High-throughput sequencing and degradome analysis reveal differential expression of miRNAs and their targets between cytoplasmic male-sterile cybrid pummelo and its fertile type (Citrus grandis)

High-throughput sRNA and degradome sequencing was applied in G1+HBP and its fertile type HBP to identify miRNAs and their targets during reproductive development. A total of 184 known miRNAs, 22 novel miRNAs and 86 target genes were identified. Some of the targets are transcription factors involved in floral development, such as auxin response factors (ARFs), SQUAMOSA promoter binding protein box (SBP-box), MYB, basic region-leucine zipper (bZIP), APETALA2 (AP2), transport inhibitor response 1 (TIR1), etc. Eight target genes were confirmed to be sliced by corresponding miRNAs using 5'RACE technology. Based on the sequencing abundance, 42 differentially expressed miRNAs between CMS line G1+HBP and fertile line HBP were discovered. Differential expression of miRNAs and their target genes was validated by quantitative RT-PCR and reciprocal expression patterns between some miRNAs and their targets were demonstrated. The regulation mechanism of miR167a was elucidated by yeast one-hybrid and dual-luciferase assay that a dehydrate responsive element binding (DREB) transcription factor bind miR167a promoter and transcriptionally repress miR167 expression. Our study revealed altered expression of miRNAs and their target genes in cytoplasmic male sterile pummelo (CMS) line and highlighted that miRNA regulatory network may be involved in nucleus-cytoplasmic cross talk of citrus CMS. Overall design: small RNA and degradome sequencing of seedy 'HBP' and its cytoplasmic male sterile and seedless line 'G1+HBP'