Molecular Species Identification of Spiny Lobster Phyllosoma Larvae of the Genus Panulirus from the Northwestern Pacific

To identify lobster phyllosoma larvae of the genus Panulirus occurring in waters adjacent to Japan, genetic variation within and between 10 Indo-Pacific lobster species was investigated using restriction fragment length polymorphism (RFLP) analysis for the 1300-base pair mitochondrial cytochrome oxidase I (COI) gene. RFLP analysis using two endonucleases (AluI and TaqI) enabled discrimination of all species, including the P. longipes complex. The diagnostic DNA markers, supplemented with nucleotide sequence analysis, were applied to 44 mid- to late-stage phyllosoma larvae (7.4 to 27.7 mm in body length) collected in the northwestern Pacific. These samples were unexpectedly variable in species composition, comprising P. japonicus (n = 16), P. longipes bispinosus (21), P. longipes longipes (1), P. “aka” (1), and P. penicillatus (5). Comparison of larval size at similar stages revealed that P. l. bispinosus larvae were significantly larger than P. japonicus.     

Community analysis of arbuscular mycorrhizal fungi in a warm-temperate deciduous broad-leaved forest and introduction of the fungal community into the seedlings of indigenous woody plants

A community of arbuscular mycorrhizal (AM) fungi was investigated in a warm-temperate deciduous broad-leaved forest using a molecular analysis method. Root samples were obtained from the forest, and DNA was extracted from the samples. Partial 18S rDNA of AM fungi were amplified from the extracted DNA by polymerase chain reaction using a universal eukaryotic primer NS31 and an AM fungal-specific primer AM1. After cloning the PCR products, 394 clones were obtained in total, which were divided into five types by restriction fragment length polymorphism (RFLP) with HinfI, RsaI, and Hsp92II. More than 20% of the clones were randomly selected from each RFLP type and sequenced. Phylogenetic analysis showed that all the obtained clones belonged to Glomus but could not be identified at species level. Topsoil of the forest containing plant roots was inoculated to nonmycorrhizal seedlings of indigenous woody plants, Rhus javanica var. roxburghii and Clethra barvinervis, to introduce the community of AM fungi into the seedlings. Among these five RFLP types, four types were detected from both seedlings, which indicates that the AM fungal community in the forest root samples was introduced at least partly into the seedlings. Meanwhile, an additional four types that were not found in the forest root samples were newly detected in the seedlings, these types were closely related to one another and close to G. fasciculatum or G. intraradices. It is expected that a community of indigenous diverse AM fungi could be introduced into target fields by planting these mycorrhizal seedlings.     

Construction of the Bac-to-Bac system of <Emphasis Type="Italic">Bombyx mori</Emphasis> nucleopolyhedroviru

To construct the Bac-to-Bac expression system of Bombyx mori nucleopolyhedrovirus (BmNPV), a transfer vector was constructed which contained an Escherichia coli (E. coli) mini-F replicon and a lacZ: attTN7: lacZ cassette within the upstream and downstream regions of the BmNPV polyhedrin gene. B. mori larvae were cotransfected with wild-type BmNPV genomic DNA and the transfer vector through subcutaneous injection to generate recombinant viruses by homologous recombination in vivo. The genomic DNA of budded viruses extracted from the hemolymph of the transfected larvae was used to transform E. coli DH10B. Recombinant bacmids were screened by kanamycin resistance, PCR and restriction enzyme (REN) digestion. One of the bacmid colonies, BmBacJS13, which had similar REN profiles to that of wild-type BmNPV, was selected for further research. To investigate the infectivity of BmBacJS13, the polyhedrin gene was introduced into the bacmid and the resultant recombinant (BmBacJS13-ph) was transfected to BmN cells. The budded viruses were collected from the supernatant of the transfected cells and used for infecting BmN cells. Growth curve analysis indicated that BmBacJS13-ph had a similar growth curve to that of wild-type BmNPV. Bio-assays indicated that BmBacJS13-ph was also infectious to B. mori larvae. Foundation items: 973 (2003CB114202); Programme Strategic Scientific Alliances between China and the Netherlands (2004CB720404); National Natural Fundation of China project (30630002)     

Community of arbuscular mycorrhizal fungi in drought-resistant plants, <Emphasis Type="Italic">Moringa</Emphasis> spp., in semiarid regions in Madagascar and Uganda

The community structure of arbuscular mycorrhizal (AM) fungi in the roots of drought-resistant trees, Moringa spp., was examined in semiarid regions in Madagascar and Uganda. Root samples were collected from 8 individuals of M. hildebrandtii and 2 individuals of M. drouhardii in Madagascar and from 21 individuals of M. oleifera in Uganda. Total DNA was extracted from the root samples, and partial nSSU rDNA of AM fungi was amplified using a universal eukaryotic primer NS31 and an AM fungalspecific primer AM1. The PCR products were cloned and divided by restriction fragment length polymorphism (RFLP) analysis with HinfI and RsaI. Some representatives in each RFLP types were sequenced, and a neighbor-joining phylogenetic analysis was conducted for the obtained sequences with analogous sequences of AM fungi. The RFLP and phylogenetic analyses showed that AM fungi closely related to Glomus intraradices or G. sinuosum were detected in many samples. The AM fungal groups frequently detected in the Moringa spp. might be widely distributed species in semiarid environments.     

Towards an integrated linkage map of common bean

Summary Two genomic libraries were established to provide markers to develop an integrated map combining molecular markers and genes for qualitative and quantitative morpho-agronomic traits in common bean. Contrasting characteristics were observed for the two libraries. While 89% of the PstI clones were classified as single-copy sequences, only 21% of the EcoRIBamHI clones belonged in that category. Clones of these two libraries were hybridized against genomic DNA of nine genotypes chosen according to their divergent evolutionary origin and contrasting agronomic traits. Eight restriction enzymes were used in this study. PstI clones revealed 80–90% polymorphism between the Andean and Middle American gene pools and 50–60% polymorphism within these gene pools. However, under the same conditions only 30% of the EcoRI-BamHI clones showed polymorphism between the Middle American and Andean gene pools. Hybridization with PstI clones to EcoRI-, EcoRV-, or HindIII-digested genomic DNA resulted in a cumulative frequency of polymorphism of approximately 80%. Hybridizations to BamHI-, HaeIII-, HinfI-, PstI-, and XbaI-digested genomic DNA detected no additional polymorphisms not revealed by the former three enzymes. In the PstI library, a positive correlation was observed between the average size of hybridizing restriction fragments and the frequency of polymorphism detected by each restriction enzyme. This relationship is consistent with the higher proportion of insertion/deletion events compared with the frequency of nucleotide substitutions observed in that library.     

Genetic variability among <Emphasis Type="Italic">Pholiota aurivella</Emphasis> isolates from a small natural population

Genetic differences between 36 Pholiota aurivella wild isolates collected from 13 decayed logs of Salicaceae trees distributed along about 1200 m of a streambed in a forest were characterized by somatic incompatibility and mating tests, and by restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA). There was a perfect correlation between somatic incompatibility and mating type groups, and isolates could be divided into 15 genets (genetically identical clones). Because the mtDNAs of the 36 wild isolates have 14 different EcoRI RFLP patterns, they likely originated from at least 14 distinct wild strains, indicating that multiple wild strains with distinct genetic compositions coexist in the forest investigated in this study. mtDNA variation of P. aurivella is apparently very high despite the close proximity of sample collection sites within the forest. The territories of single P. aurivella genets within a host log are apparently larger than other nonpathogenic wood-decaying basidiomycetes reported previously, such as Flammulina velutipes and Lentinula edodes.     

Larvae of chigger mites Neotrombicula spp. (Acari: Trombiculidae) exhibited Borrelia but no Anaplasma infections: a field study including birds from the Czech Carpathians as hosts of chiggers

Chigger mites were collected from 1,080 wild birds of 37 species at Certak (Czech Republic), in the western Carpathian Mountains, from 29 July to 24 September 2005. The prevalence of infestation with chigger larvae was 7%. A total of 325 chigger specimens from 10 bird species was identified and three chigger species were found: Neotrombicula autumnalis, N. carpathica, and N. inopinata, the latter two species being reported on new hosts. Neotrombicula carpathica is reported in the Czech Republic for the first time. A total of 509 chigger larvae found on 79 host specimens were examined by polymerase chain reaction (PCR) for the presence of Borrelia burgdorferi s.l. DNA (fragments of the rrf (5S)—rrl (23S) intergenic spacer), and Anaplasma phagocytophilum DNA (epank1 gene). A fragment of specific Borrelia DNA was amplified through PCR in one sample, and the PCR product was further analyzed by reverse line blotting assay, whereby both genospecies of B. garinii and B. valaisiana were proved. This sample pooled five chigger larvae collected from one Sylvia atricapilla on 11 August 2005. No A. phagocytophilum DNA was amplified. We conclude that larvae of the genus Neotrombicula can be infected with Borrelia genospecies originated from their present or former hosts.     

Chloroplast DNA inheritance in theStellaria longipes complex (Caryophyllaceae)

Inheritance of chloroplast DNA (cpDNA) was examined in F₁ progenies derived from three crosses and three corresponding reciprocal crosses betweenStellaria porsildii andS. longifolia. Chloroplast DNA restriction fragments were analyzed using methods of nonradioactive digoxigenin-11-dUTP labeling and chemiluminescent detection with Lumi-Phos 530. Distinct interspecific restriction fragment polymorphisms were identified and used to demonstrate the mode of cpDNA inheritance. Mode of cpDNA inheritance differed among crosses. Two crosses in whichS. porsildii, SP2920-21, was the maternal parent exhibited three different types of plastids, maternal, paternal and biparental, among the F₁ hybrids, suggesting a biparental cpDNA inheritance and plastid sorting-out inStellaria.     

<Emphasis Type="Italic">Dasytricha</Emphasis> Dominance in Surti Buffalo Rumen Revealed by 18S rRNA Sequences and Real-Time PCR Assay

The genetic diversity of protozoa in Surti buffalo rumen was studied by amplified ribosomal DNA restriction analysis, 18S rDNA sequence homology and phylogenetic and Real-time PCR analysis methods. Three animals were fed diet comprised green fodder Napier bajra 21 (Pennisetum purpureum), mature pasture grass (Dicanthium annulatum) and concentrate mixture (20% crude protein, 65% total digestible nutrients). A protozoa-specific primer (P-SSU-342f) and a eukarya-specific primer (Medlin B) were used to amplify a 1,360 bp fragment of DNA encoding protozoal small subunit (SSU) ribosomal RNA from rumen fluid. A total of 91 clones were examined and identified 14 different 18S RNA sequences based on PCR–RFLP pattern. These 14 phylotypes were distributed into four genera-based 18S rDNA database sequences and identified as Dasytricha (57 clones), Isotricha (14 clones), Ostracodinium (11 clones) and Polyplastron (9 clones). Phylogenetic analyses were also used to infer the makeup of protozoa communities in the rumen of Surti buffalo. Out of 14 sequences, 8 sequences (69 clones) clustered with the Dasytricha ruminantium-like clone and 4 sequences (13 clones) were also phylogenetically placed with the Isotricha prostoma-like clone. Moreover, 2 phylotypes (9 clones) were related to Polyplastron multivesiculatum-like clone. In addition, the number of 18S rDNA gene copies of Dasytricha ruminantium (0.05% to ciliate protozoa) was higher than Entodinium sp. (2.0 × 10⁵ vs. 1.3 × 10⁴) in per ml ruminal fluid.     

Genetic diversity of dodder (<Emphasis Type="Italic">Cuscuta</Emphasis> spp.) collected from commercial cranberry production as revealed in the<Emphasis Type="Italic">trn</Emphasis>L (UAA) intron

Swamp dodder (Cuscuta gronovii) is a parasitic plant detrimental to cranberries. Observation of emergence of dodder seeds collected from a cultivated cranberry bog in Massachusetts revealed 2 or more peak emergence times during 4 consecutive growing seasons. Molecular methods were used to investigate genetic variation among the emerging dodder seedlings. On emergence, dodder seedlings were collected and analyzed for DNA sequence diversity in thetrnL (UAA) intron, a noncoding region of chloroplast DNA. DNA sequence analysis of 87 seedlings collected during the 1999 and 2000 growing seasons revealed the presence of 2 dodder ecotypes, designated A and B. Comparative DNA sequence analysis indicated that in thetrnL (UAA) intron, the sequence of ecotype A is identical to that ofCuscuta gronovii, whereas the sequence of ecotype B is closest to that ofCuscuta attenuata (99.3% sequence identity; 293 bases considered). ABg/II restriction enzyme cut site was identified that distinguished between thetrnL (UAA) introns of ecotypes A and B. Restriction fragment length polymorphism (RFLP) was used to analyze the sequences of 100 seedlings collected during the growing seasons of 2001 and 2002. Only 10 of the 187 samples were ecotype A, all of which emerged on or before May 7 in the growing seasons. Therefore, the predominant dodder haplotype found in this study may be a close relative ofC. attenuata and notC. gronovii, the common species found in cranberry bogs.     

Infection rates of Borrelia burgdorferi in different instars of Ixodes ricinus ticks from the Dutch North Sea Island of Ameland

Between 1988 and 1993, a total of 7173 I. ricinus ticks, predominantly nymphs, were collected from the vegetation on the Dutch North Sea Island of Ameland. A proportion of the ticks (n=547) was screened for the presence of Borrelia by immunofluorescence. Infection rates of Borrelia varied, in nymphs (n=347) from 13% to 46% and in adults, (n=122) from 20% to 43%. The infection rate in larvae (n=84) collected in 1993 was 21%, showing that transovarial transmission of B. burgdorferi occurs in the I. ricinus population on Ameland. Two tick-naive sheep seroconverted for B. burgdorferi after field-collected adult or nymphal I. ricinus were allowed to feed on them. Larval progeny (n=168) of 15 female adult ticks fed on one of these sheep were free from B. burgdorferi. B. burgdorferi was isolated in culture from field-collected adult ticks. Serotyping using monoclonal antibodies against outer surface proteins A and C indicated that both isolates belonged to genospecies B. garinii, and this was confirmed by DraI restriction analysis of the variable DNA sequence between the 5S and 23S rRNA genes.     

Streptomycin resistant and sensitive somatic hybrids of Nicotiana tabacum + Nicotiana knightiana: correlation of resistance to N. tabacum plastids

Summary Protoplasts of Nicotiana tabacum SRI (streptomycin resistant) and of Nicotiana knightiana (streptomycin sensitive) were fused using polyethylene glycol treatment. From three heterokaryons 500 clones were obtained. From the 43 which were further investigated, 6 resistant, 3 sensitive, and 34 chimeric (consisting of resistant and sensitive sectors) calli were found. From eight clones, a total of 39 plants were regenerated and identified as somatic hybrids. Chloroplast type (N. tabacum = NT or N. knightiana = NK) in the plants was determined on the basis of the species specific EcoRI restriction pattern of the chloroplast DNA. Regenerates contained NT (13 plants) or NK (15 plants) plastids but only the plants with the NT chloroplasts were resistant to streptomycin. This finding and our earlier data on uniparental inheritance points to the chloroplasts as the carriers of the streptomycin resistance factor.     

Genotyping of somatic hybrids between <Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb. and <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

In order to genotype hybrid genomes of distant asymmetric somatic hybrids, we synthesized hybrid calli and plants via PEG-mediated protoplast fusion between recipient tall fescue (Festuca. arundinacea Schreb.) and donor wheat (Triticum aestivum L.). Seventeen and 25 putative hybrid clones were produced from the fusion combinations I and II, each with the donor wheat protoplast treated by UV light for 30 s and 1 min, respectively. Isozyme and RAPD profiles confirmed that ten hybrid clones were obtained from combination I and 19 from combination II. Out of the 29 hybrids, 12 regenerated hybrid plants with tall fescue phenotype. Composition and methylation-variation of the nuclear and cytoplasmic genomes of some hybrids, either with or without regenerative ability, were compared by genomic in situ hybridization, restriction fragment length polymorphism, and DNA methylation-sensitive amplification polymorphism. Our results indicated that these selected hybrids all contained introgressed nuclear and cytoplasmic DNA as well as obvious methylation variations compared to both parents. However, there were no differences either in nuclear/cytoplasmic DNA or methylation degree between the regenerable and non-regenerable hybrid clones. We conclude that both regeneration complementation and genetic material balance are crucial for hybrid plant regeneration.     

Cryptic clonal lineages and genetic diversity in the loach Misgurnus anguillicaudatus (Teleostei: Cobitidae) inferred from nuclear and mitochondrial DNA analyses

In the loach Misgurnus anguillicaudatue, the asexual lineage, which produces unreduced clonal diploid eggs, has been identified. Among 833 specimens collected from 54 localities in Japan and two localities in China, 82 candidates of other lineage(s) of cryptic clones were screened by examining RFLP (restriction fragment length polymorphism)-PCR haplotypes in the control region of mtDNA. This analysis was performed because triploid loaches arise from the accidental incorporation of the sperm nucleus into unreduced diploid eggs of a clone. The categorization of members belonging to three newly identified lineages (clones 2–4) and the previously identified clonal lineage (clone 1) was verified by evaluating the genetic identity between two or more individuals from each clonal lineage based on RAPD (random amplified polymorphic DNA)-PCR and multilocus DNA fingerprints. We detected 75 haplotypes by observing the nucleotide status at variable sites from the control region of mtDNA. Phylogenic trees constructed from such sequences showed two highly diversified clades, A and B, that were beyond the level common for interspecific genetic differentiation. That result suggests that M. anguillicaudatus in Japan is not a single species entity. Two clone-specific mtDNA sequences were included in clade A, and the loaches with such sequences may be the maternal origin of the clones. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of the genes encoding the haemolytic toxin and the mosquitocidal delta-endotoxin of Bacillus thuringiensis israelensis

Summary The crystalline parasporal inclusions (crystals) of Bacillus thuringiensis israelensis (Bti), which are specifically toxic to mosquito and black fly larvae, contain three main polypeptides of 28 kDa, 68 kDa and 130 kDa. The genes encoding the 28 kDa protein and the 130 kDa protein have been cloned from a large plasmid of Bti. Escherichiacoli recombinant clones containing the 130 kDa protein gene were highly active against larvae of Aedes aegypti and Culex pipiens, while B. subtilis recombinant cells containing the 28 kDa protein gene were haemolytic for sheep red blood cells. A fragment of the Bti plasmid which is partially homologous to the 130 kDa protein gene was also isolated; it probably corresponds to part of a second type of mosquitocidal toxin gene. Furthermore, restriction enzyme analysis suggested that the 130 kDa protein gene is located on the same Bti EcoRI fragment as another kind of Bti mosquitocidal protein gene cloned by Thorne et al. (1986). Hybridization experiments conducted with the 28 kDa protein gene and the 230 kDa protein gene showed that these two Bti genes are probably present in the plasmid DNA of B. thuringiensis subsp. morrisoni (PG14), which is also highly active against mosquito larvae.     

A quantitative analysis of shotgun cloning in Bacillus subtilis protoplasts

Summary We used the Escherichia coli-Bacillus subtilis shuttle vector pHP13, which carries the replication functions of the cryptic B. subtilis plasmid pTA1060, to study the effects of BsuM restriction, plasmid size and DNA concentration on the efficiency of shotgun cloning of heterologous E. coli DNA in B. subtilis protoplasts. In a restriction-deficient strain, clones were obtained with low frequency (19% of the transformants contained a recombinant plasmid) and large inserts (>6 kb) were relatively rare (12% of the clones contained inserts in the range of 6–9 kb). The efficiency of shotgun cloning was severely reduced in restricting protoplasts: the class of large inserts (>6 kb) was under-represented in the clone bank (4% of the clones contained inserts in the range of 6–6.1 kb). Furthermore, BsuM restriction caused structural instability of some recombinant plasmids. Transformation of protoplasts with individual recombinant plasmids showed that plasmid size and transforming activity were negatively correlated. The size effect was most extreme with cut and religated plasmid DNA. The yield of clones was independent of the DNA concentration during transformation. It is therefore unlikely that clones were not detected because of simultaneous uptake of more than one plasmid. It is concluded that shotgun cloning in B. subtilis protoplasts is inferior to that in competent cells.     

The Freshwater Animal Diversity Assessment: an overview of the results

The geographic genetic structure of two common encrusting sponges, Hymeniacidon sinapium and Hymeniacidon flavia (family Halichondriidae), was investigated using two DNA markers, Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA and NADH dehydrogenase subunit 5 (nad5) of mitochondrial DNA. In the ITS analyses, multiple sequence types were identified within each species. Geographic distribution patterns of sequence types showed higher diversity in the western than eastern areas in both species. However, intraspecific genetic diversity of the two species in Japan differed markedly. Hymeniacidon flavia had far more diverse sequence types, and several genetic differentiations between localities were detected. In contrast, H. sinapium had only four sequence types in Japan, and two Atlantic Hymeniacidon species had sequence types similar to this species. In comparison to ITS, nad5 showed very low genetic diversity in both species, with two haplotypes identified in each species. In H. flavia, frequency of haplotype changed gradually from north to south. In H. sinapium, one haplotype was predominant in most regions, and another haplotype was minor and distributed only in the Korean and Tsushima populations. Based on the unique distribution patterns of sequence types around Shikoku and Kyushu, geographical history and ocean currents were assumed to affect the generation of genetic structure. The geographic genetic structure of H. flavia suggests low dispersal ability of pelagic larvae, whereas higher larval dispersal ability and a far broader distribution range are suggested in H. sinapium. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Handling editor: C. Sturmbauer     

Cloning and structural analysis of the snap-back DNA of Pharbitis nil

We isolated and cloned DNA fragments that exist as inverted-repeat structures in the genome of Pharbitis nil. The method used exploited the fact that if inverted repeat DNA is present in the DNA fragment, intramolecular double-stranded structures can be partly formed within single-stranded DNA molecules after denaturation and rapid renaturation of the fragment. The rapidly renaturing DNA fragments (termed snap-back DNA) were isolated by hybroxylapatite column chromatography and treatment with mungbean nuclease and were cloned into the pUC9 vector. Four snap-back DNA members out of thousands of independent clones obtained were characterized with respect to the reiteration frequency and the nucleotide sequences. When used as probes in Southern hybridization experiments, some of the members identified restriction fragment length polymorphism among the cultivars, suggesting that these sequences might be fluid in the genome. One of the four clones has regions of nucleotide sequence homology to those of inverted-repeat regions in the transposon Taml of Antirrhinum majus.     

Chloroplast and mitochondrial DNA diversity in Theobroma cacao

The variability of cocoa (Theobroma cacao) cytoplasmic genomes has been investigated. A total of 177 cocoa clones was surveyed for restriction fragment length polymorphism (RFLP) in chloroplast DNA and in mitochondrial DNA using two restriction endonucleases and various heterologous cytoplasmic probes. A high level of polymorphism was found for the mitochondrial genome. This study points up a structuring of the species that fits with the distinction between the Criollo and Forastero populations. In contrast to all previous analyses, a higher level of polymorphism is found among the Criollo clones while the Forastero clones form quite a homogeneous group.     

Assessment of the degree of restriction fragment length polymorphism in Brassica

Summary The feasibility of creating a restriction fragment length polymorphism (RFLP) linkage map in Brassica species was assessed by screening EcoRI-, HindIII-, or EcoRV-digested total genomic DNA from several accessions of B. campestris, B. oleracea, and B. napus using random genomic DNA clones from three Brassica libraries as hybridization probes. Differences in restriction fragment hybridization patterns occurred at frequencies of 95% for comparisons of accessions among species, 79% for comparisons of accessions among subspecies within species, and 70% for comparisons among accessions within subspecies. In addition, species differences in the level of hybridization were noted for some clones. The high degree of polymorphism found even among closely related Brassica accessions indicates that RFLP analysis will be a very useful tool in genetic, taxonomic, and evolutionary studies of the Brassica genus. Development of RFLP linkage maps is now in progress.