Digging miRNAs Related to Citrus Somatic Embryogenesis by High-Throughput Sequencing

Somatic embryogenesis (SE) of embryogenic callus (EC) is an indispensable process in citrus in vitro germplasm preservation and genetic improvement by bio-technology. However, EC of many citrus species and varieties tend to lose their SE capability gradually during years of subculture. miRNAs play essential roles in plant development, while their  involvement in citrus SE was rarely inspected. In this study, Solexa sequencing was applied on three SE tissues of Valencia sweet orange (Citrus sinensis), a variety maintaining strong SE capability after 27 years of subculture, to obtain small RNA information. The three sequenced tissues were: non-embryogenic callus (NEC), without SE capability; EC, with strong SE capability; mixed tissue of EC exposed to embryo induction for 1-4 weeks (E1-4).  Forty-nine conserved miRNAs from 25 families and 91 novel miRNAs from 37 families were identified. The conserved miRNA families generally contain one to three members, while the novel ones are much more diversified, with one to ten members. miRNA expression module was validated by qPCR and Northern blot, some miRNAs expressed only in EC and induced EC, like csi-miR156; some expressed specifically in induced EC, like novel-csi-miR103; some accumulated most in morphologic embryos (as globular embryo), like csi-miR164; some expressed most in NEC, like csi-miR390, csi-miR2911. 35S::csi-MIR156::GUS vector was constructed and then transformed into EC of “Guoqing NO. 1” Satsuma mandarin (Citrus unshiu), which had lost SE capability.  Overexpression of miR156 promoted callus cells to accumulate significantly more amyloids and exhibited EC cell-like properties, despite its incompetence to recover SE capability.