A complete sequence of the pGA1611 binary vector

We report the nucleotide sequence of the binary vector pGA1611, which is used for the transformation of foreign DNA into rice. This vector is 13,476 bp long. The 5577- bp T- DNA region consists of a 1987- bp ubiquitine promoter region, 45 bp for the multiple cloning site, a 253- bpnos terminator region, and the 2045- bpCaMV35S- hph- T7 chimaeric gene. The vector backbone (7004 bp) carriesoriT,traJ,trfA,tetA,tetR, andoriV. An 892- bp RB region and the 489- bp LB region are also present The T- DNA possesses 15 unique sites, six of which are at the multiple cloning site. This information will be valuable for cloning foreign DNA and modifying the vector.     

Plant DNA barcode library for native flowering plants in the arid region of northwestern China

DNA barcoding is a well-established tool for rapid species identification and biodiversity monitoring. A reliable and traceable DNA barcode reference library with extensive coverage is necessary but unavailable for many geographical regions. The arid region in northwestern China, a vast area of about 2.5 million km², is ecologically fragile and often overlooked in biodiversity studies. In particular, DNA barcode data from the arid region in China are lacking. We develop and evaluate the efficacy of an extensive DNA barcode library for native flowering plants in the arid region of northwestern China. Plant specimens were collected, identified and vouchered for this purpose. The database utilized four DNA barcode markers, namely rbcL, matK, ITS and ITS2, for 1816 accessions (representing 890 species from 385 genera and 72 families), and consisted of 5196 barcode sequences. Individual barcodes varied in resolution rates: species- and genus-level rates for rbcL, matK, ITS and ITS2 were 79.9%–51.1%/76.1%, 79.9%–67.2%/88.9%, 85.0%–72.0%/88.2% and 81.0%–67.4%/84.9%, respectively. The three-barcode combination of rbcL + matK + ITS (RMI) revealed a higher species- and genus-level resolution (75.5%/92.1%, respectively). A total of 110 plastomes were newly generated as super-barcodes to increase species resolution for seven species-rich genera, namely Astragalus, Caragana, Lactuca, Lappula, Lepidium, Silene and Zygophyllum. Plastomes revealed higher species resolution compared to standard DNA barcodes and their combination. We suggest future databases include super-barcodes, especially for species-rich and complex genera. The plant DNA barcode library in the current study provides a valuable resource for future biological investigations in the arid regions of China.     

Identification of Mytilus spp. and Pecten maximus in Irish Waters by Standard PCR of the 18S rDNA Gene and Multiplex PCR of the 16S rDNA Gene

Two molecular protocols for the identification of mussel and scallop have been developed using specific primers targeting the mitochondrial 16S ribosomal DNA gene and the nuclear 18S ribosomal DNA gene. Primers for the mitochondrial 16S ribosomal DNA gene in multiplex polymerase chain reaction (PCR) protocols yielded diagnostic DNA fragments for the mussels Mytilus edulis, Mytilus galloprovincialis, and the hybrid Mytilus edulis/galloprovincialis (335 bp), the king scallop Pecten maximus (382 bp) and the black scallop Mimachlamys varia (398 bp). DNA from the queen scallop Aequipecten opercularis showed no consistent PCR amplification of the 16S rDNA gene. Primers for the nuclear 18S rDNA gene in standard PCR protocols yielded similar-sized, diagnostic DNA fragments (approx. 190 bp) for the mussels Mytilus edulis, Mytilus galloprovincialis, and the hybrid Mytilus edulis/galloprovincialis, the king scallop Pecten maximus, the black scallop Mimachlamys varia, and the queen scallop Aequipecten opercularis. Both protocols have been tested with Mytilus spp., P. maximus, and 6 other bivalve species from a wide range of locations in Irish and European waters. Cross reaction of the specific primers with DNA template from any of the 6 other bivalve species was not observed. Rapid DNA extraction using FTA Card technology and the16S rDNA primers allowed for the detection of at least 10 mussel larvae in a subsample of natural plankton.     

Development of a single base extension-tag microarray for the detection of pathogenic Vibrio species in seafood

In this study, a single base extension-tag array on glass slides (SBE-TAGS) microarray was established to detect the seven leading seafood-borne pathogens, including Vibrio parahaemolyticus, Vibrio cholerae, Vibrio vulnificus, Vibrio mimicus, Vibrio alginolyticus, Vibrio anguillarum, and Vibrio harveyi. Three multiplex PCR assays were developed to specifically target the following species with individual gene markers, which are aadS, tdh, and trh for V. parahaemolyticus; col, toxR, and vvh for V. alginolyticus, V. mimicus, and V. vulnificus; and empA, vhh1, and tcpA for V. anguillarum, V. harveyi, and V. cholerae, respectively. The purified PCR products were used as template DNA for single base extension-tag reactions, labeled with Cy3 fluorescent dye and hybridized to DNA microarrays. The detection specificity of this microarray method was 100%, with the sensitivity for pure genomic DNA at 200 fg to 2 pg per reaction. Application of the DNA microarray methodology to 55 naturally contaminated seafood samples (shrimp, fish, and oysters) revealed the presence of V. parahaemolyticus at 50.9% and V. alginolyticus at 32.7%. This corresponds with traditional assays (microbiological and biochemical tests) except one sample which was identified as negative in V. parahaemolyticus by the microarray assay but as positive by the conventional method. Therefore, a combination of multiplex PCR with DNA microarray hybridization based on SBE-TAGS ensures rapid and accurate detection of pathogenic Vibrio species in seafood, thereby providing safer seafood products for consumers at a low financial burden to the aquaculture industry.     

Mapped DNA probes from loblolly pine can be used for restriction fragment length polymorphism mapping in other conifers

A high-density genetic map based on restriction fragment length polymorphisms (RFLPs) is being constructed for loblolly pine (Pinus taeda L.). Consequently, a large number of DNA probes from loblolly pine are potentially available for use in other species. We have used some of these DNA probes to detect RFLPs in 12 conifers and an angiosperm. Thirty complementary DNA and two genomic DNA probes from loblolly pine were hybridized to Southern blots containing DNA from five species of Pinus (P. elliottii, P. lambertiana, P. radiata, P. sylvestris, and P. taeda), one species from each of four other genera of Pinaceae (Abies concolor, Larix laricina, Picea abies, and Pseudotsuga menziesii), one species from each of three other families of Coniferales [Sequoia sempervirens (Taxodiaceae), Torreya californica (Taxaceae) and Calocedrus decurrens (Cupressaceae)], and to one angiosperm species (Populus nigra). Results showed that mapped DNA probes from lobolly pine will cross-hybridize to genomic DNA of other species of Pinus and some other genera of the Pinaceae. Only a small proportion of the probes hybridized to genomic DNA from three other families of the Coniferales and the one angiosperm examined. This study demonstrates that mapped DNA probes from loblolly pine can be used to construct RFLP maps for related species, thus enabling the opportunity for comparative genome mapping in conifers.     

A study of DNA denaturation in the ultracentrifuge

The melting transition of DNA in alkaline CsCl can be followed in the analytical ultracentrifuge. Equilibrium partially denatured states can be observed. These partially denatured DNA bands have bandwidths of up to several times those of native DNA. Less stable molecules melt early and are found at heavier densities in the melting region. An idealized ultracentrifuge melting transition is described. The melting transition of singly nicked PM-2 DNA resembles the idealized curve. The DNA profile is a Gaussian band at all points in the melt. DNA's from mouse, D. Melanogaster, M. lysodeikticus, T4, and T7 also show equilibrium bands at partially denatured densities, some of which are highly asymmetric. Simple sequence satellite DNA shows an all-or-none transition with no equilibrium bands at partially denatured densities. The temperature at which a DNA denatures is an increasing function of the (G + C) content of the DNA. The T_m does not show a molecular-weight dependence in the range 1.2 × 10⁶–1.5 × 10⁷ daltons (single strand) for mouse, M. lysodeikticus, or T4 DNA. The mouse DNA partially denatured bands do not change shape as a function of molecular weight. The T4 DNA intermediate band develops a late-melting tail at low molecular weight. M. lysodeikticus DNA bands at partially denatured densities become broader as the molecular weight is decreased. Mouse DNA is resolved into six Gaussian components at each point in the melting transition.     

G2 / M checkpoint genes of Saccharomyces cerevisiae : further evidence for roles in DNA replication and / or repair

We have cloned, sequenced and disrupted the checkpoint genes RAD17, RAD24 and MEC3 of Saccharomyces cerevisiae. Mec3p shows no strong similarity to other proteins currently in the database. Rad17p is similar to Rec1 from Ustilago maydis, a 3′ to 5′ DNA exonuclease/checkpoint protein, and the checkpoint protein Rad1p from Schizosaccharomyces pombe (as we previously reported). Rad24p shows sequence similarity to replication factor C (RFC) subunits, and the S. pombe Rad17p checkpoint protein, suggesting it has a role in DNA replication and/or repair. This hypothesis is supported by our genetic experiments which show that overexpression of RAD24 strongly reduces the growth rate of yeast strains that are defective in the DNA replication/repair proteins Rfc1p (cdc44), DNA polα (cdc17) and DNA polδ (cdc2) but has much weaker effects on cdc6, cdc9, cdc15 and CDC ⁺ strains. The idea that RAD24 overexpression induces DNA damage, perhaps by interfering with replication/repair complexes, is further supported by our observation that RAD24 overexpression increases mitotic chromosome recombination in CDC ⁺ strains. Although RAD17, RAD24 and MEC3 are not required for cell cycle arrest when S phase is inhibited by hydroxyurea (HU), they do contribute to the viability of yeast cells grown in the presence of HU, possibly because they are required for the repair of HU-induced DNA damage. In addition, all three are required for the rapid death of cdc13 rad9 mutants. All our data are consistent with models in which RAD17, RAD24 and MEC3 are coordinately required for the activity of one or more DNA repair pathways that link DNA damage to cell cycle arrest. Received: 8 April 1997 / Accepted: 10 May 1997     

Patterns of genome size in the copepoda

Adult somatic nuclear DNA contents are reported for eleven cyclopoid species (Megacyclops latipes, Mesocyclops edax, M. longisetus, M. ruttneri, M. leuckarti, M. woutersi, Macrocyclops albidus, Cyclops strenuus, Acanthocyclops robustus, Diothona oculata, Thermocyclops crassus) and for the harpacticoid Tigriopus californicus and range from 0.50 to 4.1 pg DNA per nucleus. These diploid genome sizes are consistent with previously published values for four Cyclops species (0.28–1.8 pg DNA per nucleus), but are strikingly smaller than those reported for marine calanoids (4.32–24.92 pg DNA per nucleus). We discuss three explanations, none of them exclusive of another, to account for the smaller size and range of cyclopoid genome sizes relative to calanoid genome sizes: (1) higher prevalence of chromatin diminution in the Cyclopoida, (2) phylogenetic structure or older age of the Calanoida relative to Cyclopoida and (3) nucleotypic selection that may influence life history variation and fitness. Measurements of genome size were made on Feulgen stained, somatic cell nuclei, using scanning microdensitometry which is well suited to the sparse and heterogeneous populations of copepod nuclei. The importance of measuring large numbers of nuclei per specimen, possible sources of variation associated with cytophotometric measurements, and appropriate use of internal reference standards and stoichiometry of the Feulgen stained nuclei are discussed.     

Species discrimination in Sisyrinchium (Iridaceae): assessment of DNA barcodes in a taxonomically challenging genus

DNA barcoding aims to develop an efficient tool for species identification based on short and standardized DNA sequences. In this study, the DNA barcode paradigm was tested among the genera of the tribe Sisyrinchieae (Iridoideae). Sisyrinchium, with more than 77% of the species richness in the tribe, is a taxonomically complex genus. A total of 185 samples belonging to 98 species of Sisyrinchium, Olsynium, Orthrosanthus and Solenomelus were tested using matK, trnH‐psbA and internal transcribed spacer (ITS). Candidate DNA barcodes were analysed either as single markers or in combination. Detection of a barcoding gap, similarity‐based methods and tree‐based analyses were used to assess the discrimination efficiency of DNA barcodes. The levels of species identification obtained from plastid barcodes were low and ranged from 17.35% to 20.41% for matK and 5.11% to 7.14% for trnH‐psbA. The ITS provided better results with 30.61–38.78% of species identified. The analyses of the combined data sets did not result in a significant improvement in the discrimination rate. Among the tree‐based methods, the best taxonomic resolution was obtained with Bayesian inference, particularly when the three data sets were combined. The study illustrates the difficulties for DNA barcoding to identify species in evolutionary complex lineages. Plastid markers are not recommended for barcoding Sisyrinchium due to the low discrimination power observed. ITS gave better results and may be used as a starting point for species identification.     

Sex-specific methylation in <Emphasis Type="Italic">Drosophila</Emphasis>: an investigation of the <Emphasis Type="Italic">Sophophora</Emphasis> subgenus

Epigenetic phenomena have been widely characterized in the genomes of vertebrates and DNA methylation is a key mechanism of epigenetic regulation. The DNA methylation systems of invertebrates and vertebrates show several notable differences. However, the evolutionary implications of those differences only recently began to be revealed. Our study investigated the recurrence of sex-specific methylation, as previously described for the species Drosophila willistoni, in other species of the Sophophora subgenus that present close evolutionary relationship. The MSRE and Southern blot techniques were used to analyze rDNA of some species of the willistoni, melanogaster, saltans and obscura groups of Drosophila and the results suggested that differential DNA methylation between sexes only occurs in Drosophila tropicalis and D. insularis, two sibling species of the willistoni subgroup. However, only using the MSRE technique we could detect sex-specific patterns of DNA methylation in all species of willistoni subgroup. These results indicate that DNA methylation may present important differences, even between closely related species, shedding new light on this Neotropical species complex.     

Difference Analysis of ClCYC2-Like Genes Expression and DNA Methylation Between the Two Types of Florets in Chrysanthemum lavandulifolium

DNA methylation is an important epigenetic modification, that is involved in the regulation of gene expression and cell differentiation, and plays an important regulatory role in flower development in higher plants. There are two types of florets on the capitulum in the genus Chrysanthemum, the flower symmetry factor CYCLOIDEA (CYC) 2-like genes may be important candidate genes for determining the identity of the two types of florets. In this study, the diploid plant Chrysanthemum lavandulifolium was used as the research material, and qRT-PCR and bisulfite sequencing polymerase chain reaction (BSP) were used to identify the expression and DNA methylation pattern of CYC2-like genes in the two types of florets. Gene expression analysis showed that the six ClCYC2-like genes were significantly different in the two types of florets, and the expression levels of ClCYC2c, ClCYC2d, ClCYC2e and ClCYC2f in the ray florets were significantly higher than those in the disc florets. For the DNA methylation analysis of the three genes ClCYC2c, ClCYC2d, and ClCYC2e, it was found that the DNA methylation levels of these three genes were negative correlated with their expression levels, and the ways in which the three genes were regulated by the DNA methylation were different. It is speculated that the different DNA methylation of ClCYC2-like genes in the two types of florets may affect the differentiation and development of the two types of florets. This study provides new clues about epigenetics for the analysis of capitulum formation in Asteraceae.

     

Cloning and characterization of chloroplast ribosomal protein-encoding genes, rpl16 and rps3, of the marine macro-algae, Gracilaria tenuistipitata

In Gracilaria tenuistipitata, a highly differentiated multicellular member of the marine red algae, Rhodophyta, chloroplast (cp) DNA can be separated as a satellite band from the nuclear DNA in a CsCl gradient. Using a heterologous probe from Chlamydomonas, the ribosomal protein-encoding gene, rpl16, was located on a 4.5-kb EcoRI fragment of cp DNA. The fragment was cloned and a 1365-bp region around rpl16 was sequenced. The gene order around rpl16, 5′ rpl22-rps3-rpl16, is identical to that detected in the chloroplast DNA of liverwort, tobacco and maize. Both the nucleotide sequence and the amino-acid sequence of rpl16 are more conserved than that of rps3. The rpl16 gene contains no intron, a feature which shows more similarity to the unicellular green algae, Chlamydomonas, than the other land plants. Sequences that may form a stable stem-loop structure were detected within the coding sequence of rpl16.     

Agrobacterium T‐DNA integration into the plant genome can occur without the activity of key non‐homologous end‐joining proteins

Non‐homologous end joining (NHEJ) is the major model proposed for Agrobacterium T‐DNA integration into the plant genome. In animal cells, several proteins, including KU70, KU80, ARTEMIS, DNA‐PKcs, DNA ligase IV (LIG4), Ataxia telangiectasia mutated (ATM), and ATM‐ and Rad3‐related (ATR), play an important role in ‘classical’ (c)NHEJ. Other proteins, including histone H1 (HON1), XRCC1, and PARP1, participate in a ‘backup’ (b)NHEJ process. We examined transient and stable transformation frequencies of Arabidopsis thaliana roots mutant for numerous NHEJ and other related genes. Mutants of KU70, KU80, and the plant‐specific DNA LIGASE VI (LIG6) showed increased stable transformation susceptibility. However, these mutants showed transient transformation susceptibility similar to that of wild‐type plants, suggesting enhanced T‐DNA integration in these mutants. These results were confirmed using a promoter‐trap transformation vector that requires T‐DNA integration into the plant genome to activate a promoterless gusA (uidA) gene, by virus‐induced gene silencing (VIGS) of Nicotiana benthamiana NHEJ genes, and by biochemical assays for T‐DNA integration. No alteration in transient or stable transformation frequencies was detected with atm, atr, lig4, xrcc1, or parp1 mutants. However, mutation of parp1 caused high levels of T‐DNA integration and transgene methylation. A double mutant (ku80/parp1), knocking out components of both NHEJ pathways, did not show any decrease in stable transformation or T‐DNA integration. Thus, T‐DNA integration does not require known NHEJ proteins, suggesting an alternative route for integration.     

Identification of RAPD markers for 11 Hessian fly resistance genes in wheat

 The pyramiding of genes that confer race- or biotype-specific resistance has become increasingly attractive as a breeding strategy now that DNA-based marker-assisted selection is feasible. Our objective here was to identify DNA markers closely linked to genes in wheat (Triticum aestivum L.) that condition resistance to Hessian fly [Mayetiola destructor (Say)]. We used a set of near-isogenic wheat lines, each carrying a resistance gene at 1 of 11 loci (H3, H5, H6, H9, H10, H11, H12, H13, H14, H16 or H17) and developed by backcrossing to the Hessian fly-susceptible wheat cultivar ‘Newton’. Using genomic DNA of these 11 lines and ‘Newton’, we have identified 18 randomly amplified polymorphic DNA (RAPD) markers linked to the 11 resistance genes. Seven of these markers were identified by denaturing gradient gel electrophoresis and the others by agarose gel electrophoresis. We confirmed linkage to the Hessian fly resistance loci by cosegregation analysis in F₂ populations of 50–120 plants for each different gene. Several of the DNA markers were used to determine the presence/absence of specific Hessian fly resistance genes in resistant wheat lines that have 1 or possibly multiple genes for resistance. The use of RAPD markers presents a valuable strategy for selection of single and combined Hessian fly resistance genes in wheat improvement. Received: 20 March 1996 / Accepted: 6 September 1996     

Heavy metal ions affect the activity of DNA glycosylases of the Fpg family

Prokaryotic enzymes formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease VIII (Nei) and their eukaryotic homologs NEIL1, NEIL2, and NEIL3 define the Fpg family of DNA glycosylases, which initiate the process of repair of oxidized DNA bases. The repair of oxidative DNA lesions is known to be impaired in vivo in the presence of ions of some heavy metals. We have studied the effect of salts of several alkaline earth and transition metals on the activity of Fpg-family DNA glycosylases in the reaction of excision of 5,6-dihydrouracil, a typical DNA oxidation product. The reaction catalyzed by NEIL1 was characterized by values K _m = 150 nM and k _cat = 1.2 min⁻¹, which were in the range of these constants for excision of other damaged bases by this enzyme. NEIL1 was inhibited by Al³⁺, Ni²⁺, Co²⁺, Cd²⁺, Cu²⁺, Zn²⁺, and Fe²⁺ in Tris-HCl buffer and by Cd²⁺, Zn²⁺, Cu²⁺, and Fe²⁺ in potassium phosphate buffer. Fpg and Nei, the prokaryotic homologs of NEIL1, were inhibited by the same metal ions as NEIL1. The values of I₅₀ for NEIL1 inhibition were 7 μM for Cd²⁺, 16 μM for Zn²⁺, and 400 μM for Cu²⁺. The inhibition of NEIL1 by Cd²⁺, Zn²⁺, and Cu²⁺ was at least partly due to the formation of metal-DNA complexes. In the case of Cd²⁺ and Cu²⁺, which preferentially bind to DNA bases rather than phosphates, the presence of metal ions caused the enzyme to lose the ability for preferential binding to damaged DNA. Therefore, the inhibition of NEIL1 activity in removal of oxidative lesions by heavy metal ions may be a reason for their comutagenicity under oxidative stress.     

Molecular screening of Leishmania spp. infection and bloodmeals in sandflies from a leishmaniasis focus in southwestern Turkey

Leishmaniasis is an arthropod‐borne disease that affects approximately 2 million people worldwide annually. The aims of this study were to detect the presence of Leishmania (Kinetoplastida: Trypanosomatidae) DNA and the feeding preferences of probable vector species in an endemic focus of Leishmania infantum in Turkey. Entomological sampling was performed in August and October 2015 in Ayd?n province, where cases of human and canine leishmaniasis have been reported previously. A total of 1059 sandfly specimens comprising nine species belonging to two genera, Phlebotomus and Sergentomyia (both: Diptera: Psychodidae), and five subgenera of the Phlebotomus genus (Phlebotomus, Paraphlebotomus, Larroussius, Adlerius and Transphlebotomus) were collected in five villages. Among all Phlebotomus specimens, Phlebotomus neglectus (39%) was noted as the most abundant species, followed by Phlebotomus tobbi (18%). Leishmania DNA was detected in pools from P. neglectus, P. tobbi and Sergentomyia dentata by kDNA polymerase chain reaction (PCR). Leishmania DNA from Phlebotomus specimens was identified as L. infantum, but Leishmania DNA from Sergentomyia spp. could not be identified to species level by ITS‐1 real‐time PCR. The detection of Leishmania DNA in wild‐caught P. neglectus and the high percentage (24.2%) of human DNA in engorged specimens suggests that P. neglectus is probably an important vector species for L. infantum in Ayd?n province.     

Genome-wide DNA methylation studies suggest distinct DNA methylation patterns in pediatric embryonal and alveolar rhabdomyosarcomas

Rhabdomyosarcoma is the most common soft-tissue sarcoma in children. While cytogenetic abnormalities have been well characterized in this disease, aberrant epigenetic events such as DNA hypermethylation have not been described in genome-wide studies. We have analyzed the methylation status of 25,500 promoters in normal skeletal muscle, and in cell lines and tumor samples of embryonal and alveolar rhabdomyosarcoma from pediatric patients. We identified over 1,900 CpG islands that are hypermethylated in rhabdomyosarcomas relative to skeletal muscle. Genes involved in tissue development, differentiation, and oncogenesis such as DNAJA4, HES5, IRX1, BMP8A, GATA4, GATA6, ALX3, and P4HTM were hypermethylated in both RMS cell lines and primary samples, implicating aberrant DNA methylation in the pathogenesis of rhabdomyosarcoma. Furthermore, cluster analysis revealed embryonal and alveolar subtypes had distinct DNA methylation patterns, with the alveolar subtype being enriched in DNA hypermethylation of polycomb target genes. These results suggest that DNA methylation signatures may aid in the diagnosis and risk stratification of pediatric rhabdomyosarcoma and help identify new targets for therapy.     

Detection of nptII (kanamycin resistance) genes in genomes of transgenic plants by marker-rescue transformation

We have developed a novel system for the sensitive detection of nptII genes (kanamycin resistance determinants) including those present in transgenic plant genomes. The assay is based on the recombinational repair of an nptII gene with an internal 10-bp deletion located on a plasmid downstream of a bacterial promoter. Uptake of an nptII gene by transformation restores kanamycin resistance. In Escherichia coli, promoterless nptII genes provided by electroporation were rescued with high efficiency in a RecA-dependent recombinational process. For the rescue of nptII genes present in chromosomal plant DNA, the system was adapted to natural transformation, which favours the uptake of linear DNA. When competent Acinetobacter sp. BD413 (formerly A. calcoaceticus) cells containing the mutant nptII gene on a plasmid were transformed with DNA from various transgenic plants carrying nptII as a marker gene (Solanum tuberosum, Nicotiana tabacum, Beta vulgaris, Brassica napus, Lycopersicon esculentum), kanamycin-resistant transformants were obtained roughly in proportion to the concentration of nptII genes in the plant DNA. The rescue of nptII genes occurred in the presence of a more than 6 × 10⁶-fold excess of plant DNA. Only 18 ng of potato DNA (2.5 × 10³ genome equivalents, each with one copy of nptII) was required to produce one kanamycin-resistant transformant. These experiments and others employing DNA isolated from soil samples demonstrate that the system allows reliable and highly sensitive monitoring of nptII genes in transgenic plant DNA and in DNA from environmental sources, such as soil, without the need for prior DNA amplification (e.g. by PCR). Received: 20 May 1997 / Accepted: 17 October 1997