Markers For DNA Fingerprinting Research Articles

Susceptibility for breast cancer in young patients with short rare minisatellite alleles of BORIS

In this study, we characterized two blocks of minisatellites in the 5' upstream region of the BORIS gene (BORIS-MS1, -MS2). BORIS-MS2 was found to be polymorphic; therefore, this locus could be useful as a marker for DNA fingerprinting. We assessed the association between BORIS-MS2 and breast cancer by a case-control study with 428 controls and 793 breast cancers cases. Rare alleles in the younger group (age, <40) were associated with a statistically significant increased risk of breast cancer (odds ratio, 4.84; 95% confidence interval, 1.06-22.22; and P = 0.026). A statistically significant association between the short rare alleles and cancer was identified in the younger group (8.02; 1.01-63.83; P = 0.021). Kaplan-Meier estimates showed that poor prognosis was associated with patients who contained the rare alleles. Our data suggest that the short rare alleles of BORIS-MS2 could be used to identify the risk for breast cancer in young patients.

SSR-BASED DNA FINGERPRINTING OF POTATO CLONES FROM THE PACIFIC NORTHWEST POTATO VARIETY DEVELOPMENT PROGRAM

DNA fingerprinting is a valuable tool for plant cultivar identification and discrimination. The use of simple sequence repeat (SSR) markers for DNA fingerprinting of potatoes could benefit the declaration of distinctiveness of new potato clones for plant variety protection (PVP), granting the Plant Breeder's Rights and the legal protection of new cultivars. A total of 54 tetraploid potato clones, including new cultivars released by Oregon State University under the umbrella of the Pacific Northwest Tri-State Potato Variety Development Program and a set of common commercial cultivars, were analyzed using 13 SSR markers. The SSR amplification products were separated by electrophoresis on a LI-COR 4300 DNA analyzer system and then compiled using the SAGA Generation 2 software. These 13 SSRs were able to distinguish all clones; a reduced subset of 6 SSRs with the highest PIC (polymorphic information content) value were also sufficient to distinguish all 54 potato genotypes. Therefore, this subset of 6 SRRs markers could be a used for legal protection of newly released potato cultivars as part of the cultivar description for PVP.

Retrospective identification of hybridogenic walnut plants by SSR fingerprinting and parentage analysis

Juglans × intermedia (Juglans nigra × Juglans regia) is considered the prototype walnut for quality wood production in Europe. Hybridization between the parental species is rare under natural conditions and difficult using controlled pollination because of phenological and genetic incompatibilities. The identification of hybridogenic parents is the first step toward obtaining hybrid progeny. We report the application of microsatellite markers for DNA fingerprinting and parentage analysis of half-sib families collected in a natural mixed population for which no phenological and morphological data were available. Ten nuclear, neutral, simple sequence repeat markers were used to analyse 600 samples. The high levels of polymorphism detected positively influenced the exclusion and identity probabilities. The assignment analysis revealed the presence of 198 diploid J. × intermedia hybrids among the seedling progeny. Maternity checks were performed on all individuals and few errors of sampling were found. Four distinct hybridogenic mother trees were identified, each showing different reproductive success rates. The 198 diploid hybrids belonged to four open-pollinated families based on an analysis of paternity using a likelihood approach. Differential male reproductive success was observed among pollen donors within the research site. Forty-nine of the 198 diploid hybrids detected in four progenies were sired by only three J. regia genotypes. Backward selection might be used to establish new seed orchards for inter-specific F1 hybrid production using genotypes with demonstrated compatibility.

Discovery of a sexual cycle in the opportunistic fungal pathogen Aspergillus fumigatus

Aspergillus fumigatus is a saprotrophic fungus whose spores are ubiquitous in the atmosphere. It is also an opportunistic human pathogen in immunocompromised individuals, causing potentially lethal invasive infections, and is associated with severe asthma and sinusitis. The species is only known to reproduce by asexual means, but there has been accumulating evidence for recombination and gene flow from population genetic studies, genome analysis, the presence of mating-type genes and expression of sex-related genes in the fungus. Here we show that A. fumigatus possesses a fully functional sexual reproductive cycle that leads to the production of cleistothecia and ascospores, and the teleomorph Neosartorya fumigata is described. The species has a heterothallic breeding system; isolates of complementary mating types are required for sex to occur. We demonstrate increased genotypic variation resulting from recombination between mating type and DNA fingerprint markers in ascospore progeny from an Irish environmental subpopulation. The ability of A. fumigatus to engage in sexual reproduction is highly significant in understanding the biology and evolution of the species. The presence of a sexual cycle provides an invaluable tool for classical genetic analyses and will facilitate research into the genetic basis of pathogenicity and fungicide resistance in A. fumigatus, with the aim of improving methods for the control of aspergillosis. These results also yield insights into the potential for sexual reproduction in other supposedly 'asexual' fungi.

Molecular markers for biodiversity analysis of wildlife animals: a brief review

Molecular markers are indis­pensable tools for determining the genetic variation and biodiversity with high levels of accuracy and repro­ducibility. These markers are mainly classified into two types; mitochondrial and nuclear markers. The widely used mitochondrial DNA markers with decreasing order of conserved sequences are 12S rDNA &gt; 16S rDNA &gt; cytochrome b &gt; control region (CR); thus the 12S rDNA is highly conserved and the CR is highly variable. The most commonly used nuclear markers for DNA fingerprinting include random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and microsatellites. This short review narrates the application of these molecular markers for biodiversity analysis of wildlife animals.

Utility of Blueberry-derived EST-PCR Primers in Related Ericaceae Species

Expressed sequence tag-polymerase chain reaction (EST-PCR) markers for DNA fingerprinting and mapping in blueberry (Vaccinium sp.) had previously been developed from expressed sequence tags (ESTs) produced from a cDNA library, derived from RNA from floral buds of cold acclimated plants. Because EST-PCR markers are derived from gene coding regions, they are more likely to be conserved across populations and species than markers derived from random regions of DNA, such as randomly amplified polymorphic DNA (RAPD) or amplified fragment length polymorphism (AFLP) markers. In this study, we tested whether many of the EST-PCR primer pairs developed for blueberry are capable of amplifying DNA fragments in other members of the family Ericaceae. In addition, we cloned and sequenced a selection of 13 EST-PCR fragments to determine if they showed homology to the original blueberry cDNA clones from which the EST-PCR primer pairs were derived. Closely related cranberry genotypes (two wild selections of V. oxycoccus L. and two cultivars of V. macrocarpon Aiton, `Early Black' and `Stevens') and more distantly related rhododendron genotypes (one wild selection each of Rhododendron arboreum Marsh, R. maximum L., and R. ponticum L. and three complex species hybrids, `Sonata', `Grumpy Yellow', and `Roseum elegans') were used. Of 26 primer pairs tested in cranberry, 23 (89%) resulted in successful amplification and eight of those (35%) amplified polymorphic fragments among the cranberry genotypes. Of 39 primer pairs tested in rhododendron, 29 (74%) resulted in successful amplification and 21 of those (72%) amplified polymorphic fragments among the rhododendron genotypes. Approximately 50% of the 13 sequenced EST-PCR fragments were found to be homologous to the original blueberry cDNA clones. These markers should be useful for DNA fingerprinting, mapping, and assessing genetic diversity within cranberry and rhododendron species. The markers which are shown to be homologous to the blueberry cDNA clones by DNA sequencing should also be useful for comparative mapping and genetic diversity studies between some genera of the family Ericaceae.

Development of EST-PCR Markers for DNA Fingerprinting and Genetic Relationship Studies in Blueberry (Vaccinium, section Cyanococcus)

Because randomly amplified polymorphic DNA (RAPD) is the only type of molecular marker that has been used extensively in blueberry (Vaccinium spp.) for mapping and DNA fingerprinting of cultivars, there is a need to develop a new, robust marker system. Expressed sequence tags (ESTs) produced from a cDNA library, derived from RNA from floral buds of cold acclimated plants, were used to develop EST-PCR markers for blueberry. Thirty clones, picked at random from the cDNA library, were single-pass sequenced from the 5' and 3' ends. Thirty PCR primer pairs were designed from the ends of the best quality sequences that were generated and were tested in amplification reactions with genomic DNA from 19 blueberry genotypes, including two wild selections (the original parents of a mapping population), and 17 cultivars. Fifteen of the 30 primer pairs resulted in amplification of polymorphic fragments that were detectable directly after ethidium bromide staining of agarose gels. Several of the monomorphic amplification products were digested with the restriction enzyme AluI and approximately half resulted in polymorphic-sized fragments (cleaved amplified polymorphic sequences or CAPS markers). The polymorphic EST-PCR and CAPS markers developed in this study distinguished all the genotypes indicating that these markers should have general utility for DNA fingerprinting and examination of genetic relationships in blueberry. Similarity values were calculated based on the molecular marker data, and a dendrogram was constructed based on the similarity matrix. Coefficients of coancestry were calculated for each pair of genotypes from complete pedigree information. A fair correlation between similarity coefficients calculated from marker data and coefficients of coancestry was found.

Allelic diversity of simple sequence repeats among elite inbred lines of cultivated sunflower.

Simple sequence repeat (SSR) markers were developed for cultivated sunflower (Helianthus annuus L.) from the DNA sequences of 970 clones isolated from genomic DNA libraries enriched for (CA)n,, (CT)n, (CAA)n, (CATA)n, or (GATA)n. The clones harbored 632 SSRs, of which 259 were unique. SSR markers were developed for 130 unique SSRs by designing and testing primers for 171 unique SSRs. Of the total, 74 SSR markers were polymorphic when screened for length polymorphisms among 16 elite inbred lines. The mean number of alleles per locus was 3.7 for dinucleotide, 3.6 for trinucleotide, and 9.5 for tetranucleotide repeats and the mean polymorphic information content (PIC) scores were 0.53 for dinucleotide, 0.53 for trinucleotide, and 0.83 for tetranucleotide repeats. Cluster analyses uncovered patterns of genetic diversity concordant with patterns produced by RFLP fingerprinting. SSRs were found to be slightly more polymorphic than RFLPs. Several individual SSRs were significantly more polymorphic than RFLP and other DNA markers in sunflower (20% of the polymorphic SSR markers had PIC scores ranging from 0.70 to 0.93). The newly developed SSRs greatly increase the supply of sequence-based DNA markers for DNA fingerprinting, genetic mapping, and molecular breeding in sunflower; however, several hundred additional SSR markers are needed to routinely construct complete genetic maps and saturate the genome.

Development of SCAR Markers for DNA Fingerprinting and Germplasm Analysis of American Cranberry

DNA fingerprinting has been useful for genotypic classification of American cranberry (Vaccinium macrocarpon Ait.). Polymerase chain reaction (PCR) based methodologies including randomly amplified polymorphic DNA (RAPD) markers are relatively easy to use, and inexpensive as compared to other methods. However, RAPD markers have some limitations including seamless interlaboratory transferability and susceptibility to certain types of error. An alternative method, sequence characterized amplified regions (SCARs), was developed for cranberry germplasm analysis. Nine primer sets were designed from RAPD-identified polymorphic markers for use in two multiplex PCR reactions. These primer sets generated 38 markers across a cranberry germplasm collection. Estimates of genetic relatedness deduced from employment of the RAPD and SCAR methods were compared among 27 randomly chosen cranberry germplasm accessions. Although both methods produced comparable results above 0.90 coefficient of similarity, branches below this level exhibited variation in clustering. SCAR and RAPD markers can be employed for identifying closely related genotypes. However, the inferences of more distant genetic relationships are less certain. SCAR marker reactions provided more polymorphic markers on a per reaction basis than RAPD marker reactions and as such more readily separated closely related progeny. When SCAR primers were fluorescent dye-labeled for computerized detection and data collection, reduced marker intensity relative to unlabeled reactions was one problem encountered.

Macro-, micromorphological characters and DNA fingerprinting markers on three Ruprechtia species (Polygonaceae) in Egypt

Macro-,micromorphological characters and DNA fingerprinting markers have been used to differentiate the threeRuprechtia C. A. Meyer species growing in Egypt (R. exelsa, R. polystachya, and R. salicifolia). Interestingly,the macro- and micromorphological criteria of the vegetative organs showed no fundamental characters todiscriminate between the three examined species. However, the data obtained from the seed coat surface (usingLM and SEM) revealed some variability among the tested species. Seed coat surface was colliculate in (R.excelsa), pusticulate in (R. polystachya) and reticulate–scalariform in R. salicifolia. Accordingly, the seed coatsurface can be used as a fundamental criterion to discriminate between the species of this study. The RAPD–PCR electrophoretic profile, showed unique RAPD markers some of which were species-specific. Speciesspecificmarkers were recorded by using primers OPA-01, OPA-02 and OPA-05 respectively. Two RAPDmarkers specific to R. polystachya and R. salicifolia were recorded. The two markers for the former species havea molecular size of 1800 and 2600 bp using primer OPA-01. While those specific to the latter species have amolecular size of 300 and 400 bp using primers OPA-02. For R. excelsa, one specific RAPD marker wasrecorded, it has a molecular size 800 bp using primer OPA-05. The RAPD markers may be considered as validcriteria to discriminate between the three Ruprechtia species used in this investigation. However, the similarityin the banding profile of RAPD-PCR and the morphological criteria justify the maintenance of the three speciesin the same genus.

The use of DNA markers for rapid improvement of crops in Africa

Genetic engineering and biotechnology are providing new tools for genetic improvement of food crops. Molecular DNA markers are some of these tools which can be used in various fields of plant breeding and germplasm management. For example, molecular markers have been used to confirm the identity of hybrids in breeding programmes. Another application of molecular markers is in determining phylogenetic relationships in related species. Information on phylogenetic relationships is useful in facilitating introgression of desirable traits from wild relatives to cultivated crop species. Molecular markers are also being used to construct genetic maps. A genetic map is a collection of genetic markers that have been grouped according to their linkage. Breeders can use DNA maps to carry out marker-assisted selection. This technique enables plants carrying desirable traits such as pest and disease resistance to be selected while still in the seedling stage. Ultimately, this enables the cloning of the genes to be used for crop improvement. The polymerase chain reaction (PCR) has become a popular technique for molecular genome mapping and the diagnosis of plant pathogens. The technique ensures amplification of specific DNA sequences by the use of primers and the enzyme Taq DNA polymerase. Restriction Fragment Length Polymorphisms (RFLPs), Random Amplified Polymorphic DNAs (RAPDs), microsatellites and Amplified Fragment Length Polymorphism (AFLP) are some of the most useful molecular markers for DNA fingerprinting. For viral, fungal and bacterial DNA fingerprinting and diagnosis as well as strain differentiation of rhizobia, PCR-RAPD and cDNA probes can be applied alongside with monoclonal antibodies. Key Words: Crop improvement, DNA polymorphism, marker-assisted selection (African Crop Science Journal 2000 8(1): 99-108)

Genetic variation in three Ceratocystis species with outcrossing, selfing and asexual reproductive strategies

SummaryNuclear and mitochondrial markers were used to examine variation within three closely related species of tree pathogens with differing reproductive strategies. Ceratocystis eucalypti is obligately outcrossing; Ceratocystis virescens is capable of selfing due to unidirectional mating type switching; and Chalara australis is an asexual species, comprised of a single mating type. When the nuclear DNA fingerprinting markers (CAT)5 and (CAG)5 were used as probes against Pst I‐restricted DNA, isolates of C. eucalypti were found to be highly polymorphic, and Ch. australis showed very little polymorphism. The selfing C. virescens showed an intermediate level of variation in the nuclear fingerprint markers, and much of the variation appeared to be due to differences between two forms of the species, one pathogenic to Acer and Liriodendron and another less‐pathogenic form on Fagus and other hardwoods. Mitochondrial DNA (mtDNA) polymorphisms were examined by digesting total DNA with Hae III or CfoI, and C. eucalypti showed somewhat more variation in mtDNA than did C. virescens. The only polymorphism seen in the mtDNA of Ch. australis was associated with a plasmid. Selfing in C. virescens may be common and could explain an intermediate level of diversity when compared to the obligately outcrossing C. eucalypti and the asexual Ch. australis.

A novel method for estimating heritability using molecular markers

Heritability is usually estimated with individuals of known relatedness generated using a controlled breeding programme or through response to selection. In this paper, we use two single-locus VNTR DNA fingerprint markers in conjunction with a maximum likelihood method to infer relatedness among pairs of individuals in a captive population of Pacific chinook salmon (Oncorhynchus tshawytscha). Patterns of relatedness inferred from the two DNA fingerprint markers were used to estimate heritability for, and genetic correlations among, several economically and ecologically important traits (weight, length, flesh colour and precocious male maturation). Heritabilities ranged from 0.20 for weight, 0.38 for length, 0.67 for precocious male maturation (‘jacking’) to 0.76 for flesh colour, which are in good agreement with estimates for salmonids generated using classical quantitative genetic methods. This molecular marker-based method allows for the estimation of heritability in wild, long-lived species not easily manipulated for study using controlled breeding programmes.

AFLP markers for DNA fingerprinting in cattle.

This work reports on use of the recently described amplified fragment length polymorphism (AFLP) technology for DNA fingerprinting in cattle. The AFLP technology produces molecular markers through the high-stringency polymerase chain reaction (PCR)-amplification of restriction fragments that are ligated to synthetic adapters and amplified using primers, complementary to the adapters, which carry selective nucleotides at their 3' ends. While, for plants, the double digestion of genomic DNA with EcoRI and MseI is suggested, in mammals the enzyme combination EcoRI/TaqI produces clearer and more polymorphic AFLP patterns. In a sample of 47 Italian Holstein genotypes, 16 EcoRI/TaqI primer combinations identified 248 polymorphic bands in a species known for its low level of restriction polymorphism. In spite of the low information content carried by each AFLP polymorphism (average polymorphism information content = 0.31), the number of fragments revealed by each primer combination increased significantly the level of genetic information gained in each experiment. AFLP patterns are reproducible in independent experiments and polymorphic fragments segregate in cattle families according to Mendelian rules.

Relatedness measured by oligonucleotide probe DNA fingerprints and an estimate of the mating system of Sea Lavender (Limonium carolinianum)

Using DNA fingerprint markers within species and populations of wild plants requires information on the relationship between fingerprint similarity and relatedness. We identified a hypervariable marker based on oliog(GATA)4-hybridization of DpnII-cut genomic DNA from Sea Lavender (Limonium carolinianum). Banding patterns were somatically stable and highly variable among unrelated individuals. Band molecular-weight sizing errors (as a percent of band molecular weight) were estimated at 0.44%±0.003 within gels and 0.76%±0.964 between gels. Band sizing errors defined a 99% confidence bin of ±0.95% (1.90% total) of molecular weight. Band-sharing estimates were based on this bin size and on variance estimates that compensate for non-independent comparisons. Band-sharing among nine unrelated individuals (θ) was 0.198±0.O11. Experimental pollinations designed to produce selfed, fulland half-sib progeny groups led to five selfed progeny groups and no outcrossed progeny (mean band-sharing, ovS=0.468±0.074). A linear regression between band-sharing (S) and relatedness (r) assuming 17% inbreeding was r=0.006+0.914*S (R(2)=0.973) and established the maximum amount of inbreeding. ovS(0.392±0.022) estimated from wild pollinated seeds from four maternal families was intermediate to unrelated individuals and experimental selfed progeny, giving evidence for mixed mating in wild plants. More extensive plant pedigrees with known levels of inbreeding will be needed to measure variation in the relationship between S and r among populations and families.

DNA fingerprinting confirms isogenicity of androgenetically derived rainbow trout lines.

Homozygous and hybrid clonal lines of rainbow trout (Oncorhynchus mykiss) were confirmed to be isogenic using multilocus DNA fingerprinting. Homozygous clones were produced by androgenesis and gynogenesis using gametes from androgenetic male and female rainbow trout, respectively. Isogenic F1 hybrid lines were produced by crossing homozygous fish from different strains. One line of hybrid clones showed segregation for maternally inherited DNA fingerprint markers. The female from this cross, the only presumptive homozygous gynogenetic individual used in this study, was thought to have been produced by gynogenesis followed by blockage of the first cleavage division, but based on the DNA fingerprint analysis, apparently was derived by spontaneous polar body retention that maintained heterozygosity at some loci. Mutations at DNA fingerprint loci were not observed, indicating relative stability of fingerprint loci in the clonal lines. DNA fingerprinting appears to be a useful tool for identifying and genetically monitoring clonal lines of rainbow trout. Isogenic lines of rainbow trout will facilitate the production of saturated genetic maps for rainbow trout and enhance such endeavors as quantitative trait locus (QTL) analysis and loss of heterozygosity (LOH) studies in tumors.

Random amplified polymorphic DNA markers for DNA fingerprinting and genetic variability assessment of minute parasitic wasp species (Hymenoptera: Mymaridae and Trichogrammatidae) used in biological control programs of phytophagous insects

Biological control of insects that feed on our crops has become more practical in recent years by mass release of egg parasitoid microhymenoptera. Trichogramma species are now commercially reared and spread in commercial fields to control specific insect pests. Microhymenoptera species are, however, very small and morphologically indistinguishable within species, although strains of a given species differ in their efficiency to control specific insect pests. Traditional taxonomy is unable to differentiate microhymenoptera species at the strain level. It is becoming increasingly important to develop a reliable system to monitor genetic variations both within and between strains of commercially important microhymenoptera, to detect genetic drift occurring during several generations of multiplication, to protect patents, and to certify the lots of commercially released microhymenoptera. We have developed a system based on DNA markers to rapidly characterize individuals of five species of microhymenoptera from the genus Anaphes and Trichogramma including a new species of Anaphes not previously described. The main components of our system are a rapid and simple DNA micro-extraction method and fast DNA polymorphism analyses based on random amplified polymorphic DNA markers.

Development and use of striped bass‐specific RFLP probes

We sought to develop nuclear DNA (nDNA) probes which could be used to complement mtDNA and DNA fingerprinting markers in distinguishing striped bass, Morone saxatilis (Walbaum), from discrete spawning systems. Restriction endonuclease‐generated single copy, 10–20‐kb striped bass nuclear nDNA fragments were cloned into the bacteriophage vector Lambda Dash II and tested in Southern blot analyses for their abilities to reveal population‐specific polymorphisms. Three of the I7 nDNA sequences tested exhibited polymorphisms which potentially could be used to delineate striped bass populations. One probe, DSB 22, revealed significant genotypic frequency differences between Gulf of Mexico and Atlantic striped bass and among striped bass representative of some Atlantic systems. These preliminary results suggest that single copy nDNA sequences may provide sufficient polymorphisms to aid in stock identification of species which proved genetically monomorphic using other approaches.

M13 phage DNA as a universal marker for DNA fingerprinting of animals, plants and microorganisms

Hypervariable polymorphic patterns were detected with M13 phage DNA as a probe in genomic DNA of organisms belonging to different taxonomic groups including animals (vertebrates and invertebrates), plants and microorganisms. Individual-specific restriction pattern analysis (DNA fingerprinting) with this probe proved to be useful for individual identification, analysis of somatic stability and paternity testing in man. The nuclear type of inheritance indicates that the hypervariable DNA regions in question are located in the chromosomes, not in the mitochondrial DNA. The data obtained also demonstrate a potential range of M13 DNA applications as a probe for DNA fingerprinting of animals, plants and microorganisms, particularly for the determination of inbred lines, identification of bacterial strains and establishing stock, variety and strain distinctions.

Common Buckwheat-based EST Primers in the Genome of other Species of &lt;i&gt;Fagopyrum&lt;/i&gt;

If the EST primers designed for one species can be used in related species, then the cost involvedin developing markers for DNA fingerprinting, genetic relationship studies, mapping, etc. forother species is significantly reduced. We tested the applicability of 17 EST primers developedfrom common buckwheat in other wild and cultivated Fagopyrum species. A total of 18accessions consisting of 4 subspecies and 2 species were used. Sequences of 93 cDNA cloneswere used to design primers using Primer3. Amplification products were different in bandintensity. In most of the cases, the bands of F. homotropicum were with high intensity. Allprimers showed single band except in Accession C9022. Three primers 23, 31 and 69 producedvery clear singe band. All primers amplified the genomic DNA of F. homotropicum (2x). Eightprimers amplified the DNA of all accessions. Results indicated that the transferability of ESTmarkers developed for common buckwheat decreased with an increase in genetic distancebetween them.Key words: Common buckwheat; EST markers; Fagopyrum species; transferabilityDOI: 10.3126/narj.v7i0.1863Nepal Agriculture Research Journal Vol.7 2006 pp.27-36