H other species. The alignments were performed with the CLUSTALW 2.1. An * (asterisk) indicates positions which have a single, fully conserved residue. A : (colon) indicates conservation 10781694 between groups of
Viroids are plant pathogens with circular and single stranded RNA genomes. Viroid RNAs are short and not protein-coding. Crops affected by viroids may develop severe diseases, which may result in significant economic losses to the agricultural industry. Viroids can be spread worldwide via mechanical Pentagastrin web contact, seed transmission, or biological vectors. International trade also increases the chance of spreading the viroids to new territories. Thus a fast and accurate diagnostic tool is needed to control the disease and reduce the risk of the introduction of new viroids. Many diagnostic techniques have been developed for plant viroid detection. These include bioassays [1?], return-polyacrylamide gel electrophoresis (Return-PAGE) [5], reverse transcriptionpolymerase chain reaction (RT-PCR) [6?5], RT-PCR using genus degenerate primers and multiplex primers [16?0], RTPCR dot-blot hybridization (RT-PCR-DBH) [31], real time RTPCR [32?7], molecular hybridization [38?0], reverse transcription loop-mediated isothermal amplification (RT-LAMP) [41,42] and microarrays [43?7]. The aforementioned techniques can detect one or several viroid 16985061 species or genera, but fail to detect a wide range of viroid pathogens. These techniques usually need a priori knowledge of the viroids and this makes them unsuitable to detect new emergingviroids. These emerging viroids may include new viroid species, as well as established viroid species being transferred to new host species or new territories. Advances in next generation sequencing (NGS) Failure VSSA .256 .256 43 VSSA VSSA 52 VSSA VSSA 43 0.5 .256 .256 ` 42 VSSA .256 .256 0.6 III t037 ST239 F technologies have provided a powerful alternative for pathogen detection. Using small RNA (sRNA) library construction and deep sequencing, NGS is able to identify both known and novel viroid species, and de novo assemble viroid genomes. Li et al. applied sRNA deep sequencing to analyze infected tomato samples and detected Potato spindle tuber viroid (PSTVd), Pepino mosaic virus (PepMV) and an unknown potyvirus species [48]. Several computational algorithms were developed to detect the viroids quantatively using deep sequencing [49] or in a homology independent manner [50]. However, NGS is very computation intensive and much more expensive than the commonly used methods, e.g. PCR or ELISA. Thus there is a great need for a high throughput, cost effective and relative specific way of detecting plant viroid pathogens. Microarrays with genus level probes are suitable tools for this purpose. Using genus level probes, microarrays are able to detect multiple viroid genera in a single chip and are capable of detecting new viroids at the genus level. Several microarrays have been reported that detect a wide range of viruses [51?4]. However, no microarray with a similar capability of detecting plant viroids has been reported before.Microarray Detection of ViroidsIn this paper, we report a new microarray platform which detects plant viroids at the genus level. A minimal number of 40 mer genus level probes were selected for 36 species from all the 8 reported plant viroid genera and 1 unclassified viroid species. Several standard plant viroid samples were collected and validated using this microarray. The application of the microarray was tested on three infected plant samples from the field. Hop stunt viroid was detected as the major causative p.H other species. The alignments were performed with the CLUSTALW 2.1. An * (asterisk) indicates positions which have a single, fully conserved residue. A : (colon) indicates conservation 10781694 between groups of
Viroids are plant pathogens with circular and single stranded RNA genomes. Viroid RNAs are short and not protein-coding. Crops affected by viroids may develop severe diseases, which may result in significant economic losses to the agricultural industry. Viroids can be spread worldwide via mechanical contact, seed transmission, or biological vectors. International trade also increases the chance of spreading the viroids to new territories. Thus a fast and accurate diagnostic tool is needed to control the disease and reduce the risk of the introduction of new viroids. Many diagnostic techniques have been developed for plant viroid detection. These include bioassays [1?], return-polyacrylamide gel electrophoresis (Return-PAGE) [5], reverse transcriptionpolymerase chain reaction (RT-PCR) [6?5], RT-PCR using genus degenerate primers and multiplex primers [16?0], RTPCR dot-blot hybridization (RT-PCR-DBH) [31], real time RTPCR [32?7], molecular hybridization [38?0], reverse transcription loop-mediated isothermal amplification (RT-LAMP) [41,42] and microarrays [43?7]. The aforementioned techniques can detect one or several viroid 16985061 species or genera, but fail to detect a wide range of viroid pathogens. These techniques usually need a priori knowledge of the viroids and this makes them unsuitable to detect new emergingviroids. These emerging viroids may include new viroid species, as well as established viroid species being transferred to new host species or new territories. Advances in next generation sequencing (NGS) technologies have provided a powerful alternative for pathogen detection. Using small RNA (sRNA) library construction and deep sequencing, NGS is able to identify both known and novel viroid species, and de novo assemble viroid genomes. Li et al. applied sRNA deep sequencing to analyze infected tomato samples and detected Potato spindle tuber viroid (PSTVd), Pepino mosaic virus (PepMV) and an unknown potyvirus species [48]. Several computational algorithms were developed to detect the viroids quantatively using deep sequencing [49] or in a homology independent manner [50]. However, NGS is very computation intensive and much more expensive than the commonly used methods, e.g. PCR or ELISA. Thus there is a great need for a high throughput, cost effective and relative specific way of detecting plant viroid pathogens. Microarrays with genus level probes are suitable tools for this purpose. Using genus level probes, microarrays are able to detect multiple viroid genera in a single chip and are capable of detecting new viroids at the genus level. Several microarrays have been reported that detect a wide range of viruses [51?4]. However, no microarray with a similar capability of detecting plant viroids has been reported before.Microarray Detection of ViroidsIn this paper, we report a new microarray platform which detects plant viroids at the genus level. A minimal number of 40 mer genus level probes were selected for 36 species from all the 8 reported plant viroid genera and 1 unclassified viroid species. Several standard plant viroid samples were collected and validated using this microarray. The application of the microarray was tested on three infected plant samples from the field. Hop stunt viroid was detected as the major causative p.

H other species. The alignments were performed with the CLUSTALW 2.1. An

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