Intraspecific and interspecific variation in the second internal transcribed spacer (ITS-2) sequence for Metastrongylus (Nematoda: Metastrongyloidea) detected by high resolution PCR-RFLP

Metastrongylus species are important parasites of free-range pigs and wild boar, but little is known about the genetic make-up of natural populations. This study was undertaken to examine sequence variation in internal transcribed spacer 2 of ribosomal DNA within and among three species of Metastrongylus using PCR-linked restriction fragment length polymorphism analysis. In contrast to many other species of bursate nematodes, significant intraspecific variation was detected in restriction fragment length polymorphism profiles among individual worms. In spite of this, it was possible to identify the three species by their distinctive restriction profiles. The findings suggest that the internal transcribed spacer 2 region should be useful for analysing population variation within Metastrongylus species.     

Differentiation among three species of bovine Thelazia (Nematoda: Thelaziidae) by polymerase chain reaction-restriction fragment length polymorphism of the first internal transcribed spacer ITS-1 (rDNA).

Thelazia gulosa, Thelazia rhodesi and Thelazia skrjabini are nematodes transmitted by some species of Musca (Diptera: Muscidae) which cause ocular infestations in bovines. Differences in the rDNA of these species were determined by a PCR using different sets of relatively conserved oligonucleotide primers. PCR on the first internal transcribed spacer (ITS-1) revealed differences in size in Thelazia species (437 bp for T. gulosa, 370 bp for T. rhodesi and 506 bp for T. skrjabini) while the DNA control of Musca spp. was not amplified. The ITS-1 amplicons of the three species were sequenced and then analysed. The GC contents ranged from 26 to 36% and the level of differences in the nucleotide sequences of ITS-1 was lower between T. skrjabini and T. gulosa (39%) than the latter and T. rhodesi (49–56%). Restriction fragment length polymorphism (RFLP) of ITS-1 amplicons was also carried out and the restriction profiles compared. Clear genetic differences among the three Thelazia examined were demonstrated by using the enzymes HpaII, CpoI and SspI. This PCR–RFLP for the delineation of T. gulosa, T. rhodesi and T. skrjabini offers prospects as a molecular epidemiological tool to study parasite transmission patterns and prevalence.     

Differentiation among three species of bovine Thelazia (Nematoda: Thelaziidae) by polymerase chain reaction–restriction fragment length polymorphism of the first internal transcribed spacer ITS-1 (rDNA)

Thelazia gulosa, Thelazia rhodesi and Thelazia skrjabini are nematodes transmitted by some species of Musca (Diptera: Muscidae) which cause ocular infestations in bovines. Differences in the rDNA of these species were determined by a PCR using different sets of relatively conserved oligonucleotide primers. PCR on the first internal transcribed spacer (ITS-1) revealed differences in size in Thelazia species (437 bp for T. gulosa, 370 bp for T. rhodesi and 506 bp for T. skrjabini) while the DNA control of Musca spp. was not amplified. The ITS-1 amplicons of the three species were sequenced and then analysed. The GC contents ranged from 26 to 36% and the level of differences in the nucleotide sequences of ITS-1 was lower between T. skrjabini and T. gulosa (39%) than the latter and T. rhodesi (49–56%). Restriction fragment length polymorphism (RFLP) of ITS-1 amplicons was also carried out and the restriction profiles compared. Clear genetic differences among the three Thelazia examined were demonstrated by using the enzymes HpaII, CpoI and SspI. This PCR–RFLP for the delineation of T. gulosa, T. rhodesi and T. skrjabini offers prospects as a molecular epidemiological tool to study parasite transmission patterns and prevalence.     

Confirmation of cultivated Porphyra tenera (Bangiales, Rhodophyta) by polymerase chain reaction restriction fragment length polymorphism analyses of the plastid and nuclear DNA

Polymerase chain reaction restriction fragment length polymorphism (PCR‐RFLP) analysis of the plastid ribulose‐1,5‐bisphosphate carboxylase (RuBisCo) spacer region was developed for a more reliable and rapid species identification of cultivated Porphyra in combination with PCR‐RFLP analysis of the nuclear internal transcribed spacer (ITS) region. From the PCR‐RFLP analyses of the plastid and nuclear DNA, we examined seven strains of conchocelis that were used for cultivation as Porphyra tenera Kjellman but without strict species identification. The PCR‐RFLP analyses suggested that two strains, C‐32 and 90‐02, were cultivated P. tenera and that the other five strains, C‐24, C‐28, C‐29, C‐30 and M‐1, were Porphyra yezoensis f. narawaensis Miura. To identify species more accurately and to reveal additional genetic variation, the two strains C‐32 and 90‐02 were further studied by sequencing their RuBisCo spacer and ITS‐1 regions. Although RuBisCo spacer sequences of the two strains were identical to each other, each of their ITS‐1 sequences showed a single substitution. The sequence data again confirmed that the two strains (C‐32 and 90‐02) were cultivated P. tenera, and suggested that the two strains showed some genetic variation. We concluded that PCR‐RFLP analysis of the plastid and nuclear DNA is a powerful tool for reliable and rapid species identification of many strains of cultivated Porphyra in Japan and for the collection of genetically variable breeding material of Porphyra.     

Sequence differences in the internal transcribed spacer ribosomal DNA among four species of hookworm (Ancylostomatoidea: Ancylostoma)

The two ribosomal DNA internal transcribed spacers (1 and 2) of the hookworms Ancylostoma caninum, A. tubaeforme, A. ceylanicum and A. duodenale were sequenced. The sequence lengths were similar among the four species, except that A. ceylanicum had slightly longer (by 5–7 bp) internal transcribed spacer 1 and 2 sequences. The predicted secondary structure of the internal transcribed spacer 2 precursor rRNA was similar for all species, despite interspecific differences in primary sequence ranging from 0.9% to 13.2%. Interspecific differences in internal transcribed spacer 1 sequence ranged from 0.9% to 7.5%. A cladistic analysis of the sequence data, using the human hookworm Necator americanus as the outgroup, provided little resolution of the phylogenetic relationships, except that A. ceylanicum occurred on a branch external to the other three species. Nonetheless, internal transcribed spacers 1 and 2 may provide useful phylogenetic information at higher taxonomic levels within the superfamily Ancylostomatoidea.     

Rapid investigation of French sourdough microbiota by restriction fragment length polymorphism of the 16S-23S rRNA gene intergenic spacer region

The aim of the present research is to identify rapidly the lactic acid bacteria (LAB) microflora of four natural French sourdoughs (GO, BF, VB and RF), applying a biphasic (restriction length polymorphism (RFLP) and sequencing) approach for bacterial identification. For this purpose, a database with the RFLP patterns of 30 lactobacilli type strains was created. So-developed ISR-RFLP algorithm was further applied for the differentiation and identification of 134 sourdough isolates. The 16S-23S rDNA intergenic spacer region was amplified by primers t^Ala and 23S/10, and then digested by HindIII, HinfI and α-TaqI enzymes. Nucleotide sequences of the cloned 16S-23S intergenic spacer region (ISR) were determined by the dideoxynucleotide chain termination method. The T7Prom and M13rev primers flanking the multiple cloning site of pCR2.1 DNA were used to sequence both DNA strands. The RFLP profile obtained upon digestion with HindIII, HinfI and α-TaqI enzymes can be used to discriminate Lactobacillus sanfranciscensis (66%), Lactobacillus panis (17%), Lactobacillus nantensis (11%) and Lactobacillus hammesii(6%) in sourdough GO, Lactobacillus sanfranciscensis (80%), Lactobacillus spicheri (14%) and Lactobacillus pontis(6%) in sourdoughs BF. In sourdoughs VB, which differed in the process temperature, we can differentiate Lactobacillus sanfranciscensis (89%) and Leuconostoc mesenteroidessubsp. mesenteroides (11%). Lactobacillus frumenti(47%), Lactobacillus hammesii (8%), and Lactobacillus paralimentarius (45%) were differentiated in sourdough RF.     

Mitochondrial restriction fragment length polymorphism (RFLP) and sequence variation among closely related avian species and the genetic characterization of hybrid Dendroica warblers

To address several interconnected goals, we used mitochondrial DNA (mtDNA) sequences to explore evolutionary relationships among four potentially hybridizing taxa in a North American avian superspecies (Dendroica occidentalis, D. townsendi, D. virens, and D. nigrescens). We first compared the results of a previous restriction fragment length polymorphism (RFLP)-based study with 1453 nucleotides from the mitochondrial cytochrome oxidase subunit I (COI), ATP-synthase 6 (ATPase 6), and ATP-synthase 8 (ATPase 8) genes. Separate phylogenetic analyses of the RFLP and sequence data provided identical and well-supported hierarchical species-level reconstructions that grouped occidentalis and townsendi as sister taxa. We then explored several general features of mitochondrial evolution via a comparison of the RFLP and sequence data sets. Qualitative rate differences that seemed evident in highly autocorrelated comparisons of RFLP vs. sequence pairwise distances were not supported when autocorrelation was removed. We also noted a high variance in corresponding RFLP and sequence distances after the removal of autocorrelation effects. This variance suggests that caution should be used when combining RFLP and sequence-based data in studies that require the large-scale synthesis of divergence estimates drawn from sources employing different molecular techniques. Finally, we used our parallel RFLP and sequence data to design and validate a rapid and inexpensive polymerase chain reaction-RFLP (PCR-RFLP) protocol for determining species-specific mitochondrial haplotypes. This PCR-RFLP technique will be applied in ongoing studies of the occidentalis/townsendi hybrid zone, where the historic and geographical complexity of the interbreeding populations necessitates the genotyping of thousands of individual warblers.     

Ribosomal DNA spacer-length variation inSecale spp. (Poaceae)

Variation in ribosomal DNA spacer length was analysed in 23 populations of 12Secale spp. by restriction enzyme analysis. Digestion of rDNA with Taq I endonuclease enzyme yields spacer fragments that include the subrepeat array and the non-repetitive region downstream of the array. Extensive spacer length variation existed in most species with Taq I fragment lengths ranging from 0.9–3.1 kb. These length variants have been attributed to the differences in number of 134 bp spacer subrepeats within rDNA arrays.S. silvestre was the only species to exhibit a unique spacer length variant of 0.9 kb and this was shown to result from the presence of an extra Taq I site in the spacer. rDNA spacer length frequencies were determined for the species. These frequencies were used to derive phenetic relationships between the species by numerical taxonomic methods. In plots constructed fromGower's distance matrices,S. silvestre appeared well separated from the major cluster consisting of the other species. On the basis of morphological and cytogenetic criteria,S. silvestre is considered the most ancient species. The rDNA data is consistent with this interpretation as it shows a clear differentiation ofS. silvestre from all the other species based on length and nucleotide sequence composition of the spacer region.     

Genetic variation in chloroplast and nuclear ribosomal DNA in Utah juniper (Juniperus osteosperma, Cupressaceae): evidence for interspecific gene flow

Geographic patterns of genetic variation in chlorolast (cpDNA) and nuclear ribosomal (nrDNA) DNA were examined to test the hypothesis of hybridization between Juniperus osteosperma and Juniperus occidentalis in the Great Basin of western Nevada. Noncoding DNA from the trnL-trnF intergenic spacer and the trnL intron of the chloroplast genome was sequenced from seven populations of J. osteosperma and four populations of J. occidentalis sampled over a large proportion of their respective ranges. An adenine nucleotide at position 436 in the aligned sequence and within a Tru 9I restriction site was found to be present in individuals of J. osteosperma sampled from western Colorado and central Utah, but absent in sequences of J. osteosperma sampled from central and western Nevada and all sequences of J. occidentalis. Two hundred fourteen individuals from 34 populations of J. osteosperma and J. occidentalis were then screened for cpDNA haplotype by Tru 9I digestion of the trnL-trnF polymerase chain reaction (PCR) product. Two cpDNA haplotypes were evident, each consisting of restriction fragment profiles that differed solely with respect to the presence or absence of the Tru 9I site encompassing the adenine nucleotide at position 436. One of these haplotypes was monomorphic in J. occidentalis and exhibited a decreasing frequency in J. osteosperma with increasing geographic distance from J. occidentalis in west-central Nevada. Geographic patterns in nuclear ribosomal DNA (nrDNA) variation were examined by restriction fragment analysis and, although spatially more restricted, exhibited patterns of clinal variation similar to those observed in cpDNA haplotype. Genetic relationships based on DNA sequences and geographic patterns of genetic variation in chloroplast and nuclear ribosomal DNA are consistent with morphology in suggesting interspecific gene flow between J. occidentalis and J. osteosperma.     

Trypanosoma evansi: molecular homogeneity as inferred by phenetical analysis of ribosomal internal transcribed spacers DNA of an eclectic parasite

The protozoan Trypanosoma evansi is described as presenting high morphological and genetic similarities among the isolates despite its biological heterogeneity and wide geographical distribution. PCR amplification of the internal transcribed spacers of the ribosomal gene in combination with the coding region of the 5.8S ribosomal subunit further submitted to restriction enzymes digestion were carried out in DNAs extracted from 41 T. evansi strains isolated from horses, dogs, coatis and capybaras from two distinct regions of the Brazilian Pantanal. We also used one T. evansi isolate from Africa, one from Asia and one isolate of T. b. brucei from Africa. Analysis of the RFLP profiles yielded a unique "riboprinting" that does not vary intraspecifically. These results provide insights on the ribosomal gene organization of T. evansi and showed that ITS analysis by RFLP show high genetic similarity of this locus among isolates of this protozoan parasite.