Mapping And Candidate Gene Analysis Of Clustered Bud Of Main Inflorescence Gene (Bnclib) In Brassica Napus L.

Rapeseed（Brassica napus L.）is an important oil crop in China.It is the main breedinggoal of rapeseed breeders to cultivate high-yield rapeseed varieties suitable for simplified and labor saving cultivation,and mechanized harvest.In simplified and labor saving cultivation conditions,the yield of siliques in the main inflorescence of rapeseed contributed the most to the yield per plant.It is one of the ideas to develop the suitable selection of rapeseed varieties for simplified and labor saving cultivation.The mutant rapeseed 12R1402 and its derivative 16R480 showed clusters bud of main inflorescence（Bnclib）.The average number of siliques in the main inflorescence of these two varieties was 175,and the highest was 250.These materials are suitable for breeding rapeseed varieties suitable for simplified and labor saving cultivation and high density planting.In this study,combination I（12R1402×Huyou 17）and II（16R480×7P71）prepared with two main inflorescences bud clusters 12R1402 and 16R480 were used for reciprocal crosses.The clusters bud of main inflorescence morphology of F₁and F₂populations was observed,and the genetic pattern of Bnclib traits was analyzed;extreme mixing pools were constructed in the F₂population to perform whole genome resequencing;the region associated with the localization of Bnclib traits was localized.Markers closely associated with Bnclib traits were screened in the associated regions.These markers assisted the construction of Bnclib NIL Transcriptome analysis was performed on genes differentially expressed in the associated regions.Combined with the results of q PCR,candidate genes related to the trait of Bnclib were screened.The main results are shown below:1.The Bnclib trait showed clusters buds in the main inflorescence.In the fruiting stage,the main inflorescence had more siliques,more Angle density,and more main inflorescence or the top of main inflorescence bifurcated.Genetic analysis showed that the separation ratio between Bnclib and wild type in F₂generation was 3:1,which indicated that the trait was single gene dominant inheritance.2.In combination I（12R1402×Huyou 17）,the genes controlling clustered bud of main inflorescence were initially mapped to four associated regions based on BSA resquencing,which were respectively chr A03 25.37-27.58 Mb,chr A03-random 4.41-4.63 Mb,chr A03-random 5.77-5.8 Mb and chr A09-random 3.39-4.02 Mb.The combinationⅡ（16R480×7P71）was based on BSA resquencing to locate clustered bud of main inflorescence gene into three associated regions on A3 and A4,which were respectively chra03 28.30-29.68 Mb and chra03-random 2.30-6.01 Mb and chr A04-random 12.77-13.81 Mb.The clustered bud of main inflorescence traits of the main inflorescence were all derived from the common parent 12R1402.In this study,the intersection of two locale correlation intervals was selected and the gene was located in the two locale regions of A3chromosome:（1）chr A03 28301316-28492991;（2）chr A03-random 5775817-5806729,with 22 and 2 candidate genes,respectively.Transcriptome analysis was performed on Huyou 17,Bnclib material 12R1402,and the Bnclib NIL（Bnclib NIL）constructed with Huyou 17 as the cyclic parent binding molecular marker.Among the 24 candidate genes,only one gene,Bna A03g53930D,was differentially expressed between groups in Huyou 17and 12R1402 and its Bnclib NIL.（FDR≤0.05,|log₂FC|≥1）.The q PCR validation of Bna A03g53930D gene between Huyou 17 and its Bnclib NIL showed that,the expression pattern of Bna A03g53930D was consistent with that of RNA-seq,and was extremely significantly different in the shoot apical and leaf of Huyou 17 and its Bnclib NIL.NCBI annotation Bna A03g53930D gene is cytochrome P450 gene,similar to the CYP81D11 gene in Arabidopsis thaliana,and plays a role in regulating jasmonic acid synthesis.3.The determination of GA,CTK,IAA,SLs,JA and BR content in the shoot apical of Huyou 17 by Bnclib NIL and wild type showed that,all the 6 hormones,including jasmonic acid,were significantly different between the Bnclib NIL and Huyou 17,and the content of 6 hormones in the Bnclib NIL were significantly lower than that in the wild type.Relationship between jasmonic acid and other 5 hormones and Bnclib traits,and the functional verification of Bna A03g53930D gene needs further study.Bnclib gene inheritance has no cytoplasmic effect,clustered bud of main inflorescence are a single gene dominant character,controlled by a pair of major genes.The Bnclib gene was located in the two correlation regions of chr A03 28.30-28.50 Mb and chr A03-random5.77-5.81 Mb on A03 chromosome,and there were 22 candidate genes and 2 candidate genes respectively.According to the analysis results,27 pairs of primers closely linked to Bnclib gene were designed and screened.Transcriptome analysis and q PCR verification showed that the only Bna A03g53930D gene was extremely differentially expressed in the shoot apical and leaf of Bnclib mutants and wild-type plants.Functional annotation indicated that this gene is a cytochrome P450 gene,which regulates jasmonic acid synthesis in Arabidopsis thaliana.