Cytoplasmic male sterility (CMS) provides crucial breeding materials that facilitate hybrid seed production in various crops, and thus plays an important role in the study of hybrid vigor (heterosis), in plants. However, the CMS regulatory network in soybean remains unclear. MicroRNAs (miRNAs) play crucial roles in flower and pollen development by targeting genes that regulate their expression in plants. To identify the miRNAs and their targets that exist in the soybean CMS line NJCMS1A and its maintainer NJCMS1B, high-throughput sequencing and degradome analysis were conducted in this study. Two small RNA libraries were constructed from the flower buds of the soybean CMS line NJCMS1A and its maintainer NJCMS1B. A total of 105 new miRNAs present on the other arm of known pre-miRNAs, 23 new miRNA members, 158 novel miRNAs and 160 high-confidence soybean miRNAs were identified using high-throughput sequencing. Among the identified miRNAs, 101 differentially expressed miRNAs with greater than two-fold changes between NJCMS1A and NJCMS1B were discovered. The different expression levels of selected miRNAs were confirmed by stem-loop quantitative real-time PCR. A degradome analysis showed that 856 targets were predicted to be targeted by 296 miRNAs, including a squamosa promoter-binding protein-like transcription factor family protein, a pentatricopeptide repeat-containing protein, and an auxin response factor, which were previously shown to be involved in floral organ or anther development in plants. Additionally, some targets, including a MADS-box transcription factor, NADP-dependent isocitrate dehydrogenase and NADH-ubiquinone oxidoreductase 24 kDa subunit, were identified, and they may have some relationship with the programmed cell death, reactive oxygen species accumulation and energy deficiencies, which might lead to soybean male sterility. The present study is the first to use deep sequencing technology to identify miRNAs and their targets in the flower buds of the soybean CMS line NJCMS1A and its maintainer NJCMS1B. The results revealed that the miRNAs might participate in flower and pollen development, which could facilitate our understanding of the molecular mechanisms behind CMS in soybean.

Cytoplasmic male sterility (CMS) is a natural phenomenon of pollen abortion caused by the interaction between cytoplasmic genes and nuclear genes. CMS is a simple and effective pollination control system, and plays an important role in crop heterosis utilization. Circular RNAs (circRNAs) are a vital type of non-coding RNAs, which play crucial roles in microRNAs (miRNAs) function and post-transcription control. To explore the expression profile and possible functions of circRNAs in the soybean CMS line NJCMS1A and its maintainer NJCMS1B, high-throughput deep sequencing coupled with RNase R enrichment strategy was conducted. CircRNA libraries were constructed from flower buds of NJCMS1A and its maintainer NJCMS1B with three biological replicates. A total of 2867 circRNAs were identified, with 1009 circRNAs differentially expressed between NJCMS1A and NJCMS1B based on analysis of high-throughput sequencing. 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. Tissue specific expression patterns were also verified with two circRNAs by quantitative real-time PCR. Most parental genes of differentially expressed circRNAs were mainly involved in biological processes such as metabolic process, biological regulation, and reproductive process. Moreover, 83 miRNAs were predicted from the differentially expressed circRNAs, some of which were strongly related to pollen development and male fertility; The functions of miRNA targets were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), and the target mRNAs were significantly enriched in signal transduction and programmed cell death. Furthermore, a total of 165 soybean circRNAs were predicted to contain at least one internal ribosome entry site (IRES) element and an open reading frame, indicating their potential to encode polypeptides or proteins. Our study indicated that the circRNAs might participate in the regulation of flower and pollen development, which could provide a new insight into the molecular mechanisms of CMS in soybean.

DNA methylation is an important epigenetic modification. It can regulate the expression of many key genes without changing the primary structure of the genomic DNA, and plays a vital role in the growth and development of the organism. The genome-wide DNA methylation profile of the cytoplasmic male sterile (CMS) line in soybean has not been reported so far. In this study, genome-wide comparative analysis of DNA methylation between soybean CMS line NJCMS5A and its maintainer NJCMS5B was conducted by whole-genome bisulfite sequencing. The results showed 3527 differentially methylated regions (DMRs) and 485 differentially methylated genes (DMGs), including 353 high-credible methylated genes, 56 methylated genes coding unknown protein and 76 novel methylated genes with no known function were identified. Among them, 25 DMRs were further validated that the genome-wide DNA methylation data were reliable through bisulfite treatment, and 9 DMRs were confirmed the relationship between DNA methylation and gene expression by qRT-PCR. Finally, 8 key DMGs possibly associated with soybean CMS were identified. Genome-wide DNA methylation profile of the soybean CMS line NJCMS5A and its maintainer NJCMS5B was obtained for the first time. Several specific DMGs which participated in pollen and flower development were further identified to be probably associated with soybean CMS. This study will contribute to further understanding of the molecular mechanism behind soybean CMS.

MicroRNAs (miRNAs), a class of noncoding small RNAs (sRNAs), are widely involved in the response to high temperature (HT) stress at both the seedling and flowering stages. To dissect the roles of miRNAs in regulating male fertility in soybean cytoplasmic male sterility (CMS)-based F₁ under HT, sRNA sequencing was performed using flower buds from HT-tolerant and HT-sensitive CMS-based F₁ combinations (NF₁ and YF₁, respectively). A total of 554 known miRNAs, 59 new members of known miRNAs, 712 novel miRNAs, and 1145 target genes of 580 differentially expressed miRNAs (DEMs) were identified under normal temperature and HT conditions. Further integrated analysis of sRNA and transcriptome sequencing found that 21 DEMs and 15 differentially expressed target genes, such as gma-miR397a/Laccase 2, gma-miR399a/Inorganic phosphate transporter 1-4, and gma-miR4413a/PPR proteins, mitochondrial-like, were negatively regulated under HT stress. Furthermore, all members of the gma-miR156 family were suppressed by HT stress in both NF₁ and YF₁, but were highly expressed in YF₁ under HT condition. The negative correlation between gma-miR156b and its target gene squamosa promoter-binding protein-like 2b was confirmed by expression analysis, and overexpression of gma-miR156b in Arabidopsis led to male sterility under HT stress. With these results, we proposed that miRNAs play an important role in the regulation of male fertility stability in soybean CMS-based F₁ under HT stress.

Cytoplasmic male sterility (CMS) lays a foundation for the utilization of heterosis in soybean. The soybean CMS line SXCMS5A is an excellent CMS line exhibiting 100% male sterility. Cytological analysis revealed that in SXCMS5A compared to its maintainer SXCMS5B, its tapetum was vacuolated and abnormally developed. To identify the genes and metabolic pathways involving in pollen abortion of SXCMS5A, a comparative transcriptome analysis was conducted between SXCMS5A and SXCMS5B using flower buds. A total of 372,973,796 high quality clean reads were obtained from 6 samples (3 replicates for each material), and 840 differentially expressed genes (DEGs) were identified, including 658 downregulated and 182 upregulated ones in SXCMS5A compared to SXCMS5B. Among them, 13 DEGs, i.e., 12 open reading frames (ORFs) and 1 COX2, were mitochondrial genome genes in which ORF178 and ORF103c were upregulated in CMS lines and had transmembrane domain(s), therefore, identified as CMS candidate mitochondrial genes of SXCMS5A. Furthermore, numerous DEGs were associated with pollen wall development, carbohydrate metabolism, sugar transport, reactive oxygen species (ROS) metabolism and transcription factor. Some of them were further confirmed by quantitative real time PCR analysis between CMS lines with the same cytoplasmic source as SXCMS5A and their respective maintainer lines. The amount of soluble sugar and adenosine triphosphate and the activity of catalase and ascorbic acid oxidase showed that energy supply and ROS scavenging decreased in SXCMS5A compared to SXCMS5B. These findings provide valuable information for further understanding the molecular mechanism regulating the pollen abortion of soybean CMS.