DNA fingerprinting of Peronospora parasitica,a biotrophic fungal pathogen of crucifers

The fungus Peronospora parasitica (Pers. ex Fr.) Fr. is an obligate biotroph infecting a wide range of host species in the family Cruciferae. Isolates from different hosts are morphologically similar, and pathotypes are usually distinguished on the basis of host range. Random Amplified Polymorphic DNA (RAPD) fingerprints were generated from a range of P. parasitica isolates from different Brassica species. Reaction conditions, in particular DNA template, primer and Mg²⁺ concentrations, were optimized to ensure that amplifications were reproducible. Possible artefacts arising through host plant DNA were assessed by including such DNA in control reactions. Confirmation that diagnostic RAPD bands were generated from fungal DNA was also obtained by Southern hybridization of a RAPD band to genomic fungal DNA. By screening 20 decamer primers, 2 were found to detect sufficient genetic variation to allow complete differentiation between pathotypes. These results illustrate the potential value of RAPDs for detecting polymorphisms between isolates of a non-culturable plant pathogenic fungus.     

牛鞭草品种EST-SSR指纹图谱构建及遗传多样性分析

为探讨牛鞭草(Hemarthria spp.)品种间的遗传多样性,从86对EST-SSR引物中筛选出8对引物对牛鞭草属6个品种进行指纹图谱的构建及遗传多样性分析。结果表明,8对EST-SSR引物对牛鞭草属6个品种共扩增出193条清晰条带,多态性条带161条,多态性比例为83.4%。每条引物的多态信息含量(PIC)为0.480~0.695,平均为0.602。UPGMA聚类分析表明,牛鞭草属6个品种在相似系数为0.652处可分为两大类群。8对EST-SSR引物均能将6个品种完全区分开,以3对EST-SSR引物扩增的电泳图谱为基础,建立了牛鞭草属6个品种的指纹图谱标准模式图,每个品种都有唯一的指纹图谱。牛鞭草属6个品种的平均Nei’s基因多样性指数为0.333,平均Shannon信息指数为0.496,品种间的相似系数介于0.399~0.782之间。可见,牛鞭草属植物品种的遗传多样性较丰富,种间差异明显。     

Male reproductive tactics in the threespine stickleback– an evaluation by DNA fingerprinting

A random sample of 17 threespine stickleback nests was analysed using DNA fingerprinting. DNA from the guardian male and a random subsample of 10 fry per nest was probed with pYNZ132, a human single–locus VNTR probe which detects a multilocus fingerprint pattern in sticklebacks. Band–sharing indices (BSIs, the proportion of bands shared by two individuals) between the guardian male and its fry were calculated. In 147 of a total of 170 pair–wise comparisons the BSIs varied between 0.40 and 0.77. The guardian male was thought to be the true father of all these fry (p < 0.10). For the remaining 23 fry the BSIs varied between 0.09 and 0.34, suggesting that these fry were fathered by a different male (P<0.06). Once the paternal bands in each legitimate fry were determined, the remaining (i.e. maternal) bands among these fry were compared. Based on the BSIs obtained, the minimum number of females that spawned per nest was determined, and the maternal DNA fingerprints of the legitimate fry were traced back. In one nest five eggs of the sample had been fertilized by a sneaker, in two nests the guardian male had stolen eggs from a rival male, and in another nest one of the eggs was fertilized by a sneaker and three were stolen eggs.     

GENETIC VARIABILITY WITHIN A POPULATION AND BETWEEN DIPLOID/HAPLOID TISSUE OF MACROCYSTIS PYRIFERA (PHAEOPHYCEAE)1

Multi-locus DNA fingerprints using an M13 probe were obtained for eight individuals of giant kelp Macrocystis pyrifera (L.) C. Ag. collected from Monterey Bay, California. For each individual, DNA was extracted from a diploid blade and from ca. 10⁹ haploid spores that were released from four to Jive sporophylls. Viable or swimming spores from one individual were pooled and referred to as a spore group. A total of 34 bands (4–19 kb) was detected in DNA fingerprints from the eight blades and eight spore groups, with individual blade or spore groups exhibiting 7–18 bands (mean = 12.6). One band (4.5 kb) was present in all 16 samples. Eight bands were detected in 11–14 of the 16 samples. Similarity indices were calculated for all pairwise comparisons of fingerprint bands among all possible combinations of blades and spore groups. Mean similarity indices for the eight blades (0.51, SE = 0.032) and spore groups (0.56, SE = 0.031) were significantly lower than for the eight comparisons of the blade and spore groups from a single individual (0.86, SE = 0.052). The data indicate that DNA fingerprints can be used to measure genetic variation within populations of M. pyrifera because variation of DNA fingerprints associated with meiotic products (spores) of a given individual is small relative to variation observed among individuals within the population. Additionally, fingerprint variation between diploid vegetative tissue and haploid meiotic products may be a measure of genetic change due to recombination or DNA turnover mechanisms.     

GENETIC VARIABILITY AND PARENTAGE IN MACROCYSTIS PYRZFERA (PHAEOPHYCEAE) USING MULTI-LOCUS DNA FINGERPRINTING1

Multi-locus DNA fingerprints using an M13 probe were obtained for two individuals of Macrocystis pyrifera (L.) C. Ag. collected from Monterey Bay, California, and their laboratory-reared offspring. DNA was extracted from each of two field-collected individuals (= parents), their self-fertilized diploid offspring (three and seven individuals), and one diploid individual resulting from spores of the two parents. A total of 20 bands (4–19 kb) was detected among all individuals, ranging from 7 to 14 bands for any one individual. Two bands were present in all individuals, and three bands were unique to one parent and its three progeny. Ten bands were observed in the out-crossed individual, of which three were inherited from one parent, two from the other parent, and five were present in both parents. Genetic similarity between each parent and their self-fertilized offspring was significantly higher than similarity between the two self-fertilized groups. The data show that multi-locus DNA fingerprints can be used to assess parentage in the giant kelp and that there is consistent agreement between genetic similarity and known genetic relatedness among parents and offspring.     

Molecular phylogeny and DNA amplification fingerprinting of Petunia taxa

The relationship of five species of Petunia and ten cultivars of the cultivated petunia, Petunia x hybrida, were investigated using DNA-amplification fingerprinting (DAF). Reproducible banding profiles were obtained from P. parodii and P. axillaris DNA from different seed sources. In contrast, other petunias such as P. inflata, P. violacea and P. integrifolia produced variable fingerprints when different plants were examined. However, representative profiles of the variable Petunia taxa were obtained by bulking the leaf tissue from ten different individual plants. Each of ten octamer primers revealed polymorphic loci between taxa. Among a total of 201 bands produced, 146 (73%) loci were polymorphic and distinguished all species and cultivars. Phenetic and cluster analysis using DAF markers separated P. axillaris from P. parodii and distinguished between the violet-flowered species, P. inflata, P. violacea, and P. integrifolia. P. parodii grouped together with the monophyletic set of the ten cultivars of P. x hybrida examined, indicating that it had made a major contribution to the development of these cultivars. Cultivars were distributed within the dendograms by flower color. The results demonstrated the utility of DAF in establishing relationships among closely related species and cultivars of Petunia.     

皂荚种质资源SCoT遗传多样性分析及指纹图谱的构建

采用SCoT标记分析了18个皂荚种质的遗传多样性,并采用UPGMA法对18个皂荚种质进行了聚类分析。在此基础上,通过筛选出的多态性条带构建了18个皂荚种质的SCoT指纹图谱。扩增结果表明:从51个SCoT引物中筛选了15个引物进行PCR扩增,共扩增出226条带,其中多态性条带216条,多态性比率为96.61%。各引物多态性信息含量(PIC)、观测等位基因数(Na)、有效等位基因数(Ne)、Nei’s基因多样性指数(H)和Shannon’s信息指数(I)的平均值分别为0.875 9、1.964 9、1.440 1、0.272 6、0.426 1。18个皂荚种质的遗传相似系数在0.491 4~0.938 1之间,表明供试材料之间具有较丰富的遗传多样性。聚类分析结果表明:在遗传相似系数为0.60处可将18个皂荚种质分为3组,其中野皂荚单独为一组,山皂荚和皂荚-T聚为一组,其它皂荚材料聚为一组。利用3个引物扩增的8个多态性位点构建了18份皂荚种质资源的DNA指纹图谱,可以将其区分并精准鉴定。该研究结果为皂荚种质的鉴定和新品种选育提供了一定的理论依据。     

Genomic studies on killer yeasts belonging to the genusPichia

Twenty-four species belonging to the genusPichia were investigated using restriction fragment length polymorphism (RFLP) and Southern blot hybridization of their genomic DNA.Saccharomyces cerevisiae, Kluyveromyces lactis, Williopsis mrakii andCandida albicans were also included in this study. The RFLP patterns were obtained from digestion of yeast DNA with several restriction endonuclease enzymes, and showed various bands with different mobility; in most isolates, the more deeply stained bands were species-specific. This observation was confirmed by the results obtained from Southern blot hybridization of theEcoRI andXhoI RFLP patterns withP. anomala UCSC 25F DNA, digested with the same enzymes, used as probes. These bands are likely to be ribosomal DNA as shown by hybridization of digested DNA from unrelated yeast species (S. cerevisiae, K. lactis andC. albicans). However, one hybridized band, located at 3.9–4.1 Kb, seems to be peculiar to thePichia species. Our study confirms the usefulness of molecular tools in studying genetic relatedness among yeasts.     

Restriction fragment length polymorphism (RFLP) of Salmonella organisms

Genetic relatedness of 20 Salmonella isolates comprising 16 serotypes was analysed by restriction endonuclease digestion of the total DNA with six endonucleases individually. The rDNA fingerprints generated by EcoRI were more polymorphic, each serotype showed a unique fingerprint sharing several core (monomorphic) bands with several polymorphic bands. Eight characteristic NciI rDNA fingerprints were found. Similar rDNA RFLP patterns were observed in strains of Salmonella from different serotypes.S.I. Koh-Luar and E. Lau are with the Department of Chemical Process & Biotechnology, Singapore Polytechnic, 500 Doyer Road, Singapore 139651. Republic of Singapore; S. T. Chew is with the Veterinary Public Health Laboratory (VPHL), Primary Production Department (PPD), 51 Jalan Buroh, Singapore 619415. Republic of Singapore. S.B. Chua is with the Veterinary Public Health Division, Primary Production Department, 5 Maxwell Road, No. 03-00, Tower Block, MND Complex, Singapore 069110. Republic of Singapore.     

Molecular characterisation of Sargassum polycystum and S. siliquosum (Phaeophyta) by polymerase chain reaction (PCR) using random amplified polymorphic DNA (RAPD) primers

Genomic DNA was extracted from 13 samples of Sargassum polycystum and S. siliquosum collected from various localities around Peninsular Malaysia and Singapore by using four different extraction methods. The yields and the suitability of the DNA to be used as template for the polymerase chain reaction (PCR) was compared. DNA samples were subjected to PCR analysis by using random primers. Only DNA samples that were extracted using the CTAB method were successfully amplified by random amplified polymorphic DNA (RAPD)-PCR. Five of 31 random primers (OPA02, OPA03, OPA04, OPA13 and OPM10) tested amplified sequences of DNA from the DNA samples. Reproducible, amplified products were obtained using these primers and showed some potential to be useful in discriminating individual samples within the genus, in determining relationships between species within a genus and in developing individual fingerprints for individual samples.     

Identification of 27 <Emphasis Type="Italic">Porphyra</Emphasis> lines (Rhodophyta) by DNA fingerprinting and molecular markers

Twenty-seven Porphyra lines, including lines widely used in China, wild lines and lines introduced to China from abroad in recent years, were screened by random amplified polymorphic DNA (RAPD) technique with 120 operon primers. From the generated RAPD products, 11 bands that showed stable and repeatable RAPD patterns amplified by OPC-04, OPJ-18 and OPX-06, respectively were scored and used to develop the DNA fingerprints of the 27 Porphyra lines. Moreover, the DNA fingerprinting patterns were converted into computer language expressed with two digitals, 1 and 0, which represented the presence (numbered as 1) or absence (numbered as 0) of the corresponding band, respectively. Based on the above results, computerized DNA fingerprints were constructed in which each of the 27 Porphyra lines has its unique fingerprinting pattern and can be easily distinguished from others. Software named PGI (Porphyra germplasm identification) was designed for identification of the 27 Porphyra lines. In addition, seven specific RAPD markers from seven Porphyra lines were identified and two of them were successfully converted into SCAR (sequence characterized amplification region) markers. The developed DNA fingerprinting and specific molecular markers provide useful ways for the identification, classification and resource protection of the Porphyra lines.     

Genetic relationships among Mexican white pines (Pinus, Pinaceae) based on RAPD markers

Genetic relationships among Mexican white pines have not been completely resolved by DNA sequencing analyses. The use of random amplified polymorphic DNA (RAPD) markers for the study of interspecific relationships has been questioned because of the possible lack of homology of co-migrating bands between species. However, several RAPD based studies on pines have provided sufficient information to discriminate between closely related taxa. Genetic relationships among four species of Mexican white pines (Pinus ayacahuite, Pinus strobiformis, Pinus lambertiana and Pinus chiapensis) were estimated based on RAPD markers. Sixty-nine primers generated 247 bands in pooled DNA samples from ten populations. In addition, four selected primers generated 27 bands in 176 individual DNA samples. Unweighted Pair Group Method with Arithmetic Average (UPGMA) dendrograms based on Jaccard similarity indices were constructed. The results suggest that the closest pine species analyzed were P. ayacahuite and P. strobiformis, followed by P. lambertiana. The most genetically distant species was P. chiapensis. Cluster analyses did not support P. strobiformis as a distinct species from P. ayacahuite.     

Mitochondrial DNA diversity in the genera of Triticum and Aegilops revealed by southern blot hybridization

Summary Southern blot hybridization of total DNA to defined mitochondrial DNA sequences provides a sensitive assay for mtDNA variation in the genera of Triticum and Aegilops. A clear distinction between cytoplasms of tetraploid species sharing the AG haploid genome is reported for the first time. The Sitopsis section of the genus Aegilops showed the most extensive intra- and inter-specific variation, whereas no variation could be detected among the cytoplasms of polyploid Triticum species (wheats) sharing the AB haploid genome. Extensive cytoplasmic intraspecific diversity was revealed in Ae. speltoides.     

Evaluation of Comparative DNA Amplification Fingerprinting for Rapid Species Identification within the Genus Clusia

Understanding the taxonomiy of the tropical genus Clusia (Fam. Clusiaceae, Ord. Theales) has been hampered by the difficulties inherent in studying tropical dioecious, succulent, arborescent, epi- or hemiepiphytic taxa. Species identification by morphological traits often requires the terminal inflorescences and/or the succulent capsular fruits. To allow species differentiation based exclusively on vegetative tissue, a frequent necessity during ecological field studies, a procedure has been developed for rapid isolation of genomic DNA from Clusia leaf tissue followed by DNA amplification fingerprinting with a set of single arbitrary oligomer primers (23–27 mers). Fingerprints obtained with independent DNA preparations from one individual as well as DNA preparations from several individuals of the same species were identical for the major amplification products, although minor bands were somewhat variable. Polymorphic fingerprints have been obtained with 3 different primers for 3 Clusia species (C. minor L., C. alata Pl. & Tr., C. multiflora H. B. K.), and the related Oedematopus obovatus Spruce ex. PL (Clusiaceae). The interspecific Randomly Amplified Polymorphic Markers (RAPDs) thus obtained allow a rapid identification of vegetative tissue samples collected in the field, and will assist in a revision of the controversial taxonomy of the genus Clusia.     

DNA fingerprinting in reptiles: Bkm hybridization patterns in Crocodilia and Chelonia

The simple tetranucleotide repeat GATA (GACA) occurs in all eukaryotes so far studied. In many species, the arrangement of these sequences varies considerably among individuals. Thus GATA (GACA) type probes produce DNA fingerprints when hybridized with restricted DNA from different individuals within a species. Banded krait minor (Bkm) satellite DNA (related to sequences originally recovered from the W chromosome of the banded krait and consisting essentially of a series of GATA repeats) is found in a wide spectrum of vertebrates and invertebrates. We used the Bkm 2(8) clone to evaluate the occurrence of this satellite in DNA from five species of Crocodilia and six species of Chelonia, including the sea turtles Chelonia mydas and Lepidochelys kempi. Well-resolved DNA fingerprints were obtained. Among the crocodilians, fewer restriction fragments were generated and fewer of the fragments were polymorphic, than among the chelonians, consistent with the view that the crocodilians are less divergent within species. The Bkm 2(8) clone can accordingly be used to advantage in individual, familial, and population studies, and perhaps in the evaluation of taxonomic relationships in these animals. This is of potential value in endangered species such as C. mydas and L. kempi.     

Identification ofporphyra lines (rhodophyta) by AFLP DNA fingerprinting and molecular markers

Twenty-sevenPorphyra lines from 5 classes, including lines widely used in China, wild lines, and lines introduced to China from abroad in recent years, were screened by means of amplified fragment length polymorphism (AFLP) with 24 primer pairs. From the generated AFLP products, 13 bands that showed stable and repeatable AFLP patterns amplified by primer pairs M-CGA/E-AA and M-CGA/E-TA were scored and used to develop the DNA fingerprints of the 27Porphyra lines. Moreover, the DNA fingerprinting patterns were converted into computer language expressed with digitals 1 and 0, which represented the presence (numbered as 1) or absence (numbered as 0) of the corresponding band. On the basis of these results, computerized AFLP DNA fingerprints were constructed in which each of the 27Porphyra lines has its unique AFLP fingerprinting pattern and can be easily distinguished from others. Software called PGI-AFLP (Porphyra germ-plasm identification-AFLP) was designed for identification of the 27Porphyra lines. In addition, 21 specific AFLP markers from 15Porphyra lines were identified; 6 AFLP markers from 4Porphyra lines were sequenced, and 2 of them were successfully converted into SCAR (sequence characterized amplification region) markers. The developed AFLP DNA fingerprinting and specific molecular markers provide useful ways for the identification, classification, and resource protection of thePorphyra lines.     

Spawning patterns in the three-spined stickleback (Gasterosteus aculeatus L.): an evaluation by DNA fingerprinting

We analysed a random sample of 10 three-spined stickleback nests by DNA fingerprinting. DNA from the guardian male and a random subsample of LO fry per nest were probed with pYNZ 132, a human single-locus probe for VNTR-loci (variable number of tandem repeats). On average this probe produced DNA fingerprints of 12 scoreable bands. By comparing the bands present in each individual, we calculated band sharing indices (BSIs) between the guardian male and its fry. The BSls varied between 0.40 and 0.77 with an average (± S.D.) of 0.59 ± 0.09. We therefore conclude that the guardian male was the true father of the fry in all these nests. Once the paternal bands in each fry were determined, we compared the maternal bands among the fry of each nest. Based on the BSIs obtained with these comparisons, we found that one guardian male enticed three females to spawn in its nest, six enticed two females and three enticed one female.     

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.     

DNA fragments of organellar origin in random amplified polymorphic DNA (RAPD) patterns of sugar beet (Beta vulgaris L.)

The technique of random amplified polymorphic DNA (RAPD) offers a broad range of applications in the investigation of plant genomes. A promising prospect is the use of RAPD products as genetic markers. We have investigated a possible organellar source of fragments in RAPD patterns of total DNA. Two nearly-isogenic lines of cytoplasmic male-sterile and male-fertile sugar beet (Beta vulgaris L.) were subjected to RAPD analysis with six different primers. Total, nuclear, mitochondrial (mt), and chloroplast (cp), DNA from each line were investigated. Reproducible DNA fingerprints could be obtained from both organellar DNAs. Differences in band patterns of mtDNA between cytoplasmic male-sterile and -fertile lines were observed with five out of six primers, whereas different cpDNA patterns were generated by one of the primers. Consequently, the RAPD technique can be used to discriminate between different cytoplasms. Clear evidence is provided for the organellar origin of fragments in genomic (total DNA) RAPD patterns. The consequences of these results for the interpretation of RAPD analyses are discussed.     

DNA fingerprinting of red clover (Trifolium pratense L.) with Jeffrey's probes: detection of somaclonal variation and other applications

Summary DNA fingerprints generated by the Jeffreys' probes, 33.6 and 33.15, indicated the presence of minisatellite-like sequences in the red clover genome. The fingerprints generated by probe 33.6 gave less background and fewer but better defined bands than those obtained with probe 33.15. Assay of a regenerative somaclonal variant (F49R) by DNA fingerprinting with probe 33.6 detected mutation that was unlinked to the regenerative trait. The fingerprints obtained under the applied conditions also demonstrated genetic stability of consecutive generations of the regenerants in tissue culture. DNA fingerprints of F₁ plants revealed that each polymorphic band was inherited from either one or the other parent. Both probes distinguished individual-specific genotypes in seven cultivars of red clover. Greater variability in DNA fingerprints was detected between (V=0.899) than within (0.417≤V≤0.548) cultivars.