Genome-wide identification and expression profiles of phased siRNAs in a male-sterile somatic cybrid of pummelo (Citrus grandis)

Phased small interfering RNAs (phasiRNAs) are 21- or 24-nucleotide small non-coding RNAs that play important regulatory roles in various biological processes of plant growth and development. Biogenesis of phasiRNAs is typically triggered by miRNAs and involves dicer-processing. In our previous study, we constructed high-throughput sRNA and degradome sequencing libraries out of developing flower of the male sterile cybrid pummelo (G1 + HBP) and its fertile protoplast fusion parent Hirado Buntan pummelo (HBP), and identified the miRNAs involved in citrus male sterility. In this study, 280 PHAS loci generating 2235 unique 21-nucleotide phasiRNAs were identified in the flowers of the cybrid pummelo and its fertile fusion parent; of them, 210 overlapped with annotated protein coding genes, and 26 encoded transcription factors families involved in plant development, including ARF, MYB, AP2, NAC, GRF, TIR1/AFB, and AGL. Based on integration of miRNAs, phasiRNA and the degradome data, we identified nine miRNAs and eight phasiRNAs that trigger phasiRNAs production out of 29 PHAS loci, and 67 genes of broad regulatory activities were identified to be targets of phasiRNAs. We further identified nine miRNA-PHAS-phasiRNA-targets regulatory cascades, one phasiRNA-PHAS-phasiRNA-targets cascade, and 88 phasiRNAs with differential expression patterns in flowers between the cybrid and HBP. This analysis provided new information about miRNA-PHAS-phasiRNA-target networks and suggested potential roles of phasiRNAs in male sterility of pummelo and contributed to further study of phasiRNA function in citrus.