Restriction Fragment Length Polymorphism Genotyping Research Articles

Prevalence of hepatitis C genotypes among patients with chronic hepatitis C in Norway. Construct Group.

Among 116 patients with biopsy-confirmed chronic hepatitis C (Riba 2 or Riba 3 positive) in a multicenter study in southern Norway on interferon, we determined hepatitis C virus genotype by restriction fragment length polymorphism (RFLP) of the 5' NCR. The RFLP method was supplemented by and compared with a serological typing method based on the detection of type-specific antibody to peptide from the NS-4 region. A total of 102/106 (96%) patient sera showed detectable type-specific antibody to NS-4 peptides and corresponded in all cases, except two, to the genotype detected by polymerase chain reaction. Combining the results from RFLP genotyping and serotyping, genotype 1 was found in 40 (35%) (27 with 1a and 10 with 1b, 3 subtypes not determined), genotype 2 in 15 (13%) (subtype 2b in 14 and 1 subtype not determined), and genotype 3 in 58 (50%) of patients. The low mean age of the patients (34 years), the low prevalence of cirrhosis (3.5%), the short duration of the disease, and a high prevalence of intravenous-drug abusers may account for the low prevalence of infection with genotype 1b (9%). The epidemiological features of hepatitis C patients are markedly different from patient groups described in southern Europe in terms of risk factors, age, and genotype distribution.

The Evolution of Races ofPhytophthora sojaein Australia

Isolates of Phytophthora sojae collected between 1979 and 1993 from three soybean-growing regions in eastern Australia and the United States were characterized for virulence and restriction fragment length polymorphisms (RFLPs). A total of 99 isolates were characterized: 84 from Australia and 15 from the United States. In the Australian population of P. sojae, five races (1, 4, 13, 15, and X) were identified. Five RFLP probes were selected that detected 30 fragments, of which 7 were polymorphic, leading to the identification of three multilocus RFLP genotypes amongst the Australian isolates. One multilocus RFLP genotype occurred in over 95% of all samples collected throughout the soybean-growing regions of eastern Australia. The 15 isolates obtained from the United States represented 12 races; the five RFLP probes detected 31 fragments, of which 13 were polymorphic, corresponding to 12 multilocus RFLP genotypes. The low levels of genotypic diversity (2.5 to 14.3%) observed in Australian fl sojae populations compared to the high level (60%) observed in the U.S. population indicates that the Australian population was most likely established by a single introduction of the pathogen. All five races in Australia occurred in the same multilocus RFLP genotype, suggesting that new races in Australia evolve from a common genetic background through mutation.

Analysis of mosquito genome structure using graphical genotyping

AbstractRestriction fragment length polymorphism (RFLP) markers provide effective tools for establishing the genotype at many loci for individual mosquitoes. RFLP genotypes can be organized to provide information concerning parental origin or the allelic configuration of various chromosome segments. Whole genome genotypic information for individuals can be represented in a simple graphic format, termed graphical genotyping. In this study, RFLP markers were used to demonstrate the utility of using graphical genotyping to easily and quickly evaluate the entire genome of the mosquito Aedes aegypti. Two substrains selected for susceptibility and refractoriness, from a strain highly susceptible to the filarial worm parasite Brugia malayi, were compared. Graphical genotyping provided a mechanism to rapidly evaluate genome structure both within and between the two substrains with a simple visual analysis. This information was used in related studies to identify a new locus influencing B. malayi susceptibility in A. aegypti. Graphical genotyping also provided perspectives on some features of genome structure in A. aegypti. It suggested the presence and location of a genetic lethal on chromosome 3 and possible inversion polymorphisms associated with each of the three A. aegypti chromosomes.

Evaluation of a novel serotyping system for hepatitis C virus: strong correlation with standard genotyping methodologies

Direct sequencing and analysis of viral genomes are definitive methods for identifying various hepatitis C virus (HCV) genotypes. However, HCV genome sequencing methods are cumbersome and unsuitable for analyzing large numbers of clinical samples. We have developed a convenient, reliable, and reproducible RIBA strip immunoblot assay system for determining HCV serotype. Briefly, the assay consists of an immunoblot strip on which there are five lanes of immobilized serotype-specific HCV peptides from the nonstructural (NS-4) and core regions of the genomes of HCV types 1,2, and 3. HCV serotype is deduced by determining the greatest intensity of reactivity to the NS-4 serotype-specific HCV peptide band in relation to the intensity of the human immunoglobulin G internal control bands on each strip. HCV core peptide reactivity is used only in the absence of NS-4 reactivity. We used this assay to successfully serotype a high percentage of sera from well-documented HCV-infected patients. Our serotyping results correlated 99% with the findings from the standard restriction fragment length polymorphism genotyping methods. Less than 5% of the serum samples were untypeable. For a selected group of alpha interferon-treated patients we observed that the nonresponders (76.2%) and a majority of the responders who relapsed (72.2%) had type 2 HCV infection. A small population (n= 8) of complete responders was split 3:4:1 as type 1, type 2, and type 3, respectively. Our data indicate that this new serotyping assay has the potential to be a highly specific and reliable method for typing of HCV infection in patients.

Quantitative Trait Loci for Flowering, Plant and Ear Height, and Kernel Traits in Maize

The inheritance of quantitative traits is not well understood. A study was conducted to determine the number and chromosomal locations of quantitative trait loci (QTL) controlling male anthesis date; plant and ear height; kernel weight; and kernel protein, oil, and starch concentration in maize (Zea mays L.). Two hundred S1 lines were derived from a single F1 plant from a cross of Illinois High Oil (IHO) by lllinois Low Oil (Early Maturity) [ILO(EM)]. Restriction fragment length polymorphism (RFLP) analysis was performed to determine RFLP genotypes of the 200 S1 families at 80 polymorphic loci spaced on average 24 centimorgans throughout the genome. The 200 S1 families were measured for phenotypic traits in replicated field trials during 1992 and 1993. Analysis of variance detected significant (P < 0.05) associations between several RFLP loci and each phenotypic trait. A total of 16 marker loci clustered in eight chromosomal regions were significantly associated with male anthesis date, 18 marker loci clustered in II regions were significantly associated with plant height, 14 marker loci clustered in nine regions were significantly associated with ear height, 27 marker loci clustered in II regions were associated with kernel weight, 16 marker loci clustered in eight regions were associated with protein concentration, 31 marker loci clustered in II regions were associated with oil concentration, and 28 marker loci clustered in 13 regions were associated with starch concentration. A number of QTL were detected in chromosomal regions where known gene loci of biological relevance are located.

Extensive polymorphism at the Gp63 locus in field isolates of Leishmania peruviana

Genetic diversity within and between tandemly arrayed copies of the Gp63 gene occurs in laboratory isolates of Leishmania spp., but the extent to which this represents natural genetic diversity has not been assessed. Here, the Gp63 locus is examined in 58 fresh isolates of L. peruviana, and clones derived from them, collected throughout the Peruvian Andes. Extensive polymorphism is observed, both in size of Gp63 containing chromosomes, and for restriction-fragment-length polymorphisms (RFLPs) at the Gp63 locus. All clones within an isolate are identical, including those with two distinct Gp63-hybridising chromosomal-sized pulsed-field gel electrophoresis (PFGE) bands, consistent with diploidy but with size differences in homologous chromosomes. For RFLP analysis, three enzymes were selected to cut within the coding region ( PstI), in the intergenic region ( SalI) and outside ( EcoRI) the Gp63 gene cluster. PstI gave identical banding patterns across all isolates/clones. For EcoRI and SalI, all clones within an isolate were identical, but isolates were polymorphic for fragments at 13 (2–30 kb) and 8 (2.6–8.8 kb) different molecular mass locations generating 19 and 16 distinct RFLP patterns or genotypes for each enzyme, respectively. EcoRI restriction patterns, analysed by PFGE, were consistent with the presence of two clusters of Gp63 genes on each homologous chromosome, one contained within EcoRI fragments large enough to carry from 3 to 10 copies of the Gp63 gene, the second on fragments which could carry 1 or 2 copies of the gene. SalI patterns indicated variable restriction sites within clusters, but not within every intergenic region. A hierarchical analysis of variance of allele frequencies, expressed in terms of Wright's F-statistic, indicated significant barriers to gene flow at all levels, valleys within regions (north/south), villages within valleys, and individuals within villages. This extreme polymorphism at the Gp63 locus of L. peruviana demonstrates the great potential for generation of genetic diversity in parasite populations.

Genetic diversity of wild, weedy and cultivated forms of Brassica rapa

Restriction Fragment Length Polymorphisms (RFLPs) were used to study the genetic diversity within and between accessions of 'wild' and cultivated B. rapa. Two of the wild accessions were likely to be escapes from cultivation because of their geographical origins (Argentina and California). The nature of the other three wild accessions (from Turkey, Algeria and Sicily) was not known. Principal components analysis placed the Argentinian, Californian and Turkish accessions within a cluster which contained all the cultivated forms of B. rapa. The other two B. rapa accessions were genetically divergent and, on the basis of their RFLP genotypes, would have been considered to be more distant from the cultivated forms of B. rapa than accessions of B. nigra and B. montana. The implications of these results for germplasm conservation, selection of material for breeding programmes and phylogenetic studies on the origin of Brassica crops are discussed.

Multiple restriction fragment length polymorphism genotypes of Porphyromonas gingivalis in single periodontal pockets

A total of 188 Porphyromonas gingivalis strains isolated from 13 periodontal pockets of 8 periodontitis patients were investigated by means of restriction fragment length polymorphism (RFLP) analysis using the fimA gene as a pobe. A total of 5 RFLP genotypes were identified, and over half of the isolates belonged to type I. Four of the 8 patients harbored only 1 RFLP genotype of P. gingivalis, and 1 patient harbored only 2 RFLP genotypes in different sites. On the other hand, in 3 other patients, multiple RFLP genotypes were found in single periodontal pockets. Further studies will be required to clarify whether multiple genotypes of P. gingivalis colonized a single periodontal pocket simultaneously or whether a mutation occurred in the fimbrilin gene locus of P. gingivalis.

A germline TaqI restriction fragment length polymorphism in the progesterone receptor gene in ovarian carcinoma

Clinical outcome in ovarian carcinoma is predicted by progesterone receptor status, indicating an endocrine aspect to this disease. Peripheral leucocyte genomic DNAs were obtained from 41 patients with primary ovarian carcinoma and 83 controls from Ireland, as well as from 26 primary ovarian carcinoma patients and 101 controls in Germany. Southern analysis using a human progesterone receptor (hPR) cDNA probe identified a germline TaqI restriction fragment length polymorphism (RFLP) defined by two alleles: T1, represented by a 2.7 kb fragment; and T2, represented by a 1.9 kb fragment and characterised by an additional TaqI restriction site with respect to T1. An over-representation of T2 in ovarian cancer patients compared with controls in the pooled Irish/German population (P < 0.025) was observed. A difference (P < 0.02) in the distribution of the RFLP genotypes between Irish and German control populations was also observed. The allele distributions could not be shown to differ significantly from Hardy-Weinberg distribution in any subgroup. Using hPR cDNA region-specific probes, the extra TaqI restriction site was mapped to intron G of the hPR gene.

Identification of quantitative trait loci influencing wood specific gravity in an outbred pedigree of loblolly pine.

We report the identification of quantitative trait loci (QTL) influencing wood specific gravity (WSG) in an outbred pedigree of loblolly pine (Pinus taeda L.). QTL mapping in an outcrossing species is complicated by the presence of multiple alleles (> 2) at QTL and marker loci. Multiple alleles at QTL allow the examination of interaction among alleles at QTL (deviation from additive gene action). Restriction fragment length polymorphism (RFLP) marker genotypes and wood specific gravity phenotypes were determined for 177 progeny. Two RFLP linkage maps were constructed, representing maternal and paternal parent gamete segregations as inferred from diploid progeny RFLP genotypes. RFLP loci segregating for multiple alleles were vital for aligning the two maps. Each RFLP locus was assayed for cosegregation with WSG QTL using analysis of variance (ANOVA). Five regions of the genome contained one or more RFLP loci showing differences in mean WSG at or below the P = 0.05 level for progeny as grouped by RFLP genotype. One region contained a marker locus (S6a) whose QTL-associated effects were highly significant (P > 0.0002). Marker S6a segregated for multiple alleles, a prerequisite for determining the number of alleles segregating at the linked QTL and analyzing the interactions among QTL alleles. The QTL associated with marker S6a appeared to be segregating for multiple alleles which interacted with each other and with environments. No evidence for digenic epistasis was found among the five QTL.

Molecular marker analyses of powdery mildew resistance in barley

Powdery mildew, caused byEryisphe graminis f. sp.hordei, is one of the most important diseases of barley (Hordeum vulgare). A number of loci conditioning resistance to this disease have been reported previously. The objective of this study was to use molecular markers to identify chromosomal regions containing genes for powdery mildew resistance and to estimate the resistance effect of each locus. A set of 28 F1 hybrids and eight parental lines from a barley diallel study was inoculated with each of five isolates ofE. graminis. The parents were surveyed for restriction fragment length polymorphisms (RFLPs) at 84 marker loci that cover about 1100 cM of the barley genome. The RFLP genotypes of the F1s were deduced from those of the parents. A total of 27 loci, distributed on six of the seven barley chromosomes, detected significant resistance effects to at least one of the five isolates. Almost all the chromosomal regions previously reported to carry genes for powdery mildew resistance were detected, plus the possible existence of 1 additional locus on chromosome 7. The analysis indicated that additive genetic effects are the most important component in conditioning powdery mildew resistance. However, there is also a considerable amount of dominance effects at most loci, and even overdominance is likely to be present at a number of loci. These results suggest that quantitative differences are likely to exist among alleles even at loci which are considered to carry major genes for resistance, and minor effects may be prevalent in cultivars that are not known to carry major genes for resistance.

Restriction fragment length polymorphism differences among Illinois long-term selection oil strains

Restriction fragment length polymorphism (RFLP) analysis was used to characterize variability in the Illinois Long-Term Selection Experiment oil strains. Considerable polymorphism was detected within each oil strain and among oil strains. Fifty-two individual plants from each of the Illinois High Oil (IHO), Illinois Low Oil (ILO), Reverse High Oil (RHO) and Reverse Low Oil (RLO) strains were sampled to determine RFLP allele/variant frequencies. Generation 90 was sampled for IHO, RHO, and RLO whereas generation 87 was sampled for ILO. Forty-nine RFLP probes distributed throughout the maize genome were used. Chi-square analysis was performed to determine if RFLP genotypes at each of the 49 RFLP loci were significantly different among strains. Oil strains that have been separated for 90 generations showed high levels of significantly-different RFLP genotypic frequencies. The comparison of ILO vs RHO gave only significant chi-square values while the comparisons of IHO vs RLO and RHO vs RLO had 11∶1 ratios of significant to non-significant chi-square values. Strains that have been separated for only 42 generations showed a lower level of significantly-different RFLP genotypic frequencies. The comparisons of IHO vs RHO and ILO vs RLO both had only a 3∶2 ratio of significant to non-significant chi-squares values. Detection of multiple RFLP alleles/variants among the oil strains was common with 59% of the RFLP loci examined exhibiting multiple variants. A number of RFLP loci in RHO (3) and RLO (11) were associated with a trend in RFLP allele/variant frequencies consistent with a response to reverse selection for oil concentration.

Ribosomal DNA, peroxidase and extensin variation in genomic DNA of Medicago laciniata (L.) Miller from Australia and Africa

Fifty-four Medicago laciniata (L.) Miller accessions from diverse locations in eastern Australia, Africa and Israel were characterized for genotypic differences by restriction endonuclease cleavage of genomic DNA and probing with radio-labelled DNA sequences to detect restriction fragment length polymorphisms (RFLP). Digestion of the genomic DNA with the restriction endonuclease, Bam HI, and probing with the complete ribosomal DNA (rDNA) repeat unit of soybean (pGmrl) and its subclones revealed that the variable region in M. laciniata spans the Bam H1 sites of the intergenic spacer region between the 18s and 26s DNA. The length variation in the rDNA repeat unit clearly distinguished South African from Australian accessions. The rDNA variants from SA1847 (Morocco) and SA7750 (Tunisia) were the same as the Australian accessions and slightly larger than the rDNA spacer variant unique to SA3428 (Israel). When Eco R1 digested M. laciniata genomic DNA was probed with peroxidase and extensin cDNA clones, all Australian accessions were again characterized by the same RFLP genotype with further differentiation between African and Israeli accessions. An accession of unknown origin, SA3412, possessed the same RFLP genotype as the Australian accessions for all endonuclease and probe combinations. For the African accessions, differences in RFLP genotype were more frequent than differences in phenotype (leaflet laciniation and colour). However, of three South African accessions with the same RFLP genotype, SA26844, SA26847, and SA26848, SA26847 had more pronounced leaflet laciniation.

Genetic Differentiation BetweenPhaeosphaeria nodorumandP. avenariaUsing Restriction Fragment Length Polymorphisms

Genetic variation among 14 isolates of Phaeosphaeria nodarum and 10 isolates of P. avenaria that originated from diverse geographic locations was assessed by the use of restriction fragment length polymorphisms (RFLPs). Genomic DNAs were digested with the restriction enzyme EcoRI and hybridized with 38 anonymous DNA probes. Twenty of the 38 probes were isolated from P. nodorum, and the other 18 probes were isolated from P. avenaria. Most of the probes hybridized with one or two DNA bands per isolate. Each isolate was assigned a RFLP genotype, which is a combination of the banding patterns with all 38 probes. Isolates of P. nodorum showed a significantly lower degree of genetic variation than the isolates of P. avenaria [...]

Mapping oligogenic resistance to powdery mildew in mungbean with RFLPs

We have used restriction fragment length polymorphisms (RFLPs) to map genes in mungbean (Vigna radiata) that confer partial resistance to the powdery mildew fungus, Erysiphe polygoni. DNA genotypes for 145 RFLP loci spanning 1570 centimorgans of the mungbean genome were assayed in a population of 58 F2 plants. This population was derived from a cross between a moderately powdery mildew resistant ("VC3980A") and a susceptible ("TC1966") mungbean parent. F3 lines derived from the F2 plants were assayed in the field for powdery mildew response and the results were compared to the RFLP genotype data, thereby identifying loci associated with powdery mildew response. A total of three genomic regions were found to have an effect on powdery mildew response, together explaining 58% of the total variation. At 65 days after planting, two genomic regions were significantly associated with powdery mildew resistance. For both loci, the allele from "VC3890A" was associated with increased resistance. At 85 days, a third genomic region was also associated with powdery mildew response. For this locus, the allele from the susceptible parent ("TC1966") was the one associated with higher levels of powdery mildew resistance. These results indicate that putative partial resistance loci for powdery mildew in mungbean can be identified with DNA markers, even in a population of modest size analyzed at a single location in a single year.

Use of Mitochondrial DNA Polymorphisms to Estimate the Relative Contributions of the Hudson River and Chesapeake Bay Striped Bass Stocks to the Mixed Fishery on the Atlantic Coast

Restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA) was used to characterize stocks of striped bass Morone saxatilis and to estimate their relative contributions during the fall of 1989 to the mixed coastal fishery at eastern Long Island, New York. Mitochondrial DNA was obtained from reference samples of striped bass collected during the spring of 1989 from the Hudson River, New York, and four spawning areas of the Chesapeake Bay (Choptank, Rappahannock, and Potomac rivers and the upper Chesapeake Bay). Five mtDNA major length genotypes were detected in these fish, and significant differences in their frequencies were observed between the Hudson River and Chesapeake Bay samples. An mtDNA minor length genotype found in some fish (13%) from the Chesapeake Bay and absent from all Hudson River samples provided a second discriminatory character. By using a constrained generalized least squares approach, we estimated that the Hudson River and Chesapeake Bay stocks contributed 73% (95% confidence interval 50–87%) and 27% (95% confidence interval 13–51 %) respectively, of the mixed fishery sample. The probability of the Hudson River contribution exceeding the Chesapeake Bay contribution in this sample was more than 95%. These results suggest that the Hudson River contribution to the mixed coastal fishery was greater in 1989 than reported in earlier studies. We also found no differences in mtDNA major length genotype frequencies among as many as 11 year-classes within the Hudson River, Chesapeake Bay, or Roanoke River spawning systems. These results indicate that mtDNA RFLP genotypes in these striped bass stocks are temporally stable within a fisheries context. An advantage to mtDNA analysis over phenotypic approaches is that, because mtDNA genotype frequencies are not subject to environmentally induced variation, efforts subsequent to an initial survey can focus on characterizing the mixed coastal fishery.

Apolipoprotein B-gene DNA polymorphisms (XbaI and EcoRI), serum lipids, and apolipoproteins in healthy Chinese.

The frequency of restriction fragment length polymorphisms (RFLPs) of the apolipoprotein B (apo B) gene, detected by XbaI and EcoRI, and their influence on serum lipids and apolipoproteins were studied in healthy Chinese of both sexes in Singapore. A total of 221 subjects (150 males, 71 females) were investigated for the XbaI and 159 subjects for the EcoRI polymorphisms, while serum lipids and apolipoprotein levels were available for 196 subjects. The frequency of the X2 allele was found to be significantly lower in the Chinese than that reported in Caucasians from the United Kingdom (0.09 vs. 0.51, P less than 0.001). The haplotype frequencies were also significantly different between the Chinese and Caucasians with a higher frequency of X1R1 in the former compared to the latter (0.85 vs. 0.34, P less than 0.0001). The distribution of RFLP genotypes at both of the restriction sites was at Hardy-Weinberg equilibrium in all groups. The influence of the apo B RFLPs on serum lipids and apolipoprotein levels (apo AI, AII, and B) was studied by both residual and multiple regression analyses considering age, sex, body mass index (BMI), and genotypes as independent variables in all possible combinations. No association was observed between the apo B genotypes and serum lipids or apolipoprotein levels except for high density lipoprotein cholesterol (HDLC), apo AI and AII, with the X2 being associated with significantly lower levels of HDLC as well as apo AI and AII, the effect being stronger in males. These data raise the possibility that the mechanism of reported association between apo B polymorphism and coronary artery disease may be through effects on HDLC.

Molecular basis of an autoantibody-associated restriction fragment length polymorphism that confers susceptibility to autoimmune diseases.

Recently, combined serological and molecular studies of autoantibodies have revealed that these antibodies play an important role in the normal function of the immune system and in the development of the B cell repertoire. Accordingly, we hypothesized that a homozygous deletion of a critical autoantibody-associated Ig variable (V) gene may alter the immune system and thus predispose the host to autoimmune disorders. Initial experiments revealed several restriction fragment length polymorphisms (RFLP) of the Humhv3005 gene, that is likely to encode heavy chains of rheumatoid factors, and the closely related 1.9III gene. By probing EcoR1-digested DNA with the Humhv3005/P1 probe, we found that one of the four major hybridizing bands was missing in approximately 20% of patients with either rheumatoid arthritis or systemic lupus erythematosus, but only 2% of normal subjects. To delineate the genetic basis of this polymorphism, we have now employed the PCR to amplify and analyze hv3005, 1.9III, and homologous genes in individuals with characteristic RFLP genotypes. Our results indicate that the human Vh gene repertoire contains several hv3005- and 1.9III-like genes, and that a complete deletion of the hv3005-like genes is relatively restricted to a subset of autoimmune patients. These findings provide initial evidence for deletion of developmentally regulated autoreactive V genes in autoimmune diseases.

DNA Polymorphisms in Lentinula edodes , the Shiitake Mushroom

DNA restriction fragment length polymorphisms (RFLPs) were examined in Lentinula edodes strains. Genomic DNA from strain 70 was cloned in plasmid vector pUC19, and 18 random clones containing low-copy DNA sequences were used to probe seven strains in Southern DNA-DNA hybridizations. Each cloned fragment revealed DNA polymorphism. An RFLP genotype was determined for each strain and the genetic relatedness was assessed. The coefficients of genetic similarity among the seven strains ranged from 0.43 to 0.90. The inheritance of RFLP markers was examined in single spore isolates. Homokaryons displayed a loss of polymorphic bands compared with the parent dikaryon. Hybrids constructed by crossing compatible homokaryons displayed the inheritance of RFLP markers from each parent homokaryon.

Variation of molecular markers among geographically diverse accessions of Triticum tauschii

Triticum tauschii (Coss.) Schmal. (genome DD), a diploid progenitor of hexaploid wheat (Triticum aestivum L.; AABBDD), grows across large areas of southwest Asia and contains more genetic variability for disease and insect resistance, isozymes, and seed storage proteins than the D genome of T. aestivum. To study patterns of variability at a large number of loci, we determined restriction fragment length polymorphism genotypes at 25 loci in a germ-plasm collection of 102 T. tauschii accessions. All accessions were homozygous at all loci, so "alleles" and "genotypes" were equivalent. Twenty loci were polymorphic, with two to six genotypes per locus and polymorphic indexes ranging from 0.06 to 0.74. Linkage disequilibrium was widespread. On the basis of Hedrick's probability of genotypic identity, botanical varieties T. t. ssp. eusquarrosa var. typica and T. t. ssp. eusquarrosa var. anathera were very similar to each other, as were T. t. ssp. strangulata and T. t. ssp. eusquarrosa var. meyeri, with a large genetic distance between these two pairs of taxonomic groups. Genetic variability for molecular markers was highest near the Caspian Sea, intermediate in Afghanistan, and lowest in Turkey and Pakistan. Genetic and geographical distances were related and generally consistent with the hypothesis that T. tauschii originated near the southern or southwestern coast of the Caspian Sea. Unique genotypes were found in most regions. The results of this study, along with data on economically relevant traits, will provide a basis for selecting breeding parents from the T. tauschii germ-plasm collection.Key words: germ plasm, Aegilops squarrosa, diversity, restriction fragment length polymorphism.