- Of the 12 randomly selected circRNAs with different expression levels, 10 showed consistent expression patterns based on high-throughput sequencing and quantitative real-time PCR analyses. - Full list of author information is available at the end of the article. - 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Utilization of heterosis has been proved to be one of the effective methods to increase crop yield, and cytoplasmic male sterility (CMS) plays an important role in heterosis utilization [1]. - CircRNAs were first reported based on deep sequencing of RNA by Salzman et al. - Li et al. - Meanwhile, Zhao et al. - [35] found that up-regulation of circRNAs might decrease the activity of target miRNAs and increase expression of the related mRNAs.. - The insert size of the libraries was detected by Agilent 2100/. - The effective concentration of the libraries was accurately quantified by qRT-PCR, and the effective concentration was greater than 2 nM to ensure the libraries quality. - Clustering of the index-coded samples was performed on a cBot Cluster Generation System using HiSeq PE Cluster Kit v4 cBot (Illumina) according to the manufac- turer ’ s instructions. - The index of the reference genome was built using Bow- tie v2.0.6, paired-end reads were aligned to the reference genome (soybean genome version 2.0 in Phytozome) using TopHat v2.0.9. - 20-mers from 5′ and 3′ end of the unmapped reads were extracted and aligned independ- ently to reference sequences using Bowtie v2.0.6. - 2 based on the method used by Wang et al. - [30] and Liu et al. - According to the instruction of the iScript Select cDNA Synthesis Kit (BIO-RAD, USA), 1 μ g of the total RNA untreated with RNase R was reverse-transcribed with random primers. - All real-time PCR assays were performed with three biological replicates, and the expression of the housekeeping gene GADPH was used as a reference for data normalization. - To predict the IRES ele- ments in soybean circRNAs, we blasted sequences of the circRNAs to all the IRES sequences in the website (http://iresite.org/) at an E-value <. - The possible coding products of the circRNAs with protein-coding potential were used to predict the conserved domains using the Conserved Domain Database (https://www.ncbi.nlm.nih.gov/. - Identification of circRNAs in soybean. - circRNAs in A. - However, only exonic circRNAs were identified in soy- bean by Zhao et al. - The length distribution of soybean circRNAs in this study was similar to that reported by Zhao et al. - Additionally, the sequence alignment between the cir- cRNAs inform this study and previous study (Zhao et al.. - The back splicing sites and expression profiles of the 12 randomly selected from 1009 circRNAs were further experimentally validated. - Two incon- sistent expression of circRNAs candidates (gma-circ2468 and gma-circ2848) may be caused by the low expression levels of the two circRNAs.. - (a) Venn diagram shows the number of the identified circRNAs in NJCMS1A and NJCMS1B. - (b) Source statistics of the circRNAs. - (c) Sequence length distribution of the circRNAs in different samples. - (d) Circle plot shows the distribution of the identified circRNAs in soybean chromosomes and their expression levels. - The middle blue lines show the distribution of the circRNAs in soybean chromosomes, while the denser lines indicate more circRNA distribution. - The innermost green lines show expression levels of the circRNAs, and the height of the lines indicates the level of expression. - Functional categorization of parental genes of differentially expressed circRNAs. - To understand the possible functions of circRNAs in CMS of soybean, the parental genes of the differentially expressed circRNAs were predicted. - A total of 545 parental genes were obtained from the 1009 differentially expressed circRNAs. - 3 Validation of differentially expressed circRNAs in NJCMS1A and NJCMS1B by qRT-PCR. - Expression of the GADPH gene was used as the internal reference. - All qRT-PCR reactions were performed with three biological replicates, and the error bars indicate the standard errors of the means of 2 –ΔΔCt , with NJCMS1B as a control. - The relative expression levels of gma-circRNA0002 and gma-circRNA2483 were obtained from roots, stems, leaves and flower buds with three biological replicates by qRT-PCR,, and the error bars indicated the standard error of the mean of 2 –ΔΔCt , with NJCMS1B as a control.. - GO analysis of the parental genes showed that the differentially expressed circRNAs from NJCNS1A and NJCMS1B were associated with various functions in different biological processes, cellular components, and molecular function, indicating that circRNAs may play an important role in the fertility of soybean. - Putative functions of parental genes of differentially expressed circRNAs in flower development and male fertility. - Glyma.06G173500, the parental gene of gma-circRNA0717, was a homolog of the Glucan Synthase-Like 8 ( GSL8 ) gene in A. - GSL8 and Glucan Synthase-Like 10 ( GSL10 ) were two members of the A. - Glyma.13G230500, the parental gene of gma-circRNA1856, was an ortholog of the maternal effect embryo arrest 18 ( MEE18 ) gene in A. - In this study, Glyma.18G164900, the paren- tal gene of gma-circRNA2481, was a homolog of the XRI1 gene. - Characterization of binding miRNAs of differentially expressed circRNAs. - 5 Gene Ontology (GO) annotation of parental genes of the differentially expressed circRNAs between NJCMS1A and NJCMS1B. - have a similar function in soybean, we predicted potential binding sites of miRNAs of the differentially expressed circRNAs. - miR156 is one of the highly conserved miRNA families, which was first reported in A. - Previous studies showed that miR156 regulated the development stage transition [56], flowering process [57], fertility mainten- ance [58], and fruit ripening [59] in plants by regulating members of the SPL family. - an important part of the pyruvate dehydrogenase complex. - gma-circRNA0195 was predicted to target gam-miR172j, which could target Glyma.01 g188400, the homolog of the APETALA2 ( AP2 ) of A. - thaliana , miR172 could control flowering time and floral organ formation by regulating expression of the AP2 -like transcription factor. - 6 CircRNA-miRNA interaction network for differentially expressed circRNAs in NJCMS1A and NJCMS1B. - 7 A direct acyclic graph (DAG) illustrating the biological process category generated from the Gene Ontology (GO) annotation of the predicted target miRNAs. - The smaller of the term ’ s adjusted p -value, the more significant statistically, and the node ’ s color is darker and redder. - Inside the box of the significant terms, the information include: GO term, adjusted p-value, GO description, item number mapping the GO in the query list and background, and total number of the query list and background. - For example, the PET1-CMS cytoplasm in sunflower causes premature PCD of the tapetal cells, which then leads to abnormal anther development [70].. - To verify whether soybean circRNAs have a similar function, we predicted the protein-coding potential by blasting sequences of the circRNAs to all the IRES se- quences at the website. - Furthermore, conserved domains of the possible protein-coding products were predicted by Conserved Domain Database (Additional file 10) [73], which might have important functions. - The protein-coding products of these circRNAs may affect flower and pollen development in soybean by affecting the function of the parental genes.. - previous study of Zhao et al. - differentially expressed circRNAs in soybean. - The up-expressed circRNAs identified in NJCMS1A and NJCMS1B. - The down-expressed circRNAs identified in NJCMS1A and NJCMS1B. - Predicted circRNA-miRNA-mRNA connection for differentially expressed circRNAs in NJCMS1A and NJCMS1B. - Conserved domains of the predicted protein-coding products. - The collection and usage of samples followed the ethics of the People ’ s Republic of China.. - Hu J, Wang K, Huang W, Liu G, Gao Y, Wang J, et al. - Fujii S, Yamada M, Fujita M, Itabashi E, Hamada K, Yano K, et al. - Li JJ, Han SH, Ding XL, He TT, Dai JY, Yang SP, et al. - Li JJ, Ding XL, Han SH, He TT, Zhang H, Yang LW, et al. - Ding XL, Li JJ, Zhang H, He TT, Han SH, Li YW, et al. - Li YW, Ding XL, Wang X, He TT, Zhang H, Yang LS, et al. - Jeck WR, Sorrentino JA, Wang K, Slevin MK, Burd CE, Liu J, et al. - 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