) have very low variation rates, whereas atpB‐rbcL, atpF‐H, trnH‐psbA, trnL‐F and trnS‐G show more variability. The most variable loci, atpB‐rbcL and trnH‐psbA, can distinguish three of the five species. Two two‐locus combinations, atpB‐rbcL+trnL‐F and atpB‐rbcL+atpF‐H, can resolve all five species. We also find that identification powers of the loci are method‐dependent. The NeighborNet method shows higher species identification power than maximum parsimony, neighbor joining, and unweighted pair group method with arithmetic mean methods.     

Genetic Diversity and Pathogenicity of Fusarium oxysporum f.sp. melonis Races from Different Areas of Italy

Fusarium wilt caused by Fusarium oxysporum f.sp. melonis (FOM) is a devastating disease of melon worldwide. Pathogenicity tests performed with F. oxysporum isolates obtained from Italian melon‐growing areas allowed to identify thirty‐four FOM isolates and the presence of all four races. The aims of this work were to examine genetic relatedness among FOM isolates by race determination and to perform phylogenetic analyses of identified FOM races including also other formae speciales of F. oxysporum of cucurbits. Results showed that FOM race 1,2 was the most numerous with a total of eighteen isolates, while six and nine isolates were identified as race 0 and 1, respectively, and just one isolate was assigned to race 2. Phylogenetic analysis was performed by random amplified polymorphic DNA (RAPD) profiling and by translation elongation factor‐1α (TEF‐1α) sequencing. The analysis of RAPD profiles separated FOM races into two distinct clades. Clade 1, which included races 0, 1 and 1,2, was further divided into ‘subclade a’ which grouped almost all race 1,2 isolates, and into ‘subclade b’ which included race 0 and 1 isolates. Clade 2 comprised only race 2 isolates. The phylogenetic analysis based on TEF‐1α separated FOM from the other formae speciales of F. oxysporum. Also with TEF‐1α analysis, FOM races 0, 1 and 1,2 isolates grouped in one single clade clearly separated from FOM race 2 isolates which grouped closer to F. oxysporum f.sp. cucumerinum. RAPD technique was more effective than TEF‐1α in differentiating FOM race 1,2 isolates from those belonging to the closely related races 0 and 1. Both phylogenetic analyses supported the close relationship between the three different FOM races which might imply the derivation from one another and the different origin of FOM race 2.     

Impatiens nanlingensis sp. nov. (Balsaminaceae) from Guangdong,China

Impatiens nanlingensis A. Q. Dong & F. W. Xing, a new species of Impatiens (Balsaminaceae) from Guangdong, China, is described and illustrated. It is morphologically similar to I. jinggangensis Y. L. Chen, but differs mainly by its lateral united petals; basal lobes suborbicular, marked with yellow stripes; distal lobes narrowly caudate, apex tailed. It also resembles I. bicornuta, but I. bicornuta differs mainly in its longer peduncles, up to 25 cm, pale blue–purple flowers and broadly sigmoid‐curved–saccate lower sepal.     

Phylogeographical history of the widespread meadow fescue (Festuca pratensis Huds.) inferred from chloroplast DNA sequences

Aim Here we explore the variation in chloroplast DNA (cpDNA) in a widespread Eurasian diploid forage grass, meadow fescue (Festuca pratensis Huds.), to address its phylogeographical history. In particular, we aim to answer whether the post‐glacial migration routes of meadow fescue are associated with the spread of agriculture or concurrent with well‐documented natural migration pathways from glacial refugia. Location A total of 56 Eurasian accessions of F. pratensis were analysed, representing the entire native distribution area as well as non‐native areas in northernmost Europe. Methods Based on initial sequencing of 10 non‐coding cpDNA regions, three regions were sequenced for all F. pratensis accessions. For reference, three closely related species [the diploid Lolium perenne L. and the polyploids Festuca arundinacea Schreb. and Festuca gigantea (L.) Vill.] were also sequenced, as well as the more distantly related Festuca ovina L. Divergence times were estimated assuming a simple molecular clock, calibrated using a previously published estimate of 9 Myr for the divergence between fine‐leaved (F. ovina) and broad‐leaved fescues (F. pratensis, F. arundinacea and F. gigantea). Results Limited, but geographically structured, cpDNA variation was observed in F. pratensis. Three haplotypes, estimated to have diverged 0.16 Ma, were identified: one western European (A), one with a wide eastern distribution from central‐eastern Europe into Asia (B) and one Caucasian (C). The haplotypes of the polyploids and L. perenne were estimated to have diverged from haplotype A in F. pratensis 0.8–1.3 Ma. Main conclusions We found no definite evidence for migration of the diploid F. pratensis associated with the spread of agriculture from the Fertile Crescent after the last glaciation. The distinct geographical structuring of the present‐day variation in cpDNA can rather be explained by northwards expansion of the western haplotype from an Iberian refugium, expansion of the eastern haplotype from an unlocated (south‐)eastern refugium and glacial survival without subsequent expansion from a Caucasian refugium. The high level of cpDNA divergence observed between this diploid and the polyploids which have probably been derived from it may suggest that the very low level of cpDNA variation in the diploid is caused by a recent bottleneck. Today, F. pratensis is widespread in the open agricultural landscape but appears otherwise confined to naturally open habitats such as river banks, and its populations may have been decimated when dense forests dominated in the previous interglacial.