分子标记技术在蛛形学研究中的应用

DNA分子标记作为新发展起来的一种遗传标记形式,凭借其可靠有效等优点,在动植物研究中的应用已越来越广泛。简述了DNA分子标记技术的种类、原理和特点,并综述了分子标记技术在蜘蛛亲缘关系界定和系统进化等方面的研究进展,对有关问题进行了初步讨论和展望。     

Random Amplified Polymorphic DNA and Polymerase Chain Reaction Markers for the Differentiation and Detection of Stenocarpella maydis in Maize Seeds

The genetic relationship of 34 isolates of Stenocarpella maydis from different geographic regions in South Africa was analysed by random amplified polymorphic DNA (RAPD) and ribosomal DNA markers. Two genetic groups were differentiated by using three RAPD primers and correlated to the cultural morphology of the isolates. Of all the isolates tested, 79.4% were clustered into RAPD group I (RG I), which did not sporulate when cultured on potato dextrose agar (PDA) at 25°C for 10 days. The rest of the isolates designated as RG II sporulated on PDA medium and showed a higher genetic variation. Ribosomal DNA (rDNA) was amplified using polymerase chain reaction (PCR) with the universal primers, internal transcribed spacer (ITS) 1 and ITS 4. Restriction digestion of PCR products displayed three types (RF A, RF B and RF C) of profiles. RF A was in accordance with RG I. RF B was consistent with RG II except for one isolate, U5. However, U5 displayed a unique profile and had no restriction sites for Hpa II and Hae III. The results indicate that two distinct genetic groups exist among S. maydis isolates from maize in S. Africa. The ITS1 and ITS2 regions of rDNA were sequenced and primers were designed. The designed primer pair P1/P2 permitted a sensitive and specific detection of S. maydis .     

Application of polymerase chain reaction (PCR) using random amplified polymorphic DNA (RAPD) primers in the molecular identification of selected Sargassum species (Phaeophyta,Fucales)

The random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) was used for the molecular characterisation and identification of Sargassum spp. A total of 17 samples of Sargassum (Sargassaceae, Fucales) was obtained from various localities around Peninsular Malaysia and Singapore. On the basis of morphological characteristics, the samples were tentatively grouped into five species: Sargassum baccularia, S. glaucescens, S. oligocystum, S. polycystum and S. siliquosum. By RAPD-PCR, five of 31 random primers tested generated reproducible amplification products, and polymorphic loci were detected by four of them (OPA02, OPA03, OPA04, OPA13). The RAPD-PCR profiles did not correlate with the morphological grouping into five species and extensive variation was detected between different isolates of the same species. A 450 base pair fragment generated using OPA13 was detected in 12 of 17 samples of Sargassum. This fragment was also present in profiles from Turbinaria (Sargassaceae). This study suggests that RAPD-PCR is useful in discriminating individual samples of the genus Sargassum and in developing fingerprints for them.     

Two DNA sequences specific for the canine Y chromosome

Data are presented on the characterization of two nucleotide sequences found exclusively in the DNA of male dogs. In polymerase chain reactions (PCRs) of canine genomic DNA with a decanucleotide primer of arbitrary sequence (OP-W17), two nucleotide segments (650 and 990 bp) were amplified only from male samples, whereas a number of other fragments between 400 and 2500 bp in size were amplified from both male and female samples. The two male-specific segments were cloned and sequenced, and terminal 24mer oligonucleotide primer pairs were synthesized. PCR with these specific primer pairs resulted in amplification of the two male-specific sequences only from DNA samples of 34 male dogs; no product was amplified from 42 samples of females. A segment of the SRY gene previously localized on the Y chromosome could be amplified in DNA samples that had the two new sequences. Eco RI digested genomic male DNA when hybridized with the 650 bp or the 990 bp sequences, resulted in a single band for each on Southern analysis; DNA from females did not yield any bands. Comparisons between the two new sequences and the SRY gene segment revealed no homologies. We concluded that the two new sequences are specific for the canine Y chromosome and do not contain the short characterized segment of the SRY gene.     

Co-migration of RAPD-PCR amplicons from Aeromonas hydrophila

Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) uses arbitrary primers and low stringency annealing conditions to amplify anonymous DNA fragments which are then depicted in agarose gels. RAPD-PCR fingerprints have been used for typing and differentiation of bacteria and, increasingly, for the study of genetic relationships between strains and species of microorganisms, plants and animals. The analysis of such fingerprints is based upon the assumption that co-migration of amplicons does not occur and that any given band contains a single amplicon. This report shows that co-migration of fragments of nearly identical size, but different nucleotide sequences, occurs between different isolates and within single RAPD-PCR bands from Aeromonas hydrophila. The possibility of the same phenomenon occurring for other prokaryotic or eukaryotic genomes argues for caution in the interpretation of RAPD-PCR fingerprints.     

Optimization of primer screening for evaluation of genetic relationship in rose cultivars

Optimization of primer screening for evaluation of genetic relationship in 34 cultivars of rose through random amplified polymorphic DNA (RAPD) markers was investigated. Four series of decamer primers were used for screening and optimization of RAPD analysis between which A and N series performed good amplification of fragments as compared with other series. The primers OPN-07 and OPN-15 produced maximum number of DNA fragments in Rosa hybrida cv. Anuraag. Some primer either did not produce amplification or produced very poor amplification. Further, ten selected primers were used for genetic analysis of 34 rose cultivars. The primer OPN-15 amplified 21 fragments in all cultivars tested. A total of 162 distinct DNA fragments (bands) ranging from 100 to 3400 base pairs were amplified by using 10 selected random primers. The cluster analysis indicated that these rose cultivars formed nine clusters.     

RAPD标记构建家蚕分子连锁图

以大造、Ｃ１０８及其Ｆ２群体构建了１个家蚕的ＲＡＰＤ连锁框架图,该图含ＲＡＰＤ标记位点１８２个,来自大造的１０３个标记分属前２３个连锁群,来自Ｃ１０８的７９个标记分属后１６个连锁群,覆盖基因组的总长度为１１４８．３ｃＭ（ｃｅｎｔｉｍｏｒｇａｎ）,它能与本室用同一群体构建的ＳＡＤＦ图谱相整合,亦可与相应的ＲＦＬＰ图谱相互补充。     

Molecular characterisation of Sargassum polycystum and S. siliquosum (Phaeophyta) by polymerase chain reaction (PCR) using random amplified polymorphic DNA (RAPD) primers

Genomic DNA was extracted from 13 samples of Sargassum polycystum and S. siliquosum collected from various localities around Peninsular Malaysia and Singapore by using four different extraction methods. The yields and the suitability of the DNA to be used as template for the polymerase chain reaction (PCR) was compared. DNA samples were subjected to PCR analysis by using random primers. Only DNA samples that were extracted using the CTAB method were successfully amplified by random amplified polymorphic DNA (RAPD)-PCR. Five of 31 random primers (OPA02, OPA03, OPA04, OPA13 and OPM10) tested amplified sequences of DNA from the DNA samples. Reproducible, amplified products were obtained using these primers and showed some potential to be useful in discriminating individual samples within the genus, in determining relationships between species within a genus and in developing individual fingerprints for individual samples.     

Microsatellites reveal high genetic diversity within colonies of Camponotus ants

In order to characterize the sociogenetic structure of colonies in the carpenter ants Camponotus herculeanus and C. ligniperda, we have developed microsatellite markers. The three loci studied were either fixed for different alleles in the two species or showed different patterns of polymorphisms. Genotyping of workers and males showed that the broods of C. ligniperda include several matrilines, a rare phenomenon in the genus. Five alleles from a locus polymorphic in both species were sequenced from the respective PCR-products. A part of the length variation appeared to be due to changes outside the repeat sequence, and some PCR products of an equal length had a different number of dinucleotide repeats.     

Determination of RAPD Markers in Rice and their Conversion into Sequence Tagged Sites (STSs) and STS-Specific Primers

We produced 102 randomly amplified polymorphic DNA (RAPD) markersmapped on all 12 chromosomes of rice using DNAs of cultivarsNipponbare (japonica) and Kasalath (indica) and of F2 populationgenerated by a single cross of these parents. Sixty random primers10 nucleotides long were used both singly and in random pairsand about 1,400 primer-pairs were tested. Using both agarosegel and polyacrylamide gel electrophoresis enabled us to detectpolymorphisms appearing in the range from <100 bp to 2 kb.The loci of the RAPD markers were determined onto the frameworkof our RFLP linkage map and some of these markers were mappedto regions with few markers. Out of the 102 RAPD markers, 20STSs (sequence-tagged sites) and STS-specific primer pairs weredetermined by cloning, identifying and sequencing of the mappedpolymorphic fragments.     

Isolation and characterization of Legionella spp. and Pseudomonas spp. from greenhouse misting systems

AIMS: Greenhouse misting systems used for watering plants produce fine aerosols. They are a possible cause for bacterial infections. This study investigates the colonization of greenhouse misting systems with Legionella spp. and Pseudomonas spp. and evaluates a possible health hazard. METHODS AND RESULTS: Between June and September 2003, a total of 80 water samples were collected in 20 different greenhouse systems in Germany, each tested on two different occasions. Each time, water was drawn at a central tap and at the outlet of spray nozzles. Sampled greenhouses were used to cultivate various plants and trees for commercial, recreational or scientific reasons, some of them in tropical conditions. Legionella spp. were detected in 10% of the systems (two systems), but only in low numbers. On the contrary, Pseudomonas spp. were recovered from 70% of the greenhouse watering systems (14 systems), occasionally at counts greater than 10,000 CFU per 100 ml. A random amplified polymorphic DNA polymerase chain reaction typing method was used to demonstrate that each colonized greenhouse had one or several individual strains of Legionella and Pseudomonas that could not be detected in any other system. CONCLUSIONS: This study demonstrates that aerosolizing greenhouse watering systems may be contaminated with Legionella or Pseudomonas which under certain circumstances could become a potential source of infection for workers and visitors. SIGNIFICANCE AND IMPACT OF THE STUDY: The study results indicate that greenhouse misting systems should be included in Legionella and Pseudomonas monitoring and control programs.     

PCR-RFLP patterns of four isolates of Trichinella for rDNA ITS1 region

We have studied the genetic differences among four isolates of Trichinella including a new strain of Trichinella spiralis (ISS 623) recently found from a human case who took a badger in Korea. Because they have a different host origin and came from geographically separated regions, we supposed the genetic pattern of the isolates might be different as had been previously reported. It was analysed by PCR-RFLP analysis of the rDNA repeat that can readily distinguish a species or strain from others. Isolated genomic DNA of each isolate of Trichinella larvae was amplified with ITS1 specific primers and digested with restriction endonucleases. The PCR product of ITS1 was confirmed using Southern blot analysis to be a 910 bp fragment. The restriction fragments of each isolate had variable patterns when it was digested with Rsa 1 only. According to the RFLP patterns, the estimated genetic divergence between each isolate was different. In conclusion, four isolates of Trichinella including a new strain of T. spiralis obtained from a Korean patient may have genetic differences in the ITS1 region and the Shanghai isolate was genetically more similar to the Japanese unknown isolate than others in the ITS1 region.     

Molecular Detection and Exploration of Diversity Among Fungal Consortium Involved in Phosphate Solubilization

Abstract

Phosphorous (P) that upholds life become unattainable as most of them become unavailable due to the formation of insoluble complexes with cations such as Ca²⁺, Al³⁺ and Fe³⁺ forming a complex calcium phosphate (Ca₃(PO₄)₂), aluminum phosphate (AlPO) and ferrous phosphate (FePO) that results in the decrease of soluble P to a greater extent. There are several reports stating that several rhizospheric fungal species play an important role in solubilizing these insoluble phosphates into a soluble form by the excretion of enzymes like phosphatase, phytase enzymes, and organic acids. In view of this, so we have collected twenty fungal isolates having probable phosphate solubilizing efficiency from different regions of Lucknow, India. Their morphological and biochemical characteristics were tested. Among all, six efficient fungal isolates were further checked at molecular level by using 18S rRNA universal primers and by RAPD means. A dendrogram indicated 40-90% homology i.e., highest similarity was found in between species of Aspergillus flavus and A. biplanus with 33.8% similarity while minimum similarity was observed among A. flavus and Fusarium oxysporum. These findings suggest RAPD proves as, a reliable molecular tool that helps in strain specific discrimination.     

APPLICATION OF RAPD MARKERS IN AN EXAMINATION OF HETEROSIS IN GELIDIUM VAGUM (RHODOPHYTA)1

Four morphologically indistinguishable isolates of monoecious Gelidium vagum Okamura were crossed reciprocally to obtain hybrids for a study on heterosis in this alga. Approximately 50% outcrossing was achieved by adding a small fragment of the designated female parent to a much larger quantity of designated male thallus in the crossing dish. Hybrids in the mixed population of isomorphic hybrid and inbred sporelings were identified by the presence of male-specific random amplified polymorphic DNA markers. Growth performance of hybrid tetrasporophytes was compared to that of their gametophytic parents and with inbred tetrasporophytes at near-optimum and sub-optimum temperature and density. In general, the hybrids showed growth superiority over inbred lines, particularly under sub-optimum conditions. In these experiments, the hybrid plants from cross 129 × 130 exhibited a 9.5–130% higher growth rate as compared to the mid-value of the related inbred tetrasporophytes, strongly suggesting the presence of heterosis.     

弓背蚁属蚂蚁初级内共生菌Blochmannia的研究进展

昆虫与其体内细菌的互利共生关系是近十余年来生物学家关注的热点领域,也是研究物种间协同进化的理想模型。Blochmannia是蚁科弓背蚁属Camponotus蚂蚁体内的初级内共生菌,作为最早被描述的动物与细菌之间的共生关系,Blochmannia在帮助宿主蚂蚁合成食物中所缺少的必需营养物质、维持生长发育、繁殖和营养代谢等方面发挥重要作用。本文从Blochmannia的分布规律、母系传播、基因组特征及其功能等方面进行综述,以期为相关研究提供参考和借鉴。     

Genetic Variation Among Rhynchosporium secalis Populations of West Asia and North Africa as Revealed by RAPD and AFLP Analysis

In order to study genetic variation among populations of Rhynchosporium secalis, 65 isolates were sampled from the West Asian and North African regions and used for polymerase chain reaction (PCR)‐based DNA marker analyses [namely random amplified polymorphism DNAs (RAPDs) and amplified fragment length polymorphisms (AFLPs)]. The study revealed that genetic diversity among and within populations accounted for 80 and 20%, respectively, of the total genetic diversity, indicating that the local field populations of R. secalis in West Asia and North Africa originated from genetically diverse source populations. Furthermore, high genetic similarity among isolates from the same location suggests that scald populations originated from a local founder population, possibly through rain‐splash‐dispersed conidia.     

Genotypic diversity in a localized population of Ralstonia solanacearum as revealed by random amplified polymorphic DNA markers

AIMS: To assess genotypic diversity within Ralstonia solanacearum isolates of a single field. METHODS AND RESULTS: A total of 44 field isolates and 22 in vitro generated clones of R. solanacearum were studied for genotypic diversity by random amplified polymorphic DNA (RAPD) technique. Genomic DNA of these isolates and clones was extracted by proteinase-K-SDS lysis mini-prep method. RAPD analysis was done with 30 decamer primers. The data were analysed using NTSYSpc 2.02h software. Forty-two out of 44 field isolates and all the clonal isolates were identified as distinct genotypes at 70% similarity level. CONCLUSION: Very high level of genome variability was observed within the field and clonal isolates of R. solanacearum. This might be a reason for the wide host range of this bacterium and for quick breakdown of wilt resistance in host plants. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that it would be difficult to design specific diagnostic protocol for R. solanacearum even for a localized population and to breed cultivars with broad-spectrum resistance.     

Micropropagation of Tectona grandis: Assessment of Genetic Fidelity

Random amplified polymorphic DNA (RAPD) markers were used to analyze genetic fidelity of micropropagated teak (Tectona grandis L.) clones with respect to subcultural passage. Of the twenty primers screened, no variation in RAPD profiles was noticed in the in vitro clones of fifth, tenth, fifteenth and twentieth passage in comparison to the in vivo mother plants. Only one micropropagated plant of twenty-fifth subcultural passage, however, differed from the in vivo ones. It revealed the appearance of a new polymorphic DNA fragment (molecular mass 379 kb) in case of primer OPB-08. This primer, manifesting detectable variation, may be utilized as a diagnostic marker for assessing genetic fidelity of micropropagted teak plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

金龟甲基因组DNA RAPD反应条件的优化

以华北大黑鳃金龟[Holotrichia oblita (Faldermann)]和矮臀鳃金龟(H.ernesti Reitter)为供试材料,对DNA模板、Mg2+、dNTPs浓度、淬火和循环内延伸时间及循环数进行优化.提出了金龟甲RAPD分析的最佳反应体系,即25 μL体系中含DNA模板1.2 ng·μL-1、Mg2+ 2. 5 mmol·L-1、dNTPs 0. 25 mmol·L-1;并确定最适淬火时间为40 s,循环内延伸时间为2 min;最佳循环数为40.