Use of restriction fragment length polymorphism to distinguish between salmon species

Identification of 10 salmon species using DNA-based methodology was investigated. Amplification of DNA was carried out using a primer set which amplified a region of the mitochondrial cytochrome b gene. Sequences of PCR-amplified DNA from the salmon species were used to select six restriction enzymes allowing species to be uniquely classified. RFLP patterns generated following analysis with each enzyme were resolved using polyacrylamide gel electrophoresis and visualized by silver staining. Results indicate that it is possible to differentiate between all 10 salmon species and that the technique could be easily adopted by the food industry for analysis of processed salmon products.     

Evaluating the physical capture method of terminal restriction fragment length polymorphism for comparison of soil microbial communities

Terminal restriction fragment length polymorphism (T-RFLP) is a popular method of comparative microbial community analysis which is normally accomplished by tagging terminal restriction fragments (T-RFs) with a fluorescent primer. Here, we evaluate an alternative method of T-RFLP where T-RFs are physically captured using a biotinylated primer and streptavidin-coated beads. This eliminates one of the primary criticisms of T-RFLP, namely that T-RFs cannot be identified by sequence analysis, and also represents an alternative method for collecting T-RFLP profiles. Microbial communities from forest, agricultural, and turf soils were investigated using several sets of primers specific for different microbial groups. The physical capture method of T-RFLP resulted in similar profiles to those generated by fluorescent T-RFLP. The relationships among ecosystem types captured by both methods and revealed by ordination were virtually identical. The total variance in the profiles that was attributed to ecosystem type was approximately equal, or greater, when generated by the physical capture method, depending on the primers used. However, physical capture T-RFLP resolved fewer T-RFs than fluorescent T-RFLP, and this may reduce the sensitivity to changes in non-dominant populations within the community. Direct cloning and sequencing of physical capture T-RFs revealed that most bands were not comprised of sequences related to those in the database that would generate T-RFs of similar size. T-RFs should therefore be identified by sequencing, rather than by comparing the sizes of T-RFs to computer digests of database sequences. Physical capture T-RFLP should be a useful tool to identify T-RFs by sequencing, and for laboratories without economical access to equipment required to perform fluorescent T-RFLP.     

Principal component analysis and discriminant analysis (PCA-DA) for discriminating profiles of terminal restriction fragment length polymorphism (T-RFLP) in soil bacterial communities

To assess the impact of a transgenic crop on soil environment, we compared soil bacterial communities from the rhizospheres of cucumber green mottle mosaic virus (CGMMV)-resistant transgenic watermelon (Citrullus vulgaris [Twinser] cv. Gongdae) and non-transgenic parental line watermelon at an experimental farm in Miryang, Korea. Soil microbial community structure was studied using terminal restriction fragment length polymorphism (T-RFLP) using HaeIII and HhaI enzymes on products from polymerase chain amplification reactions (PCR) of total DNA from rhizosphere. We used principal component analyses (PCA) to reduce dimensionality of T-RFLP profiles before comparison. On these PCA scores, we conducted discrimination analyses to compare soil microbial communities from the rhizosphere of transgenic and non-transgenic. Discriminant analyses indicate that microbial communities from rhizosphere of transgenic and non-transgenic watermelon did not differ with significance at 95% level. Our study could be used as a model case to assess the environmental risk assessment of transgenic crops on soil microbial organisms.     

A phylogenetic analysis of the genus Carica L. (Caricaceae) based on restriction fragment length variation in a cpDNA intergenic spacer region

The phylogenetic relationships among twelve wild and cultivated species of Carica (Caricaceae) were analyzed using restriction fragment length variation in a 3.2-kb PCR amplified intergenic spacer region of the chloroplast DNA. A total of 138 fragments representing 137 restriction sites accounting for 5.8% of the amplified region were examined. Both parsimony and neighbor joining cluster analyses confirmed the close association among South American wild Carica species. However, cpDNA data did not support the traditional monophyly hypothesis for the evolution of Carica. Further, cpDNA analyses showed two basic evolutionary lineages within the genus Carica, one defined by cultivated C. papaya and another consisting of the remaining wild species from South America in a well resolved but poorly supported monophyletic assemblage. This evolutionary split in Carica strongly suggests that C. papaya diverged from the rest of the species early in the evolution of the genus and evolved in isolation, probably in Central America.     

Detection of land animal remains in fish meals by the polymerase chain reaction-restriction fragment length polymorphism technique

In the present study a technique was developed with the aim of guaranteeing the composition and security of fish meals, since it allows verification of whether these meals contain land animal remains. The method is based on polymerase chain reaction (PCR) and length polymorphism, followed by a restriction fragment length polymorphism (RFLP). Specific primers for every species were designed and calibrated, generating exclusively a PCR product with a specific size when DNA for each species was present in the sample. This technique allows the detection of land animal remains in fish meals, specifically cow, chicken, pig, horse, sheep, and goat. The identity of the PCR products can be confirmed by RFLP analysis using only one restriction enzyme. The selected restrictase generated one characteristic restriction profile for every species included in this study. The detection limit of this method was calculated by using mixtures of fish meals in different proportions and meal that exclusively contained remains of one of these land species studied. The analytical strategy herein proposed was applied to fish and meat meals, giving good results, both in the analyzed standards and in commercial samples.     

Ectomycorrhizal fungi identification in single and pooled root samples: terminal restriction fragment length polymorphism (TRFLP) and morphotyping compared

We describe here the results of a study conducted to evaluate a terminal restriction fragment length polymorphism (TRFLP) approach targeting rRNA genes for determination of ectomycorrhizal (ECM) communities colonizing the roots of loblolly pine (Pinus taeda L.). Root tips separated from soil cores were classified according to morphological characteristics and DNA was then extracted from each group of morphotyped tips. Labeled primers were used to generate terminal restriction fragments (TRF) for molecular fingerprinting of root colonizing fungi and to determine how well TRFLP could be used to discriminate between ectomycorrhizal types. Morphotypes generally correlated well with specific TRFs and sequence analysis confirmed that TRFs could be used to discriminate among fungal types. Sequence analysis indicated that important ECM fungi including Russulaceae, Thelephorales, and Tricholomataceae could be fingerprinted with TRFLP. In addition, a fixed proportion of the DNA extracted from each morphotype from the same core was used in a pooling experiment used to assess whether previously identified fungal species types could be distinguished from one another within reconstructed communities. Since some morphotypes share TRFs, dual analysis of ITS1 and ITS2 was necessary for accurate fingerprinting of fungal types. Approximately, 5.0±0.3 phylotypes were detected per core with TRFLP-corrected morphotyping as compared to 4.0±0.4 phylotypes using TRFLP on pooled community samples. TRFLP made on experimental sporocarp communities suggested that reduced ECM richness with TRFLP may be partly caused by differences in the amount of DNA available for PCR and primer bias. Nonetheless, TRFLP on pooled morphotypes accounted for more than 93% of colonized root tips. The method can be used to facilitate analysis of mycorrhizal communities using root tips collected from soil cores.     

Identification of species in animal feedstuffs by polymerase chain reaction--restriction fragment length polymorphism analysis of mitochondrial DNA

Restriction site analysis of Polymerase Chain Reaction (PCR) products of cytochrome b mitochondrial DNA was applied to identify species in meat meal and animal feedstuffs. PCR was used to amplify a variable region of cytochrome b mitochondrial DNA gene. Species differentiation was determined by digestion of the obtained 359 bp amplicon with restriction enzymes, which generated species-specific electrophoresis patterns; the sequencing of PCR products was used as confirming analysis. PCR-RFLP analysis revealed the presence of meat meal in animal feedstuffs and distinguished species of interest. The results supported the application of the method in control measures which should be adopted for meat-meal-based animal feed, as suggested by EU law. As a technical improvement, to simplify the analysis, the number of enzymes presented in this study for the detection of different species was smaller than others described in the literature; discrimination between ruminant and nonruminant species and between mammalian and poultry species was possible with few digestions.     

Community structure of the microbiota in the floodwater of a Japanese paddy field estimated by restriction fragment length polymorphism and denaturing gradient gel electrophoresis pattern analyses

The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and PCR-denaturing gradient gel electrophoresis (DGGE) patterns of 16S rDNA were studied to elucidate the effects of the type of fertilization and the growth stage of rice plants on the community structure of the microbiota in the floodwater of a Japanese paddy field under a long-term fertilizer trial. From the mid tillering stage, a higher pH and temperature were observed in the plot without fertilization (NoF plot) than in the plots supplied with chemical fertilizers (CF plot) and with compost (CM plot). DNA fragments specific to the respective plots and common to every plot were detected after the digestion of PCR products by restriction enzymes. Cluster analysis separated the RFLP and DGGE patterns of the microbiota in the floodwater into four clusters; the microbiota in (1) the NoF plot, (2) the CF plot, (3) the CM plot, and (4) the CF and CM plots in the early growing stage. The effect of fertilizer application on the community structure was more conspicuous than that of seasonal variation.     

A simplified procedure for terminal restriction fragment length polymorphism analysis of the soil bacterial community to study the effects of pesticides on the soil microflora using 4,6-dinitroorthocresol as a test case

We have studied the structural effects of application to the soil of a potentially detrimental herbicide, 4,6-dinitroorthocresol (DNOC) by analysing amplified ribosomal DNA restriction analysis (ARDRA) and terminal restriction fragment length polymorphism (T-RFLP) signatures of 16S rDNA fragments of culturable bacterial communities isolated from diluted soil suspensions. This approach has the potential to reveal changes induced by stressing the soil microflora with DNOC. This paper shows that, whereas only few changes of the ARDRA and T-RFLP profiles result from ageing of the soil, treatment of the soil with DNOC induces major modifications of these profiles. Therefore, for the practical purpose of pesticide registration, ARDRA and T-RFLP analysis performed on the dominant culturable fraction of the soil bacteria, implemented using conventional gel electrophoresis, offers the means of a routine, simple and meaningful test for detecting some of the changes affecting the structure of the soil microflora in response to pesticide application.     

玉米混合组织的EST文库的构建和部分EST序列的分析

以CLONTEECH的pDNR-LIB质粒为载体,构建了干旱和盐碱共胁迫48h和相应的无胁迫正常生长条件下耐旱玉米(ZeamaysL.)YQ7-96品系的雌雄分化期(12片叶龄)的混合组织(叶、茎和花器)EST文库。该文库容量>2×105个克隆,覆盖了预计的玉米基因组5万个基因的4倍以上。随机对3168个克隆中插入的cDNA进行测序,获得有效序列2098条,其中,2002个克隆中的cDNA进行了5'端测序,96个克隆中的cDNA的进行了5'和3'两端测序。结果表明,3'端测序的96个克隆的cDNA都含有polyA尾巴;进一步对这2098条组装分析,形成168个重叠群(Contig)和1693个单拷贝EST(Singlet),代表了共计1861条独立基因(Unigene);根据GenBank中的玉米EST数据库和BLASTn分析软件进行核苷酸同源性分析,1731条(93.01%)EST的score值大于或等于100,130条(6.99%)的EST的score值小于100,说明这130条属于新的表达基因的序列,有21条可以定位在玉米的不同染色体上;BLASTx分析表明,2098条EST中,1007条(48%)有功能注释信息,1091条(52%)无功能注释信息。该文库中的EST将会对公共数据库中现存的玉米EST数据起到补充作用。     

应用RT-PCR及RFLP技术对鸡传染性支气管炎病毒进行诊断与分型的研究进展

本文概述了近年来国内外应用ＲＴ－ＰＣＲ及ＲＦＬＰ技术对对外开放传染性支气管炎病毒（ＩＢＶ）进行诊断与分型的研究进展。介绍以ＩＢＶ通用引物的ＲＴ－ＰＣＲ进行诊断及其应用、型特异引物的ＲＴ－ＰＣＲ及其应用以及ＲＴ－ＰＣＲ结合ＲＦＬＰ分析技术进行分型的应用,而且还展望了这上结技术在临床诊断及分子流行病学研究上的应用前景。     

Molecular phylogenetic analysis of a novel strain from Neelipleona enriches Wolbachia diversity in soil biota

Eubacterial cellular endoparasites belonging to the genus Wolbachia (Rickettsiales) are extremely widespread symbionts of Arthropoda and Nematoda. Their ability to manipulate the reproductive behavior of the host is of particular importance to the fauna of the deep soil horizon, an environment in which parthenogenesis-inducing symbionts can play a crucial role in shaping population dynamics and speciation processes. In this study, three novel cases of infection in parthenogenetic Collembola (Parisotoma notabilis, Neelus murinus and Megalothorax minimus) are described. Sequences for molecular markers 16S rDNA and ftsZ were obtained for each species; their phylogenetic affinities with known Wolbachia supergroups were established using Bayesian inference. The analysis confirmed the presence of a Wolbachia strain belonging to the supergroup E, already reported from Folsomia candida and the Tullbergiidae, in the isotomid P. notabilis, while the Neelipleona M. minimus and N. murinus host a well differentiated strain which is phylogenetically distinct from supergroup E. Multiple events of Wolbachia infection in springtails as well as a richer diversity of the symbiont strains in soil arthropods were hereby confirmed.     

Assessment of genetic diversity, and phylogenetic relationships based on ribosomal DNA repeat unit length variation and Internal Transcribed Spacer (ITS) sequences in chickpea (Cicer arietinum) cultivars and its wild species

Repeat unit length variation and internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA were used to assess genetic diversity, and phylogenetic relationships in chickpea (C. arietinum) cultivars, and its related wild species. Total genomic DNAs of 76 accessions of 10 Cicer species, belonging to three sections of the genus, were restricted with seven enzymes and the restriction fragments were hybridized to heterologous ribosomal clones of wheat pTa71 and Vicia faba probes Ver 6-5 and Ver18-6. A single repeat unit length class of 11.4 kb or 10.5 kb was recognized across Cicer accessions with pTa71. The intraspecific variation was negligible in those species where more than one accession was studied, except the four C. judaicum accessions, which were different from the rest. EcoRI and DraI digests gave two and one-two fragments, respectively. All the accessions produced three and three-five bands with BamHI and SacI, respectively. Both the accessions of C. yamashitae differed in their rDNA repeat unit length as well as restriction site variation. Maximum likelihood tree with rDNA RFLP recognized five clades which were more or less congruent with the previous data. Length of ITS-1 region was more variable (235–239 bp) than the ITS-2 region (212–213 bp). Cladistic analysis of ITS data revealed two major clades, clade I consisting of C. arietinum, C. reticulatum and C. echinospermum, and clade II comprised of C. judaicum, C. chorassanicum, C. bijugum and C. cuneatum. C. microphyllum grouped with the above four species. C. pinnatifidum was present as a separate branch. C. yamashitae emerged as the most distinct species.     

Identification of anchovy (Engraulis encrasicholus L.) and gilt sardine (Sardinella aurita) by polymerase chain reaction, sequence of their mitochondrial cytochrome b gene, and restriction analysis of polymerase chain reaction products in semipreserves

A method of authenticating anchovy (Engraulis encrasicholus L.) and gilt sardine (Sardinella aurita) semipreserves (salt-cured and fillets in oil) has been developed by polymerase chain reaction (PCR) followed by sequence and restriction site analysis. The amplification of a fragment of the cytochrome b gene by universal primers produced a 376 base pairs (bp) fragment in all samples analyzed. Digestion of PCR products with XhoI, TaqI, AluI, and HinfI endonucleases yielded species-specific profiles distinguishing anchovy from gilt sardine. Therefore, the restriction length fragment polymorphism (RLFP) technique can be used to determine the species identity of anchovy and gilt sardine in semipreserves.     

Authentication of Panax ginseng and Panax quinquefolius using amplified fragment length polymorphism (AFLP) and directed amplification of minisatellite region DNA (DAMD)

AFLP profiles characteristic to Panax ginseng and Panax quinquefolius were generated using primers E-AGG/M-CAA. P. ginseng samples from different farms in China and Korea are homogeneous genetically [similarity index (SI) = 0.88-0.99], whereas samples of P. quinquefolius from different sources are much more heterogeneous (SI = 0.64-0.96). Detailed analysis of one of the polymorphic bands in P. ginseng led to the identification of a minisatellite Pg2, which contains eight repeats of 5'-AGGACTCATCACATTGTTACTC. The minisatellite DNA was consequently used in directed amplification minisatellite region DNA analysis to authenticate the two ginsengs.     

Establishment and application of a fluorescent polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for identifying porcine,caprine, and bovine meats

A method of fluorescent Polymerase Chain Reaction-restriction fragment length polymorphism (PCR-RFLP) was applied as an analytical and quantitative tool for meat identification. Following alignments of the nucleotide sequences, an oligonucleotide primer pair was designed to amplify the partial sequences within the 12S ribosomal RNA (12S rRNA) gene of mitochondrial DNA from porcine, caprine, and bovine meats. No fragment can be amplified from dog, cat, fish, duck, goose, turkey, and chicken DNA with the primer pair. Using fluorescence sensor capillary electrophoresis, the species-specific DNA fingerprints of pork, goat, and beef were generated by restriction enzyme digestion following a fluorescence-labeling PCR amplification. Species identification was conducted on the meat mixtures. The reliably semiquantitative levels were below 1% for binary mixtures of pork, goat, and beef. Cooking and autoclaving of meats did not influence the generation of the PCR-RFLP profiles or the analytical accuracy.     

Genetic diversity and species delimitation in the cultivated and wild Guinea yams (Dioscorea spp.) from Southwest Ethiopia as determined by AFLP (amplified fragment length polymorphism) markers

Yams (Dioscorea spp.) rank as the fourth most important root and tuber crop after potatoes, cassava and sweet potatoes. They are an economic crop in most of the tropics especially in West Africa, which produces over 95 % of the world output. Despite their cultural and economic importance there is taxonomic confusion regarding Guinea yams. The currently used classification scheme, which relies on vegetative and inflorescence characters, does not consistently delimit species boundaries between members of Guinea yams (D. cayenensis Lam.–D. rotundata Poir. complex), their wild relatives (D. abyssinica Kunth and D. praehensilis Benth.,) and D. sagittifolia Pax. Establishing the taxonomic identity of the germplasm and understanding the systematic relationships among crops is vital to the management of genetic resources and the utilization of accessions. In this study, amplified fragment length polymorphism (AFLP) genetic fingerprinting was used to evaluate and characterize 43 individual plants, belonging to different populations of wild and cultivated Guinea yams. The three primer combinations used in the AFLP analyses generated 158 scorable bands, with an overall polymorphism of 78 %. Ordination and cluster analyses of AFLP data failed to produce any clear species boundary between either the Guinea yam accessions or between them and their wild relatives. The average genetic similarity between the study individuals of Guinea yams and their wild relatives ranged from 60 to 100 %. The first, second and third principal coordinates axes cumulatively account for 77.45 % of the total variation. AFLP analyses also revealed a higher genetic divergence among cultivated Guinea yam accessions of the Sheko cultivars. Ordination and cluster analysis using UPGMA revealed no clear species boundaries between members of the complex. Thus, the taxonomy of these “species” needs to be revisited using other markers.