Please wait a minute...
Journal of Integrative Agriculture  2020, Vol. 19 Issue (7): 1731- 1742    DOI: 10.1016/S2095-3119(19)62780-2
Special Issue: 油料作物合辑Oil Crops
Crop Science Advanced Online Publication | Current Issue | Archive | Adv Search |
Differentially expressed miRNAs in anthers may contribute to the fertility of a novel Brassica napus genic male sterile line CN12A
Dong Yun1, 2, Wang Yi1, Jin Feng-wei1, Xing Li-juan2, Fang Yan3, Zhang Zheng-ying1, ZOU Jun-jie2, Wang Lei2, Xu Miao-yun2  
1 Crop Research Institute, Gansu Academy of Agricultural Sciences, Lanzhou 730070, P.R.China
2 National Key Facility for Crop Gene Resources and Genetic Improvement/Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China
3 Gansu Provincial Key Lab of Aridland Crop Sciences/Gansu Agricultural University, Lanzhou 730070, P.R.Chi
Download:  PDF in ScienceDirect  
Export:  BibTeX | EndNote (RIS)      
In Brassica napus L. (rapeseed), complete genic male sterility (GMS) plays an important role in the utilization of heterosis.  Although microRNAs (miRNAs) play essential regulatory roles during bud development, knowledge of how GMS is regulated by miRNAs in rapeseed is rather limited.  In this study, we obtained a novel recessive GMS system, CN12AB.  The sterile line CN12A has defects in tapetal differentiation and degradation.  Illumina sequencing was employed to examine the expression of miRNAs in the buds of CN12A and the fertile line CN12B.  We identified 85 known miRNAs and 120 novel miRNAs that were expressed during rapeseed anther development.  When comparing the expression levels of miRNAs between CN12A and CN12B, 19 and 18 known miRNAs were found to be differentially expressed in 0.5–1.0 mm buds and in 2.5–3.0 mm buds, respectively.  Among these, the expression levels of 14 miRNAs were higher and the levels of 23 miRNAs were lower in CN12A compared with CN12B.  The predicted target genes of these differentially expressed miRNAs encode protein kinases, F-box domain-containing proteins, MADS-box family proteins, SBP-box gene family members, HD-ZIP proteins, floral homeotic protein APETALA 2 (AP2), and nuclear factor Y, subunit A.  These targets have previously been reported to be involved in pollen development and male sterility, suggesting that miRNAs might act as regulators of GMS in rapeseed anthers.  Furthermore, RT-qPCR data suggest that one of the differentially expressed miRNAs, bna-miR159, plays a role in tapetal differentiation by regulating the expression of transcription factor BnMYB101 and participates in tapetal degradation and influences callose degradation by manipulating the expression of BnA6.  These findings contribute to our understanding of the roles of miRNAs during anther development and the occurrence of GMS in rapeseed.
Keywords:  Brassica napus L.        genic male sterility        miRNAs        targets        molecular regulation network  
Received: 19 February 2019   Accepted:
Fund: This project was supported by the National Key Research Program of China (2018YFD0100500), the Major Projects for New Varieties of Genetically Modified Organisms, China (2018ZX0801109B) and the Agricultural Science and Technology Innovation Project of Gansu Academy of Agricultural Sciences, China (2018GAAS04).
Corresponding Authors:  Correspondence WANG Lei, Tel: +86-10-82105317, E-mail:; XU Miao-yun, Tel: +86-10-82105317, E-mail:   
About author:  DONG Yun, E-mail:;

Cite this article: 

Dong Yun, Wang Yi, Jin Feng-wei, Xing Li-juan, Fang Yan, Zhang Zheng-ying, ZOU Jun-jie, Wang Lei, Xu Miao-yun. 2020. Differentially expressed miRNAs in anthers may contribute to the fertility of a novel Brassica napus genic male sterile line CN12A. Journal of Integrative Agriculture, 19(7): 1731- 1742.

Aya K, Ueguchi-Tanaka M, Kondo M, Hamada K, Yano K, Nishimura M, Matsuoka M. 2009. Gibberellin modulates anther development in rice via the transcriptional regulation of GAMYB. The Plant Cell, 21, 1453–1472.
Cartolano M, Castillo R, Efremova N, Kuckenberg M, Zethof J, Gerats T, Schwarz-Sommer Z, Vandenbussche M. 2007. A conserved microRNA module exerts homeotic control over Petunia hybrida and Antirrhinum majus floral organ identity. Nature Genetics, 39, 901–905.
Chan P P, Lowe T M. 2009. GtRNAdb: A database of transfer RNA genes detected in genomic sequence. Nucleic Acids Research, 37, D93–D97.
Chitwood D H, Nogueira F T, Howell M D, Montgomery T A, Carrington J C, Timmermans M C. 2009. Pattern formation via small RNA mobility. Genes, 23, 549–554.
D’Ario M, Griffiths-Jones S, Kim M. 2017. Small RNAs: Big impact on plant development. Trends in Plant Science, 22, 1056–1068.
Ding X L, Li J J, Zhang H, He T T, Han S H, Li Y W, Yang S P, Gai J Y. 2016. Identification of miRNAs and their targets by high-throughput sequencing and degradome analysis in cytoplasmic male-sterile line NJCMS1A and its maintainer NJCMS1B of soybean. BMC Genomics, 17, 24–39.
Fang Y N, Zheng B B, Wang L, Yang W, Wu X M, Xu Q, Guo W W. 2016. High-throughput sequencing and degradome analysis reveal altered expression of miRNAs and their targets in a male-sterile cybrid pummelo (Citrus grandis). BMC Genomics, 17, 591–605.
Field S, Thompson B. 2016. Analysis of the maize dicer-like1 mutant, fuzzy tassel, implicates microRNAs in anther maturation and dehiscence. PLoS ONE, 11, e0146534.
Hird D L, Worrall D, Hodge R, Smartt S, Paul W, Scott R. 1993. The anther-specific protein encoded by the Brassica napus and Arabidopsis thaliana A6 gene displays similarity to beta-1,3-glucanases. The Plant Journal, 4, 1023–1033.
Kohany O, Gentles A J, Hankus L, Jurka J. 2006. Annotation, submission and screening of repetitive elements in Repbase: RepbaseSubmitter and Censor. BMC Bioinformatics, 7, 474.
Krüger J, Rehmsmeier M. 2006. RNAhybrid: MicroRNA target prediction easy, fast and flexible. Nucleic Acids Research, 34, W451–W454.
John B, Enright A J, Aravin A, Tuschl T, Sander C, Marks D S. 2004. Human MicroRNA targets. PLoS Biology, 2, e363.
Leng N, Dawson J A, Thomson J A, Ruotti V, Rissman A I, Smits B M, Haag J D, Gould M N, Stewart R M, Kendziorski C. 2013. EBSeq: An empirical bayes hierarchical model for inference in RNA-seq experiments. Bioinformatics, 29, 1035–1043.
Liu H H, Guo S Y, Xu Y Y, Li C H, Zhang Z Y, Zhang D J, Xu S J, Zhang C, Chong K. 2014. OsmiR396d-regulated OsGRFs function in floral organogenesis in rice through binding to their targets OsJMJ706 and OsCR4. Plant Physiology, 165, 160–174.
Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(–Delta Delta C(T)) method. Methods, 25, 402–408.
Lord E M, Mollet J C. 2002. Plant cell adhesion: A bioassay facilitates discovery of the first pectin biosynthetic gene. Proceedings of the National Academy of Sciences of the United States of America, 99, 15843–15845.
Millar A A, Gubler F. 2005. The Arabidopsis GAMYB-like genes, MYB33 and MYB65, are microRNA-regulated genes that redundantly facilitate anther development. The Plant Cell, 17, 705–721.
Murray F, Kalla R, Jacobsen J, Gubler F. 2003. A role for HvGAMYB in anther development. The Plant Journal, 33, 481–491.
Nawrocki E P, Burge S W, Bateman A, Daub J, Eberhardt R Y, Eddy S R, Floden E W, Gardner P P, Jones T A, Tate J, Finn R D. 2015. Rfam 12.0: Updates to the RNA families database. Nucleic Acids Research, 43, D130–D137.
Pruitt K D, Tatusova T, Klimke W, Maglott D R. 2009. NCBI reference sequences: Current status, policy and new initiatives. Nucleic Acids Research, 37, D32–D36.
Shen Y, Zhang Z, Lin H, Liu H, Chen J, Peng H, Cao M, Rong T, Pan G. 2011. Cytoplasmic male sterility-regulated novel microRNAs from maize. Functional Integrative Genomics, 11, 179–191.
Vernoud V, Laigle G, Rozier F, Meeley R B, Perez P, Rogowsky P M. 2009. The HD-ZIP IV transcription factor OCL4 is necessary for trichome patterning and anther development in maize. The Plant Journal, 59, 883–894.
Wang Y, Sun F, Cao H, Peng H, Ni Z, Sun Q, Yao Y. 2012. TamiR159 directed wheat TaGAMYB cleavage and its involvement in anther development and heat response. PLoS ONE, 7, e48445.
Wei M, Wei H, Wu M, Song M, Zhang J, Yu J, Fan S, Yu S. 2013. Comparative expression profiling of miRNA during anther development in genetic male sterile and wild type cotton. BMC Plant Biology, 13, 66–79.
Wei X, Zhang X, Yao Q, Yuan Y, Li X, Wei F, Zhao Y, Zhang Q, Wang Z, Jiang W, Zhang X. 2015. The miRNAs and their regulatory networks responsible for pollen abortion in Ogura-CMS Chinese cabbage revealed by high-throughput sequencing of miRNAs, degradomes, and transcriptomes. Frontiers in Plant Science, 6, 894–909.
Wu M F, Tian Q, Reed J W. 2006. Arabidopsis microRNA167 controls patterns of ARF6 and ARF8 expression, and regulates both female and male reproduction. Development, 133, 4211–4218.
Xing L, Zhu M, Zhang M, Li W, Jiang H, Zou J, Wang L, Xu M. 2017. High-throughput sequencing of small RNA transcriptomes in maize kernel identifies miRNAs involved in embryo and endosperm development. Genes, 8, 274–287.
Xing S, Salinas M, Hohmann S, Berndtgen R, Huijser P. 2010. miR156-targeted and nontargeted SBP-box transcription factors act in concert to secure male fertility in Arabidopsis. The Plant Cell, 22, 3935–3950.
Xu M Y, Zhang L, Li W W, Hu X L, Wang M B, Fan Y L, Zhang C Y, Wang L. 2014. Stress-induced early flowering is mediated by miR169 in Arabidopsis thaliana. Journal of Experimental Botany, 65, 89–101.
Xue T, Liu Z, Dai X, Xiang F. 2017. Primary root growth in Arabidopsis thaliana is inhibited by the miR159 mediated repression of MYB33, MYB65 and MYB101. Plant Science, 262, 182–189.
Yan J, Zhang H, Zheng Y, Ding Y. 2015. Comparative expression profiling of miRNAs between the cytoplasmic male sterile line MeixiangA and its maintainer line MeixiangB during rice anther development. Planta, 241, 109–123.
Yang J, Liu X, Xu B, Zhao N, Yang X, Zhang M. 2013. Identification of miRNAs and their targets using high-throughput sequencing and degradome analysis in cytoplasmic male-sterile and its maintainer fertile lines of Brassica juncea. BMC Genomics, 14, 9–23.
Yang S, Poretska O, Sieberer T. 2018. ALTERED MERISTEM PROGRAM1 restricts shoot meristem proliferation and regeneration by limiting HD-ZIP III-mediated expression of RAP2.6L. Plant Physiology, 177, 1580–1594.
Yang X, Zhao Y, Xie D, Sun Y, Zhu X, Esmaeili N, Yang Z, Wang Y, Yin G, Lv S, Nie L, Tang Z, Zhao F, Li W, Mishra N, Sun L, Zhu W, Fang W. 2016. Identification and functional analysis of microRNAs involved in the anther development in cotton genic male sterile line Yu98-8A. International Journal of Molecular Sciences, 17, 1677.
Zhang W, Xie Y, Xu L, Wang Y, Zhu X, Wang R, Zhang Y, Muleke E M, Liu L. 2016. Identification of microRNAs and their target genes explores miRNA-mediated regulatory network of cytoplasmic male sterility occurrence during anther development in radish (Raphanus sativus L.). Frontiers in Plant Science, 7, 1054–1069.
Zhang Z B, Zhu J, Gao J F, Wang C, Li H, Li H, Zhang H Q, Zhang S, Wang D M, Wang Q X, Huang H, Xia H J, Yang Z N. 2007. Transcription factor AtMYB103 is required for anther development by regulating tapetum development, callose dissolution and exine formation in Arabidopsis. The Plant Journal, 52, 528–538.
Zhou L, Chen J, Li Z, Li X, Hu X, Huang Y, Zhao X, Liang C, Wang Y, Sun L, Shi M, Xu X, Shen F, Chen M, Han Z, Peng Z, Zhai Q, Chen J, Zhang Z, Yang R. 2010. Integrated profiling of microRNAs and mRNAs: microRNAs located on Xq27.3 associate with clear cell renal cell carcinoma. PLoS ONE, 5, e15224.
Zhou Z, Dun X, Xia S, Shi D, Qin M, Yi B, Wen J, Shen J, Ma C, Tu J, Fu T. 2012. BnMs3 is required for tapetal differentiation and degradation, microspore separation, and pollen-wall biosynthesis in Brassica napus. Journal of Experimental Botany, 63, 2041–2058.
Zhu M, Zhang M, Xing L, Li W, Jiang H, Wang L, Xu M. 2017. Transcriptomic analysis of long non-coding RNAs and coding genes uncovers a complex regulatory network that is involved in maize seed development. Genes, 8, 385–400.
No related articles found!
No Suggested Reading articles found!