Athogen for an infected citrus sample.Materials and Methods Viroid Standard

Athogen for an infected citrus sample.Materials and Methods Viroid Standard Samples and Nucleotide SequencesStandard viroid samples were collected by the Institute of Plant Protection, 10781694 Chinese Academy of Agricultural Sciences (Beijing, China), and purchased from the American Type Culture Collection (Manassas, VA, USA) and several other sources (Tables 1 and 2). These viroid samples were verified by sequencing. The sequence of Coleus blumei viroid collected by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences was submitted to the GenBank database with accession number of KC581915. A total of 35 full-length genomic sequences and 2,659 partial genomic sequences were downloaded from the NCBI Taxonomy Browser (http://www.ncbi.nlm.nih.gov/Taxonomy/) (Table 1). These sequences come from eight viroid genera. Nucleotide sequences shorter than 250 bp were removed. If there were more than 100 sequences in a viroid genera, nucleotide sequences with .98 identical nucleotide across .98 of the sequence were clustered and only the longest sequence was retained as the representative sequence for the Indolactam V web following analysis. This left 421 filtered sequences to be used in the probe design. An additional 943 plant viral genome sequences and 105 895 plant mRNA sequences were used to identify off-target hybridizations.The 16985061 candidate CAL 120 web probes were required to (i) have a GC content between 40 and 60 , (ii) be less than four continuous mononucleotides, and (iii) have less than six nucleotides in the inner hairpin. Probes showing potential off-target hybridization to the other viroid genera, plant viruses and plant mRNA were removed. Probes that passed the above filters were aligned to all the sequences in the viroid genus using BLASTN. A probe to viroid sequence match was defined as at least 80 of the probe matching the viroid sequence. Final microarray probes were selected by maximizing the coverage of viroid genera using a minimal number of probes. Selection of probe combination was repeated three times, thus three probe sets were generated to target most viroid sequences by three probes.Microarray ConstructionMicroarrays were synthesized using the selected 40 mer probes on 75-mm625-mm glass slides using the SmartArrayer system following the manufacturer’s instruction (CapitalBio, Beijing, China) (Table S1). All probes were spotted in triplicate on the microarray. Several control probes were used to monitor the performance of the microarray. (i) The 59 amino-linker oligonucleotide (59-TTTTTTTTTTTTTTTCTCATGCCCATGCCGATGC-39) was a random sequence which did not potentially hybridize with any viroid sequences. The targeted sequence of this probe was mixed with the samples before hybridization. This probe was used as positive control (PC) monitoring the probe hybridization efficiency. (ii) The internal control (HEX) was spotted on the microarray to monitor the probes linking to the chips. It was the 59 Hex-linker oligonucleotide (59-GTCACATGCGATGGATCGAGCTCCTTTATCATCGTTCCCACCTTAATGCA-39). (iii) Probes designed to match the highly conserved regions in plant 18 s rRNAs were used as positive controls to detect host plant rRNAs. (iv) The spotting buffer was used as the negative control (NC). In a standard hybridization result, the PC and HEX probes should be positive with high signal intensities. NC probes should be negative with signal intensities similar to the background level. In this study, the viroid sequences were amplified using species spe.Athogen for an infected citrus sample.Materials and Methods Viroid Standard Samples and Nucleotide SequencesStandard viroid samples were collected by the Institute of Plant Protection, 10781694 Chinese Academy of Agricultural Sciences (Beijing, China), and purchased from the American Type Culture Collection (Manassas, VA, USA) and several other sources (Tables 1 and 2). These viroid samples were verified by sequencing. The sequence of Coleus blumei viroid collected by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences was submitted to the GenBank database with accession number of KC581915. A total of 35 full-length genomic sequences and 2,659 partial genomic sequences were downloaded from the NCBI Taxonomy Browser (http://www.ncbi.nlm.nih.gov/Taxonomy/) (Table 1). These sequences come from eight viroid genera. Nucleotide sequences shorter than 250 bp were removed. If there were more than 100 sequences in a viroid genera, nucleotide sequences with .98 identical nucleotide across .98 of the sequence were clustered and only the longest sequence was retained as the representative sequence for the following analysis. This left 421 filtered sequences to be used in the probe design. An additional 943 plant viral genome sequences and 105 895 plant mRNA sequences were used to identify off-target hybridizations.The 16985061 candidate probes were required to (i) have a GC content between 40 and 60 , (ii) be less than four continuous mononucleotides, and (iii) have less than six nucleotides in the inner hairpin. Probes showing potential off-target hybridization to the other viroid genera, plant viruses and plant mRNA were removed. Probes that passed the above filters were aligned to all the sequences in the viroid genus using BLASTN. A probe to viroid sequence match was defined as at least 80 of the probe matching the viroid sequence. Final microarray probes were selected by maximizing the coverage of viroid genera using a minimal number of probes. Selection of probe combination was repeated three times, thus three probe sets were generated to target most viroid sequences by three probes.Microarray ConstructionMicroarrays were synthesized using the selected 40 mer probes on 75-mm625-mm glass slides using the SmartArrayer system following the manufacturer’s instruction (CapitalBio, Beijing, China) (Table S1). All probes were spotted in triplicate on the microarray. Several control probes were used to monitor the performance of the microarray. (i) The 59 amino-linker oligonucleotide (59-TTTTTTTTTTTTTTTCTCATGCCCATGCCGATGC-39) was a random sequence which did not potentially hybridize with any viroid sequences. The targeted sequence of this probe was mixed with the samples before hybridization. This probe was used as positive control (PC) monitoring the probe hybridization efficiency. (ii) The internal control (HEX) was spotted on the microarray to monitor the probes linking to the chips. It was the 59 Hex-linker oligonucleotide (59-GTCACATGCGATGGATCGAGCTCCTTTATCATCGTTCCCACCTTAATGCA-39). (iii) Probes designed to match the highly conserved regions in plant 18 s rRNAs were used as positive controls to detect host plant rRNAs. (iv) The spotting buffer was used as the negative control (NC). In a standard hybridization result, the PC and HEX probes should be positive with high signal intensities. NC probes should be negative with signal intensities similar to the background level. In this study, the viroid sequences were amplified using species spe.