MiRNA Sequencing And Analysis Of MiRNAs Differential Expression Between Self-pollinated And Cross-pollinated Style Of Citrus Grandis Var.Shatinyu

Citrus grandis var.Shatinyu,a member of the rutaceae,is a fruit species with high gametophyte self incompatibility.At present,the self-incompatible molecular mechanism of Citrus grandis var.Shatinyuhas not yet been reported.miRNA is a newly identified class of 20-24nt non-protein-coding and endogenous small RNAs,which plays an important role in plant grow and development and response to environmental stresses.Therefore,it is of great importance to search the miRNA associated self-incompatible,andexpoundsthe molecular mechanism of miRNA-mediated self-incompatibility.In this study,the Self-pollinated 1-3days,Cross-pollinatied 1-3days and non-pollinated styles were choosed to be the materials to construction of small RNA sequencing library.All Small RNAs were sequenced through high thtoughput sequencing.Then,the differentially expressed miRNAs were classified and predicted,and the target genes were annotated and the KEGG pathway was used to investigate the effect of miRNA on the self-incompatibility of Citrus maxima.The main results were as follow:1、The small RNA library of 7 samples of Shatian pomelo was sequenced by high-throughput sequencing technique.The total-reads of each sample were:SC was 13326242、YJ1 was 23674636、YJ2 was 14465682、YJ3 was 24805180、ZJ1 was 21113205、ZJ2 was 19050972、ZJ3 was 22881662.Removedlow quality reads、adapter、smallerthanl8nt reads,Get the clean-reads:SC was 11523295、YJ1 was 23046850、YJ2 was 12556786、YJ3 was 23949192、ZJ1 was 20472150、ZJ2 was 18567950、ZJ3 was 22197840respectively.The obtained clean-reads were readed map to genome,and the number of sRNAs for each sample compared to the genome was:SC was 7284921、YJ1 was 16759796、YJ2 was 8594610、YJ3 was 17855172、ZJ1 was 15341949、ZJ2 was 13545677、ZJ3 was 16132232.2、Based on miRNA similarity sequences and secondary structure prediction results show that:identified known miRNAs in 7 samples belonge to 28 families,including an undefined family.Among the seven samples,the number of miRNAs known in the non-pollinated style was 118 and the novelmiRNAs was 411.The number of known miRNAs in the Cross-pollinatied 1,2,and 3days were 232,122 and 219,respectively,and the new miRNAs were 1184,476 and 836,respectively.The number of known miRNAs in the Self-pollinated 1,2,and 3days were 178.179 and 186,respectively,and the new miRNAs were 640,739 and 801,respectively.The number of new miRNAs in each sample is greater than the number of known miRNAs.3、Cross/self-differentially expressed miRNAs,miR390a-5p regulates auxin response that stimulates the growth of pollen tubes and may play a role in pollen tube growth;miR172a-5p targets the SCF complex in the Ubiquitin mediated proteolysis,which is involved in the Ubiquitin mediated proteolysis associated with the miR5059-targeted AUX/IAA and the plant hormone signaling pathway,and the two miRNA expression patterns are essentially consistent,indicating that it may be through the regulation of two metabolic pathways involved in self-incompatibility.MiR171f continued to be down-regulated in cross samples in miRNAs that were continuously up-regulated or down-regulated in self-pollinated and cross-pollinated samples,whereas miR393-3p was continuously up-regulated in cross-pollinated samples.These two miRNAs were primarily involved in plant stress response reactions and there have overlaps between pollination and stress responses.miR6197-5p,miR414-targeted WRKY transcription factor function of the growth process of pollen tube,and have different expression in YJ/ZJ;indicating that the above miRNAs may play a role in the self-incompatibility reaction.4.Furthermore,novel_mir₆91、novel_mir₁021 targeting auxin transcription factor;novel_mir₁7 target gene annotation to cullin-like protein.novel_mir₂854 is annotated as 14-3-3 protein which is involved in a variety of plant hormonal signaling pathways,novel_mir₁770 Targeting the membrane element of the phosphatidylinositol,which is differentially expressed in YJ/ZJ,indicating that it may be involved in the self-incompatibility reaction.5、Self-pollinated having differential expressionunique miRNAs and targets,novel_mir₂344 target gene function of pectinesterase inhibitor,miR9470-3p target gene function of y-aminobutyric acid（GABA）receptors,and involved in the glycolytic pathway,miR9560a-5p target gene function is wall-related receptor kinase,miR395 target gene function for galactose metabolism and in the wall synthesis also play a role,miR2118b target gene function for heat shock protein and molecular chaperone function,miR8775 target gene function includes Ubiquitin-regulated proteasome pathway;MiR9560a-5p and novel_mir₆51 persistently up-regulated in three days of Self-pollinated,targeting the wall receptor-associated protein kinase and plant hormone signaling process receptor proteins,respectively,these miRNAs may play a negative regulatory role in self-incompatibility.The target gene of novel_mir₂651 is an actin binding protein and flavonoids that affect the growth of pollen tubes,as well as cytochrome P450 that affects the composition of sporopollenin（pollen wall）,but the role of its regulation and the role of self-incompatibility in Citrus maximaneed to be further verified.6、The 14 differential expression miRNAs were analyzed with Real-time PCR,the results showed that miRNAs were consistent with the sequencing data.