Profiling MiRNAs Involved In Male Sterility Of ‘Qianyang’ Seedless Ponkan And Cybrid HB Pummelo

Male sterility is the primary cause of seedless fruit. It provides important theoretical basis for citrus seedless breeding to explore the molecular mechanism of citrus male sterility and seedlessness. Micro RNAs play important role in plant development in various biological processes. In this study, by high-throughput sequencing, two seedless citrus varieties, ‘Qiangyang’ seedless Ponkan(Citrus reticulate Blanco) and G1+HBP(Citrus grandis Osbeck), are used to identify some potential mi RNAs and target genes which are related with citrus male sterility, and ‘Egan No.1’ Ponkan and HBP act as control materials. ‘Qianyang’ seedless Ponkan(QS) is the bud mutation of ordinary Ponkan and it is a male sterile line. G1+HBP is a cybrid gernerated by protoplast fusion with callus parent G1 and mesophyll parent HB pummelo. In addition, a large number of PHAS loci and phasi RNAs are identified by s RNA clean reads. To build the regulatory network of mi RNAs/phasi RNAs-PHAS-phasi RNAs-targets, a series of phasi RNAs targets are also identified by degradome data subsequently. This study is to investigate the role and biological function of mi RNA regulatory network in citrus flower development and male sterility. The main results are as follows:1. Identification of mi RNAs and targets in Ponkan and pummelo by s RNA sequencing and degradome analysisFor s RNA sequencing, the samples of Ponkan are divided into two developmental stages: one is flower buds at meiosis tetrad and the other is mature anther at full-bloom stage. The floral buds of G1+HBP and HBP are divided into three reproductive developmental stages from stamen primordial to the release of mature pollens. As a result, in Ponkan, 156 known mi RNAs that belong to 32 families and 24 novel mi RNAs are identified, as well as 138 target genes. In HBP and G1+HBP, 184 known mi RNAs and 22 novel mi RNAs and 86 targets are identified. The targets are analyzed that most of them are transcription factors, such as auxin response factors(ARFs), SQUAMOSA promoter binding protein box(SBP-box), MYB, basic region-leucine zipper(b ZIP), APETALA2(AP2) and transport inhibitor response 1(TIR1). Five targets in Ponkan and eight target genes in pummelo are confirmed to be sliced by corresponding mi RNAs using 5’RACE technology.2. Candidate mi RNAs related to citrus male sterilityFrom s RNA sequencing analysis, 71 known mi RNAs and 11 novel mi RNAs are differentially expressed between EG and QS, 42 mi RNAs are differentially expressed between HBP and G1+HBP. q RT-PCR and northern blot technology are used to validate the expression profiles of mi RNAs and the result shows that most of the mi RNA expression patterns are in agreement with the sequencing data, including mi R156, mi R167, mi R399, mi R172, mi R827, etc, which could be regarded as the potential mi RNAs related to citrus male sterility. q RT-PCR analysis of target genes indicate that most targets are negatively regulated by corresponding mi RNA. Among all of the differentially expressed mi RNAs, mi R167 and mi R399 show different expression between the sterile and fertile lines of both Ponkan and pummelo. Very interestingly, almost of the members of mi R167 and mi R399 families are down-regulated in G1+HBP, suggesting that they might play important role in citrus male organ development.3. Function verification of candidate mi RNAsYeast-one hybrid and dual-luciferase assays analysis reveal that one dehydrate responsive element binding(DREB) transcription factor binds to mi R167 a promoter and transcriptionally repress mi R167 a expression. Over expression vector of mi R167 a.1 is constructed and transformed to Arabidopsis and kumquat. Arabidopsis overexpressing mi R167 a.1 transgenic lines are sterile: abnormal flower development, petal can not open, siliques are very short and no seed in them. Over expression vectors of mi R156 a.1 and mi R399 a short tandem target mimic(STTM) are constructed and transformed into kumquat. Only one positive transformant line is obtained for mi R156 a.1 and 16 transgenic lines are obtained for mi R399 STTM. The phenotype characterization result can not be performed since the transgenic plants flower and bear fruit next year.4. Identification of phasi RNAs in HBP and G1+HBP280 PHAS loci are identified based on the s RNA sequencing data, among them 157 PHAS genes are located at intergenic region. 147 PHAS genes are coding genes, including 90 resistant genes NB-LRR, 12 PPR genes, 19 long non-coding RNAs and 3 transcription factors(ARF, NAC and MYB). 12 mi RNAs are identified as the triggers of 22 PHAS loci, including mi R167, mi R390, mi R3954, mi R396, mi R472, mi R482 and mi R172. 19 phasi RNAs are identified to be the triggers of 24 PHAS loci. A total of 2476 phasi RNAs are generated from the 280 PHAS loci. Based on the degradome data, 95 genes are identified to be the targets of phasi RNAs and they are involved in various cellular metabolic processes, like carbohydrate metabolism, aromatic substances and lipid metabolism, stress response and nutrient transport. In addition, two regulatory network, mi R390-TAS-phasi RNAs-ARF and PPRs by mi R472 and other phasi RNAs directly or indirectly, are discovered. Above all, mi RNA/phasi RNAs-PHAS-phasi RNAs-m RNA targets regulatory network might be involved in the citrus anther development and citrus male sterility.