Single juveniles of the potato cyst nematodes Globodera rostochiensis and G. pallida differentiated by randomly amplified polymorphic DNA

Parasitology ◽  
1993 ◽  
Vol 107 (5) ◽  
pp. 567-572 ◽  
Author(s):  
J. Roosien ◽  
P. M. Van Zandvoort ◽  
R. T. Folkertsma ◽  
J. N. A. M. Rouppe Van Der Voort ◽  
A. Goverse ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Rapd Markers ◽  
Random Amplified Polymorphic Dna ◽  
Nematode Species ◽  
Globodera Rostochiensis ◽  
Cyst Nematodes ◽  
Wide Range ◽  
Potential Basis ◽  
Polymerase Chain ◽  
Species Specific

SUMMARYRandom amplified polymorphic DNA (RAPD) offers a potential basis for the development of a diagnostic assay to differentiate the potato cyst nematode species Globodera rostochiensis and G. pallida. Nine decamer primers have been tested for their ability to amplify species-specific DNA sequences. Primer OPG-05 produced 2 discrete DNA fragments, which were consistently present in 5 G. rostochiensis populations and absent in 5 G. pallida populations. These fragments were detectable in single females as well as in single 2nd-stage juveniles. Their amplification is extremely efficient, and reproducible over a wide range of template concentrations. One-fifth of a single juvenile is sufficient to generate reproducible RAPD markers. The amplification from single juveniles requires no DNA isolation. The use of a crude homogenate does not impair the polymerase chain reaction.

Inheritance of random amplified polymorphic DNA markers in an interspecific cross in the genus Stylosanthes

Genome ◽  
1993 ◽  
Vol 36 (1) ◽  
pp. 50-56 ◽  
Author(s):  
Kemal Kazan ◽  
John M. Manners ◽  
Don F. Cameron
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Rapd Markers ◽  
Dna Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Interspecific Cross ◽  
Parental Origin ◽  
Chain Reaction ◽  
Stylosanthes Hamata ◽  
Polymerase Chain

The inheritance of random amplified polymorphic DNA (RAPD) markers generated via the polymerase chain reaction amplification of genomic DNA sequences in an F2 family of an interspecific cross between Stylosanthes hamata and S. scabra was investigated. An initial comparison between the parental species, S. hamata cv. Verano and S. scabra cv. Fitzroy, demonstrated that 34% of detected RAPD bands were polymorphic. Of 90 primers tested, 35 showed relatively simple and reliably scorable polymorphisms and were used for segregation analysis. Sixty F2 individuals were scored for the segregation of 73 RAPD markers and 55 of these markers fit a 3:1 ratio. Segregation of eight other RAPD markers deviated significantly from a 3:1 ratio. There was no bias in the inheritance of RAPD markers regarding parental origin of the segregating RAPD markers. Linkage analysis revealed 10 linkage groups containing a total of 44 RAPD loci. Another 10 RAPD markers (7 of maternal origin) that were polymorphic between the parents did not segregate in the F2 population. One of the maternally inherited RAPD bands hybridized to chloroplast DNA. Analysis of RAPD loci by DNA hybridization indicated that mainly repeated sequences were amplified. These data indicate that RAPDs are useful genetic markers in Stylosanthes spp. and they may be suitable for genetic mapping.Key words: genetic mapping, molecular markers, polymerase chain reaction, Stylosanthes hamata, Stylosanthes scabra.

scholarly journals Morphological and molecular identification of the potato cyst nematodes Globodera rostochiensis and G. pallida in Portuguese potato fields

Nematology ◽  
2017 ◽  
Vol 19 (8) ◽  
pp. 883-889 ◽  
Author(s):  
Maria J. Camacho ◽  
Filomena Nóbrega ◽  
Arlindo Lima ◽  
Manuel Mota ◽  
Maria L. Inácio
Keyword(s):  
Multiplex Pcr ◽  
Molecular Identification ◽  
Diagnostic Technique ◽  
Nematode Species ◽  
Globodera Rostochiensis ◽  
Control Measures ◽  
Potato Cyst Nematodes ◽  
Cyst Nematodes ◽  
Widespread Species ◽  
Species Specific

The potato cyst nematodes (PCN) Globodera rostochiensis and G. pallida pose one of the greatest threats to potato crops worldwide and are subject to strict quarantine regulations in many countries. The identification of these Globodera species based on morphology may be ambiguous due to the variability of the main morphological features and the overlapping of the standard parameters in these two species; thus, confirmation via molecular methods is recommended. Multiplex PCR with species-specific primers (ITS5/PITSp4 + PITSr3) allows both species to be distinguished. However, despite the development of molecular identification methods, the morphological approach remains useful as a complementary diagnostic technique. In this work, we report results of morphological and molecular analyses that were carried out in two Globodera species from Portuguese potato fields. The average morphometric values of 40 cysts and 40 second-stage juveniles were generally within the expected ranges for G. pallida and G. rostochiensis with some variations noted. Molecular analysis with multiplex PCR confirmed the morphometric identification. The present results confirmed the occurrence of two potato cyst nematode species, G. rostochiensis and G. pallida. Surprisingly, the analysis of soils from Portuguese potato fields detected a greater number of samples infested with G. pallida, which is contrary to expectation as G. rostochiensis has been considered the most widespread species in Portugal. The distinction between the two species is therefore essential in order to detect their presence in the country with a view to re-evaluating the control measures implemented so far and adopting more effective practices.

scholarly journals Clonal Stability of RAPD Markers in Three Rhododendron Species

1995 ◽  
Vol 13 (1) ◽  
pp. 43-46 ◽  
Author(s):  
M. Javed Iqbal ◽  
D. W. Paden ◽  
A. Lane Rayburn
Keyword(s):  
Dna Sequences ◽  
Rapd Markers ◽  
Cultivar Identification ◽  
Rapd Analysis ◽  
Randomly Amplified Polymorphic Dna ◽  
Plant Identification ◽  
Specific Amplification ◽  
Polymerase Chain ◽  
The Individual ◽  
Species Specific

Abstract Amplification profiles produced by polymerase chain reaction (PCR) using randomly amplified polymorphic DNA sequences (RAPD) have the potential for species and cultivar identification. Since most rhododendron plants are vegetatively propagated, it is imperative that RAPD profiles be stable during this propagation. Three species of rhododendron, Rhododendron arborescens, R. atlanticum and R. yedoense var. poukhanense were used to produce species specific amplification profiles. Stability of amplification profiles among individually cloned plants of each species were studied. Ten plants of R. atlanticum, 9 of R. arborescens, and 10 of R. yedoense var. poukhanense were studied with 10 random primers. No polymorphism was observed among individual plants of R. atlanticum and R. arborescens with all the primers. The amplification product of one plant of R. yedoense var. poukhanense showed a difference of one band with one primer. The rest of the profiles with 9 primers were identical in all plants of this species. In order to ascertain that RAPD markers can indeed reveal real genetic differences among plants, F2 plants of two hybrids were analyzed. In contrast to the clonally propagated plants, extensive polymorphisms were observed among the individual F2 plants. Thus, RAPD analysis can be used to detect genetic variability. This stability of RAPD profiles in clonally propagated rhododendron indicates the usefulness of these markers in plant identification.

Differentiation of isolates in the genus Steinernema (Nematoda: Steinernematidae).by random amplified polymorphic DNA fragments and morphological characters

Parasitology ◽  
1995 ◽  
Vol 111 (1) ◽  
pp. 119-125 ◽  
Author(s):  
J. Liu ◽  
R. E. Berry
Keyword(s):  
Dna Sequences ◽  
Random Amplified Polymorphic Dna ◽  
Pcr Amplification ◽  
Nematode Species ◽  
Morphological Characters ◽  
Dna Fragments ◽  
Undescribed Species ◽  
Chain Reaction ◽  
Rapd Pcr ◽  
Polymerase Chain

SUMMARYWe combined polymerase chain reaction (PCR) amplification of DNA sequences and important morphological characters as a technique to differentiate nematode isolates in the genus Steinernema. Five decamer oligonucleotide primers were used to generate random amplified polymorphic DNA (RAPD) fragments from 11 nematode isolates. The primers generated 8–12 fragments, ranging from 220 to 1300 bp in size. Reproducible amplified DNA fragments of 11 isolates showed obviously inter- or intra-specific polymorphisms, enabling us to differentiate easily the nematode species and isolates. Combining RAPD–PCR fragments with the examination of morphological characters of infective juveniles and 1st-generation males, we identified isolate OH1S, collected from Newport, Oregon, as S.feltiae; isolate OS21, collected from Grants Pass, Oregon, belonged to a previously undescribed species. Our study may provide a rapid and reliable method for the identification of Steinernema nematodes.

scholarly journals Integrative Morphometric and Molecular Approach to Update the Impact and Distribution of Potato Cyst Nematodes Globodera rostochiensis and Globodera pallida (Tylenchida: Heteroderidae) in Algeria

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 216
Author(s):  
Aouicha Djebroune ◽  
Gahdab Chakali ◽  
Eugénia de Andrade ◽  
Maria João Camacho ◽  
Leidy Rusinque ◽  
...  
Keyword(s):  
Nematode Species ◽  
Globodera Rostochiensis ◽  
Globodera Pallida ◽  
Morphological Identification ◽  
Potato Cyst Nematodes ◽  
Cyst Nematodes ◽  
Additional Information ◽  
Second Stage ◽  
Species Specific ◽  
The Impact

Morphological and molecular studies were conducted to characterize the specific identity of 36 isolates of potato cyst nematodes (PCNs) recovered from soil samples collected in several potato producing areas of Algeria. Morphometric data revealed that 44% of isolates contained Globodera pallida alone, 28% contained Globodera rostochiensis alone and 28% mixtures of the two species. Morphometric values of cysts and second-stage juveniles were generally distributed with slight differences in the expected ranges for both Globodera species. Inter- and intraspecific morphometric variability in nematode isolates was noted. Molecular analysis using conventional multiplex PCR with species-specific primers and TaqMan real-time PCR confirmed the morphological identification. In addition, the distribution of both potato cyst nematode species throughout various parts of the country was investigated. In the central areas, the isolates of G. pallida alone dominate, whereas isolates of G. rostochiensis alone are more frequent in the southern areas. In the eastern regions, mixed isolates are more representative. Most isolates examined in the western areas are mixtures of the two species or G. rostochiensis alone. Comparatively, G. pallida remains the most widely distributed species in its geographic range. This study confirms the presence of two PCN species, G. pallida and G. rostochiensis, in Algeria and provides additional information on their biogeographic distribution.

Species-diagnostic and species-specific DNA sequences evenly distributed throughout pine and spruce chromosomes

Genome ◽  
2010 ◽  
Vol 53 (10) ◽  
pp. 769-777 ◽  
Author(s):  
Melanie Mehes-Smith ◽  
Paul Michael ◽  
Kabwe Nkongolo
Keyword(s):  
Dna Sequences ◽  
Random Amplified Polymorphic Dna ◽  
Inter Simple Sequence Repeat ◽  
Issr Markers ◽  
Pinus Monticola ◽  
The Family ◽  
Species Specific ◽  
Rapd And Issr ◽  
Specific Sequences

Genome organization in the family Pinaceae is complex and largely unknown. The main purpose of the present study was to develop and physically map species-diagnostic and species-specific molecular markers in pine and spruce. Five RAPD (random amplified polymorphic DNA) and one ISSR (inter-simple sequence repeat) species-diagnostic or species-specific markers for Picea mariana , Picea rubens , Pinus strobus , or Pinus monticola were identified, cloned, and sequenced. In situ hybridization of these sequences to spruce and pine chromosomes showed the sequences to be present in high copy number and evenly distributed throughout the genome. The analysis of centromeric and telomeric regions revealed the absence of significant clustering of species-diagnostic and species-specific sequences in all the chromosomes of the four species studied. Both RAPD and ISSR markers showed similar patterns.

Species-specific duplex PCR for the detection of Pratylenchus penetrans

Nematology ◽  
2009 ◽  
Vol 11 (6) ◽  
pp. 847-857 ◽  
Author(s):  
Lieven Waeyenberge ◽  
Nicole Viaene ◽  
Maurice Moens
Keyword(s):  
Internal Control ◽  
Dna Sequences ◽  
Rrna Gene ◽  
Duplex Pcr ◽  
Specific Primers ◽  
5.8S Rrna Gene ◽  
5.8S Rrna ◽  
Wide Range ◽  
Pcr Products ◽  
Species Specific

Abstract ITS1, the 5.8S rRNA gene and ITS2 of the rDNA region were sequenced from 20 different Pratylenchus species. Additionally, the same region was sequenced from seven populations of P. penetrans. After purifying, cloning and sequencing the PCR products, all sequences were aligned in order to find unique sites suitable for the design of species-specific primers for P. penetrans. Since ITS regions showed variability between and even within populations of P. penetrans, only three small DNA sequences were suitable for the construction of three potentially useful species-specific primers. New species-specific primers were paired with existing universal ITS primers and tested in all possible primer combinations. The best performing primer set, supplemented with a universal 28S rDNA primer set that served as an internal control, was tested in duplex PCR. The ideal annealing temperature, Mg2+ concentration and primer ratios were then determined for the most promising primer set. The optimised duplex PCR was subsequently tested on a wide range of different Pratylenchus spp. and 25 P. penetrans populations originating from all over the world. To test the sensitivity, the duplex PCR was conducted on DNA extracted from a single P. penetrans nematode mixed with varying amounts of nematodes belonging to another Pratylenchus species. Results showed that a reliable and sensitive P. penetrans species-specific duplex PCR was constructed.

scholarly journals Molecular Diversity of Nematode Parasites in Afrotropical Reed Frogs (Hyperolius spp.)

Diversity ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 265
Author(s):  
Ulrich Sinsch ◽  
J. Maximilian Dehling ◽  
Patrick Scheid ◽  
Carsten Balczun
Keyword(s):  
Dna Sequences ◽  
Nematode Species ◽  
Sub Saharan Africa ◽  
Parasitic Nematodes ◽  
Marker Genes ◽  
Nematode Parasites ◽  
Diversity Assessment ◽  
Wide Range ◽  
Nematode Diversity ◽  
Sub Saharan

The diversity of nematodes infecting amphibians is understudied in tropical Africa and unknown in Rwanda. Diversity assessment is hampered by the fact that species descriptions refer mostly to morphological features that are unlinked to DNA sequences of marker genes available in public databases. In this paper, we explore the abundance and diversity of parasitic nematodes in reed frogs Hyperolius kivuensis (n = 115), H. parallelus (n = 45) and H. viridiflavus (n = 100) collected in Rwanda. Five nematode species were identified morphologically as Orneoascaris chrysanthemoides, O. schoutedeni, Gendria leberrei, Aplectana chamaeleonis and Rhabdias collaris. Corresponding DNA sequences of 18S and COI genes were determined and subsequently deposited in GenBank. Aplectana chamaeleonis showed the highest prevalence (8.7%), but O. chrysanthemoides the highest mean intensity of infection (6.0) and largest number (24) of individuals in H. kivuensis. To the best of our knowledge, all amphibian hosts are new records for these nematode species, which are known to infect a wide range of amphibian and reptile species. Our findings suggest that nematode diversity is probably lower than previously assumed due to low host specificity. As morphological species identification is often challenging, our data facilitate molecular identification of adult and specifically larval nematodes found in amphibians of Sub-Saharan Africa.

Variability of Echiniscus tristis Gąsiorek & Kristensen, 2018—is morphology sufficient for taxonomic differentiation of Echiniscidae?

Zootaxa ◽  
2019 ◽  
Vol 4701 (1) ◽  
pp. 1-24 ◽  
Author(s):  
TOMASZ BARTYLAK ◽  
ADAM KULPA ◽  
DARIA GROBYS ◽  
MARTA KEPEL ◽  
ANDRZEJ KEPEL ◽  
...  
Keyword(s):  
Dna Sequences ◽  
28S Rrna ◽  
Life Stages ◽  
Significant Genetic Differentiation ◽  
Wide Range ◽  
Microscope Images ◽  
Different Types ◽  
Taxonomic Confusion ◽  
Species Specific ◽  
Range Of Variation

The majority of species in the genus Echiniscus (Heterotardigrada) have been described based on differences in the chaetotaxy or dorsal sculpture. Dorsal sculpture is, in general, considered to be species-specific and not very variable; however, many problems have arisen due to various interpretations of microscope images, which has led to taxonomic confusion in the genus Echiniscus. Conversely, chaetotaxy is generally much easier to interpret, even using low-quality microscope optics. In this study, we emended the description of Madagascan population of Echiniscus tristis Gąsiorek & Kristensen, 2018 that exhibits several different types of chaetotaxy and dorsal sculpture. The analysed specimens were characterised by two types of chaetotaxy, A-C-Dd-E and A-Dd-E, but we also found a wide range of variation in appendage number, shape and length. The observed differences are partly correlated with life stages. Additionally, we analysed DNA sequences of 28S rRNA, ITS-2 and COI of the two main morphotypes, and did not find significant genetic differentiation of the two morphotypes. This highlights the importance of analysing the morphology of both immature stages and adults, as well as of DNA markers in tardigrade species identification. 

The differentiation of parasitic nematodes using random amplified polymorphic DNA

1994 ◽  
Vol 68 (2) ◽  
pp. 109-113 ◽  
Author(s):  
M.R. Chacón ◽  
E. Rodriguez ◽  
R.M.E. Parkhouse ◽  
P.R. Burrows ◽  
T. Garate
Keyword(s):  
Polymerase Chain Reaction Amplification ◽  
Random Amplified Polymorphic Dna ◽  
Nematode Species ◽  
Parasitic Nematodes ◽  
Plant Parasitic Nematodes ◽  
Plant Nematode ◽  
Isozyme Polymorphism ◽  
Reaction Amplification ◽  
Polymerase Chain ◽  
Dna Profiles

AbstractDNA from species and races of plant parasitic nematodes (Meloidogyne, Globodera and Heterodera) and a human parasitic nematode (Trichinella) were subjected to polymerase chain reaction amplification using one arbitrary primer (M-10). This technique results in relatively simple DNA profiles that include polymorphic markers known as random amplified polymorphic DNA (RAPDs). The RAPD profiles of the plant nematode species of Meloidogyne made possible the identification of M. incognita and M. hapla, but no differences were found between the patterns of M. javanica, M. arenaria and M. graminicola. Moreover, the four races of M. incognita were indistinguishable by this primer. In contrast, when races of the plant nematode Globodera rostochiensis (Ro1 and Ro2/3) were studied under the same RAPDs conditions, a race specific profile allows these two most devastating races to be differentiated. When DNAs of eight Trichinella isolates were subjected to RAPD studies, four different patterns were identified, corresponding to the four Trichinella clusters previously defined by isozyme polymorphism.

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