Assessment of genetic variation withinBrassica campestris cultivars using amplified fragment length polymorphism and random amplification of polymorphic DNA markers

Genetic relationships were evaluated among nine cultivars ofBrassica campestris by employing random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers. RAPDs generated a total of 125 bands using 13 decamer primers (an average of 9.6 bands per assay) of which nearly 80% were polymorphic. The per cent polymorphism ranged from 60–100%. AFLP, on the other hand generated a total of 319 markers, an average of 64 bands per assay. Of these, 213 were polymorphic in nature (66.8%). AFLP methodology detected polymorphism more efficiently than RAPD approach due to a greater number of loci assayed per reaction. Cultivar-specific bands were identified, for some cultivars using RAPD, and for most cultivars with AFLP. Genetic similarity matrix, based on Jaccard’s index detected coefficients ranging from 0.42 to 0.73 for RAPD, and from 0.48 to 0.925 for AFLPs indicating a wide genetic base. Cluster analyses using data generated by both RAPD and AFLP markers, clearly separated the yellow seeded, self-compatible cultivars from the brown seeded, self-incompatible cultivars although AFLP markers were able to group the cultivars more accurately. The higher genetic variation detected by AFLP in comparison to RAPD was also reflected in the topography of the phenetic dendrograms obtained. These results have been discussed in light of other studies and the relative efficiency of the marker systems for germplasm evaluation.     

Use of random amplified polymorphic DNA analysis to detect genetic variation inPyrus species

Pyrus communis L. is the most important pear species for European production. Very few cultivars satisfy standards for fruit quality and clonal fidelity; thus, accurate verification of cultivar identity for checking propagation material and patent protection is important. We evaluated the randomly amplified polymorphic DNA (RAPD) technique for its ability to identify genetic differences among standard pear (Pyrus communis L.) cultivars, William, Passa Crassana, and Conference, and three gamma-ray induced variants. To identify genotype-specific markers, we used thirty 10-mer and two 11-mer sequences, annealing temperatures from 36–45°C, 2Taq polymerases (AmpliTaq and Stoffel fragment, both from former Perkin Elmer Cetus), and 2–4 replicate amplifications. Of the 32 primers (30 from Operon Technologies, Alameda, CA, USA), very few distinguished William from Passa Crassana, and only 1 could clearly differentiate all 3 cultivars. Two primers that did not reveal polymorphisms when used singly, generated polymorphic patterns that distinguished standard from gamma-ray-treated material when used in combination. We show that RAPD analyses can discriminate pear genotypes and suggest this technique as a reliable and inexpensive method for marker-facilitated screening of propagation material and for patent protection.     

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     

Study of genetic divergence among wheat genotypes through random amplified polymorphic DNA

The degree of genetic divergence was estimated in seven wheat genotypes, six exotic genotypes and one local variety, through random amplified polymorphic DNA methodology. A total of 112 DNA fragments were generated by the 15 random primers, with an average of about 7.4 bands per primer. Among the 112, 50 fragments showed polymorphism among the seven wheat genotypes. Nei and Li's similarity matrix ranged from 86.2 to 93.0%, which indicated a narrow genetic base among the genotypes. The maximum similarity, 93.0%, was observed between 12WLRG/1-12 and WL-43. The local variety, Chenab-70, showed the lowest similarity with the exotic types. We conclude that random amplified polymorphic DNA analysis can be used for the characterization and grouping of wheat genotypes; these results will be helpful in our wheat breeding program.     

随机扩增多态DNA（RAPD）标记用于丁香品种遗传分析及品种分类

采用随机扩增多态ＤＮＡ（ＲＡＰＤ）标记分析了１５个丁香品种的ＤＮＡ扩增产物。研究选用了１６个随机引物,共扩增出９６条带,其中５５条带为可重复性条带,有价值条带大小多在５１７ｂｐ至１６３６ｂｐ之间。这些标记足以区分这些丁香品种。欧丁香（Ｓｙｒｉｎｇａｖｕｌｇａｒｉｓ）与Ｓ．×ｈｙａｃｉｎｔｈｉｆｌｏｒａ间的相似系数为６１．５％,欧丁香与Ｓ．×ｐｒｅｓｔｏｎｉａｅ间的相似系数为４７．２％,Ｓ．×ｈｙａｃｉｎｔｈｉｆｌｏｒａ与Ｓ．×ｐｒｅｓｔｏｎｉａｅ间的相似系数为４３．６％。结果表明,欧丁香与Ｓ．×ｈｙａｃｉｎｔｈｉｆｌｏｒａ亲缘关系最近。应用ＲＡＰＤ资料分析讨论了一些品种的起源。ＲＡＰＤ技术为丁香品种分类鉴定提供了可靠方法。     

Genetic variability analysis among clinical Candida spp. isolates using random amplified polymorphic DNA

The patterns of genetic variation of samples of Candida spp. isolated from patients infected with human immunodeficiency virus in Vitória, state of Espírito Santo, Brazil, were examined. Thirty-seven strains were isolated from different anatomical sites obtained from different infection episodes of 11 patients infected with the human immunodeficiency virus (HIV). These samples were subjected to randomly amplified polymorphic DNA (RAPD) analysis using 9 different primers. Reproducible and complex DNA banding patterns were obtained. The experiments indicated evidence of dynamic process of yeast colonization in HIV-infected patients, and also that certain primers are efficient in the identification of species of the Candida genus. Thus, we conclude that RAPD analysis may be useful in providing genotypic characters for Candida species typing in epidemiological investigations, and also for the rapid identification of pathogenic fungi.     

Identification of a genetic marker associated with the resistance to Schistosoma mansoni infection using random amplified polymorphic DNA analysis

In schistosomiasis, the host/parasite interaction remains not completely understood. Many questions related to the susceptibility of snails to infection by respective trematode still remain unanswered. The control of schistosomiasis requires a good understanding of the host/parasite association. In this work, the susceptibility/resistance to Schistosoma mansoni infection within Biomphalaria alexandrina snails were studied starting one month post infection and continuing thereafter weekly up to 10 weeks after miracidia exposure. Genetic variations between susceptible and resistant strains to Schistosoma infection within B. alexandrina snails using random amplified polymorphic DNA analysis technique were also carried out. The results showed that 39.8% of the examined field snails were resistant, while 60.2% of these snails showed high infection rates.In the resistant genotype snails, OPA-02 primer produced a major low molecular weight marker 430 bp. Among the two snail strains there were interpopulational variations, while the individual specimens from the same snail strain, either susceptible or resistant, record semi-identical genetic bands. Also, the resistant character was ascendant in contrast to a decline in the susceptibility of snails from one generation to the next.     

Assessment of genetic variation within Indian mustard (Brassica juncea) germplasm using random amplified polymorphic DNA markers

Genetic diversity among 45 Indian mustard (Brassica Juncea L.) genotypes comprising 37 germplasm collections, five advance breeding lines and three improved cultivars was investigated at the DNA level using the random amplified polymorphic DNA (RAPD) technique. Fifteen primers used generated a total of 92 RAPD fragments, of which 81 (88%) were polymorphic. Of these, 13 were unique to accession 'Pak85559'. Each primer produced four to nine amplified products with an average of 6.13 bands per primer. Based on pairwise comparisons of RAPD amplification products, Nei and Li's similarity coefficients were calculated to evaluate the relationships among the accessions. Pairwise similarity indices were higher among the oilseed accessions and cultivars showing narrow ranges of 0.77-0.99. An unweighted pair-group method with arithmetic averages cluster analysis based on these genetic similarities placed most of the collections and oilseed cultivars close to each other, showing a low level of polymorphism between the accessions used. However, the clusters formed by oilseed collections and cultivars were comparatively distinct from that of advanced breeding lines. Genetically, all of the accessions were classified into a few major groups and a number of individual accessions. Advanced breeding lines were relatively divergent from the rest of the accessions and formed independent clusters. Clustering of the accessions did not show any pattern of association between the RAPD markers and the collection sites. A low level of genetic variability of oilseed mustard was attributed to the selection for similar traits and horticultural uses. Perhaps close parentage of these accessions further contributed towards their little diversity. The study demonstrated that RAPD is a simple and fast technique to compare the genetic relationship and pattern of variation among the gene pool of this crop.     

Genetic diversity among cultivars of spring barley revealed by random amplified polymorphic DNA (RAPD)

RAPD (random amplified polymorphic DNA) polymorphism was studied in 23 malting and non-malting spring barley cultivars included in the official list of Polish cultivated varieties. Twenty-four 10-mer primers were tested in each cultivar, giving altogether 149 amplification products, 45% of which were polymorphic. The number of polymorphic bands revealed by one primer ranged from 1 to 6, with an average of 2.8. Genetic distance for all pairs of compared varieties was estimated and a dendrogram was constructed using unweighted pair group method of arithmetic means. The genetic distance between cultivars ranged from 0.11 for cvs. Apex and Bryl to 0.62 for cvs. Orthega and Madonna. Of the seven malting cultivars only two (Brenda and Stratus) formed one group at D = 0.25. The genetic distance between cvs. Brenda and Scarlett, especially recommended for brewery, was equal to 0.34. The detected polymorphism appeared to be sufficient for assessing genetic distances between cultivars, but on the basis of this polymorphism groups of malting and non-malting cultivars were not clearly distinguished.     

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     

Identification and genetic homogeneity of Trichophyton tonsurans isolated from several regions by random amplified polymorphic DNA

Trichophyton tonsurans is an anthropophilic dermatophyte mostly causing tinea capitis and tinea corporis. This study was carried out to identify T. tonsurans and to clarify whether it has any genetic differences depending on the phenotype or region of isolation by random amplified polymorphic DNA (RAPD) analysis with three random primers. The assay was performed in 11 Korean, 2 Japanese, 2 Taiwanese, 5 Brazilian and 1 American isolates of T. tonsurans together with the other 10 anamorphic species of dermatophytes and 3 Arthroderma spp. All tested species of dermatophytes showed distinct bands and T. tonsurans was differentiated from other dermatophytes. It was most clearly ifferentiated from T. mentagrophytes by using primer 5-GAAGGCTCCC-3 (OPAO-15). No difference was found in RAPD band patterns in all strains of T. tonsurans with these random primers. It was considered that T. tonsurans is a genetically homogeneous species regardless of its isolation regions, morphologic or physiologic characteristics.This revised version was published online in October 2005 with corrections to the Cover Date.     

Rapid identification of genetic variation and pathotype of Leptosphaeria maculans by random amplified polymorphic DNA assay.

Canadian isolates of Leptosphaeria maculans, the causal agent of blackleg of crucifers, were examined for genetic relatedness by the random amplified polymorphic DNA assay. DNA polymorphisms amplified with random decamer primers were used to distinguish three groups of isolates. Group 1 contained all isolates of the virulent pathotype, group 2 contained isolates of the avirulent pathotype from western Canada, and group 3 contained avirulent pathotype isolates from Ontario. These results agreed with other reports which showed many genetic differences between pathotypes and were consistent with the hypothesis that the virulent pathotype was recently introduced into Canada and has diverged relatively little. In contrast, the avirulent pathotype has probably been present in Canada for a longer time and has diverged with geographic isolation. In addition to establishing genetic relationships, DNA fingerprints generated by the random amplified polymorphic DNA assay have potential applications in pathotype identification and blackleg disease management.     

Rapid identification of genetic variation and pathotype of Leptosphaeria maculans by random amplified polymorphic DNA assay.

Canadian isolates of Leptosphaeria maculans, the causal agent of blackleg of crucifers, were examined for genetic relatedness by the random amplified polymorphic DNA assay. DNA polymorphisms amplified with random decamer primers were used to distinguish three groups of isolates. Group 1 contained all isolates of the virulent pathotype, group 2 contained isolates of the avirulent pathotype from western Canada, and group 3 contained avirulent pathotype isolates from Ontario. These results agreed with other reports which showed many genetic differences between pathotypes and were consistent with the hypothesis that the virulent pathotype was recently introduced into Canada and has diverged relatively little. In contrast, the avirulent pathotype has probably been present in Canada for a longer time and has diverged with geographic isolation. In addition to establishing genetic relationships, DNA fingerprints generated by the random amplified polymorphic DNA assay have potential applications in pathotype identification and blackleg disease management.     

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.     

Phylogenetic study of bisexual Artemia using random amplified polymorphic DNA

Study of polymorphisms in the eukaryotic genome is an important way to discover the evolutionary relationships between species. Artemia (Crustacea, Anostraca) offers a very interesting model for evolutionary studies. In fact the genus, distributed all over the world in hundreds of known biotopes, comprises both bisexual sibling species and parthenogenetic populations easily available from the Artemia Reference Center of Ghent. In spite of great interest in it and its extensive use in aquaculture, little is known about relationships between the different species and intraspecific populations. Recently it has been demonstrated that polymorphisms in genomic fingerprints generated by arbitrarily primed polymerase chain reaction (PCR) can distinguish between strains in many organisms. We have used this technique to estimate the phylogenetic relationships existing between 14 populations living in the American continent, in the Mediterranean area, and in China. The principal coordinate analysis (PCO) obtained from 86 random amplified polymorphic DNA (RAPD) markers indicates that the populations analyzed can be divided into homogeneous clusters representing the four known bisexual species—the American A. franciscana and A. persimilis, the Mediterranean A. salina, and the A. species from China.     

Identification and genetic mapping of random amplified polymorphic DNA (RAPD) markers to the sheep genome

The random amplified polymorphic DNA (RAPD) assay utilizes the polymerase chain reaction (PCR) and short primers of arbitrary nucleotide sequence to amplify DNA. In this study, the RAPD assay was used to identify and map polymorphic markers in the AgResearch International Mapping Flock (IMF) sheep pedigrees. Sires and dams of eight of the full-sib IMF pedigrees were screened with 131 different 10-mer oligonucleotide primers. An average of 85 RAPD polymorphisms was identified between each parental pair, and 53 markers were contributed to the AgResearch IMF collaboration. Forty-five of the RAPD markers were mapped in the AgResearch IMF genetic linkage map, and at least one marker was located on 17 of the 26 autosomes and both sex chromosomes. Three lines of evidence were used to check for the homology of scored polymorphisms in different pedigrees, pedigree evaluation, segregation analysis, and Southern blot analysis. These results demonstrate that the RAPD assay is a powerful approach for identifying polymorphisms that can be used as markers for constructing a sheep genetic linkage map. Received: 5 October 1995 / Accepted: 16 April 1996     

Differentiation of Brucella species by random amplified polymorphic DNA analysis

Random amplification of polymorphic DNA (RAPD) was used for discrimination between 46 Brucella strains and 14 representatives of the alpha-2 and alpha-1 subgroups of Proteobacteria. To evaluate a relatively quick and exact method for Brucella identification, the authors specified the most suitable conditions for RAPD amplification of Brucella DNA with two 10-mer primers, containing lower and higher percentages of G and C. The software package PHYLIP 3.1 was used for cluster analysis of the RAPD fingerprints. The optimization of RAPD conditions resulted in PCR mixes suitable for reliable typing of Brucellae. The distance-based methods (Fitch-Margoliash, UPGMA and Neighbour-joining) gave clear discrimination between Brucella species. The constructed dendrograms put Br. canis and Br. suis bv. 1 in the same cluster and differentiated Brucella strains according to their host preferences. RAPD can be useful method to distinguish related bacterial species, and under strictly established conditions the reaction appears to be a simple, quick and sensitive technique for the epidemiological investigation of brucellosis.