Barcoding of ToLCV Resistant Tomato Germplasm in Bangladesh

The current study was carried out to confirm the existence of the ToLCV resistant genes (Ty-1 to Ty-5) in the germplasm using molecular markers and to identify at the genomic level following phylogenetic relationships analysis among some local tomato germplasm using DNA barcoding. Most of the tomato germplasm (Ten out of 14) contain the dominant Ty-genes as revealed by PCR analysis. “Barcoding” of the non-coding plastid trnH-psbA intergenic spacer region, three plastidal regions: rbcL, rpoB, rpoC1, spacer region of nuclear genome ITS and a mitochondrial region matK were employed following PCR and sequence analysis of the germplasm. Among all the barcode genes, rpoB, rbcL, trnH-psbA and ITS were leading candidates for successful amplification and used for the identification of the germplasm as S. lycopersicum in multi-locus identification based on their sequences. Neighbor-Joining phylogenetic tree was constructed in which the germplasm were clustered into five main clades. The current study was successful to establish an efficient barcoding protocol for the correct identification of tomatoes and was capable of establishing elite gene source(s) for biotic stress resistance tomato varieties which would serve as potential donor plants in modern breeding programs. Plant Tissue Cult. & Biotech. 30(1): 107-117, 2020 (June)

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Erratum to “Identification of Bartonella strains isolated from wild and domestic ruminants by a single-step PCR analysis of the 16S–23S intergenic spacer region” [Vet. Microbiol. 98 (2004) 63–69]

Veterinary Microbiology ◽

10.1016/j.vetmic.2004.02.001 ◽

2004 ◽

Vol 100 (1-2) ◽

pp. 139-140

Author(s):

R Maillard ◽

M Vayssier-Taussat ◽

C Bouillin ◽

C Gandoin ◽

L Halos ◽

...

Keyword(s):

Intergenic Spacer ◽

Single Step ◽

Pcr Analysis ◽

Intergenic Spacer Region ◽

Domestic Ruminants

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VARIASI GENETIK Lactobacillus fermentum Beijerink ASAL SAYUR ASIN BERDASARKAN ANALISIS RFLP 16S-23S rDNA ISR, RAPD-PCR DAN ERIC-PCR

BERITA BIOLOGI ◽

10.14203/beritabiologi.v16i2.2772 ◽

2017 ◽

Vol 16 (2) ◽

Author(s):

Sulistiani Sulistiani ◽

Wibowo Mangunwardoyo ◽

Abinawanto Abinawanto ◽

Endang Sukara ◽

Achmad Dinoto ◽

...

Keyword(s):

Genetic Variation ◽

Random Amplified Polymorphic Dna ◽

Intergenic Spacer ◽

Molecular Techniques ◽

Lactobacillus Fermentum ◽

Pcr Analysis ◽

Intergenic Spacer Region ◽

Rapd Pcr ◽

Upgma Cluster Analysis ◽

Process Combination

Molecular analysis of Lactobacillus fermentum isolates is essential to understand their genetic variation in relations to their roles in sayur asin fermentation process. Combination of three molecular techniques which is restriction fragment length polymorphism (RFLP) of 16S23S rDNA intergenic spacer region (ISR), random amplified polymorphic DNA (RAPD-PCR) and an enterobacterial repetitive intergenic consensus (ERIC-PCR) analysis were performed to discriminate 19 representative isolates of L. fermentum isolated from sayur asin. The result showed that L. fermentum strain D11 is distantly related to other isolates based on RFLP using HhaI restriction enzyme and RAPDPCR analyses. In addition, both of RAPD-PCR and ERIC-PCR successfully determined the genetic variation among L. fermentum strains by exhibiting distinct 4-8 bands (800-2080 bp) and 4-10 bands (280-3050 bp), respectively. A dendogram generated from UPGMA cluster analysis of both RAPD-PCR and ERIC-PCR data showed two distinct genotypic groups exist among L. fermentum isolated from sayur asin in Indonesia.

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New PCR-based assay for the identification of Pectobacterium carotovorum causing potato soft rot

Plant Disease ◽

10.1094/pdis-08-21-1676-re ◽

2021 ◽

Author(s):

M. Belén Suárez ◽

Marta Diego ◽

F. J. Feria ◽

M J Martín-Robles ◽

Sergio Moreno ◽

...

Keyword(s):

Real Time ◽

Dna Sequences ◽

Real Time Pcr ◽

Bacterial Species ◽

Intergenic Spacer ◽

Soft Rot ◽

Pcr Analysis ◽

Rrna Gene ◽

Accurate Identification ◽

Intergenic Spacer Region

Soft rot on potato tuber is a destructive disease caused by pathogenic bacterial species of the genera Pectobacterium and Dickeya. Accurate identification of the causal agent is necessary to ensure adequate disease management, since different species may have distinct levels of aggressiveness and host range. One of the most important potato pathogens is P. carotovorum, a highly heterogeneous species capable of infecting multiple hosts. The complexity of this species, until recently divided into several subspecies, has made it difficult to develop precise diagnostic tests. This study proposes a PCR assay based on the new pair of primers Pcar1F/R to facilitate the identification of potato isolates of P. carotovorum according to the most recent taxonomic description of this species. The new primers were designed on a variable segment of the 16S rRNA gene and the intergenic spacer region (ITS) of available DNA sequences from classical and recently established species in the genus Pectobacterium. The results of the PCR analysis of genomic DNA from 32 Pectobacterium and Dickeya strains confirmed that the Pcar1F/R primers have sufficient nucleotide differences to discriminate between P. carotovorum and other Pectobacterium species associated with damage to potato crops, with the exception of P. versatile, which improves the specificity of the currently available primers. The proposed assay was originally developed as a conventional PCR but was later adapted to the real-time PCR format for application in combination with the existing real-time PCR test for the potato-specific pathogen P. parmentieri. This should be useful for the routine diagnosis of potato soft rot.

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Use of 16S-23S rRNA Intergenic Spacer Region PCR and Repetitive Extragenic Palindromic PCR Analyses of Escherichia coli Isolates To Identify Nonpoint Fecal Sources

Applied and Environmental Microbiology ◽

10.1128/aem.69.8.4942-4950.2003 ◽

2003 ◽

Vol 69 (8) ◽

pp. 4942-4950 ◽

Cited By ~ 46

Author(s):

Sylvie Seurinck ◽

Willy Verstraete ◽

Steven D. Siciliano

Keyword(s):

Escherichia Coli ◽

Natural Waters ◽

Average Rate ◽

Intergenic Spacer ◽

23S Rrna ◽

Pcr Analysis ◽

Factors Affecting ◽

Intergenic Spacer Region ◽

E Coli ◽

Repetitive Extragenic Palindromic

ABSTRACT Despite efforts to minimize fecal input into waterways, this kind of pollution continues to be a problem due to an inability to reliably identify nonpoint sources. Our objective was to find candidate source-specific Escherichia coli fingerprints as potential genotypic markers for raw sewage, horses, dogs, gulls, and cows. We evaluated 16S-23S rRNA intergenic spacer region (ISR)-PCR and repetitive extragenic palindromic (rep)-PCR analyses of E. coli isolates as tools to identify nonpoint fecal sources. The BOXA1R primer was used for rep-PCR analysis. A total of 267 E. coli isolates from different fecal sources were typed with both techniques. E. coli was found to be highly diverse. Only two candidate source-specific E. coli fingerprints, one for cow and one for raw sewage, were identified out of 87 ISR fingerprints. Similarly, there was only one candidate source-specific E. coli fingerprint for horse out of 59 BOX fingerprints. Jackknife analysis resulted in an average rate of correct classification (ARCC) of 83% for BOX-PCR analysis and 67% for ISR-PCR analysis for the five source categories of this study. When nonhuman sources were pooled so that each isolate was classified as animal or human derived (raw sewage), ARCCs of 82% for BOX-PCR analysis and 72% for ISR-PCR analysis were obtained. Critical factors affecting the utility of these methods, namely sample size and fingerprint stability, were also assessed. Chao1 estimation showed that generally 32 isolates per fecal source individual were sufficient to characterize the richness of the E. coli population of that source. The results of a fingerprint stability experiment indicated that BOX and ISR fingerprints were stable in natural waters at 4, 12, and 28°C for 150 days. In conclusion, 16S-23S rRNA ISR-PCR and rep-PCR analyses of E. coli isolates have the potential to identify nonpoint fecal sources. A fairly small number of isolates was needed to find candidate source-specific E. coli fingerprints that were stable under the simulated environmental conditions.

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Rapid and high throughput molecular identification of diverse mosquito species by high resolution melting analysis

F1000Research ◽

10.12688/f1000research.9224.1 ◽

2016 ◽

Vol 5 ◽

pp. 1949 ◽

Cited By ~ 9

Author(s):

Yvonne Ukamaka Ajamma ◽

Enock Mararo ◽

David Omondi ◽

Thomas Onchuru ◽

Anne W. T. Muigai ◽

...

Keyword(s):

High Resolution ◽

High Resolution Melting ◽

Mosquito Species ◽

Intergenic Spacer ◽

Rapid Identification ◽

Correct Identification ◽

Intergenic Spacer Region ◽

Species Complexes ◽

Melting Analysis ◽

Pcr Method

Mosquitoes are a diverse group of invertebrates, with members that are among the most important vectors of diseases. The correct identification of mosquitoes is paramount to the control of the diseases that they transmit. However, morphological techniques depend on the quality of the specimen and often unavailable taxonomic expertise, which may still not be able to distinguish mosquitoes among species complexes (sibling and cryptic species). High resolution melting (HRM) analyses, a closed-tube, post-polymerase chain reaction (PCR) method used to identify variations in nucleic acid sequences, has been used to differentiate species within the Anopheles gambiae and Culex pipiens complexes. We validated the use of PCR-HRM analyses to differentiate species within Anopheles and within each of six genera of culicine mosquitoes, comparing primers targeting cytochrome b (cyt b), NADH dehydrogenase subunit 1 (ND1), intergenic spacer region (IGS) and cytochrome c oxidase subunit 1 (COI) gene regions. HRM analyses of amplicons from all the six primer pairs successfully differentiated two or more mosquito species within one or more genera (Aedes (Ae. vittatus from Ae. metallicus), Culex (Cx. tenagius from Cx. antennatus, Cx. neavei from Cx. duttoni, cryptic Cx. pipiens species), Anopheles (An. gambiae s.s. from An. arabiensis) and Mansonia (Ma. africana from Ma. uniformis)) based on their HRM profiles. However, PCR-HRM could not distinguish between species within Aedeomyia (Ad. africana and Ad. furfurea), Mimomyia (Mi. hispida and Mi. splendens) and Coquillettidia (Cq. aurites, Cq. chrysosoma, Cq. fuscopennata, Cq. metallica, Cq. microannulatus, Cq. pseudoconopas and Cq. versicolor) genera using any of the primers. The IGS and COI barcode region primers gave the best and most definitive separation of mosquito species among anopheline and culicine mosquito genera, respectively, while the other markers may serve to confirm identifications of closely related sub-species. This approach can be employed for rapid identification of mosquitoes.

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Identification of Bartonella strains isolated from wild and domestic ruminants by a single-step PCR analysis of the 16S–23S intergenic spacer region

Veterinary Microbiology ◽

10.1016/j.vetmic.2003.09.022 ◽

2004 ◽

Vol 98 (1) ◽

pp. 63-69 ◽

Cited By ~ 20

Author(s):

R. Maillard ◽

M. Vayssier-Taussat ◽

C. Bouillin ◽

C. Gandoin ◽

L. Halos ◽

...

Keyword(s):

Intergenic Spacer ◽

Single Step ◽

Pcr Analysis ◽

Intergenic Spacer Region ◽

Domestic Ruminants

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Detection and Characterization of an Elm Yellows (16SrV) Group Phytoplasma Infecting Virginia Creeper Plants in Southern Florida

Plant Disease ◽

10.1094/pdis.2001.85.10.1055 ◽

2001 ◽

Vol 85 (10) ◽

pp. 1055-1062 ◽

Cited By ~ 13

Author(s):

N. A. Harrison ◽

H. M. Griffiths ◽

M. L. Carpio ◽

P. A.. Richardson

Keyword(s):

Intergenic Spacer ◽

23S Rrna ◽

Pcr Analysis ◽

Polymorphism Analysis ◽

Intergenic Spacer Region ◽

Flavescence Dorée ◽

Specific Pcr ◽

Rdna Sequences ◽

Elm Yellows ◽

Southern Florida

The polymerase chain reaction (PCR) employing phytoplasma-specific ribosomal RNA primer pair P1/P7 consistently amplified a product of expected size (1.8 kb) from 29 of 36 symptom-less Virginia creeper (Parthenocissus quinquefolia) plants growing in southern Florida. Restriction fragment length polymorphism analysis of P1/P7-primed PCR products indicated that most phytoplasmas detected in Virginia creeper were similar to phytoplasmas composing the elm yellows (16SrV) group. This relationship was verified by reamplification of P1/P7 products using an elm yellows (EY) group-specific rRNA primer pair fB1/rULWS1. rDNA products (1,571 bp) were generated by group-specific PCR from 28 phytoplasma-positive plants and 1 negatively testing plant identified by earlier P1/P7-primed PCR. Analysis of 16S rDNA sequences determined the Virginia creeper (VC) phytoplasma to be phylogenetically closest to the European alder yellows (ALY) agent, an established 16SrV-C subgroup strain. However, presence or absence of restriction sites for endonucleases AluI, BfaI, MspI, RsaI, and TaqI in the 16S rRNA and 16-23S rRNA intergenic spacer region of the VC phytoplasma collectively differentiated this strain from ALY and other 16SrV group phytoplasmas. Failure to detect the VC phytoplasma by PCR employing nonribosomal primer pair FD9f/FD9r suggests that this newly characterized agent varies from known European grapevine yellows (flavescence dorée) phyto-plasmas previously classified as 16SrV subgroup C or D strains.

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Finding Needles in a Haystack: Using Geo-References to Enhance the Selection and Utilization of Landraces in Breeding for Climate-Resilient Cultivars of Upland Cotton (Gossypium hirsutum L.)

Plants ◽

10.3390/plants10071300 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1300

Author(s):

Junghyun Shim ◽

Nonoy B. Bandillo ◽

Rosalyn B. Angeles-Shim

Keyword(s):

Allelic Diversity ◽

Potential Donor ◽

Breeding Programs ◽

Germplasm Collections ◽

Climate Shifts ◽

Potential Applications ◽

Genetic Value ◽

Modern Breeding ◽

Efficient Selection ◽

Selection Of

The genetic uniformity of cultivated cotton as a consequence of domestication and modern breeding makes it extremely vulnerable to abiotic challenges brought about by major climate shifts. To sustain productivity amidst worsening agro-environments, future breeding objectives need to seriously consider introducing new genetic variation from diverse resources into the current germplasm base of cotton. Landraces are genetically heterogeneous, population complexes that have been primarily selected for their adaptability to specific localized or regional environments. This makes them an invaluable genetic resource of novel allelic diversity that can be exploited to enhance the resilience of crops to marginal environments. The utilization of cotton landraces in breeding programs are constrained by the phenology of the plant and the lack of phenotypic information that can facilitate efficient selection of potential donor parents for breeding. In this review, the genetic value of cotton landraces and the major challenges in their utilization in breeding are discussed. Two strategies namely Focused Identification of Germplasm Strategy and Environmental Association Analysis that have been developed to effectively screen large germplasm collections for accessions with adaptive traits using geo-reference-based, mathematical modelling are highlighted. The potential applications of both approaches in mining available cotton landrace collections are also presented.

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