Development and Characterization of EST-SSR Markers via Transcriptome Sequencing in Brainea insignis (Aspleniaceae s. l.)
The mRNAs were extracted from the total RNA Using OligotexTM-dT30 (TaKaRa, Dalian, China), and ultrasonically fragmented and converted to double-stranded cDNAs. After adding an “A” nucleotide at the 30-end of the cDNAs, adapters were ligated to both ends, and the QIAquick Gel Extraction Kit (Qiagen, Hilden, German) was used to purify and collect cDNAs of approximately 215 bp in length. Finally, each amplified molecule was sequenced using Illumina sequencing technology to obtain short reads of 90 bp from both ends. A total of 23.9 million 125-bp paired-end reads were obtained and de novo assembled into 72,897 unigenes using Trinity v2.3.2 (Grabherr et al., 2011), with a minimum length of 201 bp and an average length of 799 bp.