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.

相控小干扰RNA（phasiRNA）是21或24个核苷酸的非编码小RNA，在植物生长和发育的各种生物学过程中起着重要的调节作用。phasiRNA的生物发生通常由miRNA触发，并涉及切块机加工。在我们先前的研究中，我们从雄性不育杂交种柚子（G1 + HBP）及其可育原生质体融合亲本平户本丹柚（HBP）的发育花中构建了高通量sRNA和降解组测序文库，并鉴定了与柑橘有关的miRNA。男性不育。在这项研究中，280 PHAS在杂交柚子及其可育融合亲本的花中鉴定出产生2235个独特的21个核苷酸phasiRNA的基因座；其中有210个与带注释的蛋白质编码基因重叠，还有26个与植物发育有关的编码转录因子家族，包括ARF，MYB，AP2，NAC，GRF，TIR1 / AFB和AGL。基于对miRNA，phasiRNA和降解基因组数据的整合，我们从29个PHAS基因座中鉴定出9个miRNA和8个phasiRNA触发phasiRNA的产生，并确定了67个具有广泛调控活性的基因是phasiRNA的靶标。我们进一步确定了9个miRNA- PHAS -phasiRNA-靶标调控级联，一个phasiRNA- PHAS-phasiRNA靶点级联，在杂交体和HBP之间的花中有88个具有差异表达模式的phasiRNA。该分析提供了有关miRNA - PHAS -phasiRNA-靶标网络的新信息，并暗示了phasiRNA在柚子雄性不育中的潜在作用，并为进一步研究phasiRNA在柑橘中的功能做出了贡献。