Screening And Identification Of Candidate Genes For Recessive Genic Male Sterile Twotype Line SLAB In Brassica Napus L.

Brassica napus L.is one of the most important oil crops in the world.Recessive genic male sterility,as an effective and economical pollination control system,is worldwide used for the utilization of rapeseed heterosis.To identify the critical period for anther abortion of sterile SLA,we evaluated the phenotypic features and sterility stage of SLA and SLB using anatomical analysis and scanning electron microscopy(SEM).Additionally,to elucidate the molecular mechanism governing the male sterility between SLA and SLB,we compare the RNA sequencing and whole genome sequencing analysis in SLA and SLB by high-throughput sequencing.Firstly,screening the genetic variation of genes at the genome levels between SLA and SLB;secondly,identifying the differentially expressing genes between SLA and SLB by RNA-seq and qRT-PCR;lastly,confirming the candidate genes associated with abortive anthers by comparing the results of transcriptome and genome re-sequencing using the Venny software.The main conclusions are as follows:The results of SEM analysis showed that the anthers of SLA are less than SLB,and no obviously elongation during the whole process of bud development.When the longitudinal diameter of buds were 2mm,the anthers epiderm have no significantly differences between the sterile SLA and fertile SLB.When the longitudinal diameter of buds were 4mm,the anthers epiderm were subsidence contracted,and the surface cell of anthers was unloose arranged in SLA.Apparently,the anthers of SLA are completely atrophy and degeneration,resulted in SLA failed to produce mature pollen.The toluidine blue staining of transverse sections of stamens revealed that the anthers of the SLA and SLB lines differed from each other at metaphase stage.The tapetal hypertrophy with excess vacuolation was observed,and they were not shrinked and translated to the secretory.Subsequently the microspores are defected in pollen wall formation,and later the microspores also underwent degeneration.Therefore,based on these findings,we hypothesized that the defective tapetal hypertrophy was involved in the male sterility in SLA during the tetrad stage.Comparative RNA sequencing analysis revealed that 101 genes were differentially expressed between SLA and SLB,which were identified by the qRT-PCR.With GO functional classifications,we found 25 groups in the following three main GO ontologies: “cellular component”,“molecular function”,and “biological process”.The results of gene ontology significant enrichment for 101 differentially expressed genes revealed that 55 genes were in molecular function categories,being involved in the process of iron ion binding,electron carrier activity,and energy transformation,and so on;44 genes were in the biological process,including the process of carbohydrate metabolic,proteolysis and response to oxidative stress;but only 2 genes were in the cellular component categories.These results showed that the mechanism of sterile is extremely complex in B.napus.KEGG metabolic pathways revealed that differentially expressed genes were mainly enriched in Phenylpropanoid metabolic pathway,including ACOS5(BnaC09g10950D),and three peroxidase genes(BnaA01g01280D,BnaA07g01880 D and BnaC01g02300D).This indicated that the Phenylpropanoid metabolic pathway played an important role in the abortion process of sterile SLA.High-throughput sequencing technology was used by re-sequencing to detect the variation between SLA and SLB.We found 119,863 SNP and 18,370 Indel sites by bioinformatics analysis,which were unevenly distributed in the chromosome,detected in the non-coding regions,e.g.intergenic region or introns.Based on the physical position of annotation genes on reference genome,we conferred 14,705 variant genes.Moreover,these variant genes are involved in the Nucleoside combination,the process of carbohydrate derivative,organic matter metabolism pathways,and so on.These above SNPs,Indels and structure variation would provide important markers of marker-assisted selection for B.napus breeding.Comparative RNA-seq and Whole-genome sequencing analysis,we found that 11 differentially expressed genes were simultaneously variated among them,and more than two variations were found in three genes(BnaA03Rf-4,BnaA10Rf-7 and BnaC05Rf-10).Moreover,four genes(BnaA03Rf-3,BnaA04Rf-5,BnaA10Rf-6,BnaAnn Rf-8)were detected with variation in intron region,two genes(BnaA03Rf-4,BnaC03Rf-9)were found with synonymous variations,and six genes(BnaA01Rf-1,BnaA02Rf-2,BnaA03Rf-4,BnaA10Rf-7,BnaC05Rf-10,BnaC07Rf-11)were detected with the variation in the upor down-stream regions of these genes.Moreover,8 DEGs showed the highest expression levels in Bu1(1mm buds)of SLA than in others,which were the candidate genes for furture functional characterization analysis in B.napus.