Screening And Cloning Of The Candidate Genes For Determinate Inflorescence Variation In Brassica Napus

Brassica napus?AACC,2n=38?is the main oilseed crop of China in Brassicaceae.Rapeseed inflorescence is usually indeterminate,which is important tissues containing pods and seeds for the determination of the rapeseed yield.This study reported one newly discovered natural mutant of determinate inflorescence with a terminal flower which results in early termination of flowering time and pod developmental stage,and provided one new way for plant-type regulation and also for breeding new varieties for mechanical harvesting.A combination of phenotypic analysis,genetic analysis,transcriptome analysis and gene cloning was used for studying the mechanism of determinate inflorescence,and the results are shown as follows:1.Field experiments indicated that the mutant plants displayed greater advantage than the wild plants,including plant height,plant stem,number of primary effective branches,number of secondary effective branches,primary effective branch height,number of plant effective branches,number of pods per plant,pod length,seeds number per pod,1000-seeds weight,flower size,petal size and other agronomic characters.Besides,the length of main inflorescence and number of pods per main inflorescence of mutant plants were slightly lower than that of wild plants.2.The apical meristems of wild plants and mutant plants at the 2-leaf stage,3-leaf stage,4-leaf stage,5-leaf stage,6-leaf stage and 7-leaf stage were detected by paraffin section technology.The results indicated that the determinate inflorescence of mutant plants started to vary from the wild plants between 5-leaf stage and 6-leaf stage.3.Mutant line of GD605-2?MT?and four DH lines of Ningyou 7,Bakow,97009and 97081 were crossed to produce F₁hybrids,and all of the F₁single plants showed determinate inflorescence,indicating that the determinate inflorescence mutation was recessive.In addition,the inflorescence phenotype appeared to be separated at F₂generation,and the separation ratio between wild plants and mutant plants in 2018was 13:3??2=0.073,P=0.788?;the separation ratio between orthogonal offspring of mutant plants and wild plants in 2020 was 13:3??2=0.519,P=0.471?,and the separation ratio between reciprocal offspring of mutant plants and wild plants in 2020was 13:3??2=2.169,P=0.141?,indicating that the mutant phenotype was controlled by two genes.4.Transcriptome sequencing was used to screen differentially expressed genes between determinate and indeterminate inflorescence,and the results were further verified by q RT-PCR.Through functional analysis of GO and KEGG,4100up-regulated genes and 2209 down-regulated genes were finally obtained.TERMINAL FLOWER 1?TFL1?and APETALA1?AP1?were identified as candidate genes,through published inflorescence regulatory network,Arabidopsis and the NCBI database.These two candidate genes are renamed Bn TFL1 and Bn AP1.5.Bn TFL1 and Bn AP1 were further cloned.NCBI Blast and DNAMAN 6.0 were used for sequence comparison.Bn TFL1 had 1066 bases,encoding a total of 178amino acids.The sequence analysis of Bn TFL1 indicated 99.53%DNA homology and98.88%amino acid homology between wild and mutant plants.Mutations at the 26^th?P?R?and 175^th?A?S?amino acid site were found.In addition,Bn AP1 had a sequence of 771 bases encoding 256 amino acids.The sequence analysis of Bn AP1indicated 98.96%DNA homology and 99.22%amino acid homology between wild and mutant plants.Mutations at the 122^th?S?P?and 160^th?A?S?amino acid site were detected.6.qRT-PCR analysis of Bn TFL1 and Bn AP1 in different tissues and stages of mutant and wild plants was carried out.Bn TFL1 was mainly expressed in the shoot apical meristem of wild and mutant plants,and the expression level of which was highest in the 6-leaf stage.Bn AP1 was mainly expressed in the leaves at 6-leaf stage in wild plants,while Bn AP1 was mainly expressed in the flowers?flower buds?at2-leaf stage of the mutant plants.This study confirmed that GD605-2?MT?is a novel determinate inflorescence mutant in Brassica napus.The mutant should be valuable in inflorescence morphology research,breeding new plant-type materials,breeding specific materials for tourism,and breeding varieties suitable for mechanization.