Application of DNA fingerprints for cell-line individualization.

DNA fingerprints of 46 human cell lines were derived using minisatellite probes for hypervariable genetic loci. The incidence of 121 HaeIII DNA fragments among 33 cell lines derived from unrelated individuals was used to estimate allelic and genotypic frequencies for each fragment and for composite individual DNA fingerprints. We present a quantitative estimate of the extent of genetic difference between individuals, an estimate based on the percentage of restriction fragments at which they differ. The average percent difference (APD) among pairwise combinations from the population of 33 unrelated cell lines was 76.9%, compared with the APD in band sharing among cell lines derived from the same individual (less than or equal to 1.2%). Included in this survey were nine additional cell lines previously implicated as HeLa cell derivatives, and these lines were clearly confirmed as such by DNA fingerprints (APD less than or equal to 0.6%). On the basis of fragment frequencies in the tested cell line population, a simple genetic model was developed to estimate the frequencies of each DNA fingerprint in the population. The median incidence was 2.9 X 10(-17), and the range was 2.4 X 10(-21) to 6.6 X 10(-15). This value approximates the probability that a second cell line selected at random from unrelated individuals will match a given DNA fingerprint. Related calculations address the chance that any two DNA fingerprints would be identical among a large group of cell lines. This estimate is still very slight; for example, the chance of two or more common DNA fingerprints among 1 million distinct individuals is less than .001. The procedure provides a straightforward, easily interpreted, and statistically robust method for identification and individualization of human cells.     

Preliminary study on genetic variability of Dicrocoelium dendriticum determined by random amplified polymorphic DNA

Genetic variability of adult specimens of Dicrocoelium dendriticum has been studied using random amplified polymorphic DNA (RAPD). The worms were collected from the infected livers of different sheep from several localities in León province (NW Spain). DNA fragments were amplified by means of decamer primer oligonucleotides of arbitrary sequence. Some primers produce complex and highly variable patterns of amplified DNA in D. dendriticum. Intra- and inter-population genetic variability of adult parasites were analyzed, scoring polymorphic and monomorphic reproducible bands by means of the Jaccard similarity, and dendrograms showing genetic relationships between individuals were obtained using the FITCH method. Genetic variability seems to be high in this parasite and genetic similarity within a population (worms infecting a single animal) is similar to the average similarity between worms from different sheep. These results suggest that each sheep is infected by numerous genetically different parasites from one or more populations of infected ants.     

A population genetic study of the endangered plant species Limonium dufourii (Plumbaginaceae) based on amplified fragment length polymorphism (AFLP)

Limonium dufourii ( Plumbaginaceae ) is a triploid species with obligate apomictic reproduction and is endemic to the East Mediterranean coast of Spain, where it is present in only six populations, most of which have a very low number of individuals. Genetic variation and population structure in this species was studied using amplified fragment length polymorphisms (AFLPs) as markers, using the same individuals as in a previous study with random amplified polymorphic DNA (RAPD). Three different primers provided 252 bands of which 51 were polymorphic among the 152 individuals analysed. Those polymorphic bands were able to define 65 different phenotypes, of which all but two were present in only one population. The comparative analyses of data from AFLPs with those from RAPDs show a high degree of concordance. Additionally, and given the nature of these markers, we propose the estimation of nucleotide divergences from AFLP patterns. Relationships among the different AFLP patterns and the estimates of population genetic parameters obtained with this evolutionary distance are in good agreement with previous results. These analyses show that substantial genetic variability and differentiation exist within and among populations of L. dufourii . Their higher reproducibility and the possibility of obtaining estimates of nucleotide divergence make AFLPs a much better DNA fingerprinting technique.     

Genetic segregation of random amplified polymorphic DNA in diploid cultivated alfalfa.

Polymerase chain reaction was used, with single 10-mer primers of arbitrary sequence, to amplify random regions of genomic DNA from a diploid cultivated alfalfa backcross population. Segregation of the random amplified polymorphic DNA (RAPD) fragments was analysed to determine if RAPD markers are suitable for use as genetic markers. Of the 19 primers tested, 13 amplified a total of 37 polymorphic fragments, of which 28 (76%) segregated as dominant Mendelian traits. RAPD markers appear useful for the rapid development of genetic information in species like alfalfa where little information currently exists or is difficult to obtain.     

Sexing birds using random amplified polymorphic DNA (RAPD) markers

We used random amplified polymorphic DNA (RAPD) markers to sex birds from small tissue (usually blood) samples. Arbitrarily chosen 10-mer PCR primers were screened with DNA from known-sex individuals for the production of a bright female-specific band. Suitable primers were found for seven bird species after screening about 30 primers (range 2–63), and no primer was found for three other species after screening about 50 primers for each species. Investigations into the reliability of RAPD markers for sexing great tits Parus major and oystercatchers Haematopus ostralegus show that: (i) when PCR reaction conditions for great tit DNA are varied, either the presence of the female-specific band correctly predicts the individual's sex or no DNA amplification occurs; (ii) the female-specific band in great tits can be sequenced, and subsequently amplified using specific PCR primers; (iii) null alleles of the female-specific fragment occur at an estimated frequency of 0% ( n = 241 females) in great tits and 0.6% ( n > 290 females) in oystercatchers; (iv) the female-specific fragment in great tits occurs in individuals from a wide geographical range encompassing two subspecies; and (v) the relative intensity of bands in great tit RAPD banding profiles is consistent across individual birds and scorers. The RAPD primers that we have identified are generally species specific, and the consequent time cost of screening for primers is the chief disadvantage of using RAPD markers to sex birds. However, with large sample sizes this disadvantage is outweighed by the relative technical simplicity and low cost of the technique.     

Identification of random amplified polymorphic DNA (RAPD) markers highly linked to sex determination in the red alga Gracilaria gracilis

The random amplified polymorphic DNA (RAPD) technique was employed in the haplo-diploid dioecious species Gracilaria gracilis to identify sex-linked PCR markers. Sixty-nine decamer oligonucleotide primers were tested on two bulks of DNA, one from five haploid males and the other from five haploid females. One of these primers (OPD13) generated a 430-bp fragment specific to males and a 620-bp fragment specific to females. The diploid individuals (tetrasporophytes) showed the co-occurrence of these two fragments. In order to verify the linkage between the sexual phenotypes and these markers, a progeny array of 59 haploid individuals (male and female) born on a diploid individual was analysed, in all of which the two markers produced by the OPD13 primer segregated perfectly with sex.     

三种摇蚊幼虫RAPD扩增条件的优化及在昆虫系统发育分析中的应用

为了研究摇蚊科昆虫种群遗传的多样性,以促进对其资源的合理保护,以萨摩亚摇蚊Chironomus samoensisEdwards基因组DNA为模板,对摇蚊幼虫的RAPD扩增条件进行优化,建立了摇蚊幼虫RAPD扩增反应的最佳体系:按照利用优化的RAPD扩增条件进行研究,实验有着良好的重现性。用16个随机引物对3种摇蚊幼虫类群各10个个体进行RAPD扩增,其中萨摩亚摇蚊共扩增出78个条带,多态座位率为41.03%,Shannon遗传多样性指数为0.2570,群体内相似度为0.8730;红裸须摇蚊Propsilocerus akamusi(Tokunaga)共75个条带,多态座位率为44.0%,Shannon遗传多样性指数为0.2472,群体内相似度为0.8731;刺铗长足摇蚊Tanypus punctipennis(Fabricius)共67个条带,多态座位率为41.79%,Shannon遗传多样性指数为0.1943,群体内相似度为0.9066。聚类分析结果表明,刺铗长足摇蚊与红裸须摇蚊的亲缘关系较近。     

Characterization of genetic identities and relationships of Brassica oleracea L. via a random amplified polymorphic DNA assay

Summary Effective conservation and the use of plant genetic resources are essential for future agricultural progress. Critical to this conservation effort is the development of genetic markers which not only distinguish individuals and accessions but also reflect the inherent variation and genetic relationships among collection holdings. We have examined the applicability of the random amplified polymorphic DNA (RAPD) assay for quick, cost-effective, and reliable use in addressing these needs in relation to collection organization and management. Twenty-five decamer oligonucleotide primers were screened individually with a test array composed of individuals representing a range of genetic relationships in Brassica oleracea L. (vegetable and forage cole crops). Over 140 reproducible, polymorphic fragments were generated for study. Each individual of the test array exhibited a unique molecular genotype and composites specific for accessions and botanical varieties could be established. An analysis of similarity based on amplified DNA fragments reflected the known genetic relationships among the selected entries. These results demonstrated that RAPD markers can be of great value in gene bank management for purposes of identification, measurement of variation, and establishment of genetic similarity at the intraspecific level.     

Assessing population genetic structure and variability with RAPD data: Application to Vaccinium macrocarpon (American Cranberry)

A method for estimating and comparing population genetic variation using random amplified polymorphic DNA (RAPD) profiling is presented. An analysis of molecular variance (AMOVA) is extended to accomodate phenotypic molecular data in diploid populations in Hardy-Weinberg equilibrium or with an assumed degree of selfing. We present a two step strategy: 1) Estimate RAPD site frequencies without preliminary assumptions on the unknown population structure, then perform significance testing for population substructuring. 2) If population structure is evident from the first step, use this data to calculate better estimates for RAPD site frequencies and sub-population variance components. A nonparametric test for the homogeneity of molecular variance (HOMOVA) is also presented. This test was designed to statistically test for differences in intrapopulational molecular variances (heteroscedasticity among populations). These theoretical developments are applied to a RAPD data set in Vaccinium macrocarpon (American cranberry) using small sample sizes, where a gradient of molecular diversity is found between central and marginal populations. The AMOVA and HOMOVA methods provide flexible population analysis tools when using data from RAPD or other DNA methods that provide many polymorphic markers with or without direct allelic data.     

Strain typing in Leishmania donovani by using sequence-confirmed amplified region analysis

To differentiate strains of Leishmania donovani, allelic markers at the DNA level were developed by sequence-confirmed amplified region analysis (SCAR). Homologous fragments from different strains of L. donovani were amplified by PCR using random primers and subsequently screened for single-strand conformation polymorphisms. Direct sequencing revealed 55 sequence polymorphisms in eight co-dominant DNA markers; 38 of them were single point mutations. Heterozygosity was evident for 69% and fixed heterozygosity for 25% of all polymorphisms. At most polymorphic sites one of the segregation genotypes was missing. Nineteen unique multilocus genotypes were identified among 29 strains of L. donovani. One genotype was represented by eight Sudanese strains; also two strains from Sudan as well as two strains from Kenya, respectively, shared identical genotypes. All other strains had individual multilocus genotypes. Calculation of genetic distances showed a correlation between multilocus genotypes and the geographical origin of these strains. African strains were found in one well-supported cluster with Kenyan and Sudanese strains clearly separated. SCAR markers seem to represent a random sample of neutral genetic variation present in natural populations. They are co-dominant because they can detect all possible allele combinations in a diploid organism and may, therefore, be very useful for population genetic analysis in Leishmania.     

Estimating genetic drift and effective population size from temporal shifts in dominant gene marker frequencies

Measurement of temporal change in allele frequencies represents an indirect method for estimating the genetically effective size of populations. When allele frequencies are estimated for gene markers that display dominant gene expression, such as, e.g. random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers, the estimates can be seriously biased. We quantify bias for previous allele frequency estimators and present a new expression that is generally less biased and provides a more precise assessment of temporal allele frequency change. We further develop an estimator for effective population size that is appropriate when dealing with dominant gene markers. Comparison with estimates based on codominantly expressed genes, such as allozymes or microsatellites, indicates that about twice as many loci or sampled individuals are required when using dominant markers to achieve the same precision.     

Nucleotide diversity in populations of balitorid cave fishes from Thailand

Genetic variabilities in four cave and eight surface species of balitorid freshwater fishes from Thailand were assessed using random amplified polymorphic DNA (RAPD). Cave species have consistently lower RAPD variation than surface species and it is hypothesized that this difference is a function of reduced population size in cave fishes. Indices of nucleotide diversity (pi) were calculated from the RAPD data and are four to five times higher for the surface species than the cave species: 6.4 x 10-3 vs. 1.4 x 10-3. The pi-values for cave fishes are significantly lower than those for surface balitorids and those measured in other species using RAPD/amplified fragment length polymorphism data.     

Long-distance seed dispersal in a metapopulation of Banksia hookeriana inferred from a population allocation analysis of amplified fragment length polymorphism data

There is currently a poor understanding of the nature and extent of long-distance seed dispersal, largely due to the inherent difficulty of detection. New statistical approaches and molecular markers offer the potential to accurately address this issue. A log-likelihood population allocation test (AFLPOP) was applied to a plant metapopulation to characterize interpopulation seed dispersal. Banksia hookeriana is a fire-killed shrub, restricted to sandy dune crests in fire-prone shrublands of the Eneabba sandplain, southwest Australia. Population genetic variation was assessed for 221 individuals sampled from 21 adjacent dune-crest populations of B. hookeriana using amplified fragment length polymorphism. Genetic diversity was high, with 175 of 183 (96%) amplified fragment length polymorphism markers polymorphic. Of the total genetic diversity, 8% was partitioned among populations by amova and FST. There was no relationship between genetic diversity within populations and population demographic parameters such as population size and sample size. A population allocation test on these data unambiguously assigned 177 of 221 (80.1%) individuals to a single population. Of these, 171 (77.4% of total) were assigned to the population from which they were sampled and 6 (2.7% of total) were assigned to a known population other than the one from which they were sampled. A further 9 (4.1% of total) were assigned to outside the sampled metapopulation area, and 35 individuals (15.8%) could not be assigned unambiguously to any particular population. These results suggest that both the extent [15 of 221 (6.8%) individuals originating from a population other than the one in which they occur] and distance (1.6 to > 2.5 km), of seed dispersal between dune-crest populations is greater than expected from previous studies. The extent of long-distance interpopulation seed dispersal observed provides a basis for explaining the survival of populations of the fire-killed B. hookeriana in a landscape experiencing frequent fire, where local extinctions and recolonizations may be a regular occurrence.     

Trypanosoma evansi: genetic variability detected using amplified restriction fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) analysis of Kenyan isolates

We compared two methods to generate polymorphic markers to investigate the population genetics of Trypanosoma evansi; random amplified polymorphic DNA (RAPD) and amplified restriction fragment length polymorphism (AFLP) analyses. AFLP accessed many more polymorphisms than RAPD. Cluster analysis of the AFLP data showed that 12 T.evansi isolates were very similar ('type A') whereas 2 isolates differed substantially ('type B'). Type A isolates have been generally regarded as genetically identical but AFLP analysis was able to identify multiple differences between them and split the type A T. evansi isolates into two distinct clades.     

Construction of a framework map in Pinus contorta subsp. latifolia using random amplified polymorphic DNA markers.

We report on the construction of the first random amplified polymorphic DNA (RAPD) framework map in Pinus contorta subsp. latifolia. Genomic DNA of haploid megagametophytes from 90 open-pollinated seeds originating from a single tree were amplified with 840 random decamer oligonucleotide primers by the polymerase chain reaction. Three-hundred twenty-eight RAPD markers with fragment sizes that ranged between 260 and 3080 base pairs were found segregating at 110 random decamer oligonucleotide primers. Of these 328 RAPD markers, 148 were mapped to 16 framework linkage groups and 77 were mapped as accessory markers onto the framework linkage groups, on a support interval of minimal LOD score of 3. The 16 framework maps cover a distance of 2287 cM. The estimate of genome size was 2407 cM with a 95% confidence interval of 2304-2459 cM.     

Estimating population structure in diploids with multilocus dominant DNA markers

Multilocus DNA markers [random amplified polymorphic DNA (RAPDs), amplified fragment length polymorphism (AFLPs)] are important for population studies because they reveal many polymorphic loci distributed over the genome. The markers are dominant, that is two phenotypes are distinguished at each locus, with a band and with no band. The latter one represents null-homozygotes with unamplified, recessive null-alleles. The frequency of a null-allele can be estimated by taking the square root of the fraction of individuals with no band. Lynch and Milligan (1994) have suggested a modified procedure that reduces bias introduced by the square-root transform. However, the procedure recommends to ignore those samples in which fewer than four null-homozygotes are observed. This may lead to significant bias in estimates of genetic diversity. In this study, I introduce a Bayesian approach to estimation of null-allele frequencies for dominant DNA markers. It follows from computer simulations and data on two conifer species that the Bayesian method gives nearly unbiased estimates of heterozygosity, genetic distances and F-statistics. The influence of a prior distribution and departure from Hardy-Weinberg proportions on the estimates is also considered.     

Molecular analysis of Helicobacter pylori populations in antral biopsies from individual patients using randomly amplified polymorphic DNA (RAPD) fingerprinting

Abstract In the present study, randomly amplified polymorphic DNA (RAPD) fingerprinting has been used to analyse multiple single colony isolates of Helicobacter pylori from antral biopsies in an attempt to ascertain whether or not multiple strains are present in individual patients using single biopsy samples. The RAPD fingerprints derived from single colonies obtained from the same biopsy specimen were in all cases indistinguishable. The previously noted heterogeneity between H. pylori strains from different individuals was confirmed. RAPD fingerprinting, combined with a simple method of template preparation, was shown to be an excellent method for H. pylori strain differentiation. The results of this study indicate that the H. pylori population is homogeneous in individual patients at a single gastric site.     

Statistical analysis of amplified fragment length polymorphism data: a toolbox for molecular ecologists and evolutionists

Recently, the amplified fragment length polymorphism (AFLP) technique has gained a lot of popularity, and is now frequently applied to a wide variety of organisms. Technical specificities of the AFLP procedure have been well documented over the years, but there is on the contrary little or scattered information about the statistical analysis of AFLPs. In this review, we describe the various methods available to handle AFLP data, focusing on four research topics at the population or individual level of analysis: (i) assessment of genetic diversity; (ii) identification of population structure; (iii) identification of hybrid individuals; and (iv) detection of markers associated with phenotypes. Two kinds of analysis methods can be distinguished, depending on whether they are based on the direct study of band presences or absences in AFLP profiles ('band-based' methods), or on allelic frequencies estimated at each locus from these profiles ('allele frequency-based' methods). We investigate the characteristics and limitations of these statistical tools; finally, we appeal for a wider adoption of methodologies borrowed from other research fields, like for example those especially designed to deal with binary data.     

Male-specific band in random amplified microsatellite polymorphism fingerprints of Holstein cattle

A total of 65 blood samples collected from Holstein cattle were employed for DNA extraction. Genomic DNA were amplified by means of random amplified polymorphic DNA (RAPD). One hundred and one random primers (Operon kits OPAA, OPAO, OPAV, OPC, OPE and OPA-06) were used for polymerase chain reactions (PCR). The PCR products were size fractionated by means of electrophoresis in agarose gel, transferred to a nylon membrane, and then hybridized with a alpha-32P-labeled microsatellite probe for random amplified microsatellite polymorphism (RAMPO) analysis. All the primers produced many amplification patterns in RAMPO fingerprints, but only OPE-01 (5'-CCCAAGGTCC-3') yielded a male-specific strong signal. Among the 65 cases, the specific band could be observed in the RAMPO fingerprints of the 24 male samples but not in the 41 female samples. Therefore, this specific band is peculiar to male cattle.     

Characterization of Zebu cattle breeds in Tanzania using random amplified polymorphic DNA markers

A total of 141 short primers, of arbitrary nucleotide sequence, were used singly in poly-merase chain reactions to amplify DNA fingerprints in pools of DNA representing three Zebu cattle breeds. Two primers, which discriminated between the breed-specific DNA pools were used further to amplify individual pool components in order to establish band frequencies of the amplified fingerprints. One of the primers (ILO 1127) amplified a RAPD fingerprint in 61%of TSZ animals but less than 6% in the other breeds, while another primer (ILO 1065) revealed a DNA sequence common to 89% of the Boran animals and less than 30% in the other two breeds. Bandsharing and mean average percentage difference calculated within and between the three breeds using RAPD fingerprint data showed a higher degree of homogeneity within than across the breeds and indicated measurable divergence between the three breeds. It is concluded that RAPD polymorphisms are useful as genetic markers for cattle breed differentiation.