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Talebi, R., A. M. Naji e F. Fayaz. "Geographical patterns of genetic diversity in cultivated chickpea (Cicer arietinum L.) characterized by amplified fragment length polymorphism". Plant, Soil and Environment 54, No. 10 (24 de outubro de 2008): 447–52. http://dx.doi.org/10.17221/399-pse.
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The objective of this study was to evaluate the genetic relationships of 28 chickpea accessions from diverse origin using AFLP markers. On average, 13 polymorphic bands per primer were observed in AFLP analysis. The average polymorphic information content (PIC) was 0.71, ranging from 0.48 to 0.92. The lowest and the highest PIC value were recorded for primer P-GAG/M-GC and P-AT/M-GC, respectively. The average GD, based on Fst values among the 21 accessions was 0.42, ranging from 0.61 to 0.16. From the UPGMA dendrogram, it is discernible that material taken for the analysis can be divided in four clusters. The results indicate that the greatest genetic diversity occurs in Afghanistan, Iran and Lebanon. In many cases, the diversity between individuals of an accession is as great as between individuals of different accessions. Based on DNA markers it is concluded that there are three centers of diversity for chickpea: Pakistan-Afghanistan, Iran-Turkey and Syria-Lebanon. India and Ethiopia, which were previously considered as a secondary center of diversity for chickpea, showed lower diversity than the above regions.
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Geiger, Anne, Gérard Cuny e Roger Frutos. "Two Tsetse Fly Species, Glossina palpalis gambiensis and Glossina morsitans morsitans, Carry Genetically Distinct Populations of the Secondary Symbiont Sodalis glossinidius". Applied and Environmental Microbiology 71, n.º 12 (dezembro de 2005): 8941–43. http://dx.doi.org/10.1128/aem.71.12.8941-8943.2005.
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ABSTRACT Genetic diversity among Sodalis glossinidius populations was investigated using amplified fragment length polymorphism markers. Strains collected from Glossina palpalis gambiensis and Glossina morsitans morsitans flies group into separate clusters, being differentially structured. This differential structuring may reflect different host-related selection pressures and may be related to the different vector competences of Glossina spp.
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Han, Zhi-Qiang, Gang Han, Tian-Xiang Gao, Zhi-Yong Wang e Bo-Nian Shui. "Genetic population structure of Liza haematocheilus in north-western Pacific detected by amplified fragment length polymorphism markers". Journal of the Marine Biological Association of the United Kingdom 93, n.º 2 (9 de agosto de 2012): 373–79. http://dx.doi.org/10.1017/s0025315412000872.
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Several divergent sympatry mtDNA lineages have been described in redlip mullet Liza haematocheilus, and this high inter-lineage divergence raises questions about the taxonomic status of L. haematocheilus lineages in the north-western Pacific. In this study, the amplified fragment length polymorphism technique was employed to examine genetic structure of L. haematocheilus and estimate the level of independence of the different mtDNA lineages in the north-western Pacific. A total of 186 bands were amplified from 91 individuals among 8 populations by 4 primer combinations and the percentage of polymorphic bands was 91.74%. The Unweighted Pair Group Method with Arithmetic Mean tree based on Nei genetic distance revealed two clusters (North Clade and South Clade). Molecular variance analysis and pairwise FST supported the separation of north and south populations of L. haematocheilus in the north-western Pacific. The incongruence between nuclear groups and mitochondrial lineages suggests the three distinct lineages do not represent cryptic species and the presence of divergent mitochondrial lineages in the same sample is a result of secondary contact after an extended period of isolation. The Pleistocene isolation and biological characteristics of species may be responsible for the genetic differentiation of L. haematocheilus.
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Alamalakala, L., S. R. Skoda e J. E. Foster. "Amplified fragment length polymorphism used for inter- and intraspecific differentiation of screwworms (Diptera: Calliphoridae)". Bulletin of Entomological Research 99, n.º 2 (12 de novembro de 2008): 139–49. http://dx.doi.org/10.1017/s0007485308006202.
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AbstractMorphologically, early immature stages of the economically important pest called screwworms, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), and non-pest secondary screwworms, Cochliomyia macellaria (Fabricius) (Diptera: Calliphoridae), are nearly indistinguishable. Correct identification is crucial to the ongoing eradication and exclusion program protecting the United States, Mexico and Central America from reinvasion of screwworms persistent in South America and the Caribbean. Amplified fragment length polymorphism (AFLP) polymerase chain reaction was used to differentiate populations of C. hominivorax and to discriminate them from C. macellaria. Ten primer pairs screened for interspecific discrimination of C. hominivorax from C. macellaria showed 52 discrete bands, allowing the two species to be readily distinguished; divergent branches on resulting dendrograms showed 100% bootstrap support. C. macellaria populations grouped at the 92% level; C. hominivorax populations grouped at the 68% level. Of the 52 bands, seven were monomorphic for both species, 22 were specific to C. macellaria, ten were present only in C. hominivorax and the remaining 13 bands differentiated C. hominivorax populations. Separate studies using ten strains of C. hominivorax showed a higher level of genetic similarity within than between populations. Analyses using 72 bands (19 monomorphic bands, 53 bands grouped all ten strains at the 58% similarity level) resolved seven mutant strains from Mexico (85% similarity level); all ten strains were resolved at the 72% similarity level. Diagnostic bands were identified for species and strain identification. We conclude that AFLP can be a valuable tool for studies of interspecific and intraspecific genetic variation in screwworm populations.
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Messick, Joanne B., Linda M. Berent e Sandra K. Cooper. "Development and Evaluation of a PCR-Based Assay for Detection of Haemobartonella felis in Cats and Differentiation of H. felis from Related Bacteria by Restriction Fragment Length Polymorphism Analysis". Journal of Clinical Microbiology 36, n.º 2 (1998): 462–66. http://dx.doi.org/10.1128/jcm.36.2.462-466.1998.
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The 16S rRNA gene of Haemobartonella felis was amplified by using universal eubacterial primers and was subsequently cloned and sequenced. Based on this sequence data, we designed a set ofH. felis-specific primers. These primers selectively amplified a 1,316-bp DNA fragment of the 16S rRNA gene of H. felis from each of four experimentally infected cats at peak parasitemia. No PCR product was amplified from purified DNA ofEperythrozoon suis, Mycoplasma genitalium, andBartonella bacilliformis. Blood from the experimental cats prior to infection was negative for PCR products and was greatly diminished or absent 1 month after doxycycline treatment. The overall sequence identity of this fragment varied by less than 1.0% among experimentally infected cats. By taking into consideration the secondary structure of the 16S rRNA molecule, we were able to further verify the alignment of nucleotides and quality of our sequence data. In this PCR assay, the minimum detectable number of H. felis organisms was determined to be between 50 and 704. The potential usefulness of restriction enzymes DdeI andMnlI for distinguishing H. felis from closely related bacteria was examined. This is the first report of the utility of PCR-facilitated diagnosis and discrimination of H. felisinfection in cats.
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Egert, Markus, e Michael W. Friedrich. "Formation of Pseudo-Terminal Restriction Fragments, a PCR-Related Bias Affecting Terminal Restriction Fragment Length Polymorphism Analysis of Microbial Community Structure". Applied and Environmental Microbiology 69, n.º 5 (maio de 2003): 2555–62. http://dx.doi.org/10.1128/aem.69.5.2555-2562.2003.
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ABSTRACT Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called “pseudo-T-RFs” since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.
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Spiegel, S., E. M. Kovalenko, A. Varga e D. James. "Detection and Partial Molecular Characterization of Two Plum pox virus Isolates from Plum and Wild Apricot in Southeast Kazakhstan". Plant Disease 88, n.º 9 (setembro de 2004): 973–79. http://dx.doi.org/10.1094/pdis.2004.88.9.973.
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Plum pox virus (PPV) was detected in wild apricot and cultivated plum maintained in a germ plasm collection in Kazakhstan. Both isolates were typed as D strain, with no evidence of recombination. The virus was detected by triple-antibody sandwich enzyme-linked immunosorbent assay (ELISA) utilizing the universal PPV-specific monoclonal antibody (MAb) 5B as the secondary antibody, and by reverse-transcription polymerase chain reaction (RT-PCR) assay using primers that amplified a 243-bp fragment in the C-terminus of the coat protein (CP) coding region. Immunocapture (IC) RT-PCR was used to detect PPV in nine wild apricot accessions, including eight ELISA-negative and one ELISA-positive. The plum and apricot isolates reacted positively in Western blot assay with the universal MAb 5B, and negatively with the strain-M-specific MAb-AL. Restriction fragment length polymorphism analysis applied to the amplified 243-bp fragment showed that restriction sites for AluI and RsaI were present in the were present in the plum and apricot samples. An amplified 836-bp cDNA fragment derived from the P3-6K1 coding region of both isolates had restriction profiles typical for strain D. Nucleotide identities of 99 to 100% were observed for the 243-bp fragments of the Kazakhstan isolates when compared with the corresponding regions of strain D, and 94 to 95% identity with strain M. Nucleotide sequence analysis of the entire CP coding region of the plum and apricotisolates resulted in the identification of a unique deletion of six nucleotides (two deduced proline amino acid residues) in the N-terminal region in the plum isolate. This is the first deletion of this nature observed among PPV isolates. The DAG motif was present in both isolates. Several nucleotide substitutions in the CP coding region were common to the plum and apricot isolates and appear to be unique to the Kazakstan isolates. This suggests a close relationship between the isolates.
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Collins, Alex, C. Ada N. Okoli, Anne Morton, David Parry, Simon G. Edwards e Dez J. Barbara. "Isolates of Verticillium dahliae Pathogenic to Crucifers Are of at Least Three Distinct Molecular Types". Phytopathology® 93, n.º 3 (março de 2003): 364–76. http://dx.doi.org/10.1094/phyto.2003.93.3.364.
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Diverse isolates of the soilborne wilt fungi Verticillium dahliae and V. albo-atrum were studied to understand the nature and origins of those infecting cruciferous hosts. All isolates from cruciferous crops produced microsclerotia, and the majority produced long conidia with a high nuclear DNA content; these isolates were divided into two groups by amplified fragment length polymorphism (AFLP) analysis. One group could be subdivided by other criteria such as rRNA sequences and mitochondrial DNA restriction fragment length polymorphism (RFLP) analysis. Two crucifer isolates were short spored and had a low nuclear DNA content. The results are consistent with the crucifer isolates being interspecific hybrids. The long-spored isolates are best regarded as amphihaploids (or allodiploids) with the AFLP groups probably each representing separate interspecific hybridization events. The short-spored crucifer isolates appear to be derived from interspecific hybrids and are here called ‘secondary haploids’. Molecular evidence suggests that one parent in the crosses was similar to V. dahliae. The other parent of the amphihaploids seems to have been more similar to V. albo-atrum than to V. dahliae, but was distinct from all isolates of either species so far studied. The implications for the taxonomy of crucifer isolates are discussed and the use of the name V. longisporum, proposed elsewhere for just some of these isolates, is discouraged.
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van Treuren, R., E. C. de Groot, I. W. Boukema, C. C. M. van de Wiel e Th J. L. van Hintum. "Marker-assisted reduction of redundancy in a genebank collection of cultivated lettuce". Plant Genetic Resources 8, n.º 2 (5 de janeiro de 2010): 95–105. http://dx.doi.org/10.1017/s1479262109990220.
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To reduce the level of redundancy in a collection of cultivated lettuce, data from 160 amplified fragment length polymorphism (AFLP) fragments and 10 polymorphic microsatellites were used in combination with passport data and morphological data, the latter obtained from an experimental field trial performed for verification purposes. Based on the observed distribution of the number of marker differences between and within accessions, a minimum of three AFLP differences and two microsatellite differences were regarded as levels warranting distinction between accessions in the redundancy analysis. The strategy followed in the redundancy analysis was mainly based on the confirmation of duplication by each of two independently generated data sources. The molecular data were used for the validation as well as the identification of potential duplicates, revealing a total number of 198 redundancies, corresponding to 12.9% of the total collection. Trueness to type, number of characterization and evaluation data, and collection management considerations, such as available seed quantities and germination percentages, were used as primary, secondary and tertiary criteria to decide which accession from duplication groups to maintain in the collection. Removal of accessions showed negligible effects on total collection diversity, as quantified for AFLPs and microsatellites, characterization and evaluation traits and resistance profiles against downy mildew pathotypes, indicating that the applied strategy was effective.
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Dueñas, Juan C. Rondan, Cristina N. Gardenal, Guillermo Albrieu Llinás e Graciela M. Panzetta-Dutari. "Structural organization of the mitochondrial DNA control region in Aedes aegypti". Genome 49, n.º 8 (1 de agosto de 2006): 931–37. http://dx.doi.org/10.1139/g06-053.
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The complete A+T - rich region of Aedes aegypti mitochondrial DNA has been cloned and sequenced. In Argentinean populations of the species, a polymorphism in the length of the amplified fragment was observed. Nucleotide sequence comparison of the shortest and longest A+T - rich amplified fragments detected revealed the presence of 2 types of tandemly repeated blocks. The size variation observed in natural populations is mainly due to the presence of a variable number of a 181 bp tandem repeat unit, located toward the 12S rRNA gene end. The size of the longest A+T - rich region was of 2070 bp, representing the largest control sequence reported for any mosquito species. Few relevant short blocks of primary-sequence similarity conserved in the control region of mosquitoes and other insects were detected scattered throughout the whole region. Five putative stem-loop secondary structures were found, one of them flanked by conserved sequences described in other insects. Our results suggest that there are no universal models of structure–function relations in the control region of insect mtDNA. In addition, we identified a short A+T - rich variable segment in the Ae. aegyti control region that would be suitable for population genetic studies.Key words: mitochondrial DNA, A+T - rich region, repeated elements, conserved blocks, Aedes aegypti.
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Hagidimitriou, Marianna, Andreas Katsiotis, George Menexes, Constantinos Pontikis e Michael Loukas. "Genetic Diversity of Major Greek Olive Cultivars Using Molecular (AFLPs and RAPDs) Markers and Morphological Traits". Journal of the American Society for Horticultural Science 130, n.º 2 (março de 2005): 211–17. http://dx.doi.org/10.21273/jashs.130.2.211.
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The aim of the present study was to develop a reliable reference database to discriminate between the major Greek olive (Olea europaea L.) cultivars and reveal their genetic relationships, since Greece is considered a secondary center of diversity. In order to establish genetic relationships among the 26 Greek and eight international cultivars, four amplified fragment length polymorphism (AFLP) primer pairs, 12 randomly amplified polymorphic DNA (RAPD) primers, along with measurements from 10 morphological traits, were used. A total of 576 AFLP and 113 RAPD markers were produced. Genetic similarities, estimated using the Jaccard algorithim, ranged from 0.45 to 0.83 for the AFLP data and 0.27 to 0.87 for the RAPD data. The cophenetic correlation coefficients between the genetic similarities and the unweighted pair group method of arithmetic averages (UPGMA) phenograms were 0.77 for the AFLPs, 0.81 for the RAPDs, and 0.69 for the morphological traits. However, limited clustering similarities among the phenograms derived from the three methods were observed. This was also reflected by the low correlation between the three genetic similarity matrices produced (AFLP and RAPD, r = 0.39; AFLP and morphological traits, r = 0.11; RAPD and morphological traits, r = 0.02). According to the molecular results, olive cultivars are clustered according to fruit size but not according to geographical origin. Three of the cultivars tested, `Vasilicada,' `Throumbolia', and `Lianolia Kerkiras', were found to branch distantly to the others, according to the AFLP results, and can be considered as ancient Greek cultivars.
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Canady, Michael A., Vladimir Meglic e Roger T. Chetelat. "A library of Solanum lycopersicoides introgression lines in cultivated tomato". Genome 48, n.º 4 (1 de agosto de 2005): 685–97. http://dx.doi.org/10.1139/g05-032.
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A set of introgression lines (ILs), containing individual chromosome segments from the wild nightshade Solanum lycopersicoides bred into the genetic background of cultivated tomato (Lycopersicon esculentum), has been developed. A primary group of 56 lines was selected for maximum representation of the S. lycopersicoides genome (~96% of the total map units), homozygosity, and a minimum number of introgressed segments per line. A secondary set of 34 lines provides increased map resolution in certain regions. Approximately 34% of the lines were sterile in the homozygous condition, but could be maintained by heterozygotes. To facilitate identification of segregating ILs, restriction fragment length polymorphism probes were converted to higher throughput cleaved amplified polymorphic sequence markers, which supplement allozyme and morphological loci. Strong segregation distortion was observed in F2 progeny of heterozygous ILs, with an excess of L. esculentum alleles in most regions. For introgressions on distal chromosome 1L, a preferential transmission of S. lycopersicoides alleles was observed in the male germ line. Homozygous ILs generally yielded less seed from self pollination than corresponding heterozygotes, indicating that sterility effects were recessive. This IL library provides a novel resource for genetic studies of traits found in S. lycopersicoides.Key words: Lycopersicon esculentum, Solanum lycopersicum, Solanum lycopersicoides, segregation distortion, alien introgression.
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Ney, Gideon, e Johannes Schul. "Epigenetic and genetic variation between two behaviorally isolated species of Neoconocephalus (Orthoptera: Tettigonioidea)". Journal of Orthoptera Research 28, n.º 2 (17 de maio de 2019): 11–19. http://dx.doi.org/10.3897/jor.28.28888.
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Epigenetic variation allows for rapid changes in phenotypes without alterations to nucleotide sequences. These epigenetic signatures may diverge over time among isolated populations. Epigenetic incompatibility following secondary contact between these populations could result in the evolution of reproductive isolating mechanisms. If epigenetic incompatibility drove the evolution of species isolating mechanisms, we expect to see significant epigenetic differentiation between these species. Alternatively, epigenetic variation could be the result of predominantly environmental variables and not align along species boundaries. A methylation sensitive amplified fragment length polymorphism analysis was performed on individuals of the closely related katydid species Neoconocephalusrobustus and N.bivocatus. We observed significant variation in total methylation levels between species. However, genetic differentiation remained larger than epigenetic differentiation between species groups. We measured a significant correlation between the epigenetic and genetic distance between individuals. Epigenetic differentiation is therefore likely the result of an interaction between genetic and epigenetic loci and not a mechanism for species differentiation. We therefore did not find evidence to support our hypothesis of an epigenetically mediated mechanism for speciation between N.robustus and N.bivocatus.
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Cavers, S., C. Navarro, P. Hopkins, R. A. Ennos e A. J. Lowe. "Regional and Population-scale Influences on Genetic Diversity Partitioning within Costa Rican Populations of the Pioneer Tree Vochysia ferruginea Mart". Silvae Genetica 54, n.º 1-6 (1 de dezembro de 2005): 258–64. http://dx.doi.org/10.1515/sg-2005-0037.
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Abstract The neotropical pioneer species Vochysia ferruginea is locally important for timber and is being increasingly exploited. The sustainable utilisation of this species would benefit from an understanding of the level and partitioning of genetic diversity within remnant and secondary regrowth populations. We used data from total genome (amplified fragment length polymorphism, AFLP) and chloroplast genome markers to assay diversity levels within seven Costa Rican populations. Significant chloroplast differentiation between Atlantic and Pacific watersheds was observed, suggesting divergent historical origins for these populations. Contemporary gene flow, though extensive, is geographically constrained and a clear pattern of isolation by distance was detectable when an inter-population distance representing gene flow around the central Costa Rican mountain range was used. Overall population differentiation was low (FST = 0.15) and within-population diversity high, though variable (Hs = 0.16-0.32), which fits with the overall pattern of population genetic structure expected for a widespread, outcrossed tropical tree. However genetic diversity was significantly lower and differentiation higher for recently colonised and disturbed populations compared to that at more established sites. Such a pattern seems indicative of a pioneer species undergoing repeated cycles of colonisation and succession.
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Han, Zhiqiang, Zhiyong Wang, Tianxiang Gao, Takashi Yanagimoto e Koji Iida. "Assessing the Speciation of a Cold Water Species, Japanese Sand Lance Ammodytes personatus, in the Northwestern Pacific by AFLP Markers". Animals 8, n.º 12 (28 de novembro de 2018): 224. http://dx.doi.org/10.3390/ani8120224.
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The use of molecular techniques in biodiversity research increasingly results in the recognition of multiple divergent mitochondrial DNA (mtDNA) lineages below the morphospecies level. However, the overlapping distribution of multiple divergent lineages raises the question of whether some of these lineages are in fact cryptic species. Assessing the status of these divergent lineages, delimiting evolutionarily significant units (ESUs), and identifying the dominant evolutionary and ecological drivers are critical components of successful wildlife conservation and management strategies. Amplified fragment length polymorphism (AFLP) markers were applied to characterize the phylogeography pattern of a cold water species, the Japanese sand lance Ammodytes personatus, in warm and cold ocean currents. A total of 211 individuals sampled from 12 populations through the species’ range, including samples from Kuroshio Current, Oyashio Current, Tsushima Current, and Yellow Sea, were analyzed. The Bayesian assignment probability test and Neighbor joining (NJ) analysis divided these populations into two genetically and geographically distinct clades (northern and southern clades) characterized by different sea surface temperatures. The incongruence between nuclear clades and previous mitochondrial lineages suggested that A. personatus is indeed composed of at least two genetically divergent cryptic species. Pleistocene glaciation isolation after secondary contact, local thermal adaptation, and isolation by distance may explain the observed geographic pattern of two cryptic species and genetic structure within clades.
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Lee, Ing-Ming, Kristi D. Bottner, Joseph E. Munyaneza, Robert E. Davis, James M. Crosslin, Lindsey J. du Toit e Todd Crosby. "Carrot Purple Leaf: A New Spiroplasmal Disease Associated with Carrots in Washington State". Plant Disease 90, n.º 8 (agosto de 2006): 989–93. http://dx.doi.org/10.1094/pd-90-0989.
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During the growing seasons of 2003 and 2004, a disease occurred in several carrot crops in south central Washington with symptoms suggestive of infection by phytopathogenic mollicutes (phytoplasmas and spiroplasmas). In the fall, many affected carrot plants exhibited extensive purple or yellow-purple leaf discoloration, general stunting of shoots and taproots, and formation of bunchy, fibrous secondary roots. For detection of the putative causal agents, polymerase chain reaction (PCR) assays were performed using primers specific to phytoplasmas as well as primers specific to plant-pathogenic spiroplasmas. Restriction fragment length polymorphism (RFLP) analyses of PCR-amplified 16S rDNA sequences revealed that about 81% of affected plants showing dark purple or yellow-purple leaf symptoms tested positive for Spiroplasma citri. Of affected plants showing mild purple discoloration of leaf margins, 18% tested positive for a phytoplasma strain belonging to the clover proliferation group (16SrVI), subgroup 16SrVI-A, and 11% for another phytoplasma strain belonging to the aster yellows group (16SrI), subgroup 16SrI-A. Nucleotide sequence analysis of cloned 16S rDNA confirmed the phytoplasma group affiliations. Some symptomatic plants were co-infected with S. citri and either aster yellows phytoplasma or clover proliferation group phytoplasma. To our knowledge, this is the first documentation of spiroplasma infection of carrot in the United States.
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Prohens, Jaume, José M. Blanca e Fernando Nuez. "Morphological and Molecular Variation in a Collection of Eggplants from a Secondary Center of Diversity: Implications for Conservation and Breeding". Journal of the American Society for Horticultural Science 130, n.º 1 (janeiro de 2005): 54–63. http://dx.doi.org/10.21273/jashs.130.1.54.
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Eggplant (Solanum melongena L.) was introduced by the Arabs into Spain. Since then, many local cultivars have arisen. These materials are grouped in four cultivar groups: “round,” “semi-long,” “long,” and “listada de Gandía.” We studied the morphological and molecular [amplified fragment length polymorphism (AFLP)] diversity of a collection of 28 Spanish traditional cultivars of eggplant. Four eggplant accessions from different origins were used as controls and three scarlet eggplant (Solanum aethiopicum L.) accessions as outgroups. Morphology and AFLP markers showed that S. melongena and S. aethiopicum are separate taxonomic entities, and that, compared to controls, Spanish eggplants are very variable, indicating that the Iberian Peninsula can be regarded as a secondary center of diversity. Morphological differences were found among cultivar groups in traits other than those used for the grouping although, in some cases, accessions from different cultivar groups shared a similar general morphology. Eggplant cultivar groups also showed some genetic differences, which are revealed in the gene diversity statistics (GST = 0.30). Nonetheless, no individual AFLP markers specific and universal to one cultivar group could be found. “Round” cultivars were genetically more diverse than the other cultivar groups. A positive correlation (r = 0.68) was found between morphological and molecular distances. However, correlations between geographical and either morphological or molecular distances were low. Results suggest that evolution of eggplants in Spain has involved frequent hybridizations and a frequent movement and exchange of seeds. Structure of diversity among regions indicates that most of the diversity can be collected in single selected regions. All these results have important implications in eggplant germplasm conservation and breeding.
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Sonnante, G., A. De Paolis e D. Pignone. "Relationships among artichoke cultivars and some related wild taxa based on AFLP markers". Plant Genetic Resources 1, n.º 2-3 (agosto de 2003): 125–33. http://dx.doi.org/10.1079/pgr200319.
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AbstractArtichoke, Cynara cardunculus var. scolymus is a diploid outcrossing species, originated in the Mediterranean basin, which has been much appreciated both for its tasty heads and pharmaceutical properties since ancient times. The species includes two more botanical varieties: C. cardunculus var. altilis, the cultivated leafy cardoon and C. cardunculus var. sylvestris, the presumed wild progenitor of artichoke, which are completely interfertile with the cultivated globe artichoke and all together they form the primary gene pool of artichoke. The secondary gene pool includes at least seven wild Cynara species. A high level of morphological variation, essentially in head shape and size, is observed in artichoke varieties. Amplified fragment length polymorphism (AFLP) markers were used in order to assess genetic variation and relationships among artichoke varieties and between these and some of their wild relatives. A selected group of wild and cultivated artichoke accessions belonging to different clusters detected on a morphological basis and from various geographical origins was chosen for the analysis. Twenty-four primer combinations were initially tested to evaluate their ability to detect polymorphism between samples. Nine primer combinations were chosen for further analysis on 39 cultivated artichokes, two wild progenitors, one cultivated cardoon, one sample of C. cornigera, one of C. humilis and two samples of C. syriaca. A high level of polymorphism was observed for AFLP markers. The polymorphic bands obtained were scored and used to assess genetic similarity among wild and cultivated accessions and finally to construct a UPGMA dendrogram and principal co-ordinate (PCO) analysis based on Jaccard's similarity index. The artichoke wild progenitor was quite distantly related to the cultigen and occupied a separate branch in the dendrogram. However, wild C. cardunculus was more similar to the artichoke than were the other wild species, corroborating the idea that it is the wild progenitor of cultivated artichokes. Within the cultivated artichoke, the dendrogram derived from AFLP analysis produced branches which roughly corresponded to the groups obtained on the basis of morphological and physiological characteristics. The groups were homogeneous enough, except for the ‘Romaneschi’ types, which proved to be quite genetically variable, and did not cluster in a single branch. This is interpreted on the grounds of the possible selection pathway of this more modern morpho-group.
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Bukholm, Geir, Tone Tannæs, Anne Britt Bye Kjelsberg e Nils Smith-Erichsen. "An Outbreak of Multidrug-ResistantPseudomonas AeruginosaAssociated with Increased Risk of Patient Death in an Intensive Care Unit". Infection Control & Hospital Epidemiology 23, n.º 8 (agosto de 2002): 441–46. http://dx.doi.org/10.1086/502082.
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Objective:To investigate an outbreak of multidrug-resistantPseudomonas aeruginosain an intensive care unit (ICU).Design:Epidemiologic investigation, environmental assessment, and ambidirectional cohort study.Setting:A secondary-care university hospital with a 10-bed ICU.Patients:All patients admitted to the ICU receiving ventilator treatment from December 1,1999, to September 1, 2000.Results:An outbreak in an ICU with multidrug-resistant isolates ofP. aeruginosabelonging to one amplified fragment-length polymorphism (AFLP)–defined genetic cluster was identified, characterized, and cleared. Molecular typing of bacterial isolates with AFLP made it possible to identify the outbreak and make rational decisions during the outbreak period. The outbreak included 19 patients during the study period. Infection with bacterial isolates belonging to the AFLP cluster was associated with reduced survival (odds ratio, 5.26; 95% confidence interval, 1.14 to 24.26). Enhanced barrier and hygiene precautions, cohorting of patients, and altered antibiotic policy were not sufficient to eliminate the outbreak. At the end of the study period (in July), there was a change in the outbreak pattern from long (December to June) to short Quly) incubation times before colonization and from primarily tracheal colonization (December to June) to primarily gastric or enteral Quly) colonization. In this period, the bacterium was also isolated from water taps.Conclusion:Complete elimination of the outbreak was achieved after weekly pasteurization of the water taps of the ICU and use of sterile water as a solvent in the gastric tubes.
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Dunkle, Larry D., e Morris Levy. "Genetic Relatedness of African and United States Populations of Cercospora zeae-maydis". Phytopathology® 90, n.º 5 (maio de 2000): 486–90. http://dx.doi.org/10.1094/phyto.2000.90.5.486.
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Two taxonomically identical but genetically distinct sibling species, designated groups I and II, of Cercospora zeae-maydis cause gray leaf spot of maize in the United States. Isolates of the gray leaf spot pathogen from Africa were compared with isolates from the United States by amplified fragment length polymorphism (AFLP) analysis and restriction digests of internal transcribed spacer (ITS) regions and 5.8S ribosomal DNA (rDNA), as well as by morphological and cultural characteristics. The isolates from Africa were morphologically indistinguishable from the U.S. isolates in both groups, but like isolates of group II, they grew more slowly and failed to produce detectable amounts of cercosporin in culture. Analysis of restriction fragments from the ITS and rDNA regions digested with five endonucleases indicated that all of the African isolates shared the profile of the C. zeae-maydis group II population from the eastern United States and, thus, are distinct from the group I population, which is more prevalent in the United States and other parts of the world. Cluster analysis of 85 AFLP loci confirmed that the African and U.S. group II populations were conspecific (greater than 97% average similarity) with limited variability. Among all group II isolates, only 8 of 57 AFLP loci were polymorphic, and none was specific to either population. Thus, although gray leaf spot was reported in the United States several decades prior to the first record in Africa, the relative age of the two populations on their respective continents could not be ascertained with confidence. The absence of C. zeae-maydis group I in our samples from four countries in the major maize-producing region of Africa as well as the greater AFLP haplotype diversity found in the African group II population, however, suggest that Africa was the source of C. zeae-maydis group II in the United States. The overall paucity of AFLP variation in this sibling species further suggests that its origin is recent or that the ancestral population experienced a severe bottleneck prior to secondary migration.
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Zeller, Kurt A., Robert L. Bowden e John F. Leslie. "Diversity of Epidemic Populations of Gibberella zeae from Small Quadrats in Kansas and North Dakota". Phytopathology® 93, n.º 7 (julho de 2003): 874–80. http://dx.doi.org/10.1094/phyto.2003.93.7.874.
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Gibberella zeae (anamorph Fusarium graminearum) causes Fusarium head blight (FHB) of wheat and barley and has been responsible for several billion dollars of losses in the United States since the early 1990s. We isolated G. zeae from the top, middle, and bottom positions of wheat spikes collected from 0.25-m2 quadrats during severe FHB epidemics in a single Kansas (KS) field (1993) and in a single North Dakota (ND) field (1994). Three amplified fragment length polymorphism (AFLP) primer pairs were used to resolve 94 polymorphic loci from 253 isolates. Members of a subset of 26 isolates also were tested for vegetative compatibility groups (VCGs). Both methods indicated high levels of genotypic variability and identified the same sets of isolates as probable clones. The mean number of AFLP multilocus haplotypes per head was approximately 1.8 in each population, but this value probably underestimates the true mean due to the small number of samples taken from each head. Isolates with the same AFLP haplotype often were recovered from different positions in a single head, but only rarely were such apparently clonal isolates recovered from more than one head within a quadrat, a pattern that is consistent with a genetically diverse initial inoculum and limited secondary spread. The KS and ND samples had no common AFLP haplotypes. All G. zeae isolates had high AFLP fingerprint similarity (>70%, unweighted pair group method with arithmetic means similarity) to reference isolates of G. zeae lineage 7. The genetic identity between the KS and ND populations was >99% and the estimated effective migration rate was high (Nm ≈70). Tests for linkage disequilibrium provide little evidence for nonrandom associations between loci. Our results suggest that these populations are parts of a single, panmictic population that experiences frequent recombination. Our results also suggest that a variety of population sampling designs may be satisfactory for assessing diversity in this fungus.
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Yue, H. N., Y. F. Wu, Y. Z. Shi, K. K. Wu e Y. R. Li. "First Report of Paulownia Witches'-Broom Phytoplasma in China". Plant Disease 92, n.º 7 (julho de 2008): 1134. http://dx.doi.org/10.1094/pdis-92-7-1134a.
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Paulownia witches'-broom (PaWB) is one of the most important diseases affecting Paulownia tomentosa trees in China. According to 2006 statistics, the disease has affected 880,000 ha of trees for timber production causing billions of dollars in economic losses. During the spring and summer of 2006, a survey was done in Shaanxi Province to confirm phytoplasma infection of paulownia trees exhibiting symptoms of witches'-broom, stunting, yellowing, and proliferating secondary shoots. Foliage samples were collected from 24 symptomatic and 8 symptomless paulownia plants in eight different production fields. Total DNA was extracted from 0.5 g of leaf midrib and stem phloem tissue with a modified cetyltrimethylammoniumbromide (CTAB) method (3). Resulting DNA extracts were analyzed by a nested PCR assay using phytoplasma 16S rRNA gene primer pairs R16mF2/R16mR1 followed by R16F2n/ R16R2 (1), which amplified a 1.4-kb and a 1.2-kb product, respectively, from symptomatic plants. Restriction fragment length polymorphism (RFLP) analysis of the nested 1.2-kb 16S rDNA products with AluI, MseI, HhaI, HpaI, RsaI, BfaI, HinfI, and TaqI endonuclease (2) indicated that all symptomatic plants were infected by a phytoplasma belonging to aster yellows group (16SrI) subgroup D (16SrI-D) phytoplasma strains. A 1.2-kb 16S rDNA sequence (GenBank Accession No. DQ851169) derived from representative strain PaWB-Shaanxi was identical (100%) to that of PaWB phytoplasma (L27033), a known subgroup 16SrI-D strain from Taiwan (2). The agreement between the RFLP analysis and sequence data confirms that PaWB from Shaanxi is a member of subgroup 16SrI-D. To our knowledge, this is the first report of PaWB disease being present in China and of its association with the 16SrI-D subgroup. References: (1) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (2) I.-M. Lee et al. Inst. J. Syst. Bacteriol. 48:1153, 1998. (3) Y. Qi et al. Biotechnol. Bull. 4:44, 2004.
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Duarte, V., E. G. Silva, I. C. R. Hass, I. P. Bedendo e E. W. Kitajima. "First Report of a Group 16SrIII-B Phytoplasma Associated with Decline of China Tree in Brazil". Plant Disease 93, n.º 6 (junho de 2009): 666. http://dx.doi.org/10.1094/pdis-93-6-0666b.
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China tree (Melia azedarach L.), originally from Asia, is an exotic deciduous species in Brazil and is used as an ornamental shade tree in the southern region of the country. Since 2005, plants displaying yellowing, little leaves, witches' broom, and decline have been observed in the State of Rio Grande do Sul. In the streets and avenues of the capital city of Porto Alegre, there are approximately 173 tree species and China tree (6.57% of all trees) is among the top 10 (80,000 China trees and most are symptomatic). Plants with those symptoms are very distinctive and have been found also in the cities of Livramento, Rio Grande, Santa Maria, and Vacaria, places located in seashore areas, and along highways everywhere in the state. The high incidence seems to be related to drought during the last few years. These symptoms are typical of a disease identified by yellowing or decline of China tree associated with phytoplasma and previously reported in the neighboring countries of Argentina, Paraguay, and Bolivia (2). To demonstrate the presence of phytoplasma in diseased trees and to confirm its identity, total DNA was extracted from China tree leaf midribs collected from 10 symptomatic and three asymptomatic plants. Nested PCR was performed with the P1/P7 primer pair in the primary PCR to amplify a 1.8-kb fragment encompassing the 16S rRNA gene, the 16S-23S spacer region, and the 5′ end of the 23S rRNA gene, while the secondary PCR was primed by the R16F2n/R16R2 primer pair to amplify a 1.2-kb fragment of the 16S rRNA gene from the 1.8-kb fragment (3,4). DNA fragments of 1.2 kb amplified from nested PCR were analyzed by restriction fragment length polymorphism with restriction enzymes AluI, HhaI, HpaII, KpnI, MboI, MseI, and RsaI, revealing identical profiles for each amplicon and demonstrating that a phytoplasma belonging to group 16SrIII, subgroup B (16SrIII-B) (1) was associated consistently with all symptomatic plants. BLAST analysis revealed 99% identity among these cloned 1.2-kb sequences and representative sequences of phytoplasmas affiliated with group 16SrIII (GenBank Accession Nos. AY081817 and AF147706). A majority consensus sequence representing the phytoplasma found in China trees was selected and deposited in GenBank (Accession No. FJ404775). These results were confirmed by observation with transmission electron microscopy of pleomorphic bodies 400 to 2,000 nm in diameter in the phloem sieve tubes of all symptomatic trees. No phytoplasma was detected or visualized in asymptomatic samples. These results corroborate those from studies conducted in neighboring countries that demonstrated the association between phytoplasmas of group 16SrIII and decline of China trees (1). In conclusion, the current study revealed that a phytoplasma affiliated with group 16SrIII-B is associated with the decline of China tree in Brazil, a disease previously described based solely on symptoms (2). The incidence and severity of the disease are enough to prevent further use of these trees as landscape plants in southern Brazil. References: (1) J. D. Arneodo et al. J. Phytopathol. 155:70, 2007. (2) M. Dalbosco et al. Fitopatol. Bras. 30(Suppl.):177, 2005. (3) S. Deng and C. Hiruki. J. Microbiol. Methods 14:53, 1991. (4) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.
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Viprakasit, Vip, Alison T. Merryweather-Clarke, Yingyong Chinthammitr, Lisa Schimanski, Hal Drakesmith, Somdet Srichairatanakool, Chanin Limwongse, Alain Townsend e Kathryn J. H. Robson. "Molecular Diagnosis of the First Ferroportin Mutation (C326Y) in the Far East Causing a Dominant Form of Inherited Iron Overload." Blood 104, n.º 11 (16 de novembro de 2004): 3204. http://dx.doi.org/10.1182/blood.v104.11.3204.3204.
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Abstract Genetic hemochromatosis (HH) is a common inherited disorder in populations of European origin in which different types of genetic hemochromatosis (type 1–4) have been characterized. Most hemochromatosis-type 1 patients are homozygotes or compound heterozygotes for two HFE mutations C282Y and H63D. Studies of several non-HFE iron overload families led to identification of mutations in hemojuvelin and hepcidin (juvenile form-HFE2A and B), transferrin receptor 2 (HFE3) and ferroportin (HFE4) as a cause of different forms of hemochromatosis. In the Far East, inherited hemochromatosis has rarely been reported and may have been misdiagnosed due to the high prevalence of secondary iron loading from hemoglobin disorders. This report describes, for the first time, non-HFE iron overload in patients from Southeast Asia. The affected Thai family presented with a distinctive clinical phenotype including macrocytosis and elevated transferrin saturation (>95%), increased non-transferrin bound iron (NTBI) as well as raised serum ferritin and marked hepatic hemochromatosis. Our patients tolerated therapeutic phlebotomy well. DNAs from peripheral blood leukocytes were firstly analyzed for three common HFE mutations (C282Y, H63D and IVS5+1 G→A). Subsequently, we screened all coding sequences, promoters and exon/intron boundaries of the HFE, HAMP, TfR2, HJV and SLC40A1 genes using denaturing high performance liquid chromatography (DHPLC). The entire coding region and splice sites of these genes were amplified and directly sequenced. We identified a novel mutation (C326Y) in ferroportin (SLC40A1, IREG-1, MTP-1), a membrane iron transport protein due to a G→A substitution at nucleotide 1281 in exon 7. This mutation was confirmed by restriction fragment length polymorphism (RFLP) analysis using Sfa NI. Six hundred Thai and two hundred Vietnamese chromosomes were analyzed for the C326Y mutation by RFLP analysis and it was not detected in any of the healthy controls studied. This result suggested that the G→A substitution is not a common polymorphism and is likely to be the causative mutation for the phenotype in this family. Previous reported mutations of ferroportin, including A77D and V162del, which lead to type IV hemochromatosis, were characterized by increased serum ferritin despite normal transferrin saturation, in contrast to our patients’ phenotype. These autosomal dominant mutants are postulated to lead to disease due to loss of iron exporting function. Preliminary in vivo assay using transient transfection of wild-type and ferroportin mutants in HeLa or 293T cells revealed, as expected, a loss of function and diminished surface membrane localisation in A77D and V162del mutants. Surprisingly, the C326Y mutant was indistinguishable from wt ferroportin in both iron status of the cell and protein localization suggesting different pathophysiology leading to iron overload in our patients.
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Frary, Anne, Hasan Özgür Şığva, Ayfer Tan, Tuncer Taşkın, Abdullah İnal, Sevgi Mutlu, Mehmet Haytaoğlu e Sami Doğanlar. "Molecular Genetic Diversity in the Turkish National Melon Collection and Selection of a Preliminary Core Set". Journal of the American Society for Horticultural Science 138, n.º 1 (janeiro de 2013): 50–56. http://dx.doi.org/10.21273/jashs.138.1.50.
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Turkey is a secondary center of diversity for melon (Cucumis melo) and is home to a variety of regional morphotypes. This diversity is housed in a national germplasm repository with more than 500 accessions. Molecular genetic variability of 209 melon genotypes from 115 accessions of this collection was characterized using amplified fragment length polymorphisms (AFLPs). Ten AFLP primer combinations yielded 279 reproducible fragments, which were used for dendrogram and principal coordinate analyses. These analyses showed two major clusters of Turkish melons: one group contained highly similar genotypes (maximum Dice dissimilarity coefficient of 0.18), whereas the other group was genetically more diverse (maximum dissimilarity 0.41). Although average dissimilarity was low (0.13), a broad range of genetic diversity was observed in the collection. A marker allele richness strategy was used to select a core set of 20 genotypes representing the allelic diversity of the AFLP data. The core set had double the average diversity (0.26) of the entire set and represented the major morphotypes present in the collection. Molecular genetic diversity of the core set was further validated using simple sequence repeat marker data (116 polymorphic fragments), which confirmed that the selected core set retained high levels of molecular genetic diversity.
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Antonishyn, Nick A., Ryan R. McDonald, Edward L. Chan, Greg Horsman, Carla E. Woodmansee, Pamela S. Falk e C. Glen Mayhall. "Evaluation of Fluorescence-Based Amplified Fragment Length Polymorphism Analysis for Molecular Typing in Hospital Epidemiology: Comparison with Pulsed-Field Gel Electrophoresis for Typing Strains of Vancomycin-Resistant Enterococcus faecium". Journal of Clinical Microbiology 38, n.º 11 (2000): 4058–65. http://dx.doi.org/10.1128/jcm.38.11.4058-4065.2000.
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Fluorescence-based amplified fragment length polymorphism (fbAFLP) is a novel assay based on the fluorescent analysis of an amplified subset of restriction fragments. The fbAFLP assay involves the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The ligation of adapters with primer-specific sites coupled with primers containing selective nucleotides allowed the full potential of PCR to be realized while maintaining the advantages of restriction endonuclease analysis. Fluorescence-based fragment analysis with polyacrylamide gel electrophoresis provides the accurate band sizing required for homology assessment. The large number of phylogenetically informative characters obtained by fbAFLP is well suited for cluster analysis and database development. The method demonstrated excellent reproducibility and ease of performance and interpretation. We typed 30 epidemiologically well-characterized isolates of vancomycin-resistant enterococci from an outbreak in a university hospital by fbAFLP. Clustering of fbAFLP data matched epidemiological, microbiological, and pulsed-field gel electrophoresis data. This study demonstrates the unprecedented utility of fbAFLP for epidemiological investigation. Future developments in standardization and automation will set fbAFLP as the “gold standard” for molecular typing in epidemiology.
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Viana, F. M. P., J. E. Cardoso, H. A. O. Saraiva, M. A. S. V. Ferreira, R. L. R. Mariano e L. C. Trindade. "First Report of a Bacterial Leaf and Fruit Spot of Cashew Nut (Anacardium occidentale) Caused by Xanthomonas campestris pv. mangiferaeindicae in Brazil". Plant Disease 91, n.º 10 (outubro de 2007): 1361. http://dx.doi.org/10.1094/pdis-91-10-1361c.
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In 2003 and 2004, leaves and young fruits of cashew nut plants showing an undescribed disease symptom were observed on plants of an early-dwarf clone in a commercial orchard in Ceará and Piauí states in northeastern Brazil. Initial symptoms consisted of angular, water-soaked, dark-to-black spots on the leaf and at the mid-rib vein surrounding the leaf veins. Eventually, lesions also extended from the mid-rib to the secondary veins, delineating the vein system of the leaf. In young, green fruits, symptoms were large, dark, oily spots surrounded by conspicuous water-soaked areas. A yellow-pigmented colony was consistently recovered from the lesions on nutrient yeast-extract dextrose agar medium (3 g of meat extract, 5 g of peptone, 10 g of dextrose, 5 g of yeast extract, and 18 g of agar per liter). Physiological tests revealed colonies that were gram negative, strictly aerobic, oxidase negative, catalase positive, lacking fluorescent pigmentation on King's B medium, urea hydrolase negative, and able to grow on yeast dextrose calcium carbonate medium yielding yellow colonies. These tests indicated that the bacterium belonged to the genus Xanthomonas. PCR amplification of bacterial DNA using RST2 (1) and Xcv3R (3) primers resulted in identical band patterns to mango isolates Xanthomonas campestris pv. mangiferaeindicae. Restriction fragment length polymorphism analysis of PCR-amplified products of six isolates of X. campestris pv. mangiferaeindicae was conducted with HaeIII and showed different profile patterns on agarose gel, indicating genetic variability among these isolates. Pathogenicity was demonstrated by gently piercing and misting cashew leaves with a bacterial suspension adjusted to 106 CFU/ml. Inoculated plants were enclosed in plastic bags for 24 h and then incubated in a greenhouse (29 ± 1°C). Control plants were misted with sterile water and treated the same way. After 8 days, foliar symptoms similar to those observed in the field developed on all inoculated plants, and reisolated bacteria were characterized and found to be X. campestris pv. mangiferaeindicae. Control plants remained symptomless. To our knowledge, this is the first description of commercially grown cashew plants as host to X. campestris pv. mangiferaeindicae in Brazil. This disease may pose a serious problem to the cashew-growing industry in Brazil. This bacterial pathogen has been reported on mangoes (Mangifera indica) and cashew in India (2) under the former name of Pseudomonas mangiferae-indicae. References: (1) R. P. Leite, Jr. et al. Appl. Environ. Microbiol. 60:1068, 1994. (2) M. K. Patel et al. Curr. Sci. 17:189, 1948. (3) L. C. Trindade et al. Summa Phytopathol. 33:16, 2007.
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Levy, Laurene, Lisa A. Castlebury, Lori M. Carris, Robert J. Meyer e Guillermo Pimentel. "Internal Transcribed Spacer Sequence-Based Phylogeny and Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Differentiation of Tilletia walkeri and T. indica". Phytopathology® 91, n.º 10 (outubro de 2001): 935–40. http://dx.doi.org/10.1094/phyto.2001.91.10.935.
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A polymerase chain reaction-restriction fragment length polymorphism assay to distinguish Tilleita walkeri, a rye grass bunt fungus that occurs in the southeastern United States and Oregon, from T. indica, the Karnal bunt fungus, is described. The internal transcribed spacer (ITS) region of the ribosomal DNA repeat unit was amplified and sequenced for isolates of T. indica, T. walkeri, T. horrida, and a number of other taxa in the genus Tilletia. A unique restriction digest site in the ITS1 region of T. walkeri was identified that distinguishes it from the other taxa in the genus. Phylogenetic analysis of the taxa based on ITS sequence data revealed a close relationship between T. indica and T. walkeri, but more distant relationships between these two species and other morphologically similar taxa.
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Huys, G., L. Rigouts, K. Chemlal, F. Portaels e J. Swings. "Evaluation of Amplified Fragment Length Polymorphism Analysis for Inter- and Intraspecific Differentiation ofMycobacterium bovis, M. tuberculosis, andM. ulcerans". Journal of Clinical Microbiology 38, n.º 10 (2000): 3675–80. http://dx.doi.org/10.1128/jcm.38.10.3675-3680.2000.
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The usefulness of amplified fragment length polymorphism (AFLP) analysis was evaluated for the discrimination of Mycobacterium bovis (17 strains), M. tuberculosis (15 strains), andM. ulcerans (12 strains) at the inter- and intraspecific level. The AFLP technique is a whole-genome coverage genotypic fingerprinting method based on the selective PCR amplification of modified restriction fragments obtained through a double enzymatic digest and subsequent ligation of double-stranded restriction site-specific adapter oligonucleotides. Selective amplification ofApaI/TaqI templates with primer combination A02-T02 (both having an additional C at their 3′ end) generated autoradiographic AFLP fingerprints that were grouped by numerical analysis in two main AFLP clusters allowing clear separation ofM. ulcerans (cluster I) from the M. tuberculosis complex members M. bovis and M. tuberculosis (cluster II). Calculation of similarities using the band-based Dice correlation coefficient instead of the Pearson product-moment correlation coefficient revealed a further subgrouping in cluster II. The two resulting subclusters corresponded with the phenotypic identity of M. bovis and M. tuberculosis, respectively, and could also be visually identified by two AFLP marker bands. Because of the relatively low degree of genotypic variation among the AFLP band patterns of the latter two taxa, no correlation could be found with previously reported molecular typing data or with geographical origin. The use of primer combination A02-T01 (the latter having an A as selective base) did not increase the resolving power within the M. tuberculosis complex but resulted in a visual subgrouping of the M. ulcerans strains that was not observed with primer combination A02-T02. Based on the presence or absence of a single AFLP marker band, the M. ulcerans isolates could be unambiguously classified in two continental types corresponding with the African and Australian origin of the strains, respectively. In conclusion, the radioactive AFLP method proved to be a reproducible and reliable taxonomic tool for the differentiation of the three mycobacterial species under study and also demonstrated its potential use for typing of M. ulceransstrains when employing multiple primer combinations.
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Vandamme, P., L. Debruyne, E. De Brandt e E. Falsen. "Reclassification of Bacteroides ureolyticus as Campylobacter ureolyticus comb. nov., and emended description of the genus Campylobacter". International Journal of Systematic and Evolutionary Microbiology 60, n.º 9 (1 de setembro de 2010): 2016–22. http://dx.doi.org/10.1099/ijs.0.017152-0.
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The protein profiles, genomic amplified fragment length polymorphism patterns and 16S rRNA and cpn60 gene sequences of a diverse collection of 26 Bacteroides ureolyticus strains, along with published data on their DNA base, respiratory quinone and cellular fatty acid compositions, were used to reassess the taxonomy of this bacterial species. The results demonstrate that this organism is most appropriately allocated in the genus Campylobacter. The presence of much higher amounts of 18 : 1ω7c in its cellular fatty acid profile and its ability to digest gelatin and casein are the characteristics that differentiate it from present species of the genus Campylobacter. Therefore we propose to reclassify this species incertae sedis into the genus Campylobacter as Campylobacter ureolyticus with strain LMG 6451T (=CCUG 7319T =NCTC 10941T) as the type strain.
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PAUL, CATHERINE J., SHULIN TRAN, KEVIN J. TAM e JOHN W. AUSTIN. "A Unique Restriction Site in the flaA Gene Allows Rapid Differentiation of Group I and Group II Clostridium botulinum Strains by PCR–Restriction Fragment Length Polymorphism Analysis". Journal of Food Protection 70, n.º 9 (1 de setembro de 2007): 2133–39. http://dx.doi.org/10.4315/0362-028x-70.9.2133.
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Clostridium botulinum produces the potent botulinum neurotoxin, the causative agent of botulism. Based on distinctive physiological traits, strains of C. botulinum can be divided into four groups: however, only groups I and II are associated with human illness. Alignment of the flaA gene sequences from 40 group I and 40 group II strains identified a single BsrG1 restriction cut site that was present at base pair 283 in all group II flaA sequences and was not found in any group I sequence. The flaA gene was amplified by rapid colony PCR from 22 group I strains and 18 group II strains and digested with BsrGI restriction enzyme. Standard agarose gel electrophoresis with ethidium bromide staining showed two fragments, following restriction digestion of group II flaA gene amplicons with BsrGI, but only a single band of uncut flaA from group I strains. Combining rapid colony PCR with BsrGI restriction digest of the flaA gene at 60°C is a significant improvement over current methods, such as meat digestion or amplified fragment length polymorphism, as a strain can be identified as either group I or group II in under 5 h when starting with a visible plated C. botulinum colony.
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Fajardo, Diego, Don R. La Bonte e Robert L. Jarret. "553 Genetic Diversity in Papua New Guinea Sweetpotato Germplasm". HortScience 35, n.º 3 (junho de 2000): 491B—491. http://dx.doi.org/10.21273/hortsci.35.3.491b.
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The USDA gene bank currently maintains 668 accessions of cultivated sweetpotato and 219 accessions of related Ipomoea species. Information on the genetic diversity of the collection does not exist due to funding constraints. The development of a core collection would provide a subset of accessions that represent the genetic diversity of the main collection with a minimum of repetitiveness. The small size of the core collection would facilitate the evaluation of the accessions for economically important traits. The objective of this research is to develop a core collection of Papua New Guinea sweetpotato germplasm using the Amplified Fragment Length Polymorphisms (AFLPs) marker system. This approach to quantifying genetic diversity would later serve as a model for the development of a USDA sweetpotato germplasm core collection. The germplasm choosen for this study was collected from this crop's secondary center of genetic diversity based on its potential as a source of new traits. All genotypes were fingerprinted using four primer combinations that generated 224 markers. The molecular data was then analyzed using NTSYSpc 2.0 program to determine the relatedness of the genotypes. The molecular analysis showed a homogeneous genetic constitution. The extent of diversity among accessions was correlated with the geographic origin of the plant material.
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Fajardo, Diego, Don R. La Bonte e Robert L. Jarret. "Genetic Diversity in Papua New Guinea Sweetpotato Germplasm". HortScience 35, n.º 4 (julho de 2000): 551C—551b. http://dx.doi.org/10.21273/hortsci.35.4.551c.
Texto completo da fonteResumo:
The USDA gene bank currently maintains 668 accessions of cultivated sweetpotato and 219 accessions of related Ipomoea species. Information on the genetic diversity of the collection does not exist due to funding constraints. The development of a core collection would provide a subset of accessions that represent the genetic diversity of the main collection with a minimum of repetitiveness. The small size of the core collection would facilitate the evaluation of the accessions for economically important traits. The objective of this research is to develop a core collection of Papua New Guinea sweetpotato germplasm using the Amplified Fragment Length Polymorphisms (AFLPs) marker system. This approach to quantifying genetic diversity would later serve as a model for the development of a USDA sweetpotato germplasm core collection. The germplasm choosen for this study was collected from this crop's secondary center of genetic diversity based on its potential as a source of new traits. All genotypes were fingerprinted using four primer combinations that generated 224 markers. The molecular data was then analyzed using NTSYSpc 2.0 program to determine the relatedness of the genotypes. The molecular analysis showed a homogeneous genetic constitution. The extent of diversity among accessions was correlated with the geographic origin of the plant material.
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Lucardi, Rima D., Lisa E. Wallace e Gary N. Ervin. "Patterns of Genetic Diversity in Highly Invasive Species: Cogongrass (Imperata cylindrica) Expansion in the Invaded Range of the Southern United States (US)". Plants 9, n.º 4 (31 de março de 2020): 423. http://dx.doi.org/10.3390/plants9040423.
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The spatial expansions of invasive organisms in the novel range are generally expected to follow an isolation-by-distance relationship (IBD) if the invasion is biologically driven; however, many invasions are facilitated anthropogenically. This research focused on the extant expansion patterns of cogongrass (Imperata cylindrica). Cogongrass is a widespread invasive species throughout the southern United States (US). Patterns of infestation vary among US states. Cogongrass is pyrogenic, and its invasion threatens softwood (Pinus spp.) plantations, a substantial economic market for this US region. Over 600 individuals were sampled from seven invaded US states, using amplified fragment length polymorphisms (AFLPs) to assess genetic diversity and population structure. We suspected that differences in historical management efforts among US states influenced differences in genetic diversity and structure. We detected two genetic lineages at the highest level of analysis. One genetic lineage was locally restricted, whereas the other was found throughout the study region. Admixed individuals were found in all US states and consistently co-occurred with the dominant lineage, suggesting that secondary contact and hybridization may have facilitated expansion. The widespread prevalence of only one of the two detected genetic lineages suggests a primary genetic lineage responsible for on-going population expansion in the US.
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Richmond, Jonathan Q., e Elizabeth L. Jockusch. "Body size evolution simultaneously creates and collapses species boundaries in a clade of scincid lizards". Proceedings of the Royal Society B: Biological Sciences 274, n.º 1619 (8 de maio de 2007): 1701–8. http://dx.doi.org/10.1098/rspb.2007.0364.
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Speciation is generally viewed as an irreversible process, although habitat alterations can erase reproductive barriers if divergence between ecologically differentiated species is recent. Reversed speciation might also occur if geographical contact is established between species that have evolved the same reproductive isolating barrier in parallel. Here, we demonstrate a loss of intrinsic reproductive isolation in a clade of scincid lizards as a result of parallel body size evolution, which has allowed for gene flow where large-bodied lineages are in secondary contact. An mtDNA phylogeny confirms the monophyly of the Plestiodon skiltonianus species complex, but rejects that of two size-differentiated ecomorphs. Mate compatibility experiments show that the high degree of body size divergence imposes a strong reproductive barrier between the two morphs; however, the strength of the barrier is greatly diminished between parallel-evolved forms. Since two large-bodied lineages are in geographical contact in the Sierra Nevada Mountains of California, we were also able to test for postzygotic isolation under natural conditions. Analyses of amplified fragment length polymorphisms show that extensive gene exchange is occurring across the contact zone, resulting in an overall pattern consistent with isolation by distance. These results provide evidence of reversed speciation between clades that diverged from a common ancestor more than 12 Myr ago.
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Volpini, Lays Paula Bondi, Jerusa Araújo Dias, Luciana Bueno de Freitas, Maria Carmen Lopes Ferreira Silva, Angélica Espinosa Miranda e Liliana Cruz Spano. "Viral load and high prevalence of HR-HPV52 and 58 types in black women from rural communities". BMC Infectious Diseases 21, n.º 1 (17 de abril de 2021). http://dx.doi.org/10.1186/s12879-021-06042-6.
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Abstract Background The high-risk human papillomavirus (HR-HPV) infection is the main cause of cervical cancer development, and the most common types were included in the last approved nonavalent vaccine (9vHPV). Geographical, socioeconomic and ethnic barriers in developing countries challenge primary and secondary prevention measures of cervical cancer. We aimed to determine the prevalence of HPV infection and the viral load of HR-HPV 9vHPV-related types black women resident in rural semi-isolated communities. Methods A descriptive study was conducted with 273 cervical samples of women from rural communities of Southeastern Brazil. Viral DNA was amplified by PCR, the genotype was identified by Reverse Line Blot (RLB) and Restriction Fragment Length Polymorphism (RFLP), and real-time PCR was applied to determine the viral load. Results HPV frequency was 11.4% (31/273), associated with the presence of cytological abnormalities (32.3%; p < 0.001). Thirty-one distinct genotypes were detected; HR-HPV occurred in 64.5% (20/31) of the samples and the most prevalent type were HPV52 > 58, 59. Multiple infections occurred with up to nine different genotypes. The viral load of HR-HPV 9vHPV-related types was higher in lesions than in normal cytology cases (p = 0.04); “high” and “very high” viral load occurred in HSIL and LSIL, respectively (p = 0.04). Conclusions We highlight that despite the low HPV frequency in the black rural women population, the frequency of HR-HPV was high, particularly by the HR-HPV52 and 58 types. Moreover, the HR-HPV viral load increased according to the progression from normal to lesion, being a potential biomarker to identify those women at higher risk of developing cervical lesions in this population.
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Suga, Haruhisa, Mitsuhiro Arai, Emi Fukasawa, Keiichi Motohashi, Hiroyuki Nakagawa, Hideaki Tateishi, Shin-ichi Fuji, Masafumi Shimizu, Koji Kageyama e Mitsuro Hyakumachi. "Genetic Differentiation Associated with Fumonisin and Gibberellin Production in JapaneseFusarium fujikuroi". Applied and Environmental Microbiology 85, n.º 1 (19 de outubro de 2018). http://dx.doi.org/10.1128/aem.02414-18.
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ABSTRACTFusarium fujikuroiis a pathogenic fungus that infects rice. It produces several important mycotoxins, such as fumonisins. Fumonisin production has been detected in strains of maize, strawberry, and wheat, whereas it has not been detected in strains from rice seedlings infested with bakanae disease in Japan. We investigated the genetic relationships, pathogenicity, and resistance to a fungicide, thiophanate-methyl (TM), in 51 fumonisin-producing strains and 44 nonproducing strains. Phylogenetic analyses based on amplified fragment length polymorphism (AFLP) markers and two specific genes (a combined sequence of translation elongation factor 1α [TEF1α] and RNA polymerase II second-largest subunit [RPB2]) indicated differential clustering between the fumonisin-producing and -nonproducing strains. One of the AFLP markers, EATMCAY107, was specifically present in the fumonisin-producing strains. A specific single nucleotide polymorphism (SNP) between the fumonisin-producing and nonproducing strains was also detected inRPB2, in addition to an SNP previously found inTEF1α. Gibberellin production was higher in the nonproducing than in the producing strains according to anin vitroassay, and the nonproducing strains had the strongest pathogenicity with regard to rice seedlings. TM resistance was closely correlated with the cluster of fumonisin-nonproducing strains. The results indicate that intraspecific evolution in JapaneseF. fujikuroiis associated with fumonisin production and pathogenicity. Two subgroups of JapaneseF. fujikuroi, designated G group and F group, were distinguished based on phylogenetic differences and the high production of gibberellin and fumonisin, respectively.IMPORTANCEFusarium fujikuroiis a pathogenic fungus that causes rice bakanae disease. Historically, this pathogen has been known asFusarium moniliforme, along with many other species based on a broad species concept. Gibberellin, which is currently known as a plant hormone, is a virulence factor ofF. fujikuroi. Fumonisin is a carcinogenic mycotoxin posing a serious threat to food and feed safety. Although it has been confirmed thatF. fujikuroiproduces gibberellin and fumonisin, production varies among strains, and individual production has been obscured by the traditional appellation ofF. moniliforme, difficulties in species identification, and variation in the assays used to determine the production of these secondary metabolites. In this study, we discovered two phylogenetic subgroups associated with fumonisin and gibberellin production in JapaneseF. fujikuroi.
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TORUAN-MATHIUS, Nurita, ENDANG-YUNIASTUTI, Ridwan SETIAMIHARJA e Murdaningsih H. KARMANA. "Analisis genotip normal dan abnormal pada klon kelapa sawit (Elaeis guineensis Jacq.) dengan Amplified Fragment Length Polymorphism (AFLP) Analysis normal and abnormal genotypes of oil palm clones (Elaeis guineensis Jacq.) by Amplified Fragment Length Polymorphism (AFLP)". E-Journal Menara Perkebunan 73, n.º 1 (12 de março de 2016). http://dx.doi.org/10.22302/iribb.jur.mp.v73i1.159.
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SummaryTissue culture-derived plants of oil palmmay develop abnormal flowers in whichprimordial stamens are converted into carpel-liketissue or mantled fruits, and sterile male flowers.This abnormality can be heritable, individualpalm may show variation in mantling andreversion to the normal phenotype over time hasbeen observed. The aim of these experiments wasto analyze the differences between normal andabnormal genotypes by DNA-AFLP. DNA wasisolated from young fruits of three clones,MK152, MK209, and MK 212 each of themconsisted of normal fruits, abnormal fruits andsterile male flowers. The research consisted of (i)selection of AFLP primer which can producepolymorphic bands, (ii) genetic similaritiesanalysis, UPGMA, principal component analysisand specific DNA bands between normal orabnormal genotypes. For primers selection, 20AFLP primers with DNA from MK 152 normaland abnormal genotypes were used. The selectedprimers were then used to amplify DNA of ninegenotypes. The results show that 10 primer com-binations EcoRI/MseI produced polymorphicbands. Each primer from 10 primer producedonly one or two DNA bands indicates that thedifferences between normal and abnormalgenotypes in the same clone. However, nopolymorphism was consistently found betweennormal and abnormal clones in all the sets.Genetic similarity analysis shows that betweengenotype had high genetic similarities, around92-99%. The results of UPGMA found thedifferent clustering between normal fruit,abnormal male and abnormal fruits. The resultsshow same as clustering based on first, secondand third component. This suggest that, whilstAFLP method is an effective way of detectingvariation in tissue culture-derived plants,different approaches are required to identify thecasual basis of the mantled fruit abnormality.RingkasanTanaman kelapa sawit yang dihasilkan darikultur jaringan, umumnya dalam perkembangan-nya akan memiliki organ reproduktif yangabnormal. Abnormalitas berupa primordialstamen berkembang menjadi bentuk jaringanseperti karpel, buah mantel, atau bunga jantanmandul. Penelitian ini bertujuan untukmendapatkan pembeda DNA-AFLP antaragenotip normal dan abnormal pada klon-klonkelapa sawit. DNA diisolasi dari buah muda klonMK 152, MK 209, dan MK 212 yang masing-masing terdiri atas genotip normal, berbuahabnormal, dan berbunga jantan steril. Percobaanmencakup (i) seleksi primer AFLP yang mampumenghasilkan pita yang polimorfis, (ii) analisiskemiripan genetik, UPGMA, komponen utamadan pita pembeda antar genotip normal danabnormal. Seleksi primer dilakukan terhadap 20primer AFLP menggunakan DNA dari genotipMK 152 yang normal dan abnormal. Selanjutnyaprimer terpilih digunakan untuk mengamplifikasiDNA dari kesembilan genotip yang diuji. Hasilyang diperoleh menunjukkan bahwa 10 kombi-nasi primer EcoRI/MseI mampu menghasilkanpita yang polimorfis. Dari 10 primer yang diuji,masing-masing hanya menghasilkan satu ataudua pita DNA yang mampu membedakan genotipnormal dan abnormal dalam klon yang sama.Namun, tidak ada pita DNA spesifik yangmampu membedakan genotip normal denganabnormal untuk seluruh klon yang diuji. Analisiskemiripan genetik menunjukkan bahwa antargenotip memiliki kemiripan genetik yang sangattinggi, yaitu 92-99%. Dari hasil UPGMAdiperoleh pengelompokan yang terpisah antargenotip normal, abnormal jantan dan buahabnormal. Hasil tersebut didukung olehpengelompokan berdasarkan komponen utamasatu, dua dan tiga. Dapat disimpulkan bahwa,teknik AFLP tidak efektif untuk mendeteksipembeda antar genotip tanaman yang diperolehdari kultur jaringan, pendekatan lainnyadiperlukan untuk mengidentifikasi abnormalitas.
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TORUAN-MATHIUS, Nurita, ENDANG-YUNIASTUTI, Ridwan SETIAMIHARJA e Murdaningsih H. KARMANA. "Analisis genotip normal dan abnormal pada klon kelapa sawit (Elaeis guineensis Jacq.) dengan Amplified Fragment Length Polymorphism (AFLP) Analysis normal and abnormal genotypes of oil palm clones (Elaeis guineensis Jacq.) by Amplified Fragment Length Polymorphism (AFLP)". E-Journal Menara Perkebunan 73, n.º 1 (12 de março de 2016). http://dx.doi.org/10.22302/ppbbi.jur.mp.v73i1.159.
Texto completo da fonteResumo:
SummaryTissue culture-derived plants of oil palmmay develop abnormal flowers in whichprimordial stamens are converted into carpel-liketissue or mantled fruits, and sterile male flowers.This abnormality can be heritable, individualpalm may show variation in mantling andreversion to the normal phenotype over time hasbeen observed. The aim of these experiments wasto analyze the differences between normal andabnormal genotypes by DNA-AFLP. DNA wasisolated from young fruits of three clones,MK152, MK209, and MK 212 each of themconsisted of normal fruits, abnormal fruits andsterile male flowers. The research consisted of (i)selection of AFLP primer which can producepolymorphic bands, (ii) genetic similaritiesanalysis, UPGMA, principal component analysisand specific DNA bands between normal orabnormal genotypes. For primers selection, 20AFLP primers with DNA from MK 152 normaland abnormal genotypes were used. The selectedprimers were then used to amplify DNA of ninegenotypes. The results show that 10 primer com-binations EcoRI/MseI produced polymorphicbands. Each primer from 10 primer producedonly one or two DNA bands indicates that thedifferences between normal and abnormalgenotypes in the same clone. However, nopolymorphism was consistently found betweennormal and abnormal clones in all the sets.Genetic similarity analysis shows that betweengenotype had high genetic similarities, around92-99%. The results of UPGMA found thedifferent clustering between normal fruit,abnormal male and abnormal fruits. The resultsshow same as clustering based on first, secondand third component. This suggest that, whilstAFLP method is an effective way of detectingvariation in tissue culture-derived plants,different approaches are required to identify thecasual basis of the mantled fruit abnormality.RingkasanTanaman kelapa sawit yang dihasilkan darikultur jaringan, umumnya dalam perkembangan-nya akan memiliki organ reproduktif yangabnormal. Abnormalitas berupa primordialstamen berkembang menjadi bentuk jaringanseperti karpel, buah mantel, atau bunga jantanmandul. Penelitian ini bertujuan untukmendapatkan pembeda DNA-AFLP antaragenotip normal dan abnormal pada klon-klonkelapa sawit. DNA diisolasi dari buah muda klonMK 152, MK 209, dan MK 212 yang masing-masing terdiri atas genotip normal, berbuahabnormal, dan berbunga jantan steril. Percobaanmencakup (i) seleksi primer AFLP yang mampumenghasilkan pita yang polimorfis, (ii) analisiskemiripan genetik, UPGMA, komponen utamadan pita pembeda antar genotip normal danabnormal. Seleksi primer dilakukan terhadap 20primer AFLP menggunakan DNA dari genotipMK 152 yang normal dan abnormal. Selanjutnyaprimer terpilih digunakan untuk mengamplifikasiDNA dari kesembilan genotip yang diuji. Hasilyang diperoleh menunjukkan bahwa 10 kombi-nasi primer EcoRI/MseI mampu menghasilkanpita yang polimorfis. Dari 10 primer yang diuji,masing-masing hanya menghasilkan satu ataudua pita DNA yang mampu membedakan genotipnormal dan abnormal dalam klon yang sama.Namun, tidak ada pita DNA spesifik yangmampu membedakan genotip normal denganabnormal untuk seluruh klon yang diuji. Analisiskemiripan genetik menunjukkan bahwa antargenotip memiliki kemiripan genetik yang sangattinggi, yaitu 92-99%. Dari hasil UPGMAdiperoleh pengelompokan yang terpisah antargenotip normal, abnormal jantan dan buahabnormal. Hasil tersebut didukung olehpengelompokan berdasarkan komponen utamasatu, dua dan tiga. Dapat disimpulkan bahwa,teknik AFLP tidak efektif untuk mendeteksipembeda antar genotip tanaman yang diperolehdari kultur jaringan, pendekatan lainnyadiperlukan untuk mengidentifikasi abnormalitas.
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Li, Jiao-Sheng, Luo-Yu Wu, Hui Zhang, Xiu-Shi Song, Jian-Xin Wang, Mingguo Zhou e Yi-ping Hou. "PCR-RFLP for detection of Fusarium graminearum genotypes with resistance to phenamacril". Plant Disease, 12 de outubro de 2020. http://dx.doi.org/10.1094/pdis-06-20-1156-re.
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Phenamacril is a cyanoacrylate fungicide that provides excellent control of Fusarium head blight (FHB) or wheat scab, which is caused predominantly by Fusarium graminearum and Fusarium asiaticum. Previous studies revealed that codon mutations of the myosin-5 gene of Fusarium spp. conferred resistance to phenamacril in vitro lab experiments. In this study, PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) was developed to detect three common mutations (A135T, GCC to ACC at codon 135; S217L, TCA to TTA at codon 217, and E420K, GAA to AAA at codon 420) in F. graminearum induced by fungicide domestication in vitro. PCR products of 841 bp (for mutation of A135T), 802 bp (for mutation of S217L) or 1649 bp (for mutation of E420K) in myosin-5 gene were amplified respectively by appropriate primer pairs. Restriction enzyme KpnⅠ, TasⅠ or DraⅠ was used to distinguish phenamacril-sensitive and -resistant strains with mutation genotypes of A135T, S217L and E420K, respectively. KpnⅠ digested the 841 bp PCR products of phenamacri-resistant strains with codon mutation A135T into two fragments of 256 bp and 585 bp. In contrast, KpnⅠ did not digest the PCR products of sensitive strains. TasⅠ digested the 802 bp PCR products of phenamacril-strains with codon mutation S217L into three fragments of 461 bp, 287bp and 54 bp. In contrast, TasⅠ digestion of the 802 bp PCR products of phenamacril-sensitive strains resulted in only two fragments of 515bp and 287bp. DraⅠ digested the 1649 bp PCR products of phenamacril-resistant strains with codon mutation E420K into two fragments of 932 bp and 717 bp, while the PCR products of phenamacril-sensitive strains was not digested. The three genotypes of resistance mutations were determined by analyzing electrophoresis patterns of the digestion fragments of PCR products. The PCR-RFLP method was evaluated on 48 phenamacril-resistant strains induced by fungicide domestication in vitro and compared with the conventional method (mycelial growth on fungicide-amended agar). The accuracy of the PCR-RFLP method for detecting the three resistant mutation genotypes of F. graminearum to phenamacril was 95.12% compared with conventional method. Bioinformatics analysis revealed that the PCR-RFLP method could also be used to detect the codon mutations of A135T and E420K in F. asiaticum.