Search results for "Specific primers"
showing 9 items of 9 documents
Directional high-throughput sequencing of RNAs without gene-specific primers.
2018
Ribosomal RNA analysis is a useful tool for characterization of microbial communities. However, the lack of broad-range primers has hampered the simultaneous analysis of eukaryotic and prokaryotic members by amplicon sequencing. We present a complete workflow for directional, primer-independent sequencing of size-selected small subunit ribosomal RNA fragments. The library preparation protocol includes gel extraction of the target RNA, ligation of an RNA oligo to the 5′-end of the target, and cDNA synthesis with a tailed random-hexamer primer and further barcoding. The sequencing results of a phytoplankton mock community showed a highly similar profile to the biomass indicators. This method…
Analysis of S-allele genetic diversity in Sicilian almond germplasm comparing different molecular methods
2015
Italian almond germplasm is characterized by a wide diversity in several growing areas among which Sicily is one of the most important. Analysis with consensus and specific primers and DNA sequencing was performed to investigate S-RNase genetic diversity and to elucidate the homology rate within a genetic pool of 27 Italian accessions. Interestingly, some of the self-compatible cultivars did not show the presence of Sf allele. Amplicons from consensus and allele-specific PCR primers revealed a high level of variability. Sequencing of all the S-RNase amplicons derived from consensus primers allowed the identification of two new S-RNase alleles (S51 and S52). Surprisingly, despite the AA repl…
A molecular method to assess Phytophthora diversity in environmental samples
2012
Current molecular detection methods for the genus Phytophthora are specific to a few key species rather than the whole genus and this is a recognized weakness of protocols for ecological studies and international plant health legislation. In the present study a molecular approach was developed to detect Phytophthora species in soil and water samples using novel sets of genus-specific primers designed against the internal transcribed spacer (ITS) regions. Two different rDNA primer sets were tested: one assay amplified a long product including the ITS1, 5.8S and ITS2 regions (LP) and the other a shorter product including the ITS1 only (SP). Both assays specifically amplified products from Phy…
METAGENOMIC ANALYSIS OF PHYTOPHTHORA DIVERSITY IN NURSERIES OF POTTED ORNAMENTAL SPECIES.
2015
The genetic diversity of Phytophthora was investigated in soil and root samples of potted ornamental and fruit tree plants collected in nurseries located in Apulia and Calabria (Southern Italy) using metagenomic approaches based on Phytophthora genus specific primers. PCR amplicons containing the ITS1 region of the rDNA were sequenced using both a conventional cloning and Sanger sequencing approach and a 454 pyrosequencing protocol. All sequences were accurately analyzed with an appropriate software and used as barcode for species identification utilizing a validated ITS database. The cloning/Sanger sequencing approach enabled the identification of nine different Phytophthora taxa (P. nicot…
Molecular analysis ofPhytophthoradiversity in nursery-grown ornamental and fruit plants
2015
The genetic diversity of Phytophthora spp. was investigated in potted ornamental and fruit tree species. A metabarcoding approach was used, based on a semi-nested PCR with Phytophthora genus-specific primers targeting the ITS1 region of the rDNA. More than 50 ITS1 sequence types representing at least 15 distinct Phytophthora taxa were detected. Nine had ITS sequences that grouped them in defined taxonomic groups (P. nicotianae, P. citrophthora, P. meadii, P. taxon Pgchlamydo, P. cinnamomi, P. parvispora, P. cambivora, P. niederhauserii and P. lateralis) whereas three phylotypes were associated to two or more taxa (P. citricola taxon E or III; P. pseudosyringae, P. ilicis or P. nemorosa; and…
MOLECULAR ANALYSIS OF PHYTOPHTHORA DIVERSITY IN ORNAMENTAL NURSERIES.
2013
A molecular approach based on the use of genus-specific nested-PCR primers (Scibetta et al., 2012; J. Microbiol. Meth. 88: 356-368) was utilized to detect Phytophthora species in soil and root samples of potted ornamentals, collected across Apulia and Calabria, Southern Italy. Analyzed samples comprised many plant species with different levels of symptoms of decline on the canopy and root rots. Extraction protocols were optimized to obtain DNA samples of appropriate quality from soil and roots. The analysis of sequences after cloning of nested-PCR amplicons enabled the identification of different Phytophthora species including P. nicotianae, P. cinnamomi, P. cryptogea, P. palmivora and P. n…
Identification of Phytophthora species infecting citrus in Vietnam
2013
In a survey aimed at identifying Phytophthora species infecting fruit crops in Viet Nam, P. citrophthora and P. nicotianae were the species most commonly recovered from soil and trees in citrus plantations. P. nicotianae was prevalent in citrus groves in the Ben Tre and Tien Giang provinces (southern Viet Nam). Interestingly, both A1 and A2 mating types of the latter species were found, while in other citrus-growing areas of the world, such as the Mediterranean region, South Africa, Australia and the Americas, A1 is the only mating type occurring in citrus groves, suggesting southern-east Asia could be a centre of origin of this Phytophthora species. Mixed infections of three Phytophthora s…
MOLECULAR ANALYSIS OF COLLETOTRICHUM SPECIES ASSOCIATED TO OLIVE IN CALABRIA.
2013
A new molecular approach based on the use of genus-specific primers targeting the internal transcribed spacer (ITS) regions of rDNA, was developed and used to study the diversity of Colletotrichum species associated with the olive canopy in the Gioia Tauro plain (Calabria, southern Italy). Representative symptomatic and symptomless samples of leaves, flowers and fruits were collected during 2012 and analyzed by extracting total DNA and amplifying the target region with the genus-specific primers. Amplicons were cloned and sequenced in order to use the ITS as a barcode gene. No Colletotrichum species were detected in the first sampling period (May 28, 2012), whereas around 15% of the analyze…