Greenhouse cage studies were conducted to determine the influence of shoot morphology and genetic variation on establishment of Spurgia esulae gall midge on seven leafy spurge genotypes. The genotypes were collected from South Dakota, North Dakota, Nebraska, Montana, Wyoming, Manitoba, and Austria. Genotypes from South Dakota and Nebraska were most susceptible to gall formation and had the highest larvae survival, while the genotypes from Montana and Manitoba were most resistant. Morphological characteristics of the leafy spurge stem tips, such as stem diameter, leaf length, width, and area did not correlate with gall formation or larvae survival. Chloroplast DNA restriction fragment length polymorphism analysis of the genotypes identified six chloroplast types among the seven leafy spurge genotypes. The two genotypes most resistant to galling by S. esulae, Manitoba and Montana, had the same chloroplast genotype, but also were closely related to the two most susceptible genotypes. Because eggs were laid on all genotypes, it appears that adult females were not preferentially selecting appropriate host genotypes, but that egg and larvae survival was strongly influenced by genotype.

The DNA sequence of a 196 base pair (bp) region of the acetolactate synthase (ALS) genes of three weed species, kochia, prickly lettuce, and Russian thistle, was determined. This region encompasses the coding sequence for Domain A, a region of the amino acid sequence previously demonstrated to play a pivotal role in conferring resistance to herbicides that inhibit ALS. The Domain A DNA sequence from a chlorsulfuron-resistant (R) prickly lettuce biotype from Idaho differed from that of a chlorsulfuron-susceptible (S) biotype by a single point mutation, which substituted a histidine for a proline. The Domain A DNA sequence from an R kochia biotype from Kansas also differed from that of an S biotype by a single point mutation in the same proline codon. This point mutation, however, conferred substitution of threonine for proline. Two different ALS-homologous sequences were isolated from an R biotype of Russian thistle. Neither sequence encoded amino acid substitutions in Domain A that differed from the consensus S sequence. The DNA sequence variation among the R and S kochia biotypes was used to characterize six Ada County, Idaho, kochia collections for correlation between phenotypic chlorsulfuron susceptibility and restriction digest patterns (RFLPs) of polymerase chain reaction amplification products. Most collections showed excellent correspondence between the RFLP patterns and the phenotypic response to chlorsulfuron application. However, one entirely R collection had the RFLP pattern of the S biotype, suggesting that resistance was not due to mutation in the proline codon.

Goosegrass, wide spread throughout the tropics and subtropics, is one of the most noxious weeds known. Recently, biotypes of goosegrass have been found resistant to the dinitroaniline herbicides. An alteration in the structure/composition of a tubulin protein has been postulated as an explanation for the hyperstable microtubules and the resistant phenotype. Our study was initiated to investigate the structure of the alpha (α)-, beta (β)- and gamma (γ)-tubulin related gene sequences in resistant, intermediately resistant, and susceptible biotypes. Heterologous tubulin gene clones were used as probes of restriction endonuclease-digested genomic DNA from each biotype, to determine gene size and copy number and to screen for restriction fragment length polymorphisms. The tubulin genes are organized into gene families. There are three to five α-tubulin genes, four to seven β-tubulin genes, and four to eight γ-tubulin genes. There was no evidence of multiple copies or tandem repeats of any individual gene sequence. Although RFLPs were observed, no significant difference in the banding pattern between the resistant and the susceptible biotypes was found for either α-, β-, or γ-tubulin gene families. Therefore, it is unlikely that the difference between the herbicide-response phenotypes can be attributed to large deletions or insertions in a tubulin gene.

Technological advances in molecular biology have contributed substantially to our understanding of plant genetic diversity. Early studies of allozyme variation employing protein electrophoresis revealed that plant populations have high levels of genetic diversity, most of the variation at polymorphic loci is found within populations, and geographic range and breeding system explain the largest proportion of variation in genetic diversity. With the discovery of restriction endonucleases, the first DNA-based markers allowed the detection of variation in DNA sequences in plant population studies. More recently, techniques that utilize the polymerase chain reaction have allowed a more representative assessment of genetic variation in plants by screening multiple loci distributed throughout the genome. The analyses reveal sufficient polymorphism for the examination of fine-scale genetic differences among individuals. Information on plant genetic diversity is also emerging from studies of plant genome structure. Comparative genetic mapping studies of members of the Brassicaceae, Poaceae, and Solanaceae show that gene content is highly conserved between closely related species, although gene order on a chromosomal segment may differ between species. Comparative sequencing studies reveal higher degrees of diversity at the microstructural (less than 1 million base pairs) level than predicted at the genetic map level and suggest that genes are densely packed in gene-rich regions, rather than randomly distributed along chromosomes in species with large genomes. Sequencing of the entire genomes of rice and Arabidopsis thaliana will help identify genes controlling agronomically important traits, improve our understanding of genetic variation for fitness-related traits in wild plant populations including weed species, resolve evolutionary relationships among plant taxa, and potentially revolutionize current ideas on plant diversity and evolution.

Planthoppers are important worldwide crop pests as well as vectors of numerous diseases. Different species transmit Mal de Río Cuarto virus, which causes the most economically important corn disease in central Argentina. Epidemiological studies rely on the accurate identification of the species present in the field. Presently, morphological identification of planthoppers requires taxonomic expertise and there are no taxonomic keys for females and nymphs. Nevertheless, no molecular protocols are available for accurate species identification of most frequent delphacid species from central Argentina. In this context, the aim of this study was to evaluate the utility of the cytochrome oxidase I gene (COI) as a DNA barcode and its digestion with restriction enzymes (Restriction Fragment Length Polymorphism, RFLP) for the identification of the most common species of planthoppers in central Argentina. We amplified and sequenced a 843 bp fragment of the COI gene of taxonomically identified specimens and evaluated its use as a DNA barcode. Restriction enzymes were also selected for digesting the COI fragment via RFLP. The high interspecific variability (20.79%; ± 2.32%) and low intraspecific divergence (0.12%; ± 0.17%) observed in the studied species, demonstrate the effectiveness of the COI gene for species identification of major vector delphacids affecting corn crops in Argentina. Moreover, the digestion of this COI gene fragment with Bfa I and Apo I enzymes allows a fast and cost-effective species identification method when numerous specimens need to be processed. Both molecular techniques developed here, allow the accurate identification of planthopper species at regional scale. These new tools would assist traditional identification of these insects, especially for aiding non-experts in morphological taxonomy.

Aimed at assessing the circulation of Mycobacterium tuberculosis in a highly endemic prison, this 13-month prospective study was performed on strains isolated from tuberculosis (TB) cases detected passively and actively. We used X-ray screening of newly admitted inmates and mass screening at the beginning of the study and again 1 year later. Of the 94 strains genotyped by restriction fragment-length polymorphism, 79 (84·0%) belonged to one of the 12 identified clusters (2–21 strains each), including two main clusters (18 and 21 cases, respectively). A history of TB treatment was reported in 22/79 (27·8%) clustered cases. Time–space distribution of clustered cases was predominantly consistent with transmission, in micro-epidemics. Given the dominant pattern of exogenous infection and the extensive strain circulation, effective TB control should emphasize reduction of overcrowding and improvement of environmental measures as a complement to detection and treatment of cases.

A DNA probe PCsv4 and a subclone thereof PCsv4.1, hybridize specifically to rodent malaria DNA. DNA purified from a small volume (10 μ1) of infected mouse blood was used to determine the composition of the parasite population present. The hybridization signal following PCsv4 probing of slot-blotted DNA correlated directly with parasitaemia. The hybridization pattern and Intensity, resulting from probing restriction enzyme digested and Southern-blotted genomic DNA, determined the identity of the infecting parasite line(s), and provided a semi-quantitative measure of parasite burden. Fifteen parasite lines representative of all four Plasmodium species infecting rodents can be differentiated in this way.

The entire ribosomal DNA repeat unit of a steinernematid species (Nashes isolate) was cloned as three separate EcoR I fragments in the plasmid pUC18. An equimolar cocktail of these three clones was used to identify Steinernema species on Southern blots as each species displays its own unique restriction fragment length polymorphisms. The clones also identified two new species isolated in a soil survey of coastal regions of Britain. One of the clones (pSn4.0) can detect length heterogeneities in the rDNA repeat unit of various isolates of some of the species, particularly the most common in the United Kingdom, S. feltiae. These differences in the rDNA repeat unit length remained constant over several years for one isolate of S. feltiae, but were different for each of the geographical isolates studied to date.

Trypanosoma (Nannomonas) congolense comprises morphologically identical but genetically heterogeneous parasites infective to livestock and other mammalian hosts; three different genotypes of this parasite have been described previously. Restriction enzyme fragment length polymorphisms (RFLPs) in both kinetoplast DNA minicircle and nuclear DNA sequences, and randomly amplified polymorphic deoxyribonucleic acid (RAPD) patterns have been used here to demonstrate the existence of another type of T. (N.) congolense that is genotypically distinct from those that have so far been characterized at the molecular level. A highly repetitive, tandemly arranged DNA sequence and oligonucleotide primers, for use in polymerase chain reaction (PCR) amplification are described, which can be used for specific identification of the trypanosome and its distinction from others within the Nannomonas subgenus.

The small subunit ribosomal RNA (srRNA) gene was amplified from Gyrodactylus salaris using the polymerase chain reaction (PCR), cloned, and the complete gene sequence of 1966 bp determined. The V4 region of the srRNA gene was identified and amplified from single specimens of G. salaris, G. derjavini and G. truttae. Comparison of the V4 sequences from these three species revealed sequence differences from which restriction fragment length polymorphisms (RFLPs) were predicted and an oligonucleotide probe (GsV4) specific to G. salaris designed. Digestion of the amplified V4 region of the srRNA gene with Hae III and either Alw I, BstY I, Dde I or Mbo I provided a means of discriminating between G. salaris, G. derjavini and G. truttae. The GsV4 probe was used to detect the srRNA gene from G. salaris in Southern and dot blots of the amplified V4 region. The nucleotide sequences reported in this paper have been submitted to the EMBL Data Library under accession numbers Z26942 (G. salaris), Z35128 (G. derjavini) and Z35129 (G. truttae).

Since 1993, strains of Entamoeba histolytica sensn lato have been assigned to 2 species on the basis of clinical, biochemical, immunological and genetic evidence: the pathogenic strains to E. histolytica sensu stricto, the non-pathogenic strains to Entamoeba dispar. Analysis of the gene encoding for the small subunit ribosomal RNA (SSU rDNA) supports the existence of 2 species. However, while 3 whole SSU rDNA sequences are available in the data bases for E. histolytica, only a partial sequence has been published for E. dispar. Here we report a SSU rDNA sequence for E. dispar. Compared to those of E. histolytica, this sequence shows 1·7 % nucleotide substitutions. On the basis of our rDNA data, 2 primers were designed to produce polymerase chain reaction (PCR) amplification from both E. histolytica and E. dispar. Primer specificity for the 2 amoebae was assessed both theoretically against the data bases, and experimentally against a collection of eukaryotic and prokaryotic DNAs. The amplified stretch encompasses a polymorphic Dde I restriction site which allows, after cleavage of the fragment, E. histolytica and E. dispar to be distinguished. The reliability of this method of identification was assessed comparing the results with those based on classic isoenzyme analysis.

A high level of genetic purity in crop varieties must be achieved and maintained for agronomic performance as well as to encourage investment and innovation in plant breeding and to ensure that the improvements in productivity and quality imparted by breeders are delivered to the farmer and, ultimately, to the consumer. Traditionally, morphological comparisons have formed the basis for genetic purity evaluations. However, replicated field observations are time-consuming, expensive and unreliable. Morphology cannot provide information on the purity of specific genetic attributes that relate to grain quality or to pest or herbicide resistance bred into varieties. Biochemical assays, including isozymes, can distinguish varieties within several species. Isozymes have been routinely used in checking seed-lot purity in maize (Zea mays L.) for the past 20 years. Newer DNA-based technologies such as restriction fragment length polymorphisms and more recently developed methods that use the polymerase chain reaction can allow even more discriminative and faster identification of varieties. However, none of the DNA methods have replaced biochemical methods for seed purity assays, other than in a relatively select group of crops with very high seed value, due to their high datapoint cost. It will require further miniaturization, automation and enhanced capabilities to process numerous samples simultaneously before newly developed methods supplant biochemical methods for routine usage in purity testing. New varieties that have major genes for herbicide or insect resistance incorporated within them require purity assays during product development and following seed production of the commercial variety. Immunological or DNA sequence assays can be developed and automated systems are required to process hundreds of thousands of individuals. Ultra-high, micro-array technologies and single-molecule detection systems are now under development. These technologies offer the promise that adequate distinction and high sample throughput will be combined. New methods may eclipse the capabilities of biochemical methodologies, thereby potentially raising genetic purity standards and enabling farmers and consumers better to utilize and benefit from increasingly productive varieties that are bred from a more diverse base of genetic resources.

Wild relatives of pigeonpea (Cajanus cajan L.) possess many useful genes that can be utilized for crop improvement, most importantly genes for resistance to Helicoverpa armigera, the legume pod borer. The present study aimed at quantifying diversity in a collection of Cajanus scarabaeoides, Cajanus sericeus, Cajanus reticulatus and C. cajan species selected from a wide geographic range using two PCR-based marker systems, amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSRs), and the hybridization-based restriction fragment length polymorphism (RFLP). Polymorphism was higher among the wild accessions than among the cultivated genotypes. Wild and cultivated Cajanus accessions belonging to different species clustered into four distinct major groups largely based on the interspecific differences. C. scarabaeoides accessions derived from same geographical origins formed one group reflecting similar genetic makeup of these accessions. Dendrograms generated using AFLP, RFLP and SSR marker data were comparable with minor clustering differences, which suggests that either method, or a combination of both can be applied to expanded genetic studies in Cajanus. Mantel testing confirmed the congruence between the genetic distances of three markers, indicating that the markers segregated independently, giving similar grouping patterns of all accessions having similar genetic origin.

Coagulase gene restriction fragment length polymorphism (RFLP), six-locus variable-number tandem-repeat analysis patterns (MLVA) and detection of enterotoxin genes (se) (sea, sec, sed, seg, seh, sei, sej and sel) were used to determine the phylogenetic relationship among isolates of Staphylococcus aureus isolated from dairy products from different regions of Italy. A total of 25 Staph. aureus were subtyped into 16 coagulase genotypes by RFLP, and MLVA revealed marked genomic variability. Furthermore, 17 of the isolates harboured at least one toxin gene, with the predominance of sea, sed and sej among cow isolates and sec-sel among the goat and sheep strains. Combined RFLP, MLVA polymorphism and se genes were found to be useful techniques for discriminating several genetic variants in Staph. aureus isolates.

The existence of two Pisaster ochraceus subspecies has been proposed; one located northwards (P. ochraceus ochraceus) and the other southwards (Pisaster ochraceus segnis) from Point Conception. We used polymerase chain reaction–restriction fragment length polymorphism of the CO I and CO III mitochondrial genes to assess the degree of population structure from 126 samples collected along the western coast of North America, from Vancouver, Canada to Punta San Carlos, of Baja California, Mexico. The genetic structure was tested through molecular analysis of variance and by Monte Carlo simulations of the original data set. The phylogeographical pattern was deduced from a minimum spanning network analysis. No genetic structure was detected. Instead, a high degree of genetic homogeneity along the species distribution was evident from haplotype frequencies at each location. Two haplotypes, Po1 and Po5, were predominant along the distribution and were considered ancestral because of their central position in the minimum spanning network. Since Pisaster ochraceus depicts a planktotrophic larval stage with very long duration before settlement, it seems to be able to surpass the biogeographical boundary that limits other species around Point Conception, thereby maintaining homogeneity of its genetic pool. Results of this study recognize P. ochraceus as a single species.

The Antarctic toothfish (Dissostichus mawsoni) exhibits a circumpolar distribution in coastal waters south of the Antarctic Polar Front. For a preliminary evaluation of global population structure in this species, we examined four mitochondrial regions and 13 nuclear gene fragments in samples from four CCAMLR Subareas in the Southern Ocean (Australian Antarctic Territory (Subarea 58.4.2), Ross Dependency (Subareas 88.1 and 88.2) and the South Shetland Islands (Subarea 48.1). Significant genetic differentiation within and among locations was observed for both mitochondrial and nuclear loci. The single nucleotide polymorphism (SNP) markers developed here will be useful for more extensive analyses of population structure in this species.

To prevent the decline of the brown hare Lepus europaeus, especially as a result of hunting pressure, restocking programmes, using hares from elsewhere, were carried out in Greece up to 2001. Using diagnostic RFLP mitochondrial DNA markers we traced the origins of released captive-bred brown hares. We provide evidence that released animals survived long enough to have at least one reproductive cycle and to transmit their genome. If, in the long-term, introgressed foreign genes survive, forming new genotypes with indigenous genes, this would demonstrate that they are successful in terms of competition. As nuclear gene pools are not markedly divergent between Greek and other European hares foreign nuclear genes should not be a serious handicap. Hence, in certain situations release programmes may be appropriate.

The allelic distributions of the CSN1S1 (αs1-casein) in the Indian goats are quite different from European goat breeds. Majority of Indian goat breeds and non-descript goats carry A, B, E and F alleles at αs1-casein locus, as found by analysing both DNA and protein levels. However, A and B alleles, known to be associated with better casein yield, were observed in the highest proportion in all the Indian goat breeds. Gene frequency and breed heterozygosity were computed for the CSN1S1 gene. The gene frequency of allele A in Indian goats varies from 0.68 to 1.00 and allele B varies from 0.098 to 0.23. Allele F was observed in Beetal, Marwari, Chegu and non-descript goats of MP (Local MP) in less than 1% of population. The expected heterozygosity (He) varied from 0.141 to 0.506 over the population. The Beetal breed showed the highest gene diversity (0.506) followed by Jamunapari (0.395), Chegu (0.383) and Jakhrana (0.381) breeds. Therefore, the variability at CSN1S1 locus can be utilised for conservation as well as for genetic improvement of Indian goat breeds for increasing both the quality and quantity of milk production.

The second internal transcribed spacer (ITS2) of the ribosomal RNA gene array of 6 species of diplozoids; Eudiplozoon nipponicum, Paradiplozoon bliccae, P. homoion, P. megan, P. pavlovskii and P. sapae, was amplified by PCR and sequenced. These sequences clearly demonstrate discrimination at the species level and confirm the validity of species determined by morphological identification. No intraspecific variation was found in the ITS2 sequences. There were no differences in the ITS2 sequences of P. homoion from populations parasitizing different host species. The length of the PCR product allowed discrimination of E. nipponicum from the Paradiplozoon species. Digestion of the amplified ITS2 fragment with enzymes AluI, HaeIII and HinfI provided useful genetic markers for species identification. The genetic relationships between diplozoids again demonstrated that E. nipponicum was the most genetically distinct species, whereas P. bliccae and P. sapae were the species most closely related. This represents the first molecular taxonomic study of these interesting parasites and demonstrates the utility of these methods for addressing questions of systematics.

Traditional methods alone often make it difficult to identify isolates to species level. Armillaria and Cylindrocladium are examples where new technologies could achieve this quickly and reliably. Rapid identification of pathogens is of great importance in their control, especially when dealing with quarantine organisms where detection of pathogens at early stages is critical.