High-Throughput Sequencing Uncovers miRNAs and siRNAs Involved in Citrus Somatic Embryogenesis

Somatic embryogenesis (SE) of embryogenic callus (EC) is a requisite step in citrus in vitro germplasm preservation and biotechnology based genetic improvement. However, EC of many citrus species and varieties tend to lose their SE capability gradually during years of subculture. As miRNA biogenesis and pathway genes proved to be essential for plant zygotic embryogenesis (ZE), we wondered if miRNAs are involved in SE of citrus.

In this study, Illumina sequencing was applied on small RNA (sRNA) libraries of non-embryogenic and embryogenic tissues of Valencia sweet orange (Citrus sinensis), a variety with strong SE capability maintained even after 28 years of subculture. A total of 191 sRNA embedded stem-loop structures were identified, but only 70 of them were annotated as miRNA precursors, emanating 50 known and 45 novel miRNAs. The other 121 stem-loop structures generate 126 miniature inverted-repeat transposable elements (MITEs) derived small interfering RNAs (siRNAs) and 94 other siRNAs. Along with degradome sequencing, which is also referred to as Parallel Analysis of RNA Ends (PARE), we identified 235 phased siRNAs (phasiRNAs) from 23 miRNA-cleaved PHAS genes, and a total of 203 miRNA/siRNA-cleaved targets were also identified. For most of the abundantly expressed miRNAs/siRNAs, their abundance is much higher in non-embryogenic callus (NEC) than in embryognic tissue or during SE process, and they target a substantial number of developmentally essential genes and stress response genes, which may be derepressed and release their action in SE. Seventeen abundantly expressed miRNAs/siRNAs were grouped as embryonic tissue, SE process, globular embryo (GE) or NEC enriched sRNAs respectively. Csi-miR164b and 171c proved to be positively related to SE capability of citrus varieties. Overexpression of csi-miR156a in EC of Hongkong kumquat (Fortunella hindsii Swingle), a species which is phylogenetically close to citrus and carries rather weak SE capability, significantly promoted embryo formation during SE induction, meanwhile, transformed EC cells accumulate significantly more amyloids than the wild type.