Fine-mapping Of Indeterminate Inflorescence Gene BnDM1 And Improving Clubroot Resistance Of Restorer Lines In Brassica Napus

Canola plants with determinate inflorescence exhibit improved traits in field production,such as lower plant height,better lodging resistance,and more consistent maturity,which are more favorable for mechanized harvesting than those of indeterminate inflorescence plants,and it could be a new germplasm for breeding rapeseed varieties suitable for mechanized operations.Until now,there are few studies on determinate inflorescence in Brassica napus,and the research on the formation mechanism of determinate inflorescence is even less known.In this study,we took a natural mutant 6138 with determinate inflorescence as our research object,and analyzed the genetic control loci of determinate inflorescence formation by cytological observation,fine-mapping and transcriptome analysis.The main results were as follows:1.Phenotypic description of determinate inflorescence mutants.The determinate inflorescence mutant showed lower plant height without negative effect on yield,and had more secondary branches than plants with indeterminate inflorescences.During the reproductive growth period,the apical meristem(SAM)of the determinate inflorescence transformed into floral meristem(FM),which eventually formed a variant flower structure,forming determinate inflorescence.2.Mapping of candidate genes and gene function analysis.The determinate inflorescence trait of mutant 6138 is controlled by two recessive genes(Bndm1 and Bndm2).The Brassica 60 K Illumina Bead Chip Array and Brassica 50 K Illumina Bead Chip Array combined with BSA was used to map Bn DM1 and Bn DM2.Bn DM1 and Bn DM2 were located in the 128 Kb and 2Mb intervals of C02 and A02 linkage groups,respectively,and Bna C02.KNU was identified as the target gene of Bn DM1 by genetic complementation.The expression level of Bna C02.KNU in SAM of determinate inflorescence was significantly higher than that of indeterminate inflorescence,and the Bna C02.KNU protein was localized in the nucleus.3.Promoter analysis of candidate genes.Compared with ZS11,in the mutant 6138,there is a 623 bp deletion in the promoter region of Bna C02.knu,which contains a Helitron transposon.The deletion in the promoter region enhanced the promoter activity of Bna C02.knu by more than two times and the expression of Bna C02.knu gene was significantly increased.This 623 bp deletion is not widely distributed in natural populations.Among,253 materials,there are 198 lines with this fragment and their phenotypes are indeterminate inflorescence.4.Comparative analysis of transcriptome data.Compared with the indeterminate inflorescenc e,DEGs were significantly enriched in biological processes such as the formation of floral organs at the T1 stage,that is,before the formation of the determinate inflorescence.In addition,it is mainly enriched in biological processes such as the formation of stamens,the morphology of post-embryonic plant organs,the development and formation of carpels and photosynthesis.AG,KNU,CRC were significantly up-regulated in both periods,while WUS,SUP,LFY,CLV3 were opposite.Other genes may participate in the WUS regulatory network together with KNU to regulate FM activity and produce determinate inflorescence phenotype.Ogura CMS is one of the effective ways to use heterosis in Brassica napus,and the breeding of excellent restorers has become the difficulties in the utilization of this system.In recent years,clubroot has become the main disease oilseed rape in China,and breeding excellent restorer lines with clubroot resistance is beneficial to the development of rapeseed production.In this study,using Bing 409 R,Hua Shuang 5R as Donor parents and 18 radish cytoplasmic male sterility restorer lines without the clubroot disease resistance locus as receptor parent,and the molecular marker-assisted selection technique and backcross breeding were combined to select excellent restorer line with clubroot disease resistance.The main results obtained were as follows:The two dominant clubroot resistance loci,Pb Ba8.1 and CRb,were introduced into eighteen excellent restorer lines without clubroot resistance,and the excellent restorer lines with the genome background recovery rates as high as 99% were obtained.The new clubroot resistant cultivars Kenyouza 741 R and Huayouza 706 R were bred by using the excellent clubroot-resistant restorer line R2163 R as the male parent,and the male sterile line 116 A and the sterile line Z11 A as the female parents,respectively.Kenyouza 741 R and Huayouza 706 R showed good clubroot resistance and agronomic traits in field performance.