| As an important oil crop,Brassica napus is not only the main source of edible oil and feeding values,but also widely used in industry.Therefore,it is necessary to increase the production for meeting the growing demand.Heterosis was the universal phenomena in agriculture,which could increase the production and improve the quality of crops.Of which,the male sterile is a useful tool for the complete male sterility and no negative cytoplasmic effects,which has been widely used in the production of hybrid varieties in rice,mazie,and rapeseed,respectively.Numerous results showed that the mechanism extremely complex,which are involved in stamen development,diversion of pollens with many genes and metabolic pathways during the process of pollination,but the mechanism of male sterility was unclear.In present study,analysis of the crucial stages of pollen abortion between the D3 A and D3 B by morphological observation and cytological analysis.Then detecting the variation and expression levels of DEGs between D3 A and D3 B through the high-throughput sequencing technologies.These works will provide the foundation for the functional study of the male sterility genes in D3 A.Meanwhile,it will enhance the genetic resources of heterosis utilization,which is very useful for accelerating the breeding of the Genetic Male Sterile Line and agronomic traits improvement in the future.The main results are as follows:1.The phenotypic characterization of the flowers and floral development appeared normal by the stereoscopic analysis between the D3 A and D3 B,but the ovary stigmas of D3 A were higher than those of the D3 B lines.Meanwhile,the anthers were gradually degenerated during the late development of flowers,which resulted in abnormal releasing of pollen.The results of scanning electron microscopy analysis showed that the base anthers were shrinked and the epidermal cells were tightly arranged at the early stages of anthers in D3 A lines.However,with the development of flower buds,the pollen wall of D3 A were obviously shrinked until loss of activity,eventually inactivity anthers cannot normally crack and release pollen.Whereas the anthers are normally cracked and release a large mature pollens in D3 B.In addition,pollen mother cells were normally formed at the early flower stages by the paraffin section analysis,but the pollen mother cell structure in D3 A was loose and the tapetum had begun to appear vacuole state.In tetraploid stages,tapetum cells showed highly vacuolization with no tetrad structures in D3 A lines,indicating that the tapetum cells were degraded in advance and not transformed to the secretory type tapetum,and no enough nutrients for the microspores can not be formed to release pollens led to sterile of D3 A line.Therefore,we hypothesized that the abortion of D3 A may be the abnormal type of tapetum.2.Comparative RNA sequencing analysis revealed that 374 genes were differentially expressed between young buds(≤1 mm)and middle buds(1-2 mm)in D3 A,and 1458 in D3 B,respectively.Meanwhile,679 differentially expressed genes were detected in young buds of D3 A and D3 B,and 1423 in the middle buds,respectively.However,based on the results of cytological analysis,90 DEGs were specifically expressed in young flower buds,of which,23 genes were up-regulated and 67 were down-regulated,respectively.According to the Brassica napus genome database,these genes were annotated and mainly involved in catalytic activity and protein binding,including 1 with meiosis of pollen mother cells and 5 transcription factors.In addition,GO function and KEGG Pathway showed that we obtained 135 significant GO terms and 9 significantly enriched pathways between the D3 A and D3 B,respectively.Among the 9 pathways,Wnt signaling pathway,TGF-beta signaling pathway and mRNA monitoring pathway had been found and significantly associated with fertility.In addition,we found the genes BnaC02g17290 D and BnaC05g01600 D,which were the candidate genes associated with the Wnt signaling pathway and the mRNA monitoring pathway,respectively.3.Whole genome resequencing technology was used to detect the variations between D3 A and D3 B.We found 2176589 and 2163868 SNPs,448107 and 444573 InDels,118415 and 117250 SVs,37005 and 36146 CNVs between the D3 A and D3 B respectively.Howerer,the SNPs and InDels were uniformly distributed on A subgenomes of B.napus,but unevenly on the C subgenomes,especially in the C02,C03,C05,C07 and C09 chromosomes.About 50% of C05 chromosome was the continuously unmutated regions.Among the SVs,the number of chromosomal rearrangements was much higher than others variations,and the rearrangements in D3 A was slightly more than that in D3 B,which might lead to the differentially expressed genes between the D3 A and D3 B,respectively.The deletion numbers of gene copy is much higher than their duplications in CNVs,and the deletions numbers in D3 A were obviously more than that in D3 B.Whereas the genes deletion could affect the gene coding area or shorten the Open Reading Frame of genes to alter their expression levels with different phenotypic variations among individuals,the majority of differentially expressed genes could be down-regulated in D3 A.In all,these variations were usually occurred in non-coding regions such as intergenic regions and introns,while the semantic variations could be detected in exons and other regions in D3 A that were more than in D3 B.Therefore,comparison of the semantic variation sites in D3 A and D3 B will provide selecting the molecular markers with steriles in the future.4.Comparision between RNA-seq and Whole-genome sequencing analysis,10 genes with the variations in exons were annotated between the D3 A and D3 B.Using the qRT-PCR analysis,8 genes showed obviously lower expression levels in young buds of D3 A than that in D3 B,consistent with the RNA-seq analysis.However,the gene BnaA03g44650 D showed the reverse expression between the D3 A and D3 B,indicating that the transcriptome analysis is more sensitive than qRT-PCR in detecting the expression levels of the low abundance transcripts genes. |
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