Restriction Enzyme Site Polymorphisms Research Articles

Prenatal Diagnosis of β-Thalassemias by Amniocentesis: Linkage Analysis Using Multiple Polymorphic Restriction Endonuclease Sites

In order to assess the applicability of multiple restriction endonuclease analyses of amniocyte DNA to the prenatal diagnosis of beta-thalassemias in general, we studied 12 consecutive couples at risk. DNA of both members of the 12 couples and a previous offspring of each was analyzed for the presence of 4 polymorphic restriction endonuclease sites: the Hpa I site 3' to the beta-globin gene, the Hind III site in the G gamma gene, the Hind III site in the A gamma gene, and the Bam HI site 3' to the beta-gene. Linkage disequilibrium between these sites and beta A or beta thal genes was not found, presumably due to the heterogeneity of beta thal genes. However, the high frequency of polymorphism at these sites allowed differentiation of beta A-bearing chromosomes from beta thal or beta S-bearing chromosomes in both members of 6 couples. In these couples, complete prenatal diagnosis by linkage analysis of amniocyte DNA would be possible. In the remaining 6 couples, beta A and beta thal chromosomes could be discriminated in one member. In about 50% of the pregnancies of these couples, exclusion of beta-thalassemia is possible by this analysis. These data suggest that when linkage analysis of polymorphic restriction endonuclease sites is carried out, prenatal diagnosis of beta-thalassemia states can be accomplished by amniocentesis alone in 75% of pregnancies at risk.

\u03b2-globin gene cluster haplotypes in ethnic minority populations of southwest China

The genetic diversity and relationships among ethnic minority populations of southwest China were investigated using seven polymorphic restriction enzyme sites in the β-globin gene cluster. The haplotypes of 1392 chromosomes from ten ethnic populations living in southwest China were determined. Linkage equilibrium and recombination hotspot were found between the 5′ sites and 3′ sites of the β-globin gene cluster. 5′ haplotypes 2 (+−−−), 6 (−++−+), 9 (−++++) and 3′ haplotype FW3 (−+) were the predominant haplotypes. Notably, haplotype 9 frequency was significantly high in the southwest populations, indicating their difference with other Chinese. The interpopulation differentiation of southwest Chinese minority populations is less than those in populations of northern China and other continents. Phylogenetic analysis shows that populations sharing same ethnic origin or language clustered to each other, indicating current β-globin cluster diversity in the Chinese populations reflects their ethnic origin and linguistic affiliations to a great extent. This study characterizes β-globin gene cluster haplotypes in southwest Chinese minorities for the first time, and reveals the genetic variability and affinity of these populations using β-globin cluster haplotype frequencies. The results suggest that ethnic origin plays an important role in shaping variations of the β-globin gene cluster in the southwestern ethnic populations of China.

Sequencing and annotation of the chloroplast DNAs and identification of polymorphisms distinguishing normal male-fertile and male-sterile cytoplasms of onion

Male-sterile (S) cytoplasm of onion is an alien cytoplasm introgressed into onion in antiquity and is widely used for hybrid seed production. Owing to the biennial generation time of onion, classical crossing takes at least 4 years to classify cytoplasms as S or normal (N) male-fertile. Molecular markers in the organellar DNAs that distinguish N and S cytoplasms are useful to reduce the time required to classify onion cytoplasms. In this research, we completed next-generation sequencing of the chloroplast DNAs of N- and S-cytoplasmic onions; we assembled and annotated the genomes in addition to identifying polymorphisms that distinguish these cytoplasms. The sizes (153 538 and 153 355 base pairs) and GC contents (36.8%) were very similar for the chloroplast DNAs of N and S cytoplasms, respectively, as expected given their close phylogenetic relationship. The size difference was primarily due to small indels in intergenic regions and a deletion in the accD gene of N-cytoplasmic onion. The structures of the onion chloroplast DNAs were similar to those of most land plants with large and small single copy regions separated by inverted repeats. Twenty-eight single nucleotide polymorphisms, two polymorphic restriction-enzyme sites, and one indel distributed across 20 chloroplast genes in the large and small single copy regions were selected and validated using diverse onion populations previously classified as N or S cytoplasmic using restriction fragment length polymorphisms. Although cytoplasmic male sterility is likely associated with the mitochondrial DNA, maternal transmission of the mitochondrial and chloroplast DNAs allows for polymorphisms in either genome to be useful for classifying onion cytoplasms to aid the development of hybrid onion cultivars.

A New Paradigm for Genus Mycobacterium Population Structure

The genus Mycobacterium contains the pathogenic species tuberculosis and leprae, and additionally at least 148 sometimes opportunistic pathogenic species that occupy environmental niches. The genus Mycobacterium is in the order Actinomycetes along with other acid-fast genera with mycolic acids in their cell walls, such as Corynebacterium, Nocardia, and Rhodococcus. The DNA-dependent RNA polymerase subunit b gene rpoB, is relatively conserved among these bacteria. Sequence polymorphism in a 342bp fragment of rpoB is sufficient to differentiate among most species of Mycobacterium. However, construction of a robust and testable phylogeny or population structure based on this polymorphism, or polymorphism in other genomic regions, has remained elusive. This study presents a new paradigm for the resolution of genus Mycobacterium population structure. A minimum spanning tree (MST) was constructed on restriction enzyme site polymorphisms detected in silico for 5 enzymes, on the 342bp rpoB fragment obtained from GenBank data for 47 Mycobacterium species, and one species each from Corynebacterium, Nocardia and Rhodococcus. The MST was empirically divided into 3 regions. All the rapidly growing mycobacteria (RGM), with a halo of slowly growing mycobacteria (SGM), were found in MST-region 1. The correctness of the MST-regions model of genus Mycobacterium population structure was validated by statistically confirmed linkage disequilibrium of certain restriction site alleles. For certain alleles, SGM in MST-region 1 resembled the RGM of MST-region 1 more than the SGM of MST-region 3. The model provided a framework consistent with published genotypic and phenotypic observations, including expectations from comparative genomics and ribosomal RNA (rRNA) gene properties’ distribution among species, and reported cladistic groupings of species. Corynebacterium diphtheriae, Nocardia nova and Rhodococcus equi belonged to MSTregions 2, 3, and 1 respectively. In conclusion, the MST-regions model of genus Mycobacterium population structure was robust, unambiguous, transparent for alleles, consistent with genotypes and phenotypes, and statistically testable.

Novel single‐nucleotide change in GYP*A in a person who made an alloantibody to a new high‐prevalence MNS antigen called ENEV

Alloantibodies that define some high-prevalence MNS antigens are made by people with glycophorin A (GPA) altered by a single-amino-acid change or replacement of amino acids from part of the Pseudoexon 3 of GYP*B. The finding of a patient whose plasma contained a novel alloanti-En(a)FR prompted this study. The patient's serum contained an alloantibody to a high-prevalence antigen, resistant to papain, ficin, trypsin, alpha-chymotrypsin, or dithiothreitol. The antibody was strongly reactive with all panel red blood cells (RBCs) tested, showed reduced reactivity with ENEP- and ENAV- RBCs, and was nonreactive with M(k)M(k), En(a-), GP.Hil/GP.Hil, and GP.JL/M(k) RBCs. The patient's RBCs typed M+N-S+s-, Wr(a-b+(w)), ENEP-, and ENAV-. These results indicated that the antibody recognized a new high-prevalence antigen in the MNS system. Sequencing of DNA prepared from the patient's white blood cells revealed a GYP*A nucleotide substitution of 242T>G (predicted to change Val62 of GPA to Gly). This change ablates an RsaI restriction enzyme site and polymerase chain reaction-restriction fragment length polymorphism confirmed that the proband was homozygous for Nucleotide 242G. We describe a novel high-prevalence MNS antigen, characterized by Val62 in GPA and named ENEV. The absence of the antigen is associated with Gly62. The change explains the weakened reactivity of the patient's serum with ENEP- and ENAV- RBCs and nonreactivity with anti-ENEP and anti-ENAV against her RBCs. The ENEV antigen has been assigned the ISBT number MNS45.

MitoVariome: a variome database of human mitochondrial DNA

BackgroundMitochondrial sequence variation provides critical information for studying human evolution and variation. Mitochondrial DNA provides information on the origin of humans, and plays a substantial role in forensics, degenerative diseases, cancers, and aging process. Typically, human mitochondrial DNA has various features such as HVSI, HVSII, single-nucleotide polymorphism (SNP), restriction enzyme sites, and short tandem repeat (STR).ResultsWe present a variome database (MitoVariome) of human mitochondrial DNA sequences. Queries against MitoVariome can be made using accession numbers or haplogroup/continent. Query results are presented not only in text but also in HTML tables to report extensive mitochondrial sequence variation information. The variation information includes repeat pattern, restriction enzyme site polymorphism, short tandem repeat, disease information as well as single nucleotide polymorphism. It also provides a graphical interface as Gbrowse displaying all variations at a glance. The web interface also provides the tool for assigning haplogroup based on the haplogroup-diagnostic system with complete human mitochondrial SNP position list and for retrieving sequences that users query against by using accession numbers.ConclusionMitoVariome is a freely accessible web application and database that enables human mitochondrial genome researchers to study genetic variation in mitochondrial genome with textual and graphical views accompanied by assignment function of haplogrouping if users submit their own data. Hence, the MitoVariome containing many kinds of variation features in the human mitochondrial genome will be useful for understanding mitochondrial variations of each individual, haplogroup, or geographical location to elucidate the history of human evolution.

GENETIC VARIATION AMONG STRAINS OF PSEUDOPFIESTERIA SHUMWAYAE AND PFIESTERIA PISCICIDA (DINOPHYCEAE)(1).

The putatively toxic dinoflagellates Pseudopfiesteria shumwayae (Glasgow et J. M. Burkh.) Litaker, Steid., P. L. Mason, Shields et P. A. Tester and Pfiesteria piscicida Steid. et J. M. Burkh. have been implicated in massive fish kills and of having negative impacts on human health along the mid-Atlantic seaboard of the USA. Considerable debate still remains as to the mechanisms responsible for fish mortality (toxicity vs. micropredation) caused by these dinoflagellates. Genetic differences among these cultures have not been adequately investigated and may account for or correlate with phenotypic variability among strains within each species. Genetic variation among strains of Ps.shumwayae and P.piscicida was examined by PCR-RFLP analysis using cultures obtained from the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP), as well as those from our own and other colleagues' collection efforts. Examination of restriction digest banding profiles for 22 strains of Ps.shumwayae revealed the presence of 10 polymorphic restriction endonuclease sites within the first and second internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene of the rDNA complex, and the cytochrome oxidase subunit I (COI) gene. Three compound genotypes were represented within the 22 Ps.shumwayae strains. Conversely, PCR-RFLP examination of 14 strains of P.piscicida at the same ITS1, 5.8S, and ITS2 regions revealed only one variable restriction endonuclease site, located in the ITS1 region. In addition, a dinoflagellate culture listed as P.piscicida (CCMP 1928) and analyzed as part of this study was identified as closely related to Luciella masanensis P. L. Mason, H. J. Jeong, Litaker, Reece et Steid.

Single‐copy gene markers isolated from the Caribbean coral, Montastraea annularis

AbstractWe report the isolation of seven single‐copy nuclear DNA loci from the Caribbean reef‐building coral, Montastraea annularis. All loci are polymorphic at the nucleotide level, and three loci have readily screened restriction enzyme site polymorphisms that can help to rapidly assess population genetic structure. Use of these markers can provide baseline genetic information concerning the population dynamics of dominant stony corals and fuel the implementation and management of appropriate conservation strategies.

Beauveria bassiana haplotype determination based on nuclear rDNA internal transcribed spacer PCR-RFLP

DNA sequence alignment of the nuclear 5.8S ribosomal RNA (rRNA) gene and internal transcribed spacers (ITS) from Beauveria bassiana demonstrated that 6.62% sequence variation existed between nine isolates. A higher level of mutation was observed within the ITS regions, where 8.39% divergence occurred. Polymerase chain reaction restriction fragment length polymorphism, PCR-RFLP, and DNA sequence alignment of the ITS1 and ITS2 regions identified seven polymorphic restriction endonuclease sites, Alu I, Hha I, Hinf I, Sin I, Tru 9AI and two Tha I restriction sites. The allelic frequency of each genetic marker was determined from 96 isolates. PCR-RFLP defined 24 B. bassiana genotypes within the sample set, from which eight phylogenetic clusters were predicted to exist. AMOVA and Fst (θ) indicated that no significant correlation existed between B. bassiana haplotype and insect host range as defined by insect order from which each isolate was derived.

Mitochondrial DNA polymorphism: its role in longevity of the Irish population.

The mtDNA genome has been implicated as playing a pivotal role in determining the longevity and success of the human lifespan. A PCR-RFLP methodology was used to identify polymorphic restriction enzyme sites within a 2643 bp region of the mtDNA genome and a table of genetic haplotypes for a healthy aged and a younger control cohort of patients was constructed. Forty-six different mtDNA haplotypes and 11 groups of related haplotypes were identified across the two age groups but statistical analysis failed to show any significant associations. The European J haplogroup, previously reported to be associated with longevity, was not found at an increased frequency within the Irish aged population (P=0.36). However, the haplotypes comprising the J haplogroup could be differentiated into two distinct branches by the presence or absence of the two polymorphic restriction sites, 16,389g and 16,000g. The branch of haplotypes defined by 16,389g displayed a significant increased frequency in the aged samples (8%) compared to the controls (1%), P=0.015. Inversely, the branch of haplotypes defined by 16,000g displayed a significant decreased frequency in the aged samples (4%) compared to the controls (13%), P=0.011. The polymorphism (mt5178A) associated with longevity in the Japanese was not found in the Irish population, while the polymorphism (mt9055A) associated with successful ageing in the French centenarians was found at an increased frequency in the Irish aged population (9%) compared to the younger control group (5%), but failed to reach a level of statistical significance, P=0.164.

Acquired Homozygosity (Isodisomy) of Chromosome 3 in Uveal Melanoma

Cytogenetic investigation of uveal melanoma (UM) has revealed that monosomy 3 is the most frequent karyotypic abnormality, present in approximately 60% of cases. We investigated a cohort of 41 cases of UM, 19 of which retained two apparently normal copies of chromosome 3. Investigation of loss of heterozygosity (LOH) status was undertaken in an attempt to detect subcytogenetic loss of genetic material in those cases with two copies of chromosome 3. DNA from peripheral blood lymphocytes and fresh frozen or paraffin-embedded tumor tissue from 19 patients was amplified by the polymerase chain reaction for polymorphic loci on chromosome 3, including dinucleotide repeats, a tetranucleotide repeat, and polymorphic restriction enzyme sites. Three tumors showed LOH at multiple informative loci on both short and long arms of chromosome 3. Two additional tumors showed localized LOH on 3q, which corresponded to large deletions seen by cytogenetic analysis. The remaining 16 tumors showed retention of heterozygosity at all informative loci. This study did not detect the presence of cryptic deletions but revealed instead complete chromosomal homozygosity or functional monosomy, which probably occurred by loss and then duplication of the remaining chromosome 3. The demonstration of acquired isodisomy (functional monosomy) in a subset of UM increases the percentage of cases with monosomy 3 and provides further evidence for a central role of chromosome 3 loss in the molecular pathogenesis of uveal melanoma.

Molecular markers reveal differentiation among isolates of Coccidioides immitis from California, Arizona and Texas.

Coccidioides immitis causes coccidioidomycosis, a fungal disease of both immuno-compromised and otherwise healthy people; it is capable of causing large epidemics and the disease is often refractory to chemotherapy. To quantify the magnitude of population differentiation and estimate levels of gene flow in C. immitis, multilocus genotypes were scored for 20-25 clinical isolates from each of Bakersfield (California), Tucson (Arizona), and San Antonio (Texas). The molecular markers used were PCR products with polymorphic restriction endonuclease sites, found and characterized in a previous study of the Tucson population. The data show very highly significant differences in allele frequencies between all three populations, and suggest very low levels of migration between populations. One isolate in the San Antonio sample was an outlier, showing the California-specific allele at all four of the loci distinguishing the two populations, and subsequent inquiries indicated that the infection had indeed been acquired in California. Thus, genetic information can be used to infer the geographical origin of a fungal infection.

No evidence of association between dopamine D3 receptor gene and bipolar affective disorder

A recent study reported a possible association between allele 1 of the dopamine D3 receptor gene and bipolar affective disorder using the haplotype relative risk approach. In attempt to replicate these findings, we used similar family-based methods, such as the Haplotype-Based Haplotype Relative Risk method and the Transmission Disequilibrium Test, in a sample of 44 bipolar probands from Sardinia with both parents available. Using the Bal I restriction enzyme site polymorphism of Lannfelt et al. (1992), no differences were found between transmitted and non-transmitted alleles and no evidence of linkage disequilibrium was observed.

The Australian Electrophoresis Society annual conference at Chromatography '96: Separation Sciences. Rosehill, NSW, Australia, July 9-11, 1996.

A set of eleven biallelic and three multiallelic molecular markers have been developed to analyze populations of Histoplasma capsulatum. All markers are amplified by polymerase chain reaction (PCR) and can be readily scored using minimal amounts of template DNA. The 11 biallelic loci have polymorphic restriction endonuclease sites or small insertions or deletions which may be assessed by agarose gel electrophoresis. These markers are inherited in an unambiguous manner and are ideal for assessing structure and gene flow within US populations of H. capsulatum, but are monomorphic in non-US populations. Both length and sequence variation are present in the multiallelic loci, which can be scored by direct sequencing, polyacrylamide gel electrophoresis, or single-strand conformation polymorphism (SSCP): As they are hypervariable, the multiallelic loci can be used to type isolates and to assess the level of genetic variation within populations. Preliminary results indicate that the three multiallelic markers presented are sufficient to distinguish isolates at the individual level and are polymorphic in both US and non-US populations. This collection of molecular markers will be a useful tool in population and epidemiology studies of H. capsulatum.

Genotypes of the vitamin-D-receptor gene and bone mineral density in Caucasoid postmenopausal females.

To evaluate the relation of bone mineral density (BMD) to inherited polymorphisms within the gene coding for the vitamin D receptor (VDR) in postmenopausal women. DNA samples from 163 post-menopausal women and from 112 controls were genotyped for common allelic determinants within the VDR gene as defined by the restriction enzyme sites for BsmI, ApaI, and TaqI. BMD was evaluated at the lumbar spine (L2-L4) by dual energy X-ray absorptiometry. Single restriction enzyme site polymorphisms as well as their joint genotypes were evaluated for their association with BMD in postmenopausal women. The three restriction site polymorphisms showed a close association indicating linkage disequilibrium (P < 0.0001). There was no relationship between any of the single restriction site polymorphisms and BMD. The distribution of the five most common joint genotypes among postmenopausal women and controls was similar. There was no relation of BMD and any of the five most common VDR genotype groups. Females more than 5 years after menopause had a significantly lower BMD than those less than 5 years after menopause (P < 0.001). This age-related decrease of BMD was also decernible within genotype groups. In our population, the postmenopausal period has a major influence on BMD, but BMD is not controlled significantly by any of the common allelic variations within the VDR gene.

Genotyping human and bovine isolates of Cryptosporidium parvum by polymerase chain reaction-restriction fragment length polymorphism analysis of a repetitive DNA sequence

In order to define transmission routes of cryptosporidiosis and develop markers that distinguish Cryptosporidium parvum isolates, we have identified 2 polymorphic restriction enzyme sites in a C. parvum repetitive DNA sequence. The target sequence was amplified by polymerase chain reaction from 100 to 500 oocysts and the amplified product was subjected to restriction enzyme digestion. Typing of 23 isolates showed that 10 10 calf isolates had the same profile. In contrast, 2 patterns were observed among human isolates: 7 13 displayed the calf profile, and 6 13 presented another pattern. The PCR-RFLP assay described here is a sensitive tool to distinguish C. parvum isolates.

Genotyping human and bovine isolates of Cryptosporidium parvum by polymerase chain reaction-restriction fragment length polymorphism analysis of a repetitive DNA sequence.

In order to define transmission routes of cryptosporidiosis and develop markers that distinguish Cryptosporidium parvum isolates, we have identified 2 polymorphic restriction enzyme sites in a C. parvum repetitive DNA sequence. The target sequence was amplified by polymerase chain reaction from 100 to 500 oocysts and the amplified product was subjected to restriction enzyme digestion. Typing of 23 isolates showed that 10/10 calf isolates had the same profile. In contrast, 2 patterns were observed among human isolates: 7/13 displayed the calf profile, and 6/13 presented another pattern. The PCR-RFLP assay described here is a sensitive tool to distinguish C. parvum isolates.

Restriction Fragment Length Polymorphisms Reveal Considerable Nuclear Divergence within a Well-Supported Maternal Clade in Allium Section Cepa (Alliaceae)

Maternal phylogenies estimated by restriction fragment length polymorphisms (RFLPs) in the chloroplast DNA (cpDNA) delineated a well-supported clade containing Allium altaicum, A. cepa (bulb onion), A. fistulosum (Japanese bunching onion), A. galanthum, and A. vavilovii. Few polymorphic restriction-enzyme sites were detected among the wild and cultivated species within this clade, and relationships could not be confidently estimated. Random nuclear RFLPs revealed considerable variation among these species and three distinct groups were identified (Altaicum, Cepa, and Galanthum). Relationships were estimated using principal components and cluster analyses of the Jaccard's similarity matrix. For five out of six analyses, nuclear phylogenies estimated by UPGMA and neighbor joining of the Jaccard's similarity matrix produced a weakly supported monophyletic lineage for A. altaicum, A. fistulosum, and A. galanthum, disagreeing with maternal phylogenies that produced a weakly supported monophyletic lineage for A. altaicum, A. fistulosum, A. cepa, and A. vavilovii. Allium oschaninii was closely related to A. galanthum and these two species may represent the progenitor types. Overall, restriction-enzyme analyses of the nuclear and cpDNA produced few synapomorphies among closely related species in Allium section Cepa.

Frequency of haplotypes in the beta globin gene cluster in a selected sample of the mexican population.

Five polymorphic restriction enzyme sites in the beta globin gene cluster (HindIII Gγ-Hind III Aγ-, Ava IIINV-2 β-and Hpa I and Bam HI 3'β-globin gene) were studied in individuals from 13 families: 13 homozygote patients for sickle cell anemia, two double heterozygotes (one SC and one S/βThal ), 35 AS heterozygotes (23 parents and 12 siblings), one father (A/βThal ), and three normal siblings. In addition, 17 normal unrelated Mexican subjects were studied. All subjects were from the state of Veracruz on the coast of the Gulf of Mexico. The Southern blot technique was used. Fifteen haplotypes were identified in the 142 chromosomes. Five were the most frequent: two haplotypes, (+-+++) (52.4%) and (--+-+) (19.0%) were associated with βS chromosomes; two haplotypes, (--+++) (38.2%) and (---++) (19.7%), were linked with βA chromosomes, and the fifth (--++-) was present in both types of chromosomes. Haplotype (+-+++) corresponded to the Bantu or Senegal type. With Hinc II analysis after PCR amplification in both the 5' and 3' regions of the ψβ-globin gene, it was possible to distinguish between these African types, as in the former both restriction sites are absent. This analysis was done in 23 βS and 10 βA subjects. All βS chromosomes disclosed the Bantu type, while βA were similar to Caucasians. Bantu and Benin haplotypes have been found with high frequency in African populations, indicating the great influence of African genes in the population of the Mexican coasts. In addition, two previously unidentified haplotypes were found: (++--+) and (-++++). These can be explainded by crossing-over events and/or by new mutations. © 1995 Wiley-Liss, Inc.

Evidence for two single copy units in Theileria parva ribosomal RNA genes

Bacteriophage clones containing ribosomal RNA genes of Theileria parva were isolated from genomic DNA libraries. Physical mapping studies revealed 2 ribosomal DNA units, which were distinguishable by restriction enzyme site polymorphisms in flanking sequences. The cloned ribosomal DNA units were mapped to 2 separate T. parva chromosomes. Analysis of sequences contained in λEMBL3 recombinants, together with Southern blot analysis of genomic DNA and data on the copy number of the rRNA genes, suggested that the rDNA units were not tandemly repeated. This organisation of ribosomal transcription units is similar to that described for other genera of apicomplexan protozoa, but 2 rDNA units, each containing single copies of the rRNA coding genes, would be the lowest copy number described for any eukaryote in which amplification of rRNA genes is not known to occur, EcoRI restriction fragment length polymorphisms, which were revealed using rRNA gene probes, separated T. parva stocks into 2 categories. Nucleotide sequence analysis of polymerase chain reaction-amplified internal transcribed spacer DNA revealed 2 different ITS sequences derived from rDNA transcription units within the genome of a cloned T. parva parasite. Polymorphism was also observed between ITS sequences amplified from the DNA of different T. parva stocks. A synthetic oligonucleotide derived from T. parva Uganda ribosomal ITS DNA sequences hybridised to DNA from the T. parva Uganda stock, but not to the DNA of the T. parva Muguga stock. This oligonucleotide is potentially useful as a marker for the T. parva Uganda stock.