scholarly journals Discovery of a New Self-incompatibility Allele in Apple

HortScience ◽  
2000 ◽  
Vol 35 (7) ◽  
pp. 1329-1332 ◽  
Author(s):  
Shogo Matsumoto ◽  
Kentaro Kitahara
Keyword(s):  
Preliminary Investigation ◽  
Pcr Amplification ◽  
Rflp Analysis ◽  
Unknown Origin ◽  
Analysis Method ◽  
Apple Cultivars ◽  
Total Dna ◽  
Polymerase Chain ◽  
Or History ◽  
Allele Identification

A polymerase chain reaction (PCR)-based method for identifying the S-alleles in the Asian pear [Pyrus pyrifolia (Burm) Nak.] was applied to apple (Malus ×domestica Borkh.) cultivars. With minor modifications in one of the primers, the fragments from S-genes (S-RNases) with introns were amplified from total DNA of apple cultivars possessing S2-, S3-, S5-, S7-(=Sd-), S9-(=Sc-), Sf- and Sg-allele genotypes. S-genes within S24-(=Sh-) and S26-alleles were also amplified. The PCR amplification step of this method appears to be useful for preliminary investigation of apple S-genotypes, especially for species or cultivars of unknown origin or history. Using the primers, which are a part of a new S-allele, the Se-allele encoding Se-RNase with an intron in the Se-allele was amplified. We cloned the cDNA of Se-RNase, and developed a PCR-restriction fragment length polymorphism (RFLP) analysis method for Se-allele identification. S-allele genotypes of seven apple cultivars were investigated.

scholarly journals Complete Sequences of the S-genes, Sd- and Sh-RNase cDNA in Apple

HortScience ◽  
2000 ◽  
Vol 35 (4) ◽  
pp. 712-715 ◽  
Author(s):  
Kentaro Kitahara ◽  
Junichi Soejima ◽  
Hiromitsu Komatsu ◽  
Hirokazu Fukui ◽  
Shogo Matsumoto
Keyword(s):  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Analysis Method ◽  
Chain Reaction ◽  
Specific Polymerase Chain Reaction ◽  
Complete Sequences ◽  
Allele Specific ◽  
Apple Cultivars ◽  
Polymerase Chain ◽  
S Genes

The S-locus genes in the pistil (S-RNases) were cloned from the apple (Malus ×domestica Borkh.) cultivar Akane (S-genotype SdSh from pollination analysis). The Sd- and Sh-RNase corresponded to S7- and S24-RNase, which have been cloned from `Idared' and `Braeburn', respectively. Sh-RNase was very similar to Sf- and Sg-RNases at the deduced amino acid-sequence levels (93%). We developed an S-allele specific polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis method for distinguishing the Sh from Sf and Sg, and the Sh-alleles of `Akane', `Touhoku 2', `Vista Bella', and `Worcester Pearmain' were identified. We also identified the S-allele genotypes of 16 apple cultivars.

scholarly journals Responses of Six Eurasian Ulmus Cultivars to a North American Elm Yellows Phytoplasma

Plant Disease ◽  
2000 ◽  
Vol 84 (12) ◽  
pp. 1266-1270 ◽  
Author(s):  
W. A. Sinclair ◽  
A. M. Townsend ◽  
H. M. Griffiths ◽  
T. H. Whitlow
Keyword(s):  
Pcr Amplification ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Phloem Necrosis ◽  
Fluorescence Test ◽  
Polymerase Chain ◽  
Elm Yellows ◽  
Control Tree ◽  
Eurasian Species ◽  
Dapi Fluorescence

Elms (genus Ulmus) of six clonal cultivars representing Eurasian species and hybrids were grafted when 2 to 3 years old with bark patches from U. rubra infected with an elm yellows phytoplasma or were left untreated as controls. The cultivars were U. glabra × minor ‘Pioneer’, U. minor × parvifolia ‘Frontier’, U. parvifolia ‘Pathfinder’, U. wilsoniana ‘Prospector’, and the complex hybrids ‘Homestead’ and ‘Patriot’. Trees were evaluated for infection and symptoms 1 or 2 years after inoculation. Infection was detected via the 4′,6-diamidino-2-phenylindol e·2HCl (DAPI) fluorescence test in 26 of 86 grafted trees representing five cultivars. Infection of selected trees was confirmed by polymerase chain reaction (PCR) amplification of a fragment of phytoplasmal rDNA, and the phytoplasma was identified by restriction fragment length polymorphism (RFLP) analysis of the amplified DNA using restriction enzymes AluI, RsaI, and TaqI. Elm yellows phytoplasma was also identified by nested PCR and RFLP analysis in two of seven inoculated, healthy-appearing, DAPI-negative trees and one noninoculated control tree. All RFLP profiles were identical to that of reference strain EY1. Phytoplasma-associated symptoms, observed in five cultivars, included suppressed growth, progressive size reduction of apical shoots and leaves, chlorosis, foliar reddening, witches'-brooms, and dieback. Phyto-plasma was not detected in cv. Homestead. Possible resistance of this cultivar to elm yellows phytoplasma was indicated by localized phloem necrosis in stems below inoculum patches.

scholarly journals Identification of Staphylococcus species isolated from preputium of Aceh cattle based on 16S rRNA gene sequences analysis

2019 ◽  
Vol 12 (10) ◽  
pp. 1540-1545
Author(s):  
Muhammad Hambal ◽  
Masda Admi ◽  
Safika Safika ◽  
Wahyu Eka Sari ◽  
Teuku Reza Ferasyi ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Phylogenetic Tree ◽  
Bacterial Isolate ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Multiple Alignments ◽  
Total Dna ◽  
Sequences Analysis ◽  
Polymerase Chain

Aim: This research aimed to identify Staphylococcus species isolated from preputial swabs of healthy Aceh cattle, based on 16S ribosomal RNA gene analysis. Materials and Methods: The bacterium was isolated from preputial swabs of healthy Aceh cattle. The total DNA from the isolated bacteria was extracted using the Genomic DNA Mini Kit followed by polymerase chain reaction (PCR) amplification of the 16S rRNA gene. The product of PCR amplification was then sequenced and aligned to the known sequences in the GenBank database by multiple alignments and was also analyzed by bioinformatics software to construct a phylogenetic tree. Results: The results revealed that the bacterial isolate 3A had genetically closed relation to Staphylococcus pasteuri with <97% maximum identity. Data derived from the phylogenetic tree revealed that the bacterial isolate 3A was also related to Staphylococcus warneri, yet, it shows a different evolutionary distance with the ancestors (S. pasteuri). Conclusion: The results of this research suggested that the bacterium 3A, isolated from preputial swabs of healthy Aceh cattle, is a Staphylococcus species.

Localization of the Br Polymorphism on a 144 bp Exon of the GPIa Gene and Its Application in Platelet DNA Typing

1994 ◽  
Vol 71 (05) ◽  
pp. 651-654 ◽  
Author(s):  
Rainer Kalb ◽  
Sentot Santoso ◽  
Katja Unkelbach ◽  
Volker Kiefel ◽  
Christian Mueller-Eckhardt
Keyword(s):  
Genomic Organization ◽  
Fragment Length Polymorphism ◽  
Human Platelet ◽  
Dna Typing ◽  
Rflp Analysis ◽  
Serological Typing ◽  
Allele Specific ◽  
Polymerase Chain ◽  
Alloimmune Thrombocytopenia ◽  
Rflp Typing

SummaryAlloimmunization against the human platelet alloantigen system Br (HPA-5) is the second most common cause of neonatal alloimmune thrombocytopenia (NAIT) in Caucasian populations. We have recently shown that a single base polymorphism at position 1648 on platelet mRNA coding for GPIa results in an aminoacid substitution at position 505 on the mature GPIa which is associated with the two serological defined Br phenotypes.Since DNA-typing of platelet alloantigens offers possibilities for useful clinical applications, we designed genomic DNA-based restriction fragment length polymorphism (RFLP) typing for Br alloantigens. To establish this technique we analyzed the genomic organization of GPIa adjacent to the polymorphic base. Using the polymerase chain reaction (PCR) of blood cell DNA we have identified two introns (approximately 1.7 and 1.9 kb) flanking a 144 bp coding sequence of the GPIa gene encompassing the polymorphic base 1648. Based on the in- tron sequence, a PCR primer was constructed to amplify a 274 bp fragment which was used for allele-specific RFLP to determine the Br genotypes. The results of RFLP analysis using Mnll endonuclease obtained from 15 donors (2 Br37*, 2 Br^ and 11 Brb/b) correlate perfectly with serological typing by monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay.

Characterization of a cDNA for Rhesus Monkey Protein C Inhibitor – Evidence for N-Terminal Involvement in Heparin Stimulation

1995 ◽  
Vol 74 (04) ◽  
pp. 1079-1087 ◽  
Author(s):  
Klaus-P Radtke ◽  
José A Fernández ◽  
Bruno O Villoutreix ◽  
Judith S Greengard ◽  
John H Griffin
Keyword(s):  
Rhesus Monkey ◽  
Protein C ◽  
Activated Protein C ◽  
Pcr Amplification ◽  
Amino Acid Sequences ◽  
Heparin Binding ◽  
Reactive Center ◽  
Protein C Inhibitor ◽  
Polymerase Chain

SummarycDNAs for protein C inhibitor (PCI) were cloned from human and rhesus monkey 1 liver RNAs by reverse transcription and polymerase chain reaction (PCR) amplification. Sequencing showed that rhesus monkey and human PCI cDNAs were 93% identical. Predicted amino acid sequences differed at 26 of 387 residues. Pour of these differences (T352M, N359S, R362K, L3631) were in the reactive center loop that is important for inhibitory specificity, and two were in the N-terminal helix (M8T, E13K) that is implicated in glycosaminoglycan binding. PCI in human or rhesus monkey plasma showed comparable inhibitory activity towards human activated protein C in the presence of 10 U/ml heparin. However, maximal acceleration of the inhibition of activated protein C required 5-fold lower heparin concentration for rhesus monkey than for human plasma, consistent with the interpretation that the additional positive charge (E13K) in a putative-heparin binding region increased the affinity for heparin.

scholarly journals Identification of Phytoplasmas Representing Multiple New Genetic Lineages from Phloem-Feeding Leafhoppers Highlights the Diversity of Phytoplasmas and Their Potential Vectors

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 352
Author(s):  
Wei Wei ◽  
Valeria Trivellone ◽  
Christopher H. Dietrich ◽  
Yan Zhao ◽  
Kristi D. Bottner-Parker ◽  
...  
Keyword(s):  
Host Range ◽  
Quantitative Polymerase Chain Reaction ◽  
Rflp Analysis ◽  
Natural Habitats ◽  
Genetic Lineages ◽  
Genetic Subgroup ◽  
Polymerase Chain ◽  
Phloem Feeding ◽  
Fresh Insight

Phytoplasmas are obligate transkingdom bacterial parasites that infect a variety of plant species and replicate in phloem-feeding insects in the order Hemiptera, mainly leafhoppers (Cicadellidae). The insect capacity in acquisition, transmission, survival, and host range directly determines the epidemiology of phytoplasmas. However, due to the difficulty of insect sampling and the lack of follow-up transmission trials, the confirmed phytoplasma insect hosts are still limited compared with the identified plant hosts. Recently, quantitative polymerase chain reaction (qPCR)-based quick screening of 227 leafhoppers collected in natural habitats unveiled the presence of previously unknown phytoplasmas in six samples. In the present study, 76 leafhoppers, including the six prescreened positive samples, were further examined to identify and characterize the phytoplasma strains by semi-nested PCR. A total of ten phytoplasma strains were identified in leafhoppers from four countries including South Africa, Kyrgyzstan, Australia, and China. Based on virtual restriction fragment length polymorphism (RFLP) analysis, these ten phytoplasma strains were classified into four distinct ribosomal (16Sr) groups (16SrI, 16SrIII, 16SrXIV, and 16SrXV), representing five new subgroups (16SrI-AO, 16SrXIV-D, 16SrXIV-E, 16SrXIV-F, and 16SrXV-C). The results strongly suggest that the newly identified phytoplasma strains not only represent new genetic subgroup lineages, but also extend previously undiscovered geographical distributions. In addition, ten phytoplasma-harboring leafhoppers belonged to seven known leafhopper species, none of which were previously reported insect vectors of phytoplasmas. The findings from this study provide fresh insight into genetic diversity, geographical distribution, and insect host range of phytoplasmas. Further transmission trials and screening of new potential host plants and weed reservoirs in areas adjacent to collection sites of phytoplasma harboring leafhoppers will contribute to a better understanding of phytoplasma transmission and epidemiology.

scholarly journals DMSO Improves the Ski-Slope Effect in Direct PCR

2021 ◽  
Vol 11 (4) ◽  
pp. 1943
Author(s):  
Joo-Young Kim ◽  
Ju Yeon Jung ◽  
Da-Hye Kim ◽  
Seohyun Moon ◽  
Won-Hae Lee ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Pcr Amplification ◽  
Analytical Techniques ◽  
Direct Pcr ◽  
Dna Profile ◽  
Novel Technologies ◽  
Specific Amplification ◽  
Polymerase Chain ◽  
Dna Profiles

Analytical techniques such as DNA profiling are widely used in various fields, including forensic science, and novel technologies such as direct polymerase chain reaction (PCR) amplification are continuously being developed in order to acquire DNA profiles efficiently. However, non-specific amplification may occur depending on the quality of the crime scene evidence and amplification methods employed. In particular, the ski-slope effect observed in direct PCR amplification has led to inaccurate interpretations of the DNA profile results. In this study, we aimed to reduce the ski-slope effect by using dimethyl sulfoxide (DMSO) in direct PCR. We confirmed that DMSO (3.75%, v/v) increased the amplification yield of large-sized DNA sequences more than that of small-sized ones. Using 50 Korean buccal samples, we further demonstrated that DMSO reduced the ski-slope effect in direct PCR. These results suggest that the experimental method developed in this study is suitable for direct PCR and may help to successfully obtain DNA profiles from various types of evidence at crime scenes.

scholarly journals Characterization of Xanthomonas axonopodis pv. phaseoli isolates

2008 ◽  
Vol 34 (3) ◽  
pp. 228-231 ◽  
Author(s):  
Willian Mário de Carvalho Nunes ◽  
Maria Júlia Corazza ◽  
Silvana Aparecida Crestes Dias de Souza ◽  
Siu Mui Tsai ◽  
Eiko Eurya Kuramae
Keyword(s):  
Xanthomonas Campestris ◽  
Random Amplified Polymorphic Dna ◽  
Hypersensitive Reaction ◽  
Chain Reaction ◽  
Xanthomonas Axonopodis ◽  
Paulo State ◽  
Total Dna ◽  
Polymerase Chain ◽  
Bacterial Plant Pathogen

A simple, quick and easy protocol was standardized for extraction of total DNA of the bacteria Xanthomonas axonopodis pv. phaseoli. The DNA obtained by this method had high quality and the quantity was enough for the Random Amplified Polymorphic DNA (RAPD) reactions with random primers, and Polymerase Chain Reaction (PCR) with primers of the hypersensitivity and pathogenicity gene (hrp). The DNA obtained was free of contamination by proteins or carbohydrates. The ratio 260nm/380nm of the DNA extracted ranged from 1.7 to 1.8. The hrp gene cluster is required by bacterial plant pathogen to produce symptoms on susceptible hosts and hypersensitive reaction on resistant hosts. This gene has been found in different bacteria as well as in Xanthomonas campestris pv. vesicatoria (9). The primers RST21 and RST22 (9) were used to amplify the hrp gene of nine different isolates of Xanthomonas axonopodis pv. phaseoli from Botucatu, São Paulo State, Brazil, and one isolate, "Davis". PCR amplified products were obtained in all isolates pathogenic to beans.

scholarly journals Easy and Efficient DNA Extraction from Woody Plants for the Detection of Phytoplasmas by Polymerase Chain Reaction

Plant Disease ◽  
1999 ◽  
Vol 83 (5) ◽  
pp. 482-485 ◽  
Author(s):  
Margaret J. Green ◽  
Dan A. Thompson ◽  
Donald J. MacKenzie
Keyword(s):  
Polymerase Chain Reaction ◽  
Plant Material ◽  
Woody Plant ◽  
Leaf Roll ◽  
Chain Reaction ◽  
Efficient Procedure ◽  
Identical Manner ◽  
Total Dna ◽  
Polymerase Chain ◽  
Selection Of

A simple and efficient procedure for the extraction of high-quality DNA from phytoplasma-infected woody and herbaceous plants for polymerase chain reaction (PCR) detection is described. This procedure does not require phenol, chloroform, or alcohol for the precipitation of nucleic acids. Herbaceous and woody plant material are extracted in an identical manner with no additional purification or enrichment steps required. The method utilizes commercially available microspin-column matrices, and the extraction of total DNA can be achieved in less than 1 h. The method has been used to successfully purify phytoplasma DNA from whole leaves, leaf petioles and midribs, roots, and dormant wood from a diverse selection of plant material. The phytoplasmas detected by PCR include pear decline, western X-disease, peach yellow leaf roll, peach rosette, apple proliferation, Australian grapevine yellows, and Vaccinium witches'-broom.

scholarly journals Amplification Refractory Mutation System – Polymerase Chain Reaction for Rapid Detection of rpoB Gene Mutations in Mycobacterium tuberculosis

2017 ◽  
Vol 5 (1) ◽  
pp. 81-85 ◽  
Author(s):  
Hemanta Kumari Chaudhary ◽  
Mitesh Shrestha ◽  
Prakash Chaudhary ◽  
Bal Hari Poudel
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Rpob Gene ◽  
Amplification Refractory Mutation System ◽  
Chain Reaction ◽  
Arms Pcr ◽  
Mutation System ◽  
Mdr Tb ◽  
Total Dna ◽  
Polymerase Chain ◽  
Rifampin Resistance

Multidrug-resistant tuberculosis (MDR-TB) has become a serious worldwide threat including in Nepal. MDR-TB refers to the pathological condition whereby Mycobacterium tuberculosis becomes resistant to the first line of drug treatment i.e. rifampin and isoniazid. Resistance to rifampin (RIF) is mainly caused by the mutations in the rpoB gene which codes for the β-subunit of RNA polymerase. In this study, Amplification Refractory Mutation System – Polymerase Chain Reaction (ARMS – PCR) technique has been used to detect mutations in the rpoB gene of Mycobacterium tuberculosis. Total DNA samples of 34 phenotypic MDR-TB were subjected to ARMS – PCR using three different codon specific primers (516, 526 and 531). These three codons occupy large portion of total mutation responsible for rifampin resistance. Out of the total DNA samples, all were bearing mutation in at least one of the three codons mentioned. Of those bearing mutation, the highest number had mutation in codon 531 (97.05 %) followed by codon 516 (17.64 %) and finally in codon 526 (11.76%) respectively. Hence, ARMS – PCR may be used as an alternative diagnostic technique for detection of rifampin resistance in Mycobacterium tuberculosis strains, especially for a developing country like Nepal.Int. J. Appl. Sci. Biotechnol. Vol 5(1): 81-85

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