Identification Of MiRNAs Involved In The Development And Differentiation Of Fertile And Sterile Flowers In Viburnum Macrocephalum F.Keteleeri

As the reproductive structures of the most successful group of flowering plants,flowers are crucial to the reproductive success and continuity of these species over time.Flowers come in a variety of colors,structures,shapes,and sizes,which are the evolutionary consequences of selective pressure by biotic and abiotic environmental factors.Flowers can be classified as fertile or sterile according to their abilities to reproduce sexually and form available gametes.Fertile flowers have the ability to generate normal stamens and pistils.In some species,sterile flowers evolved from fertile flowers that developed abnormal stamens and/or pistils.Although sterile flowers cannot produce fruits or seeds for the next generation,they play a crucial role in enhancing reproductive success by effectively attracting pollinators.MicroRNAs?miRNAs?are small,non-coding RNAs produced by Dicer-catalyzed excision from stem-loop precursors which regulate gene expression at the post-transcriptional level by directing RNA cleavage or inhibiting translation of target transcripts.Numerous studies have demonstrated the critical role of miRNAs in controlling flower development-related processes.However,little is known about the regulatory roles of miRNAs in fertile and sterile flower differentiation and development.Viburnum macrocephalum f.keteleeri,a Chinese wild shrub in the Adoxaceae family,has two kinds of flowers in an inflorescence.The type distributed in the internal position of the inflorescence is fertile,whereas the sterile flowers are distributed around the exterior.Fertile flowers are morphologically smaller than sterile flowers,but the number of fertile flowers is much greater than that of sterile flowers.In this study,by digital camera and environmental scanning electron microscopy,we observed the development of Viburnum macrocephalum f.keteleeri on the morphology and cytology,and found in the middle stage of the flowers,the fertile and sterile flower area can significantly increase,but because of the different cell size and cell number changes,the final area of sterile flower petals can be more than 30 times the size of fertile flower petals.Through a thin section observation,we found sterile flower have poor fertility with shoot stamens and hypogenetic embryo sac while fertile flowers have normal flower structures.On the basis of research on structure and morphology,this research choose fertile and sterile flowers in middle stage,we used high-throughput sequencing to construct two small RNA database.The main results are as follows.?1?A total of 11,447,459 and 12,365,813 raw sRNA reads were generated in fertile and sterile flowers,respectively.After removing low-quality reads,adapter sequences,poly A sequences,sequences of<18 nt,and other artifacts,7,562,443 and 7,373,905 high-quality clean reads between 18 nt and 30 nt in length,respectively,remained for further analyses?2?After using a BLASTN search for sequence similarity in the miRBase database,49 known and 67 novel miRNAs were obtained in flower.The precursor sequences of the 28 known and novel miRNA candidates were further validated by PCR amplification and sequencing.17 of the 28 validated sequences were identical to those obtained by sRNA sequencing.In these 17 sequences,11 were known miRNA and 6 were novel miRNAs.?3?To identify putative targets for known and novel miRNAs,the web-based programs psRobot and psRNATarget were employed to hunt for potential miRNAs target genes.The results showed that a total of 630 and 1,209 genes were predicted to be targets of known and novel miRNAs,respectively.30 known and 39 novel miRNA sequences were differentially expressed.Compared with the comparable data for fertile flowers,in sterile flowers,14 known miRNAs were upregulated and 16 were downregulated,whereas 21 novel miRNAs were upregulated and 18 were downregulated.?4?Combined with the transcriptome data,five AP2-EREBP genes are predicted as the targets of miR12 and several novel miRNAs.miR159 was predicted to target 6 MYB genes.In addition,GRF,ARF,NAC,bHLH,CAMTA were all contained in targets.The results demonstrated that miRNA could regulate fflower growth and development through the regulation of hormonal signal transduction,biosynthesis,disease resistance and stress responses.?5?Members of miR156 family and miR157 targeted many same target genes.miR156a,miR156g,miR156j expressed higher in sterile flowers,and had a significant difference in fertile and sterile flowers.They targeted multiple SPL transcription factors family members.Combining previous research in structure,we hypothesized that miR156 may play an important role in the stamen development.?6?We analyzed the expression patterns of six known miRNAs?miR172a,miR319a,miR166a,miR390a,miR394a,and miR396b?and three novel miRNAs?novel₁,novel₁4,and novel₁12?with known precursor sequences by qRT-PCR at the three stage of flower development to validate the dynamic expression patterns of the miRNAs and their putative targets and to determine the possible functions of the miRNAs.Our study used fertile and sterile flowers of V.macrocephalum f.keteleeri as material,which have same genetic background.By comparing miRNA expression of the two RNA small libraries,we screened differentially expressed miRNAs.With the transcriptome data and target gene functional annotation,we explored the relationship between miRNA and prediction target genes,and analyzed the function miRNA in the process of flower development.