Mapping And Preliminary Identification Of Sterile Gene In Recessive Homozygous Two-type Lines In Brassica Napus L.

Heterosis is an important ways to increase crop yield and improve the quality of crop varieties.While having a great pollination control system and parental materials are the key to making full use of heterosis.At present,9012A bred by An Hui Academy of Agricultural being belong to the recessive nuclear sterile system is one of the best pollination control ways in Brassica napus L.The recessive completely nuclear sterile system can produce seeds efficiently,which avoids pulling out 50%fertile plants.Breeding recessive homozygous two type and temporary maintainer lines are critical to the pollination control system.Therefore,locating sterile gene and developing molecular markers are important to breed recessive homozygous two type and temporary maintainer lines.In this study,two DNA pools with 30 fertile plants and 30 sterile plants of recessive homozygous two type WSLAB were constructed by mixing an equal amount of DNA,30×depth of re-sequencing was conducted.Mutmap+and QTL＿seq R pipelines were used to locate the candidate interval of the sterile gene,and SSR and InDel markers related to sterile gene were also developed.Transcription data of bud of sterile and fertile plants were analyzed to deduce candidate sterile gene using Darmor-bzh based as reference sequence.GO enrichment,KEGG enrichment and q RT-PCR were applied to analysis candidate sterile genes,then cloning and bioinformatics analysis for candidate sterile gene were aslo done.The main findings are as follows.1.Analysis of the genetic pattern of fertilityWe had conducted three sib-crossing generation crossed between WSLA and WSLB,counting the number of sterile and fertile plants in these generations.A total of 98 plants were counted in 2019,including 44 sterile plants and 54 fertile plants.In 2021,a total of134 plants were counted,including 73 sterile plants and 61 fertile plants.In 2022,a total of 658 plants were counted,including 343 sterile plants and 315 fertile plants.The fertility segregation ration of datas in the three years were consistent with the theoretical value of1:1(χ_c²<χ2_0.05),and we observed that the fertility was not afferected by other environmental factors.Segregation analysis showed that the sterile trait of recessivenomozygous two-type lines should belong to the quality trait attributing to single gene and its genetic pattern meet to accord with Mendel’s law.2.Chromosomal localization of sterile gene candidate intervalsBased on BSA analysis,DNA of a pool of sterile offspring and a pool of fertile offspring in WSLAB population were extracted respectively,re-sequenced at a depthof30×.Darmor-bzh and yellow-seeded DH cultivated being as the referencegenome,the re-sequencing data of the sterile parents WSLA,the fertile parents WSLB.sterilet and fertile offspring pools were preprocessed,and the sterile gene was located in the 43-46Mb of C09 chromosome using Mutmap+and QTL＿seq R method.3.SSR molecular markerMISA software was used to identify microsatellite and complex microsatellite sequences in the 43-46 Mb of C09 chromosome.In combination with the primer design software Primer3,a total of 42 pairs of SSR primers were designed rapidly and efficiently in the candidate interval.These SSR primers were used to amplify the DNA of sterile plants and fertile plants by PCR respectively.After polyacrylamide gel electrophoresis and silver staining developed,two SSR markers closely linked to sterile traits were obtained.4.InDel molecular markerVisualize the InDel variation in the candidate interval between the parental pools and the sterile and fertile offspring pools by IGV software,insertion sites and deletion sites larger than 5 bases were screened.The IGV software was applied to target the start and end positions of InDel in this interval and to design InDel primers.A total of 21 pairs of InDel primers were designed.These InDel primers were used to amplify the DNA of sterile plants and fertile plants by PCR respectively.After polyacrylamide gel electrophoresis and silver staining had been finished,InDel markers closely linked to sterile traits were obtained.5.Analysis of candidate geneThe RNA mixed pool of recessive homozygous two-type lines of sterile and fertile buds was constructed and two biological repeats were set for transcriptome sequencing.The result shows that there are 287 differentially expressed genes,and 28 of these genes are located in the candidate interval.Analysis of 28 differentially expressed genes by cluster analysis,homologous sequence aligment with Arabidopsis,and functional annotation,it was found that BnaC09g45810D was homologous with AT5G10650,and the functional annotation was RING/U-box superfamily protein.In Arabidopsis,this family protein involved many processes of cells,such as programmed cell death,cell cycle regulation,protein degradation,etc.By GO enrichment and KEGG analysis of 28differentially expressed genes in the candidate interval,BnaC09g45810D was enriched into autophagy and participated in the autophagy process of cells.Analysis of BnaC09g45810D by q RT-PCR,the results showed that the expression level of the gene in sterile buds and stamens was significantly lower than that in fertile buds and stamens.Based on the above analysis,this study speculated that BnaC09g45810D was sterile candidate gene in recessive homozygous two-type lines in Brassica napus L.6.Cloning and bioinformatics analysis of candidate geneBnaC09g45810D was cloned and done to multiple alignment with the two sequences between sterile and fertile materials.There were only one base differences being responsible for one amino acid.Bioinformatics analysis showed that the proteins encoded by BnaC09g45810D in both sterile and fertile plants were hydrophilic.The two proteins have either not membrane or secretory.BnaC09g45810D encodes 46 protein phosphorylation sites in sterile and ferile plants.The secondary structure of the protein encoded by BnaC09g45810D in sterile plants includes 19.07%ofαSpiral structure,23.28%of extended chain structure,46.78%of the irregular curly structure and 10.86%β-Corner structure.While the secondary structure of the protein encoded by BnaC09g45810D in fertile includes 19.25%ofαSpiral structure,24.12%of extension chain,46.46%of irregular crimp structures and 10.18%β-Corner structure.The tertiary structure of BnaC09g45810D encoding protein are basically the same between sterile and ferile plants.