Amplified Fragment Length Polymorphism (AFLP) as a source of genetic markers for red algae

A recent PCR-based fingerprinting technique, amplified fragment length polymorphism (AFLP), was successfully applied to the red alga Chondrus crispus Stackh. This is apparently the first account to describe the application of AFLP methodology to an alga. Six isolates of C. crispus were analyzed by AFLP. A total of twenty-five primer pairs were screened and six primer pairs were selected for further investigation. Both conservative and variable markers were identified within and between populations; some markers were unique to individuals. As such, AFLP should prove useful as a source of genetic markers in algae for applications as diverse as genome mapping to population genetic investigations. This revised version was published online in June 2006 with corrections to the Cover Date.     

Using Amplified Fragment Length Polymorphisms (AFLP) to Study Genetic Variability in Several Freshwater Rotifer Species

We have used amplified fragment length polymorphisms (AFLP) to investigate the potential of this technique as a tool to measure genetic variability in eight species of freshwater rotifers: Brachionus calyciflorus, Lecane bulla, L. luna, L. quadridentata, Plationus patulus, Philodina acuticornis odiosa, Rotaria neptunia, and R. rotatoria. We used nine combinations of oligonucleotides. We observed a total of 806 amplified bands, 798 polymorphic and 8 monomorphic. The data were analyzed using cluster analysis with UPGMA, first within each set of oligonucleotide combination and finally using all nine combinations. Our best dendrogram clearly separated monogononts from digononts, and grouped the species of monogononts in the two genera. However, it grouped R. neptunia with P. acuticornis odiosa rather than with R. rotatoria. These results are discussed in view of recent works in the literature measuring genetic variability and discussing the phylogeny of the Rotifera.     

Amplified Fragment Length Polymorphisms (AFLPs) detected with non-radioactive digoxigenine labelled primers in three plant species

A protocol for the detection of AFLPs with the non radioactive digoxigenine labelling is presented. The protocol has been tested on DNA samples from three different plant species. The AFLP technique was used for the first time in these species. The sensitivity and reliability of the digoxigenine labelled primers in the AFLP technique was of the same order as the sensitivity and reliability of the radioactive assay. No major adjustments of the current standard AFLP protocols is necessary to use the digoxigenine labelled primers.     

Utility of Amplified Fragment Length Polymorphisms (AFLP) to analyse genetic structures within the Alexandrium tamarense species complex

Phylogenetic analyses of the Alexandrium tamarense species complex using ribosomal RNA sequences show a differentiation of ribotypes/clades into geographic areas and not into the three morphotypes/species A. tamarense, A. fundyense and A. catenella. Different parts of the rRNA operon have proven informative in revealing the existence and the relationships of these geographic clades, whereas even internal transcribed spacer (ITS) regions lack the resolution required to gain a deeper insight into the population structure of the species complex. Here, the utility of the DNA fingerprinting technique Amplified Fragment Length Polymorphism (AFLP) as a possible tool for such purposes was tested. A mixed sampling strategy was used in order to assess the amount of variation of AFLP banding patterns at the level of populations and geographic clades. We also describe optimized methods to achieve a good reproducibility. Our results suggest that AFLPs can provide useful information at the population level using clonal samples from a certain bloom, whereas the amount of variation that we found is too high to allow for meaningful comparisons of a few strains collected from different localities at different time points even though they belong to one geographic clade.     

Randomly Amplified Polymorphic DNA tests (RAPDs) cannot be used for paternity testing in South African cranes

The three South African crane species — the Blue Crane (Anthropoides paradisea), the Wattled Crane (Bugeranus carunculatus) and the Grey Crowned Crane (Balearica regulorum regulorum) — are listed as threatened by the IUCN. This study investigated the suitability of Randomly Amplified Polymorphic DNA markers in paternity testing in these species. RAPD primers were tested for polymorphism and RAPD profiles were scored and screened for sex linkage. The average Band Sharing Coefficient (BSC) of unrelated individuals was 0.665 (±0.103) for Blue Cranes, 0.745 (±0.060) for Grey Crowned Cranes and 0.736 (±0.056) for Wattled Cranes. Comparisons of these BSC values for unrelated individuals with BSC values of parent:offspring combination within the Blue Crane and the Grey Crowned Crane gave inconsistent results, with some parent:offspring BSC values being lower than the BSC of unrelated individuals. The results indicate that RAPDs are inappropriate for use in paternity testing in South African cranes. In the future, microsatellites should be investigated as an alternative paternity-testing technique to RAPD analysis.     

Development of DNA microsatellite markers in the multiband butterflyfish (Chaetodon multicinctus)

Twelve polymorphic microsatellite loci were developed in the multiband (pebbled) butterflyfish Chaetodon multicinctus. The loci were scored in 45 individuals from Hawaii. There were five to 21 alleles per locus with observed heterozygosity ranging from 0.419 to 0.883. Four of the primer sets also reliably amplified polymorphic loci in Chaetodon quadrimaculatus. We expect these markers to be useful for studies of genetic population structure and kinship, for example to determine whether new recruits settling onto reefs are related.     

Intergeneric and Interspecific Relationships in Araceae tribe Caladieae and Development of Molecular Markers using Amplified Fragment Length Polymorphism (AFLP)

Since amplified fragment length polymorphism (AFLP) analysishas proved useful in distinguishing cultivars of Caladium, itwas used to assess the status of species of Caladium vs. Xanthosoma,both in tribe the Caladieae, and to reassess the position ofHapaline in the same tribe. AFLP analysis using three primercombinations was carried out on four species of Caladium(C.bicolor, C. humboldtii, C. lindenii and C. schomburgkii). Resultsshowed that AFLP can distinguish between the different speciesby their unique and different banding patterns. AFLP analysisconfirmed that C. humboldtii is a species distinct from C. bicolorand that C. lindenii is a true Caladium species and does notbelong to Xanthosoma. UPGMA cluster analysis showed that C.bicolor and C. schomburgkii are most similar and that C. humboldtiiis closer to the C. bicolor / C. schomburgkii cluster comparedwith C. lindenii. Genetic relationships between Caladium, Xanthosoma,Hapaline, Alocasia and Protarum were also examined by AFLP analysisusing eight primer combinations. Several useful molecular markerswere specific either to Caladium orXanthosoma , so that AFLPcan be used to distinguish species of these two genera. Geneticanalysis of the genera examined confirms that the Caladieaeand Colocasieae tribes are distinct and that Hapaline fallswithin the tribe Caladieae and that Protarum is most distantfrom all the genera examined. Copyright 2000 Annals of BotanyCompany Araceae, Caladium, Xanthosoma, Hapaline, Alocasia, Protarum, AFLP DNA fingerprinting, diversity, AFLP markers     

Restriction Fragment Length Polymorphism (RFLP) of Genomic DNA of Moraxella (Branhamella) catarrhalis Isolates in a Hospital

Epidemiological typing, based on restriction fragment length polymorphism (RFLP) by pulsed-field gel electrophoresis (PFGE), was attempted for the 38 clinical isolates of Moraxella catarrhalis obtained at Shinshu University Hospital during the years 1987 and 1993. Digestion with SmaI or NotI generated well separable, 12 to 5 genomic DNA fragments ranging from 1,000 kb to 30 kb and the strains could be classified into 14 or 13 types, respectively. The electrophoretic profile differed with the strain in most of them and was hence useful to distinguish the each strain. Investigation for their RFLP have, however, suggested that majority of them, including the type strain ATCC25238, may have derived from a common ancestor.     

Characterization of polymorphic microsatellite DNA markers in Hakea oldfieldii Benth. (Proteaceae)

A genomic library was constructed and 13 polymorphic microsatellite markers were developed for Hakea oldfieldii, a woody shrub endemic to southwest Western Australia. Polymorphism was investigated for these markers in 28 individuals from a single population located in restricted habitat at the base of the Whicher Range south of Busselton. Expected heterozygosity ranged from 0.279 to 0.770 and averaged 0.633. Observed heterozygosity ranged from 0.321 to 0.786 and averaged 0.598. The number of alleles per locus ranged from 2.0 to 6.0 and averaged 4.5. These markers will be used to assay genetic diversity and pollen dispersal in this species.     

Identification and Mapping of Amplified Fragment Length Polymorphism Markers Linked to Shell Color in Bay Scallop, Argopecten irradians irradians (Lamarck, 1819)

Amplified fragment length polymorphisms (AFLP) were used to study the inheritance of shell color in Argopecten irradians. Two scallops, one with orange and the other with white shells, were used as parents to produce four F₁ families by selfing and outcrossing. Eighty-eight progeny, 37 orange and 51 white, were randomly selected from one of the families for segregation and mapping analysis with AFLP and microsatellite markers. Twenty-five AFLP primer pairs were screened, yielding 1138 fragments, among which 148 (13.0%) were polymorphic in two parents and segregated in progeny. Six AFLP markers showed significant (P < 0.05) association with shell color. All six loci were mapped to one linkage group. One of the markers, F1f335, is completely linked to the gene for orange shell, which we designated as Orange1, without any recombination in the progeny we sampled. The marker was amplified in the orange parent and all orange progeny, but absent in the white parent and all the white progeny. The close linkage between F1f335 and Orange1 was validated using bulk segregation analysis in two natural populations, and all our data indicate that F1f335 is specific for the shell color gene, Orange1. The genomic mapping of a shell color gene in bay scallop improves our understanding of shell color inheritance and may contribute to the breeding of molluscs with desired shell colors.     

Cleavase Fragment Length Polymorphism (CFLP): A Methodology to Further Exploit Polymorphisms from PCR Products of Plastid DNA (ptDNA) in Phaseolus

The CFLP methodology was applied for Cleavase I site detection within ptDNA intergenic regions (atpB-rbcL and rps14-psaB) at both interspecific and intraspecific levels in the genus Phaseolus. Optimal Cleavase I reaction temperature was 55°C and the semi-dry electrophoretic transfer was more efficient than the original capillary one. Cleavase reactions yield a high number of fragments as compared to PCR-RFLP and allowed differentiation within and between landraces and wild forms of the Lima bean (Phaseolus lunatus L.) originating from Andean and Mesoamerican regions of Latin America. From sequencing data and using stemloop program (GCG, Madison), congruent numbers of hairpins/fragments were identified within the sequences, highlighting the robustness of the Cleavase I. Our results pointed out the ubiquity of short conserved motifs amongst a geographically localized group of species. In the vicinity of these motifs, synapomorphic-like substitutions were frequently observed. A phylogenetic tree based on these sequences is congruent with the CFLP pattern as well as with the widely accepted phylogeny of the genus. The usefulness of this new tool as alternative and/or complementary to PCR-RFLP technology on ptDNA is suggested and discussed.     

Amplification of the major satellite DNA family (FA-SAT) in a cat fibrosarcoma might be related to chromosomal instability

Most mammalian chromosomes have satellite DNA sequences located at or near the centromeres, organized in arrays of variable size and higher order structure. The implications of these specific repetitive DNA sequences and their organization for centromere function are still quite cloudy. In contrast to most mammalian species, the domestic cat seems to have the major satellite DNA family (FA-SAT) localized primarily at the telomeres and secondarily at the centromeres of the chromosomes. In the present work, we analyzed chromosome preparations from a fibrosarcoma, in comparison with nontumor cells (epithelial tissue) from the same individual, by in situ hybridization of the FA-SAT cat satellite DNA family. This repetitive sequence was found to be amplified in the cat tumor chromosomes analyzed. The amplification of these satellite DNA sequences in the cat chromosomes with variable number and appearance (marker chromosomes) is discussed and might be related to mitotic instability, which could explain the exhibition of complex patterns of chromosome aberrations detected in the fibrosarcoma analyzed.     

Characterization of microsatellite DNA markers in the white‐bearded manakin (Manacus manacus)

Eight microsatellite DNA markers were isolated and characterized from white bearded manakin (Manacus manacus) using an enrichment cloning procedure. A large number of alleles (range 9–25), and high levels of observed heterozygosity (mean 0.67) were resolved in 236 individuals. No evidence for linkage disequilibrium or the presence of null alleles was found, indicating that these markers will be useful for examining genetic relatedness, parentage and population structure in manakins.     

Isolation and characterization of polymorphic microsatellite DNA markers in the rock bream (Oplegnathus fasciatus)

From a (GT)₁₃-enriched genomic library of Oplegnathus fasciatus, 14 polymorphic microsatellite were isolated and characterized in a test population with alleles ranging from two to nine, the observed and expected heterozygosities from 0.0000 to 1.0000, and from 0.1726 to 0.8507, respectively. Five loci deviated from the Hardy–Weinberg equilibrium, and linkage disequilibrium between two loci was significant. Two loci were also polymorphic in Pagrosomus major assessed for cross-species amplification. These polymorphic microsatellites will be useful for genetic diversity analysis and molecule-assisted breeding for O. fasciatus. Changwei Shao contributed equally.     

Mapping of a Novel Race Specific Resistance Gene to Phytophthora Root Rot of Pepper (Capsicum annuum) Using Bulked Segregant Analysis Combined with Specific Length Amplified Fragment Sequencing Strategy

Phytophthora root rot caused by Phytophthora capsici (P. capsici) is a serious limitation to pepper production in Southern China, with high temperature and humidity. Mapping PRR resistance genes can provide linked DNA markers for breeding PRR resistant varieties by molecular marker-assisted selection (MAS). Two BC₁ populations and an F₂ population derived from a cross between P. capsici-resistant accession, Criollo de Morelos 334 (CM334) and P. capsici-susceptible accession, New Mexico Capsicum Accession 10399 (NMCA10399) were used to investigate the genetic characteristics of PRR resistance. PRR resistance to isolate Byl4 (race 3) was controlled by a single dominant gene, PhR10, that was mapped to an interval of 16.39Mb at the end of the long arm of chromosome 10. Integration of bulked segregant analysis (BSA) and Specific Length Amplified Fragment sequencing (SLAF-seq) provided an efficient genetic mapping strategy. Ten polymorphic Simple Sequence Repeat (SSR) markers were found within this region and used to screen the genotypes of 636 BC₁ plants, delimiting PhR10 to a 2.57 Mb interval between markers P52-11-21 (1.5 cM away) and P52-11-41 (1.1 cM). A total of 163 genes were annotated within this region and 31 were predicted to be associated with disease resistance. PhR10 is a novel race specific gene for PRR, and this paper describes linked SSR markers suitable for marker-assisted selection of PRR resistant varieties, also laying a foundation for cloning the resistance gene.     

Vancomycin-Resistant Enterococcus faecium (VREF) from Norwegian Poultry Cluster with VREF from Poultry from the United Kingdom and The Netherlands in an Amplified Fragment Length Polymorphism Genogroup

The genetic relationship between 197 vancomycin-resistant Enterococcus faecium (VREF) isolates and 21 vancomycin-susceptible E. faecium isolates from Norwegian poultry was analyzed by amplified fragment length polymorphism (AFLP). The isolates were compared to 255 VREF isolates from various sources and countries. The Norwegian isolates constituted a relatively homogeneous population of E. faecium and clustered in a previously defined poultry AFLP genogroup.     

Genetic Variation of Nuclear Ribosomal DNA in Cockroaches (Order Blattaria): Phylogenetic Analysis of Restriction Fragment Length Polymorphism

Species-specific characteristics of nuclear ribosomal DNA (rDNA) have been determined for the first time for six insect species of order Blattaria (Insecta, Dictyoptera) with the use of restriction analysis and Southern blotting. Probes containing highly conserved fragments of 18S- and 28S-like genes of the ribosomal RNA (rRNA) of Tetrahymena pyriformis were tested in this study. The genetic similarity tree constructed for the species studied agrees with evidence from classical taxonomy based on morphological, ecological, anatomical, and physiological characters.     

Patterns of population structure at microsatellite and mitochondrial DNA markers in the franciscana dolphin (Pontoporia blainvillei)

The franciscana dolphin, Pontorporia blainvillei, is an endemic cetacean of the Atlantic coast of South America. Its coastal distribution and restricted movement patterns make this species vulnerable to anthropogenic factors, particularly to incidental bycatch. We used mitochondrial DNA control region sequences, 10 microsatellites, and sex data to investigate the population structure of the franciscana dolphin from a previously established management area, which includes the southern edge of its geographic range. F‐statistics and Bayesian cluster analyses revealed the existence of three genetically distinct populations. Based on the microsatellite loci, similar levels of genetic variability were found in the area; 13 private alleles were found in Monte Hermoso, but none in Claromecó. When considering the mitochondrial DNA control region sequences, lower levels of genetic diversity were found in Monte Hermoso, when compared to the other localities. Low levels of gene flow were found between most localities. Additionally, no evidence of isolation by distance nor sex‐biased dispersal was detected in the study area. In view of these results showing that populations from Necochea/Claromecó, Monte Hermoso, and Río Negro were found to be genetically distinct and the available genetic information for the species previously published, Argentina would comprise five distinct populations: Samborombón West/Samborombón South, Cabo San Antonio/Buenos Aires East, Necochea/Claromecó/Buenos Aires Southwest, Monte Hermoso, and Río Negro. In order to ensure the long‐term survival of the franciscana dolphin, management and conservation strategies should be developed considering each of these populations as different management units.     

Isolation and characterization of polymorphic microsatellite DNA markers in topmouth gudgeon,Pseudorasbora (Teleostei: Cyprinidae)

Due to accidental introductions, the distribution of the east Asian topmouth gudgeon, Pseudorasbora parva, has rapidly expanded over recent years, thereby threatening the endangered Japanese species, P. pumila pumila. Sixteen microsatellite loci were isolated and characterized for P. parva, 14 being highly polymorphic (mean H_E = 0.58, mean number of alleles = 4.4). Successful characterization of 10 of these loci was also achieved for P. pumila pumila. These markers should be useful in future investigation of colonization of P. parva and the development of conservation strategies for P. pumila pumila.     

Verification of meiotic gynogenesis in Siberian sturgeon (Acipenser baeri Brandt) using microsatellite DNA and cytogenetical markers

Diploid gynogenesis was induced in Siberian sturgeon Acipenser baeri using ultra violet (UV)-irradiated bester ( Huso huso × Acipenser ruthenus ) sperm. The higher survival rate of meiotic diploids was noted after 1350 ergs mm⁻² UV irradiation. A total of 80 meiotic diploids of known parentage from two different experimental treatments were screened using microsatellite DNA and cytogenetical analysis, and uniparental transmission in meiotic diploids was confirmed.