Unraveling The Genetic Basis Of Fertility Restoration For Cytoplasmic Male Sterile Line WNJ01A Originated From Brassica Juncea In Brassica Napus

Using cytoplasmic male sterility（CMS）to produce hybrids is one of the main ways to utilize the heterosis of rapeseed（Brassica napus L.）,and it can significantly increase rapeseed yield.At present,the cytoplasmic simplification of rape hybrids in China is prominent.The development of new stable sterile lines/restorer lines for rapeseed hybrid production is conducive to broaden the genetic basis for the utilization of of current rapeseed heterosis.The map-based cloning of restorer genes and the analysis of fertility restoration mechanism can speed up the application of the sterile lines/restorer lines in rapeseed hybrid breeding.In this study,the morphological observation,map-based of cloning of restorer genes and transcriptome sequencing of the F₂ population obtained from the cross of the WNJ01A CMS originated from Brassica juncea and restorer line Hui01 in Brassica napus were carried out to analyze the fertility restoration mechanism by restorer gene regulated.The relevant results are as follows:1.The morphological observation of the floral organs of WNJ01A and Hui01 showed that they were only significantly different in anther structure.The anthers of Hui01 were plump and covered with a lot of yellow pollen on the surface,and the length of the filaments was slightly longer than that of the pistils.However,the anther of WNJ01A was obviously short,albino and without pollen,and its filament length was significantly shorter than that of the pistil.In addition,the results of the fertility phenotype survey at multiple points（2010-2020）in the spring and winter rapeseed areas showed that WNJ01A maintained complete pollen abortion without being affected by temperature or photoperiod.2.To elucidate the genetic characteristics of fertility restoration of WNJ01A CMS by Hui01,the fertility of the F₁ and F₂ populations of a WNJ01A CMS×Hui01 cross was evaluated.It was found that all F₁plants produced normally fertile pollen.In the F₂population,the number of fertile and sterile plants were fitted the expected 3:1 ratio（χ_c²=0.5012,P=0.46）.Taken together,the results indicated that Hui01 can completely restore the fertility of WNJ01A CMS and the fertility restoration of WNJ01A CMS is controlled by a dominant gene.It was designated Restorer of fertility for WNJ01A（Rfw）.F-and S-pools were constructed by subjecting extremely fertile and sterile F₂ plants to high-throughput sequencing.Based on comparison with the reference genome,theΔ（SNP/In Del-index）analysis preliminarily mapped Rfw to a 27.62-33.87 Mb region（6.25Mb）on chromosome A09.Meanwhile,BSR-seq of F₂ population was used to analyze the flower bud transcriptome data,and the restored gene Rfw was mapped to a 29.7-33.2Mb region（3.5 Mb）with a 95%confidence interval value on chromosome A09.3.A total of 4454 sterile F₂ plants were genotyped by linked markers using map-based cloning,and the candidate region of Rfw gene was reduced from 0.8c M to0.05c M.In the BAC library constructed by Hui01,the positive clones M36A7E6 and M16A6E7（full coverage of the candidate region sequence）screened by the co-segregation marker TY21 were subjected to Illumina Hi Seq and Pac Bio sequencing,and the sequence information of 99.75kb was obtained.4.The genes in the candidate interval of Hui01 were analyzed and predicted through the Fgenesh gene-finder.Among the 28 predicted genes in this candidate region,there were two PPR genes[PPR-Nd1:（ORF19）and PPR-Nd2:（ORF20）].PPR-Nd1corresponded to Bna A09g46030D,Bra026882 and Bju A044087 while PPR-Nd2corresponded to Bra026884 and Bju A044085 in the reference genomes（A_n subgenomes）.In the CDS sequence and amino acid sequence alignment,it was found that the 2 PPR genes detected significant differences in multiple SNPs/In Dels in both non-PPR motif and PPR motif.The constructs of p CAMBIA3301-（PPR-Nd1,PPR-Nd2 and PPR-Nd1+PPR-Nd2）（Hyg）were introduced into Agrobacterium GV3101,which were used to infect using the sterile line WNJ01A hypocotyls.The pollen fertility results showed that the positive transgenic plants containing PPR-Nd1 all showed a fertile phenotype,confirming that PPR-Nd1 was the fertility restorer gene Rfw of WNJ01A CMS.5.RT-q PCR analysis of 32 known protein-coding genes showed that 18 genes involved in the mitochondrial electron transport chain（mt ETC）pathway were significantly upregulated in fertile buds.However,mt ETC regulates the energy metabolism pathway in mitochondria,which indicates that the mitochondria of CMS lines cannot produce the energy required for normal pollen development,resulting in the male-sterile phenotype.6.Transcriptome sequencing and proteome sequencing showed that there were 1679DEGs（1023 upregulated and 656 downregulated）in fertile compared to sterile F₂ buds.The upregulated differentially expressed genes（DEGs）were enriched in the Kyoto Encyclopedia of Genes and Genomes（KEGG）lysine degradation pathway and phenylalanine metabolism pathway,and the downregulated DEGs were enriched in cutin,suberine,and wax biosynthesis pathway.There were 261 specifically expressed DEGs（9and 252 in sterile and fertile buds,respectively）.Regarding the fertile bud-specific upregulated DEGs,the ubiquitin–proteasome pathway was enriched.17 DEGs with common up-regulated expression were identified in the combined transcriptome and proteome analysis.Among them,Bna C04g00180D are involved in mitochondrial respiratory metabolism.7.In transcriptome data,44 DEGs were confirmed to be involved in pollen and anther development.such as the development of tapetum,microspore and pollen wall.With the exception of a few genes such as POE1,35 genes were up-regulated in fertile buds.PPI analysis of DEGs showed that the four hub genes with the highest degree scores of interactions（Bna A09g56400D,Bna A10g18210D,Bna A10g18220D and Bna C09g41740D）were all RAD23d genes,which provides an essential connection between ubiquitylated proteins and the 26S proteasome.