| Oilseed rape（Brassica napus L.）is one of the oil crops in China’s agricultural production.Selecting and breeding high quality varieties are important to ensure a steady improvement in the quality of agricultural products as the agricultural industry moves to modernization.Hybrid oilseed rapes have some characteristics such as good quality and adaptation,which also can effectively increase the yield of oilseed rape per unit.Male sterility in oilseed rape has been being a constant goal of plant breeding since1930s.In this study,SLAB—a recessive heterozygous two-type line of Brassica napus L.—was used as experimental material.Two pools with 30 sterile plants and 30 fertile plants of SLAB were constructed by mixing an equal amount of DNA,then 30×depth of genome-sequencing was conducted.The Mutmap+and QTL-seq R pipelines were used to locate the candidate interval of the recessive suppressor gene and SSR and In Del markers linked with recessive suppressor gene within this interval were developed by IGV and MISA methods.Transcription data of bud of sterile and fertile plants were analyzed to deduce candidate sterile gene as Darmor-bzh 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 also done.The main findings are as follows.1.Analysis of the genetic pattern of fertilityThe progeny segregation population crossed between SLA and SLB,and counting the number of sterile and fertile plants in segregation generation.A total of 238 plants were counted in 2020,including 125 sterile plants and 113 fertile plants.In 2021,a total of 537 plants were counted,including 259 sterile plants and 278 fertile plants.Through Chi-square test,the fertility segregation ratio of the two years was consistent with the theoretical value of 1:1(χc2<χ20.05),and it was observed that the fertility segregation of the population was not affected by the environment.Segregation analysis showed that the sterile trait of recessive heterozygous two-type lines was in accordance with Mendel’s law of genetic pattern and belonged to the quality trait attributing to single gene.2.Localization of recessive epistatic suppressor geneBased BSA analysis,the progeny segregation generation segregating population was constructed by the SLA and SLB parents,which were extracted leaf DNA to construct a pool of sterile progeny and a pool of fertile progeny,being re-sequenced at a depth of 30×.Darmor-bzh is cultivated by my research team being as the reference genome,the resequencing data of the sterile parents SLA,the fertile parents SLB,sterile progeny pools and fertile progeny pools were preprocessed,and the sterile gene was located in the 5.81-6.86Mb of A07 chromosome using Mutmap+and QTL-seq R method in Brassica napus L.3.SSR molecular markerMISA software was used to identify microsatellite and complex microsatellite sequences in the 5.81-6.86Mb of A07 chromosome.Combined with Primer3,a software for designing primers,a total of 34 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,4 SSR markers closely linked to sterile traits were obtained.4.In Del molecular markerVisualize the In Del variation in the candidate interval between the parental pools and the sterile and fertile progeny 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 In Del in this interval and to design In Del primers.A total of 37pairs of In Del primers were designed.These In Del 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,9 In Del markers closely linked to sterile traits were obtained.5.Candidate gene analysisThe RNA mixed pool of recessive heterozygous 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 7 differentially expressed genes in the candidate interval.Cluster analysis,functional annotation and enrichment analysis were performed on 7DEGs.In the KEGG homology analysis,the Bna A07g05460D gene was detected to be located in entry K03100,enriched in the production pathway of ta-si RNAs involved in RNA interference and important for the expression regulation of plant growth and development.Analysis of Bna A07g05460D by q RT-PCR 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 Bna A07g05460D was a sterile candidate gene in recessive heterozygous two-type lines in Brassica napus L.6.Candidate gene cloning and bioinformatics analysisUsing the recessive heterozygous two type line SLAB as the research object,Bna A07g05460D was cloned and done to multiple alignment with the two sequences between sterile and fertile materials.It was found that there were 75 base differences in the nucleic acid sequences of the Bna A07g05460D gene in the sterile and fertile traits,with an amino acid commonality of 79.6%.In addition,the results of sequence analysis of 05460D-F and 05460D-S showed that both showed hydrophilicity and instability,with the highest phosphorylation of serine;Protein conservative domain structures belong to Peptidase＿S26 gene family.10.37%of the secondary structure of 05460D-F protein isαHelical structure,27.09%in extended chain and 62.54%in irregular curl structure.28.26%of the secondary structure of 05460D-S protein isαHelical structure,20.19%in extended chain and 51.55%in irregular curl structure.The tertiary structure prediction results showed that there were significant differences between the 05460D-F and 05460D-S protein structure models. |
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