QTL Mapping Of Flowering Time And Cloning Of Wax Related Gene BnaZGLs In Brassica Napus

In plants,flowering marks the transition to reproductive growth,thereby facilitating reproduction and formation of crop yields.Moreover,the flowering characteristics of different crop varieties contribute to determining suitable planting areas.The aboveground parts of terrestrial plants are covered with cuticles that function as natural protective barriers,with wax in the outermost layer of the cuticle enabling plants to resist abiotic and biotic stresses.In this study,we had two main objectives,the first of which was to use parents with different photoperiodic sensitivities,we constructed a BZ-DH population to map flowering time QTLs and performed leaf rhythmic transcriptome sequencing to identify candidate genes for the detected QTL intervals.The second objective,a waxdeficient mutant(zgl)produced by EMS mutagenesis and normal rapeseed material were used to construct segregating populations,and the wax-related genes on chromosomes A09 and C05 were mapped and cloned.And through transgene complementation and gene editing to verify that BnaKCS5s was the target gene.The main findings are as follows:1 QTL mapping and candidate gene analysis of flowering time in Brassica napus1.1 QTL analysis of flowering time in a BZ-DH populationIn this study,we reconstructed a genetic linkage map of BZ-DH population.Using flowering time data obtained from six environments,we detected 14 QTLs associated with flowering time,and on the basis of subsequent QTL meta-analysis,we obtained five consensus QTLs.Among these,cqFT.A03-1 was only detected in four environments belonging to semi-winter rapeseed area,whereas cqFT.C08-1 was detected in all six environments.1.2 Analysis of parental transcriptome dataTranscriptome sequencing was performed by sampling seven times from parents at 4 h intervals within 24 hours.In total,11,888 genes were differentially expressed in at least one of the time points.GO enrichment analysis was performed on the differentially expressed genes(DEGs),for which we obtained 123 significantly enriched and representative terms.KEGG enrichment analysis of the 11,888 DEGs revealed 36 significantly enriched pathways,among which,plant hormone signal transduction and plant circadian rhythm pathways were related to the regulation of plant flowering.1.3 Analysis of differentially expressed genes related to flowering timeAmong DEGs,175 genes were found to be associated with flowering time,among which,the largest number were involved in the photoperiod pathway.On the basis of an analysis of the expression patterns of these genes,we identified differences in the expression patterns of genes between materials,and there were also diversity in expression patterns of paralogous genes in the same material.1.4 Analysis of candidate flowering-time genes in QTL intervalsThere were four flowering time candidates genes in the QTL intervals.Three of these genes were homologous to EMF1 {EMBRYONIC FLOWER 1),NF-YA1 {NUCLEAR FACTOR Y,SUBUNIT A1),and COL9(CONSTANS-LIKE 9)in Arabidopsis,all of which function as negative regulators in regulation of the photoperiodic pathway.The fourth candidate gene was annotated as a PHD-finger protein in Arabidopsis,which may be associated with chromatin modification of the FLC protein.This study revealed the genetic structure of flowering time and laid foundation for genetic improvement of flowering time in Brassica napus(B.napus).At the same time,the constructed gene rhythmic expression profile provided data support for the dynamic expression regulation network of genes and the functional differentiation of paralogous genes.2 Mapping and cloning of the wax-related gene BnaZGLs in B.napus2.1 Phenotypic analysis of zgl mutantsCompared with the normal material 7-567,the epidermis of zgl glossy leaf mutants is characterized by a deficiency in wax accumulation,which results in the production of plant parts,including leaves,stems,and siliques,with smooth shiny surfaces,an obvious brilliant green.Cryo-scanning electron microscopic observations of leaves and stems,revealed a clear deficiency of epidermal wax crystals in the zgl mutant.The leaf permeability and water loss rate test results show that compared with Zhongshuang 11(ZS11),which is the material before EMS mutagenesis,zgl had stronger leaf epidermis permeability and higher water loss rate.2.2 Mapping and cloning of wax-related genes BnaZGLsThrough the genetic pattern analysis of zgl mutant,revealed that the glossy leaf trait was recessive and controlled by two pairs of nuclear genes.Using segregation populations,BnaC05.ZGL and BnaA09.ZGL genes were mapped within the 326.6 kb and 1.23 Mb intervals,respectively.In the respective intervals of C05 and A09 chromosomes,there was only one gene associated with the glossy leaf phenotype respectively.These two genes were found to be homologous to the condensing enzyme KCS5 in Arabidopsis.Cloning and sequence alignment analysis of the two genes in zgl and 7-567,revealed that conversion of a single base from G to A in both genes resulted in mutations in the conserved amino acids in the FAE1_CUT1_RppA domain.2.3 Expression analysis of BnaC05.ZGL and BnaA09.ZGLWe performed qRT-PCR analysis to determine the expression of BnaC05.ZGL and BnaA09.ZGL in eight tissues in rapeseed,and accordingly found that with the exception of roots,the two genes were highly expressed in other assessed tissues,among which,leaves were characterized by the highest expression levels.Moreover,in all examined tissues,the levels of BnaA09.ZGL expression were found to be higher than those of BnaC05.ZGL.These findings were consistent with result in ZS11 expression database.2.4 Functional verification of BnaZGLsA positive plant with phenotypic restoration was obtained through transgene complementation of BnaC05.ZGL.Using CRISPR/Cas9 technology to knock out these two genes at the same time,it was found that simultaneous mutation of four alleles of BnaZGLs resulted in the same glossy leaf phenotype as zgl.Moreover,we further verified that BnaKCS5s were the target genes.In this study,we mapped and cloned the wax-related genes BnaZGLs,and created materials for functional study of BnaZGLs genes,laying a foundation for research on wax synthesis pathway in B.napus.