中国常见仓储书虱PCR-RFLP快速识别

应用PCR-RFLP技术,对我国4种常见仓储书虱即嗜虫书虱Liposcelis entomophila(Enderlein)、嗜卷书虱L.bostrychophila(Badonnel)、无色书虱L.decolor(Pearman)和小眼书虱L.paeta(Pearman)开展快速识别方法的研究。采用CTAB法从上述4种书虱体内提取基因组DNA,选用1对引物扩增16S rDNA基因区域,分别用5种限制性内切酶对PCR产物进行酶切。结果表明,PCR扩增片段大小约为500bp,用限制性内切酶DraI酶切得到的片段可将供试书虱区分开。该方法不受4种供试书虱地理种群、虫态(成虫、若虫)和性别的影响,可用于4种书虱的快速识别。     

Discrimination between closely related Triticeae species using genomic DNA as a probe

Summary Labelled total genomic DNA was used as a probe in combination with blocking DNA to discriminate between taxonomically closely related species in the genera Hordeum and Secale. Discrimination was possible both by Southern hybridization to size-fractionated restriction enzyme digests of genomic DNA and by in situ hybridization to chromosome preparations. To distinguish between two species (e.g. H. vulgare and H. bulbosum), genomic DNA from one species was used as the labelled probe, while unlabelled DNA from the other species was applied at a much higher concentration as a block. The blocking DNA presumably hybridized to sequences in common between the block and the labelled probe, and between the block and DNA sequences on the membrane or chromosomes in situ. If so, mainly species-specific sequences would remain as sites for probe hybridization. These species-specific sequences are dispersed and represent a substantial proportion of the genome (unlike many cloned, species-specific sequences). Consequently, rapid nonradioactive methods detected probe hybridization sites satisfactorily. The method was able to confirm the parentage of hybrid plants. It has potentially wide application in plant breeding for the detection of alien DNA transfer, and it can be easily adapted to many species.     

利用mtCOI PCR-RFLP技术鉴别草地贪夜蛾与其他3种形态相近昆虫

【目的】田间调查发现草地贪夜蛾与甜菜夜蛾、斜纹夜蛾、粘虫常混合发生,传统的形态学鉴定方法不能快速鉴别出该虫,当前亟需快速鉴别该虫的方法。【方法】本研究分析了草地贪夜蛾与甜菜夜蛾、斜纹夜蛾、粘虫mtCOI基因序列的酶切位点,根据目的片段设计上游引物并进行PCR-RFLP验证。【结果】草地贪夜蛾个体在mtCOI片段的556~561 bp处均存在Sbf I内切酶酶切位点,斜纹夜蛾、甜菜夜蛾、粘虫均无Sbf I酶切位点。草地贪夜蛾PCR产物经过Sbf I内切酶酶切,可出现420 bp左右的特征带,斜纹夜蛾、甜菜夜蛾、粘虫种群均不能被Sbf I内切酶酶切。【结论】基于新设计引物扩增的mtCOI片段的PCR-RFLP方法可有效鉴别草地贪夜蛾与其他3个形态相近昆虫,研究结果为草地贪夜蛾的快速鉴别提供了方法。     

利用mtCOI PCR-RFLP技术鉴定中国境内九个烟粉虱隐种

烟粉虱Bemisia tabaci (Gennadius)是一个物种复合体, 包括31个以上形态上无法区分的隐种, 其中少数隐种是世界性入侵害虫。目前, 在中国境内分布有2个入侵隐种和13个土著隐种。快速、 高效的鉴别方法对掌握烟粉虱田间发生规律及制定相关防控策略具有重要意义。然而到目前为止, 除了mtCOI基因测序比对外, 尚未有一种简便的方法可以有效地区分多个烟粉虱隐种。本研究采用mtCOI PCR-RFLP技术, 单独或组合使用TaqI, VspI, Van91I, NcoI和FokI这5种限制性内切酶, 酶切烟粉虱mtCOI的PCR扩增片段, 鉴别分布在中国境内的9个烟粉虱隐种。结果表明, 单独使用TaqI酶切, 可鉴别出MEAM1和China 1两个隐种, 使用TaqI+NcoI分步酶切可鉴定出MED和Asia 1两个隐种, 使用TaqI+Van91I分步酶切可鉴定出Asia II 3和Asia II 9两个隐种, 使用TaqI+VspI+FokI分步酶切可鉴定出Asia II 1, Asia II 6和Asia II 7 3个隐种。本研究为高效鉴别中国境内的多个烟粉虱隐种提供了方法。     

A comparison of the rate of drying of four nematode species using a liquid paraffin technique

A technique of immersion refractometry has been employed to compare the rates at which four species of nematodes lose water during desiccation at 0% r.h. Panagrellus redivivus and Ditylenchus myceliophagus showed little ability to control water loss, although in the latter species coiling helped slow the rate of drying. Ditylenchus dipsaci and Anguina tritici exhibited a much greater degree of control over water loss during desiccation, A. tritici being more successful. D. dipsaci revived from ‘wool’ showed a marked decrease in ability to control water loss when compared with freshly extracted worms. The results obtained have been compared with those from work using interference microscopy, and a critical assessment of the liquid paraffin technique is presented.     

Identification of fish-parasitic Myxobolus (Myxosporea) species using a combined PCR-RFLP method

Polymerase chain reaction (PCR) with primers specific for the family Myxobolidae was used to amplify a part of the 18S ribosomal RNA gene of Myxobolus species. The length of the amplified fragments was approximately 1600 base pairs. Six Myxobolus species identified on the basis of morphological features were compared using a combined PCR-RFLP method. The cleavage patterns generated by 2 frequent cutter restriction enzymes (HinfI and MspI) were suitable for the differentiation of the examined Myxobolus species.     

A PCR-RFLP based diagnostic technique to rapidly identify Seiridium species causing cypress canker

Seiridium cardinale, S. cupressi and S. unicorne represent three distinct species of fungi that cause cankers on Cupressus species and the disease collectively known as cypress canker. These fungi cannot be distinguished reliably from each other using morphological characters or ribosomal DNA sequence data. Here we describe a RFLP assay based on digesting β-tubulin amplicons with a single endonuclease, HaeIII, which easily can be used to distinguish among these three species. This RFLP assay provides an inexpensive and simple means of identifying Seiridium species, which include some of the most serious threats to trees in Cupressaceae.     

Genetic distances,geographic distances and migration between four villages of a single region in Slovakia

Genetic microdifferentiation has been studied among four endogamous villages of the Bystrica Valley in the Kysuce region of northwest Slovakia, which arose from a single ancestral population about 15 generations ago. Genetic distances and sample kinship between the villages were estimated from the gene frequencies of seven serum-group and isozyme genetic markers. The genetic distance was found to correlate positively (though insignificantly) with the geographic distance, and negatively with the intensity of migration between villages; the sample kinship correlates negatively with geography as well as with the genetic distance. This pattern of genetic structure within the area indicates that the genetic variation among the villages is attributable to the genetic drift. Thus, drift has brought about a detectable differentiation in the area within a limited period of approximately 300 years, in spite of the fact that the villages were not completely genetically isolated from each other. The finding of a negative correlation between the sample kinship and geographic distance indicates that the recent breakdown of genetic isolates in Slovakia is likely to be accompanied with an overall increase of heterozygosity.     

Characterization of Sarcocystis species in domestic animals using a PCR-RFLP analysis of variation in the 18S rRNA gene: a cost-effective and simple technique for routine species identification

Thirteen restriction endonucleases were used to investigate nuclotide sequence variation in the 18S rRNA DNA of 88 individuals from ten Sarcocystis taxa collected as cysts from their intermediate hosts, swine, cattle and water buffalo. A DNA sequence of approximately 900 bp was used. A total of 26 electromorphs were detected. The electromorphs were sorted into seven different haplotypes that coincided with the six named species and an unidentified species from cattle. These findings support those of our morphological examinations, which suggested that the taxa resembling Sarcocystis hirsuta, S. hominis, both found in water buffalo, and S. sinensis found in cattle, are not new species but are in fact S. hirsuta and S. hominis as found in cattle, and S. sinensis as found in water buffalo; this finding supports the idea that these species can utilize both cattle and water buffalo as intermediate hosts and are not restricted to one or the other host group as previously thought. PCR-RFLP resolved by agarose gel electrophoresis is shown to be an easy and rapid method of discriminating between these species.     

Phylogenetic analysis of Aspergillus species using DNA sequences from four loci

DNA sequences were determined for beta tubulin (BT2), calmodulin (CF), ITS and lsu rDNA (ID) and RNA polymerase II (RPB2) from ca. 460 Aspergillus isolates. RPB2 and rDNA sequences were combined and analyzed to determine relationships in the genus and in the family Trichocomaceae. Eupenicillium species form a statistically supported clade with origins among the Aspergillus clades. A. crystallinus, A. malodoratus and H. paradoxus are members of the Eupenicillium clade. A. zonatus, A. clavatoflvus and W. spinulosa occur in a clade along with Hamigera sp. Other than these exceptional species, Aspergillus species and sections occur on three strongly supported clades that descend from a polytomy. Section Versicolores as a monophyletic group includes only A. versicolor and A. sydowii and is superfluous. The other sections were retained but modified. All four loci were used in genealogical concordance analysis of species boundaries. Fennellia flavipes and F. nivea are not conspecific with their supposed anamorphs A. flavipes and A. nivea. Synonymies were found for some species and more than 20 undescribed taxa were identified in genealogical concordance analysis. Newly discovered taxa will be described elsewhere. Possibly paralogous gene fragments were amplified with the BT2 primers in sections Nidulantes, Usti and Nigri. Use of nonhomologous sequences in genealogical concordance analysis could lead to false conclusions and so BT2 sequences were not used in analysis of those sections.     

Flavonoids from four compositae species

The isolation of umbelliferone from Eupatorium angustifolium, of alnustin, 5,7,8-trimethoxyflavone, 7-hydroxy-3,5,8-trimethoxyflavone and 3,5,7,8-tetramethoxyflavone from Achyrocline satureoides var. albicans; of cirsimaritin from Baccharis eleagnoides; and of genkwanin from Symphyopappus polystachyus is reported.     

Sterols from four species of liliaceae

Fractionation of the sterol mixture from Tristagma uniflorum, Nothoscordum gramineum var. philippianum, Nothoscordum inodorum, and Nothoscordum montevidense were achieved by means of preparative TLC. Analysis of the fractions by GC and GC-MS allowed the identification of Δ⁰, Δ⁵, and Δ⁷ sterols. The unusually high proportion of cholestan-3β-ol seems to be biogenetically related to the C₂₇ steroidal sapogenins contained in those plants.     

Discrimination between Acartia (Copepoda: Calanoida) species using their diffraction pattern in a position, rotation invariant digital correlation

Digital images of Acartia discaudata, A. clausi, A. margalefiand A. tonsa were processed to obtain their diffraction pattern.To discriminate between species and sex all diffraction patternswere correlated with a spatial filter, invariant to positionand rotation, for each Acartia male and female. This filterwas made up using a combination of different images of eachspecies and sex. Considering the great similarity between thecopepod species used in this work and between the male and femaleof each species the results obtained are very good. It is concludedthat the method used to discriminate between species of thiscongeneric group can be very useful for the development of anautomated system for the identification of copepods.     

Characterization of protamines from four avian species

No data are available on the protamines of birds, with the exception of galline. We have characterized the protamines from four species of birds belonging to four different orders. All of them have very similar properties. They have been purified by carboxymethylcellulose chromatography and analyzed with respect to amino acid composition and electrophoretic behaviour. They are very arginine-rich proteins (63.4-67.3%) but do not contain lysine. Serine (12.0-18.2%), tyrosine (5.8-9.0%) and glycine (4.5-7.1%), along with arginine, make up the bulk of the amino acid residues in these molecules. The electrophoretic mobility of bird protamines in acetic acid-urea-polyacrylamide gels is intermediate between that of somatic histones and salmine. The molecular size, estimated from amino acid analysis and electrophoretic migration, is 65 +/- 5 amino acid residues.     

New species of the genusHypericum sect.Hypericum (Guttiferae) from Slovakia

A new species of the genusHypericum, Hypericum carpaticum Mártonfi, is described from Slovakia. It is pentaploid (2n=40) with morphological and chemical features that are most similar to the tetraploid speciesH. dubium Leers from western Europe. The species is most likely of hybridogenous origin, arising as a result of hybridization betweenH. maculatum Crantz andH. perforatum L. It seems that consequent speciation processes and stabilization by inherited apomixis (fromH. perforatum) allowed it to reproduce successfully and to enlarge its distribution area. According to recent knowledge of its distribution, it is necessary to consider it endemic to the Volovské vrchy Mts. in the Carpathians. With regard to its scarcity it merits EN status according to the IUCN criteria.     

Alternaria jesenskae sp. nov., a new species from Slovakia on Fumana procumbens (Cistaceae)

Alternaria jesenskae sp. nov. recovered from seeds of a shrubby perennial plant Fumana procumbens (Cistaceae) in Slovakia is described and illustrated. The new taxon can be clearly separated from the other related large-spored and filament-beaked Alternaria species based on sequences of the ITS1, 5.8S and ITS2 region as well as by its distinctive morphology. Even though the molecular data have shown close relatedness with A. multirostrata, the new species is morphologically most similar to A. tomatophila distinguished primarily by the pronounced colony pigmentation, conidial septation and beak branching.