Genetic variation in agronomically important species of Stylosanthes determined using random amplified polymorphic DNA markers

Summary Random amplified polymorphic DNA (RAPD) markers were generated from 20 cultivars and accessions representing four agronomically important species of Stylosanthes, S. scabra, S. hamata, S. guianensis, and S. humilis. Approximately 200 fragments generated by 22 primers of arbitrary sequence were used to assess the level of DNA variation. Relatively low levels of polymorphism (0–16% of total bands in pairwise comparisons) were found within each species, while polymorphisms between the species were much higher (up to 46%). Very few polymorphisms (0–2%) were detected between the individuals of the same cultivar or accession. A phenogram of relationships among the species was constructed based on band sharing. Four main clusters corresponding to each species were readily distinguished on this phenogram. The allotetraploid species S. hamata and its putative diploid progenitor, S. humilis, were more similar to each other than to S. scabra and S. guianensis. No variation in RAPD markers was found between the two commercial S. hamata cvs Verano and Amiga. Cultivar Oxley in S. guianensis was considerably different from the other cultivars and accessions of this species. The phylogenetic distinctions obtained with RAPDs were in agreement with other studies from morphology, cytology, and enzyme electrophoresis. The low level of polymorphisms observed within each species suggested that interspecific crosses may be a better vehicle for the construction of RAPD linkage maps in Stylosanthes.     

Genetic relatedness among Trypanosoma evansi stocks by random amplification of polymorphic DNA and evaluation of a synapomorphic DNA fragment for species-specific diagnosis.

In this study we employed randomly amplified polymorphic DNA patterns to assess the genetic relatedness among 14 Brazilian Trypanosoma evansi stocks from domestic and wild hosts, which are known to differ in biological characteristics. These akinetoplastic stocks were compared with one another, to three Old World (Ethiopia, China and Philippines) dyskinetoplastic stocks of T. evansi, and also with Trypanosoma equiperdum, Trypanosoma brucei brucei, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. Randomly amplified polymorphic DNA analysis showed limited heterogeneity in T. evansi stocks from different hosts and geographical regions of the world, or in other species of the subgenus Trypanozoon. However, minor variations generated random amplification of polymorphic DNA analysis disclosed a pattern consisting of a unique synapomorphic DNA fragment (termed Te664) for the T. evansi cluster that was not detected in any other trypanosome species investigated. Pulsed field gel electrophoresis analysis demonstrated that the Te664 fragment is a repetitive sequence, dispersed in intermediate and minichromosomes of T. evansi. Based on this sequence, we developed a conventional PCR assay for the detection of T. evansi using crude preparations of blood collected either on glass slides or on filter paper as template DNA. Our results showed that this assay may be useful as a diagnostic tool for field-epidemiological studies of T. evansi.     

Detection of somaclonal variation in cultured rice cells using digoxigenin-based random amplified polymorphic DNA

A procedure for the non-radioactive detection of random amplified polymorphic DNA (RAPD) was developed and designated as digoxigenin (DIG)-based RAPD. Using this procedure, we analyzed somaclonal variation in cultured cells of rice. Somaclonal variation was found to increase with culture age. More than 50 polymorphic fragments were identified with the four primers tested. Random sequencing of 10 clones generated one intron, one 5′-noncoding, and eight non-redundant expressed sequences. A database search for homology showed that the eight exon sequences displayed a significant similarity to sequences already stored in EMBL, GenBank and DDBJ. The sources of the known genes ranged from microorganism to human, including three rice genes. The results showed that somaclonal variation might have occurred in transfer RNA, ribosomal protein, and other genes during cell culture. Received: 14 November 1997 / Revision received: 21 August 1998 / Accepted: 31 August 1998     

Diversity of DNA sequences among Vibrio cholerae O139 Bengal detected by PCR-based DNA fingerprinting

Abstract Vibrio cholerae O139, a causative agent of a large epidemic of cholera-like illness, has suddenly emerged and spread widely over several months. To investigate the characteristics unique to O139, traditional typing techniques for V. cholerae , such as biochemical characteristics, antibiotic susceptibility and detection of toxin production, were performed, with the result that 145 O139 strains, except for two O139 strains isolated from Argentina and Germany, were indistinguishable from O1 strains. Thus, in order to clarify the genetical relatedness among O139 strains, and between O139 and O1 strains, the RAPD (random amplified polymorphic DNA) DNA fingerprinting method was undertaken. Although the RAPD arrays in five O139 isolates from Vellore with one arbitrary primer were slightly different from the other O139 strains, the RAPD patterns of the 145 forty-five O139 strains except for two O139 strains from Argentina and Germany were quite similar to each other, but were different from those of O1 strains, indicating that those O139 epidemic strains are closely related to each other regardless of their place of isolation. Furthermore, the RAPD patterns of the O139 strains resembled those of E1 Tor strains rather than classical strain, and a small change in the RAPD pattern of O139 strains occurred during subculture for 200 generations. These results taken together suggested that O139 V. cholerae have emerged from a common origin associated with the E1 Tor strain.     

RAPD-based genetic linkage map of blueberry derived from a cross between diploid species (Vaccinium darrowi and V. elliottii)

An initial genetic linkage map for blueberry has been constructed from over 70 random amplified polymorphic DNA (RAPD) markers that segregated 11 in a testcross population of 38 plants. The mapping population was derived from a cross between two diploid blueberry plants: the F₁ interspecific hybrid (Vaccinium darrowi Camp x V. elliottii Chapm.) and another V. darrowi plant. The map currently comprises 12 linkage groups (in agreement with the basic blueberry chromosome number) and covers a total genetic distance of over 950cM, with a range of 3–30cM between adjacent markers. The use of such a map for identifying molecular markers linked to genes controlling chilling requirement and cold hardiness is discussed.     

Genotyping of Ganoderma species by improved random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis

Species of Ganoderma are used in traditional medicines. An improved random amplified polymorphic DNA (RAPD) analysis, where the RAMP time is prolonged, has been used to characterize the genetic variation in some well known species of Ganoderma. The DNA materials were collected from ten Ganoderma strains, amplified with randomly selected 24 RAPD primers and evaluated by agarose gel electrophoresis. A cluster dendrogram was constructed for genetic analysis on the basis of amplification results. The improved RAPD amplified DNA with consistent and clear banding patterns. A total of 316 bands were found with 93% polymorphism. There was a significant genetic distance between the different strains of Ganoderma, with an index of similarity coefficient in the range of 0.52–0.74. The inter-simple sequence repeat (ISSR) analysis of the Ganoderma DNA samples showed similar trend results to the RAPD analysis with 0.49–0.81 similarity coefficients. This study reports the high level of genetic differences between different species or strains of a single species of Ganoderma and confirms the significance of the improved RAPD method in genetic characterization of organisms. Therefore, the improved RAPD combined with ISSR techniques might be used for the genetic characterization of organisms.     

Relationships among genetic distance,forage yield and heterozygosity in isogenic diploid and tetraploid alfalfa populations

Isogenic diploid and tetraploid alfalfa (Medicago sativa L.) was studied with molecular markers to help understand why diploid performance and breeding behavior does not always predict that of tetraploids. In a previous study of partially heterozygous alfalfa genotypes, we detected a low correlation between yields of isogenic diploid (2x) and tetraploid (4x) single-cross progenies, and genetic distances were more highly correlated with yields of tetraploids than diploids. These differences may be related to the level of RFLP heterozygosity expected among progenies derived from heterozygous parents at the two ploidy levels. The objectives of this study were to determine the relationships among genetic distance, forage yield and heterozygosity in isogenic 2 x and 4 x alfalfa populations. Four diploid genotypes were chromosome doubled to produce corresponding isogenic autotetraploids, and these genotypes were mated in 4 × 4 diallels to produce 6 single-cross families at each ploidy level for field evaluation. Allele compositions of parents were determined at 33 RFLP loci by monitoring segregation of homologous restriction fragments among individuals within progenies, and these were used to estimate RFLP heterozygosity levels for all single-cross progenies at both ploidy levels. RFLP heterozygosity rankings were identical between progenies of isogenic diploid and tetraploid parents; but significant associations (P < 0.05) between estimated heterozygosity levels and forage yield were detected only at the tetraploid level. Since tetraploid families were nearly 25% more heterozygous than the corresponding diploid families, inconsistencies in the association between molecular marker diversity and forage yields of isogenic 2 x and 4 x single crosses may be due to recessive alleles that are expressed in diploids but masked in tetraploids. The gene action involved in heterosis may be the same at both ploidy levels; however, tetraploids benefit from greater complementary gene interactions than are possible for equivalent diploids. Present address: AgResearch Grasslands, New Zealand Pastoral Agriculture Research Institute, Palmerston North, New Zealand     

Effects of topography on dispersal of black-billed magpie Pica pica sericea revealed by population genetic analysis

This study was conducted using random amplified polymorphic DNA analysis to examine whether the effects of topographical structure on the dispersal of black-billed magpie Pica pica sericea are reflected in its population genetic structure. The black-billed magpie is typically seen in the lowlands and is remarkably sedentary in Korea and Japan. The unweighted pair-group method of clustering analysis showed two main clusters: five populations in the western region of the Bekdudegan mountains (WRBM) in one cluster and five populations in the eastern region of the Bekdudegan mountains (ERBM), including the Japanese population in the other cluster. The populations in WRBM appeared to be diverged from the populations in ERBM. Analysis of molecular variance revealed that the populations in ERBM were more genetically divergent from each other than were those in WRBM. The high-rising mountains, very rugged topographical features and the sea in ERBM may have resulted in a lower dispersal rate and larger genetic variation among populations in ERBM compared to those in WRBM. These results suggest that the topographical structure may have an influence on the dispersal and population genetic structure of the black-billed magpie. Electronic Publication     

Staphylococcus pasteuri-specific oligonucleotide probes derived from a random amplified DNA fragment

Abstract A rapid polymerase chain reaction method was developed to differentiate Staphylococcus pasteuri from other staphylococcal species, especially the phenotypically similar S. warneri . The oligonucleotide probes used as primers were designed from the sequence of a S. pasteuri random amplified polymorphic DNA fragment.     

Typing of Staphylococcus epidermidis strains by random amplification of polymorphic DNA

Abstract The polymerase chain reaction was used to obtain randomly amplified polymorphic DNA profiles for typing of Staphylococcus epidermidis strains. Epidemiologically unrelated S. epidermidis isolates were screened with randomly amplified polymorphic DNA analysis. The discriminating ability of 45 randomly designed 10-mer primers was assessed. The highest discriminatory power was obtained with the 10-mer oligonucleotide OPAM-12. In typing a total of 13 unrelated S. epidermidis strains with OPAM-12,11 different banding profiles were obtained reproducibly by agarose gel electrophoresis. The discriminatory power of the method with OPAM-12 was estimated using the D value of Hunter and Gaston (1988) to be 0.961. A reproducibility index of 1 was obtained after typing a total of 40 cultures including 12 triplicates and one quadruplicate of the 13 unrelated strains. Following the described procedure, the randomly amplified polymorphic DNA method provided a rapid, simple and reproducible alternative to other S. epidermidis typing systems.     

Random amplified polymorphic DNA (RAPD) markers in hop, Humulus lupulus: level of genetic variability and segregation in F1 progeny

The level of genetic variation in 24 hop genotypes was studied using the recently developed technique for producing random amplified polymorphic DNAs (RAPDs). Of the 60 primers screened, eight produced polymorphic RAPD bands, 38 produced bands that were monomorphic for all genotypes and 19 did not produce any amplification product. It appeared that the level of polymorphism among the genotypes was generally low. Three of the primers, A11, A17 and C9, were used to determine the stability and segregation of RAPD markers in five families with a total of 182 F₁ progeny. The segregation ratios of these markers in the f₁ progeny suggested that they were inherited in a Mendelian manner. RAPD markers were stable and may be useful for the construction of linkage maps in hop.     

Strain typing of Lentinula edodes by random amplified polymorphic DNA assay

Abstract Single 10-base primers were used to generate randomly amplified polymorphic DNA (RAPD) markers in the shiitake mushroom, Lentinula edodes . Seven primers produced polymorphisms in all 15 strains tested, producing 12–19 bands ranging from 0.34 to 2.52 kb. Thirteen of the 15 strains had unique DNA fingerprints, whereas L. edodes ATCC 28759 and ATCC 28760 exhibited identical RAPD profiles for all the primers. Molecular-genetic markers obtained with the RAPD assay can be used to differentiate strains of L. edodes and have potential applications in mushroom breeding and strain improvement programs.     

Evaluation of genetic relationships in Dalbergia species using RAPD markers

Conservation of identified germplasm is an important component forefficient and effective management of plant genetic resources. Traditionally,species identification has relied on morphological characters like growth habit,floral morphology like flower colour, and agronomic characteristics of the plant.Dalbergia species are important wind-dispersed tropicaltimber trees which exhibit high intrafruit seed abortion because of intensesibling competition for maternal resources. Studies were undertaken foridentification and genetic relationships in five species ofDalbergia and to evaluate genetic diversity withinpopulations of Dalbergia sisso, D.latifolia, D. paniculata, D.assamica and D. spinosa by using randomamplified polymorphic DNAs (RAPD) markers. Analysis was started by using 30decamer primers that allowed to distinguish five species and to select a reducedset of primers. The selected primers were used for identification and forestablishing a profiling system to estimate genetic relationships and toevaluate the genetic variability among the individuals in a population ofDalbergia species. A total of 120 distinct DNA fragments(bands), ranging from 0.3 to 4.0 kb, were amplified byusing nine selected random decamer primers. The genetic similarity was evaluated onthe basis of presence or absence of bands, which revealed a wide range ofvariability within the species. The cluster analysis indicated that five speciesof Dalbergia formed two major clusters. The first clusterconsisted of D. spinosa, D. latifolia and D.sisso. The second cluster was represented by two species, i.e.D. paniculata and D. assamica.A maximum similarity of 60% was observed in D. paniculata andD. assamica and they formed a minor cluster.Dalbergia latifolia and D. sissoformed another minor cluster with more than 50% similarity. Dalbergiaspinosa shared up to 40% similarity with D.latifolia and D. sisso. All the species sharemore than 20% similarity among themselves. The closest genetic distance existedwithin populations of different Dalbergia species. Thus,these RAPD markers have the potential for conservation of identified clones andcharacterization of genetic relatedness among the species. This is also helpful intree breeding programs and provides an important input into conservation biology.     

Varietal identification and assessment of genetic relationships in Hippeastrum using RAPD markers

The National Botanical Research Institute (NBRI) in Lucknow, India, maintains germplasm of Hippeastrum, a beautiful summer blooming ornamental. Germplasm collections comprise NBRI hybrids developed through selective breeding, hybrids with unknown parentage, local species, and Dutch hybrids for research purposes. Considering the importance of protecting plant breeders’ rights for commercial exploitation of hybrids, a PCR-based technique (random amplified polymorphic DNA—RAPD) was used to correctly identify known and unknown hybrids and to determine cultivar relatedness. RAPD profiles were used very successfully to trace and confirm the parentage of all the hybrids tested and to determine clear molecular relationships among varieties.     

Using randomly amplified polymorphic DNA for evaluating genetic relationships among papaya cultivars

Summary We have applied the recently developed technique of random amplification of polymorphic DNA (RAPD) to the analysis of the relationships among ten cultivars of papaya (Carica papaya L.). Eleven ten-base synthetic oligonucleotides were chosen that gave multiple PCR amplification products using papaya DNA as template. These 11 primers amplified a total of 102 distinct fragments. Cultivars were scored for presence or absence of RAPD fragments and grouped by cluster analysis using simple matching coefficients of similarity. A dendrogram of the ten cultivars was constructed. Of the ten cultivars seven were of the Hawaiian type, and all of these grouped to one branch of the tree. Divisions within the Hawaiian, branch were mostly consistent with the known genetic background of these cultivars. Three non-Hawaiian, cultivars were also analyzed. The minimum similarity detected was 0.7 suggesting that the domesticated papaya germ plasm is quite narrow. Our results show that RAPD technology is a rapid, precise and sensitive technique for genomic analysis.Journal series No. 3692 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Rapid identification of Streptococcus pneumoniae by PCR amplification of ribosomal DNA spacer region

Abstract Streptococcus pneumoniae is one of the important human pathogens in clinical microbiology. A polymerase chain reaction assay was designed to detect and identify S. pneumoniae through amplification of the ribosomal DNA spacer regions between the pneumococcal 16S-23S ribosomal RNA genes. Thirty-two Streptococcus and non- Streptococcus strains were tested to verify the specificity of the assay, and only S. pneumoniae strains gave a positive reaction. This method is a powerful technique for the rapid identification of S. pneumoniae .     

Optimization of primer screening for evaluation of genetic relationship in rose cultivars

Optimization of primer screening for evaluation of genetic relationship in 34 cultivars of rose through random amplified polymorphic DNA (RAPD) markers was investigated. Four series of decamer primers were used for screening and optimization of RAPD analysis between which A and N series performed good amplification of fragments as compared with other series. The primers OPN-07 and OPN-15 produced maximum number of DNA fragments in Rosa hybrida cv. Anuraag. Some primer either did not produce amplification or produced very poor amplification. Further, ten selected primers were used for genetic analysis of 34 rose cultivars. The primer OPN-15 amplified 21 fragments in all cultivars tested. A total of 162 distinct DNA fragments (bands) ranging from 100 to 3400 base pairs were amplified by using 10 selected random primers. The cluster analysis indicated that these rose cultivars formed nine clusters.     

Reproducibility of the differential amplification between leaf and root DNAs in soybean revealed by RAPD markers

 Random amplified polymorphic DNA (RAPD) was used to determine whether such markers can be employed for detecting genomic modification during plant development or under certain stress environments. Pairwise comparisons in RAPD patterns of leaf and root DNA amplifications were studied for 11 soybean accessions representing different origins. Hydroponic culture was used for the ease of harvesting roots. From a total of 40 primers screened, it was found that 16 can detect leaf DNA polymorphism, 19 for root DNA polymorphism, while 10 show a greater consistency for detecting polymorphism between leaf and root (L/R) DNAs. Nevertheless, problems were encountered when the newly synthesized oligo-primers and different thermal cyclers were used to check the data. Several factors were then tested for their reproducibility. The results indicated that the amplified differences between root and leaf DNAs are mostly not affected by template DNA concentrations. The addition of DMSO (dimethyl sulphoxide) or TMAC (tetramethyl-ammonium chloride) also did not mask the L/R differences. However, DNA polymerase and oligo-primers synthesized from different manufacturers, as well as the thermal cyclers, reacted differently sometimes. Regardless of the general problems of reproducibility in RAPD patterns, some amplified differences remain between the L/R DNAs. The most distinct patterns involve differences in the relative intensity of amplified bands. Differential amplification might have occurred during plant leaf and root development. Southern hybridization of the eluted polymorphic bands against restriction digestion of total genomic DNA confirms their being homologous to soybean DNA fragments. Polymorphism of these specific L/R differences also exists among varieties. RAPD should be a useful tool in detecting genomic alterations during plant development or under certain stress environments, as long as the factors affecting the reproducibility of RAPD patterns can be properly controlled. An additional cycle of selection would be possible if such a type of polymorphism is proved to be correlated with certain developmental characters. Received: 7 October 1996 / Accepted: 20 May 1997     

A simple method to estimate the percentage of hybridity in canola (Brassica napus) F1 hybrids

We have developed an efficient PCR-based system that uses RAPD markers for the certification of F₁ hybrids of canola. These markers were selected by screening five parental lines used in three crosses X, Y and Z with 131, 131 and 322 primers respectively. Stable DNA fragments that were homozygous and specific to the male inbreds were used to certify F₁ hybrid populations. The hybrid production system was based on self-incompatibility (SI) alleles that prevent self-pollination of the female parent. The efficiency of two S-alleles was compared under both field and greenhouse conditions. The percentage of hybridity was estimated in different F₁ populations. We found a significant difference between the two alleles for their efficiency in controlling selfing; both alleles were stable under greenhouse conditions, one allele appeared less reliable under field conditions.     

Characterization of Thinopyrum bessarabicum chromosome segments in wheat using random amplified polymorphic DNAs (RAPDs) and genomic in situ hybridization

Ten random amplified polymorphic DNA (RAPD) markers specific to chromosome 5E^b of Thinopyrum bessarabicum were detected. Genomic in situ hybridization and standard cytological observations revealed that six of the markers are located on the 5E^b short arm and four are located on the 5E^b long arm. These RAPD markers have been used to confirm the identity of putative 5E^b (5A) and 5E^b (5D) substitution individuals. The potential of RAPDs for the detection of wheat/alien recombinants is discussed.