Deep sequencing combined with microarray hybridization to identify novel and conserved microRNAs during somatic embryogenesis of hybrid yellow-poplar (Liriodendron chinense (Hemsl.) Sarg. X L. tulipifera Linn.)

Background Hybrid yellow-poplar was a man-made cross between Liriodendron tulipifera Linn.× L. chinense(Hemsl.) Sarg.. Heterosis was strong both on growth and adaptation, no matter what, the crosses or the reciprocals. During the last 12 years, somatic embryogenesis systems were successfully established in our lab, in order to meet the needs of planting materials for expanding plantations in Southern China. Synchronous development of high quality embryos was always required both for the study of embryo development, and the supply of commercial emblings. The functions of plant microRNAs (miRNAs) in post-transcriptional gene regulation by targeting mRNAs for degradation, or the translational repression by hybridizing to complementary sequences within target mRNA molecules were well reported [1]. One of the major roles of miRNAs in regulating plant development has been obtained from a number of studiesbut molecular mechanisms regulating the development of embryos are not well understood, especially during the early stages of somatic embryos [2]. Here we present some results on the discovery of novel and conserved miRNAs during somatic embryogenesis of hybrid yellow-poplar using Illumina sequencing and miRNA microfluidic chip in order to understand how the embryogenic somatic mass is developed into embryos and plants. Materials and methods The pre-embryo mass (PEM) and different stages of somatic embryos (SE) (Figure 1) were collected sequentially from the hybrid yellow-poplar somatic embryogenesis system. All the callus and embryo tissues were staged by microscope and immediately forzen in liquid nitrogen and stored at -80C until used. Total RNA was isolated from different developmental stages of somatic embryogenesis using Total RNA Purification Kit (Norgen Biotek Corporation, Canada). All the RNA samples from different somatic embryo tissues were mixed equally to form a single RNA pool. Following the instruction of LC Science, small RNA sample was prepared and then sequenced using Illumina Technology. Processing and analysis of raw data was done following Sunkar et al [3,4]. Conserved and candidate novel miRNAs in hybrid yellow-poplar were identified . A mixed small RNA microarray hybrid was used to validate the sequencing result and detect new miRNAs in hybrid yellow-poplar; 299 sequences detected by sequencing and 1,450 plant miRNAs sequences in miRBase 15.0 were ordered to serve as probes. A well-developed stem-loop strategy [5]and SYBR Green master mix were used to perform real time PCR to validate the miRNAs expressed in both sequencing and microarray analysis. The potential targets of miRNAs were predicted using the psRNATarget program (http://bioinfo3.noble.org/ psRNATarget/) with default parameters. The biology and molecular functions of these predicted targets were categorized by the Gene Ontology program (http://www. Geneontology.org/). * Correspondence: [email protected] The key laboratory of forest genetics and gene engineering of the ministry of education of China Nanjing Forestry University Nanjing 210019, China Tingting et al. BMC Proceedings 2011, 5(Suppl 7):P74 http://www.biomedcentral.com/1753-6561/5/S7/P74