Exploring miRNAs involved in citrus nucellar embryo initiation by high-throughput small RNA sequencing

To identify miRNAs related nucellar embryo intiation (NEI) in citrus, we performed small RNA sequencing in ovules of two pairs of poly- and mono-embryonic cultivars right prior to and at NEI stages. Nearly 150 miRNAs including ~90 conserved and ~60 novel miRNAs were identified in ovules of each cultivar pair. Overall design: Two pairs of poly- and mono-embryonic citrus cultivars were used. One pair are mandarins, the polyembryonic Ponkan ‘Huagan No.2’ (PK, Citrus reticulata Blanco) and the monembryonic Nour clementina (CM, C. Clementine). The other pair are pummelo/grapefruit, the polyembryonic Cocktail grapefruit (GF, C. paradisi Osbeck) and the monoembryonic Huanong red pummelo (PU, C. grandis (L.) Osbeck). Histological analysis indicated that the NEI cells began to emerge at 3 DAF in ovules of grapefruit but at 7 DAF in ovules of Ponkan mandarin, so we took 0 DAF for grapefruit and 3 DAF for Ponkan as the stage just before NEI cells formed, i.e. the ‘pre-NEI’ stage; whereas 3 DAF for grapefruit and 7 DAF for Ponkan were taken as the stage when NEI cells emerged, i.e. the ‘NEI’ stage. Ovules were collected in two continuous years as biological replicates for high throughput sequencing. The overlapped differentially expressed miRNAs between two pairs were considered as the key miRNAs involved in nucellar embryo initiation.