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The length of the overlapping primers varied from 27 bp to 39 bp, with a minimal overlapping region of 15 bases.
Sequence data were quality trimmed using the sliding windows of 50 bp with a minimal average Phred score of 20.
Trimmomatic was also used for quality trimming of the 3' end of the reads using a sliding window (4 bp with a minimal Phred quality of 30).
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To check if the 130 bp region (-667 to -538) identified in the TsVP1 promoter was sufficient for the salt stress response, we incorporated this 130 bp region with a minimal CaMV35S promoter (-46 to +10) to produce the promoter of P-130-mini35S and detected its activity under normal and salt stress conditions by Agrobacterium-mediated GUS transient assay in tobacco leaves.
A total of 26.95 million 90-nucleotide paired-end reads were obtained and de novo assembled into 33,522 contigs using Trinity (Grabherr et al., 2011) with a minimal length of 300 bp and an average length of 834 bp.
Both perfect and compound SSRs were selected with a minimal acceptable length of 10 bp (dinucelotide motifs) and 12 bp (tri- and tetranucletide motifs).
Short-sequenced reads were trimmed with a minimal of 35-bp read and error probability <0.05.
16,476 quality EST sequences with a minimal length of 50 bp were saved for downstream analysis.
To construct the IRB, the NCBI assembly was first analyzed to find all the intrachromosomal and interchromosomal identical repeat pairs with a minimal length of 300 bp.
The remaining TCAST transposon-like elements were shorter, with a minimal size of 314 bp (sequence no. 27), and usually lacking part of, or one or both, TIRs.
Concerning the selected genomic features, CpG islands were defined as regions with a minimal length of 500 bp, a GC content of 50%% or above and an observed CpG/expected CpG ratio of 0.60 or higher [ 52].
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