2017-04-20
Abstract:
11Plant somatic embryos are widely used in the fields of germplasm conservation, breeding for genetic 12engineering and artificial seed production. MicroRNAs (miRNAs) play pivotal roles in somatic 13embryogenesis (SE) regulation.However, their regulatory roles during various stages of SE remain 14unclear. In this study, six types of embryogenic samplesofLilium pumilumDC. Fisch., including 15organogeniccallus, embryogenic callus induced for 4 weeks, embryogenic callus induced for 6 16weeks, globular embryos, torpedo embryos and cotyledon embryos, were prepared for small RNA 17sequencing.The results revealed a total of 2,378,760 small RNA reads, among which the most 18common size was 24 nt. Four hundred and fifty-twoknown miRNAs,belonging to more than 86 19families,57 novel miRNAsand40 miRNA*swere identified.The 86 known miRNA families were 20sortedaccording to an alignment with their homologs across 24 land plants into the following four 21categories: 23 highly conserved, 4 moderately conserved, 15 less conserved and 44 species-specific 22miRNAs. Differentially expressed known miRNAs were identified during various stages of SE. 23Subsequently, the expression levels of 12differentially expressed miRNAs and 4 targets were 24validated using qRT-PCR. In addition, six samples were mixed in equal amounts for transcript 25sequencing, and the sequencing data were used as transcripts for miRNA target prediction. A total of 2666,422 unigenes with an average length of 800 bp were assembled from 56,258,974 raw reads. Gene 27Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment indicated that 2838,004 and 15,497 unigenes were successfully assigned to GO terms and KEGG pathways, 29respectively. Among the unigenes, 2,182 transcripts were predicted to be targets for 396 known 30miRNAs. The potential targets of the identified miRNAs were mostly classified into the following 31GO terms: cell, binding and metabolic process. Enriched KEGG analysis demonstrated that 32carbohydrate metabolism was the predominant pathway in LiliumSE. Thus, we performed systemic 33 characterization, homology comparisons and profiling of miRNA expression, and we constructed an34Provisional miRNA in LiliumSE2miRNA-target network during LiliumSE for the first time. Our findings establish a foundation for the 35 further exploration of critical genes and elucidation of SE in Lilium.
