Polymorphic Amplification Patterns Research Articles

Identification of AFLP and SSR markers linked with the male fertility restorer gene of CMS 06J45 in heading Chinese cabbage (Brassica rapa L. ssp. pekinensis)

AbstractIn this study, AFLP and SSR techniques were combined with the bulk segregant analysis (BSA) method to map the restorer gene BrRfp using an F2‐segregating population comprising 258 individuals developed by crossing the polima (pol)‐like cytoplasmic male sterility (CMS) line 06J45 and the restorer line 01S325 of heading Chinese cabbage. A survey of 2048 AFLP primer pairs identified 21 polymorphic fragments, approximately half of which exhibited high similarity with the A09 chromosome sequence of Brassica rapa in the Brassica database (BRAD). Based on the genome sequence, three specific AFLP fragments linked with BrRfp were successfully converted into sequence‐characterized amplified region (SCAR) markers, named SC1233, SC2673 and SC2141. Subsequently, 178 pairs of SSR primers were redesigned for further screening, with five producing polymorphic amplification patterns. Linkage analysis showed that these markers were distributed along both sides of the BrRfp gene, with two markers, SSR03 and SSR2528, co‐segregating with the BrRfp locus in the F2 population. These results may be valuable for marker‐assisted selection and map‐based cloning in heading Chinese cabbage.

Genetic Variability of Citrinin-Producing Penicillium Citrinum Strains as Occupational Health Hazards in Northern Iran / Genska Varijabilnost Citrinin-Toksinogenih Sojeva Penicillium Citrinum Kao Izvor Opasnosti Za Zdravlje Profesionalno Izloženih Populacija U Sjevernom Iranu

AbstractWe evaluated the ability of randomly amplified polymorphic DNA (RAPD) to type citrinin-producing Penicillium citrinum (P. citrinum) strains recovered from the forest’s air in northern Iran. A total of 12 P.citrinum strains (P1-P12) were characterised by citrinin production and random amplification of polymorphic DNA (RAPD) technique. All the strains produced citrinin with levels ranging from 1.5 μg mL-1 to 39.6 μg mL-1 (average value: 12.68 μg mL-1). Of 11 primers tested, eight primers produced polymorphic amplification patterns. These primers generated a total of 105 reproducible RAPD bands, averaging to 13.1 bands per primer. Dendrogram for each primer indicating the distance of the strains to each other was constructed. RAPD results showed that the collected strains constituted four different clusters. The first cluster included two isolates (P1 and P3). The second cluster included seven isolates (P2, P4, P5, P6, P7, P8, and P10). The third and fourth clusters included one isolate (P9) and two isolates (P11 and P12), respectively. We concluded that RAPD analysis might be used in providing genotypic characters for toxigenic P. citrinum strains typing in epidemiological investigations and public health related risk assessment.

Genotyping of Fusarium verticillioides strains producing fumonisin B1 in feed associated with animal health problems

Summary Fusarium verticillioides (F. verticillioides) is not only a primary pathogen of maize, but also can cause disease in other crops such as sorghum. Pathogenicity is related to mycotoxin production such as fumonisin. In the present study, 24 isolates of F. verticillioides, which were previously identified by phenotype based methods, were re-identified using restriction fragment length polymorphism (RFLP) analysis. Digestion of the polymerase chain reaction (PCR) products with the restriction enzyme TasI allowed identifying four non- verticillioides strains that were discarded from our study. The genetic variations among the remaining 20 strains of F. verticillioides were analysed by random amplified polymorphic DNA (RAPD)-PCR method with 4 primers. Of the four primers tested, two primers produced polymorphic amplification patterns. Dendrogram for each primer indicated the distance of the strains to each other. Using primers of A, B, C and D, the isolates were divided to 8, 9, 7 and 7 groups, respectively. The results of this study indicated genetic relationship among DNA polymorphic patterns with geographic regions and the severity of fumonisin B1 (FB1) production. It seems that RAPD analysis is a suitable technique for strain typing of F. verticillioides.

Twenty-four additional microsatellite markers derived from expressed sequence tags of the endangered tropical tree Shorea leprosula (Dipterocarpaceae)

We developed microsatellite markers from newly isolated expressed sequence tags of an endangered tropical tree species, Shorea leprosula (Dipterocarpaceae). Twenty-four loci exhibited clear, polymorphic amplification patterns among 52 individuals from a population in Indonesia, with two to 16 alleles per locus. No locus showed significant departure from Hardy–Weinberg equilibrium, and no significant genotypic disequilibrium was detected for any pair of loci after sequential Bonferroni correction.

Cross‐species amplification at microsatellite loci in Australian quolls including the description of five new markers from the Chuditch (Dasyurus geoffroii)

AbstractThe quolls are the largest native predators remaining on mainland Australia. We describe the cross‐species testing of all available microsatellite loci for representative across the entire distribution of quolls, with the additional characterization of five new microsatellite loci from the Chuditch. All primers produced clear and polymorphic amplification patterns containing between nine and 17 alleles and with high levels of diagnostic variability. These highly polymorphic primers make them useful additional tools in planning conservation strategies across related conspecifics, many of which are under threat.

Identification of AFLP markers linked to the male fertility restorer gene of CMS (Moricandia arvensis) Brassica juncea and conversion to SCAR marker

We have developed a cytoplasmic male sterile (CMS) line of Brassica juncea through somatic hybridization with Moricandia arvensis and introgressed the fertility restorer gene into B. juncea. This fertility restorer locus is unique in that it is capable of restoring male fertility to two other alloplasmic CMS systems of B. juncea. As a first step toward cloning of this restorer gene we attempted molecular tagging of the Rf locus using the amplified fragment length polymorphism (AFLP) technique. A BC(1)F(1) population segregating for male sterility/fertility was used for tagging using the bulk segregant analysis method. Out of 64 primer combinations tested in the bulks, 5 combinations gave polymorphic amplification patterns. Further testing of these primers in individual plants showed four amplicons associated with the male fertility trait. Polymorphic amplicons were cloned and used for designing SCAR primers. One of the SCAR primers generated amplicons mostly in the fertile plants. Linkage analysis using MAPMAKER showed two AFLP and one SCAR markers linked to the male fertility gene with a map distance ranging from 0.6 to 2.9 cM. All the markers are located on one side of the Rf locus.

Isolation and characterization of microsatellites in the sea hibiscus (Hibiscus tiliaceus L., Malvaceae) and related hibiscus species

AbstractMicrosatellite loci were isolated from the sea hibiscus (Hibiscus tiliaceus L., Malvaceae), a pantropical plant with sea‐drifted seeds. This study describes six dinucleotide microsatellite loci for which the primers produced clear and polymorphic amplification patterns with different levels of variability (between three and nine alleles). Six markers were amplified in four other species of hibiscus, greatly increasing the utility of these markers.

Characterization of polymorphic microsatellite markers for the Carnaby's cockatoo (Calyptorhynchus latirostris) and related black cockatoo species

AbstractMicrosatellite loci were isolated from Carnaby's black cockatoo (Calyptorhynchus latirostris: Aves), a highly valued, endangered, and endemic species of bird from Western Australia. This study describes three dinucleotide and one tetranucleotide microsatellite loci for which the primers produced clear and polymorphic amplification patterns with between two and nine alleles and moderate levels of variability. Two additional dinucleotide markers which were monomorphic in the Carnaby's cockatoo were able to amplify and were polymorphic in two other species of black cockatoo, greatly increasing the utility of these markers.

English

  Random amplified polymorphic DNA (RAPD) analyses was used in combination with pathogenicity assays to study the taxonomic kinships among five Fusarium  species. A total of 46 isolates of Fusarium spp. obtained from diseased cotton seedlings showing typical root rot and dampping-off symptoms were characterized. Of 10 primers tested, four primers produced polymorphic amplification patterns with taxon-specific bands, in addition to individual-specific bands. Genetic analysis indicated into 2 main clusters, with the minor cluster included all F. moniliforme and F. solani  at the genetic similarity of GS=57.82%. The major cluster consisted of all F. oxysporum, F. avenaceum and F. chlamydosporum clustered at 71% similarity. There was no clear-cut relationship between clustering in the RAPD dendrogram, pathogenicity test and geographic origin of tested isolates. The results suggest that RAPD-PCR is a useful method for analysing genetic variation within and between Fusarium spp.   Key words: DNA-fingerprinting, Fusarium chlamydosporum, genetic homology, RAPD-PCR.

Microsatellite primers for the Western Pebble‐mound Mouse (Pseudomys chapmani) that show cross amplification for other species of Australian rodent

AbstractWe describe 12 dinucleotide and one tetranucleotide microsatellite loci for the Western Pebble‐mound Mouse (Pseudomys chapmani) that can be amplified with the polymerase chain reaction. All primers produced clear and highly polymorphic amplification patterns containing between seven and 14 alleles and with high expected heterozygosities. The amplification of these primers across seven related conspecifics makes them useful for population genetic studies and conservation work in several of these species.

Molecular characterisation of sweet cherry (Prunus avium L.) genotypes using peach [Prunus persica (L.) Batsch] SSR sequences.

A total of 76 sweet cherry genotypes were screened with 34 microsatellite primer pairs previously developed in peach. Amplification of SSR loci was obtained for 24 of the microsatellite primer pairs, and 14 of them produced polymorphic amplification patterns. On the basis of polymorphism and quality of amplification, a set of nine primer pairs and the resulting 27 informative alleles were used to identify 72 genotype profiles. Of these, 68 correspond to unique cultivar genotypes, and the remaining four correspond to three cultivars that could not be differentiated from the two original genotypes of which they are mutants, and two very closely related cultivars. The mean number of alleles per locus was 3.7 while the mean heterozygosity over the nine polymorphic loci averaged 0.49. The results demonstrate the usefulness of cross-species transferability of microsatellite sequences allowing the discrimination of different genotypes of a fruit tree species with sequences developed in other species of the same genus. UPGMA cluster analysis of the similarity data divided the ancient genotypes studied into two fairly well-defined groups that reflect their geographic origin, one with genotypes originating in southern Europe and the other with the genotypes from northern Europe and North America.

Taxonomic relationships among the toxigenic speciesFusarium acuminatum,Fusarium sporotrichioidesandFusarium tricinctumby isozyme analysis and RAPD assay

Isozyme and random amplified polymorphic DNA (RAPD) analyses have been used in combination with numerical taxonomy to study the taxonomic relationships among the toxigenic species Fusarium acuminatum Ellis & Everh. sensu Gordon (subsp. acuminatum and subsp. armeniacum), Fusarium sporotrichioides Sherb. and Fusarium tricinctum (Corda) Sacc. Eight enzymes, selected among 23 enzyme systems tested initially for activity, resolution, and consistent formation of bands, were used for isozyme analysis of 75 strains. Both cluster analysis grouping by average linkage method and correspondence analysis of the isozyme data set resulted in an arrangement of the four taxa inconsistent with their classical taxonomic classification in the Fusarium sections Gibbosum and Sporotrichiella. Isolates of F. acuminatum subsp. acuminatum were more closely related to F. tricinctum than to F. acuminatum subsp. armeniacum. Correspondingly, the F. acuminatum subsp. armeniacum group was closer to F. sporotrichioides than to F. acuminatum subsp. acuminatum. Twenty-seven strains of the four taxa, representative of the variability found by isozyme analysis, were studied using a RAPD analysis with five different decamer primers. All the primers produced polymorphic amplification patterns with taxon-specific bands, in addition to individual-specific bands. Correspondence analysis of the RAPDs distinguished four compact groups corresponding to the four taxa studied. These data support the separation of the varieties of F. acuminatum into two different species, and suggest a revision of the Fusarium sections Gibbosum and Sporotrichiella may be necessary. Key words: Fusarium, isozymes, RAPDs, numerical taxonomy, mycotoxins.