Comparative Study On The Molecular Mechanisms Of Chemical Induced Male Sterility And Cytoplasmic Male Sterility In Brassica Napus

Rapeseed(Brassica napus)is an important oilseed crop in the Brassicaceae family.Chemical Induced Male Sterility and Cytoplasmic Male Sterility(CMS)are widely used for hybrid production in B.napus.SX-1 is a kind of chemical hybridization agents(CHA),which is effective,low poisonous and suitable for various B.napus lines for breeding.Identification of the key genes and pathways that are involved in SX-1 induced male steriliy are important to understand the underlying molecular mechanism.CMS is mainly caused by the rearrangements of mitochondrial DNA.A kind of restorer-of-fertility(Rf)genes in the nucleus can rescue the sterility,and most of them encode the pentatricopeptide repeat(PPR)proteins.Shaan2 A CMS is one of the most important CMS system in China and has been used for decades;however,the male sterility mechanism underlying Shaan2 A CMS remains unknown.In the present report,RNA-seq and miRNA-seq,combined with physiological analysis were conducted on SX-1 induced male sterility.Transcriptomic analysis and morphological observation were performed in the Shaan2 A CMS.Then comparative analyses of SX-1 induced male sterility and Shaan2 A CMS were conducted.Additionally,genome-wide identification of the restorer-of-fertility-like(RFL)gene family was performed in Brassica napus.The main findings were as follows:(1)The deficiencies in protein processing in endoplasmic reticulum and flavonoids biosynthesis occurred at early stage in the SX-1 treated materials.Then,genes involved in plant hormone signal transduction,biosynthesis of amino acids,fatty acids and steroid were down-regulated after SX-1 treatment.144 transcript factors(TFs)were also down-regulated at early stage,which suggested that the early regulation in anther and pollen wall development were disordered in CHA treated B.napus.In addition,7 important miRNAs were identified and 2 predicted target genes of miRNAs were PPR genes.(2)The sporogenous cells,endothecium,middle layer,and tapetum could not be clearly distinguished in Shaan2 A anthers.The lamella structure of chloroplasts in Shaan2 A anther wall cells were obviously aberrant.Transcriptomic analysis revealed differentially expressed genes(DEGs)mainly related to carbon metabolism,lipid and flavonoid metabolism,and the mitochondrial electron transport/ATP synthesis pathway.Importantly,9 gene ontology(GO)categories associated with anther and pollen development were enriched among down-regulated genes at the YB stage,including microsporogenesis,sporopollenin biosynthetic process,and tapetal layer development.Additionally,464 down-regulated TFs were identified at the YB stage,including some related to early anther differentiation genes.(3)176 and 379 common down-regulated DEGs(Fold Change ? 2)were identified in the two types of male sterility at YB and SA stage respectively,and these genes were annotated in Flavonoids and Phenylpropanoids biosynthesis pathways.18 GO categories associated with ?Development process? and ?Localization? were identified at YB stage with the 176 DEGs.Only 5 GO categories related to ?Response to stimulus? were identified at SA stage with the 379 DEGs.Additionally,3 candidate genes were further transgenic to B.napus by RNA interference and CRISPR system to identify their functions.(4)53 BnRFL genes were found,and most of them were distributed as gene clusters on chromosomes A9 and C8.Tissue specific expression study and RNA-seq analysis between the restorer line KC01 and the sterile line Shaan2 A indicated that,BnRFL1,BnRFL5,BnRFL6,BnRFL8,BnRFL11,BnRFL13 and BnRFL42 that located on the gene clusters of A9 and C8 had higher expression in KC01.(5)Comparative transcriptomic analysis of the three lines in Shaan2 A CMS system were conducted,after excluding the common DEGs between Shaan2 A versus KC01 and the maintainer line Shaan2 B versus KC01,the specific DEGs between Shaan2 A and KC01 were annotated to ?Regulation process?,?Developmental process?,?Response to stimulus?,?Transport?,?Cellular process? and ?Metabolic process?.In conclusion,an interaction network of candidate genes and the putative metabolism pathways were constructed based on the integrative analysis,it provided a new insight into the male sterility induced by CHA of SX-1 in B.napus.Additonally,these results suggested that the sterility gene in the Shaan2 A mitochondrion might suppress expression of these TF genes in the nucleus,affecting early anther development.The present study provides new insights into the molecular mechanism of Shaan2 A CMS in B.napus.The identification of RFL gene family might provide new insight into the study of the Rf genes in other CMS systems,and the comparative transcriptomic study might also provide a new method for the comparative analysis between the restorer line and the sterile line.