Comparative analysis reveals dynamic changes in miRNAs and their targets and expression during somatic embryogenesis in longan (Dimocarpus longan Lour.).
To identify potential conserved and novel miRNAs from longan somatic embryo, a pooled sRNA library was generated from the synchronized embryogenic cultures (friable-embryogenic calli, globular embryos, heart-shaped embryos, torpedo-shaped embryos, and cotyledonary embryos) and sequenced on a Solexa system (Illumina). First,small (16–30 nt) RNA fragments were isolated from a 15% PAGE gel and purified. The small RNAs were then sequentially ligated to a59 RNA adapter (59-GUUCAGAGUUCUACAGUCCGACGAUC-39) and a 39 RNA adapter (59-pUCG UAUGCCGUCUUCUGCUUGidT-39; p, phosphate; idT, inverted deoxythymidine) using T4 RNA ligase. The resulting samples were reversetranscribed to cDNA with an RT primer (59-CAAGCAGAAGACGGCATACGA-39) using Superscript II reverse transcriptase (Invitrogen) and PCR-amplified. Finally, Solexa sequencing was used to sequence the sRNAs in the pooled longan samples (Beijing Genomics Institute, China).