| In recent years,driven by rural revitalization strategy,rapeseed flower has become more and more popular as an important tourist resource.Sightseeing and tourism projects such as rapeseed flower festivals held around the country have gradually become popular industries in rural outings.The flower color of traditional rapeseed is yellow strains represented by golden yellow,dark yellow,earthy yellow and yellow.Through distant hybridization with closely related species,many other strains are gradually bred,such as white strains represented by milky white and white,the red strains represented by deepred,orange-red,red and peachblow,as well as the pinkpurple strains represented by light pink,pink and purple,etc.,but the genetic and molecular mechanism of rapeseed flower color variation are still not fully resolved,so it is necessary to carry out the problem in-depth study.In this study,the representative varieties orange-red petal(OrP),white petal(WP),Zhongshuang 11(ZS11)and other yellow-flowered varieties WYA47 and DHM42 of Brassica napus L.were used as research materials.Through phenotypic observation,genetics,metabolome,transcriptome and genome-resequencing,the key components and genetic rules for the production of orange-red and white petals were determined,the key candidate genes controlling orange-red and white petals were located and cloned,and the transgenic function verification revealed the genetic and molecular mechanisms of the orange-red and white petals in rapeseed,and provided theoretical basis for the flower color molecular breeding of Brassica napus.The main conclusions are as follows:1 Study on the molecular mechanism of the main gene BnaA07.PAP2 regulating the formation of orange-red petals1.1 Accumulation of anthocyanins and carotenoids in OrP petals causing orangered colorThe hypocotyls,cotyledons and young leaves of OrP all showed red color in varying degrees,and the petals began to show red after B5(bud length 5.0?5.9 mm).The red colorimetric value ?a of petals in S3 stage(petals just fully expanded and anthers indehiscent)is a positive value,indicating that OrP petals are reddish.Metabolomic comparative analysis showed that the relative contents of total anthocyanins in OrP petals were 5.420 and 3.345 times higher than those in ZS11 in S2(the flower was half-opened in a cylindrical shape)and S4(petals fully expanded to the maximum and anthers withered),respectively;the contents of total carotenoids were 0.880 and 0.555 times than that of ZS11,respectively.The total contents of yellow pigments(lutein and zeaxanthin)were 1.927 and 0.948 times than that of ZS11,respectively.Therefore,anthocyanins and carotenoids accumulated in OrP petals make the petals appear orange red color.1.2 Comparative transcriptomics analysis of OrP and ZS11For the transcriptomics comparative analysis,the materials included the buds in B3(bud length 3.0?3.9 mm)and B5(bud length 5.0?5.9 mm)stages,the petals in B5,B8(bud length 8.0?8.9 mm),S1(petals protruding more than 1/2 of the sepals but not expanded)and S3 stages,as well as the mixed pool petals in S1,S3 stages,which from the extreme phenotype individuals with orange-red and yellow flowers in the F2 population derived from OrP×DHM42.Taking ZS11 as control,a total of 13,047 differentially expressed genes(DEGs)were screened.KEGG enrichment indicated that the metabolic pathways related to anthocyanin biosynthesis,such as phenylpropane biosynthesis,flavonoid biosynthesis and isoflavone biosynthesis,were significantly enriched.Among the 139 genes related to the anthocyanin metabolism pathway,46 genes were significantly differentially expressed in OrP and ZS11.The qRT-PCR results indicated that Bna.DFR and Bna.ANS genes were significantly up-regulated expressed in the buds and petals in B5 stage of OrP in B5 stage.1.3 Genetic analysis and fine mapping of candidate genes for orange-red flowerThe F2 population was constructed by OrP and different yellow-flowered varieties.The petals of F1 individuals were orange red color,and the segregation ratio of orangered and yellow-flowered individuals in F2 population was 3:1;and the red colorimetric value ?a of petals in S3 and S4 stages,which from the individuals of F2 population derived from OrP×DHM42,showed multi peak distribution.These results indicated that the characters of orange-red petals were controlled by a pair of dominant major nuclear genes,and might be affected by minor genes.Two parents and the mixed DNA pools,which from the extreme phenotype individuals with orange-red and yellow flowers in the F2 population,were resequenced by 30 × whole genome.The results showed that the whole genome coverage of each sample ranged from 91.37% to 95.73%;and the BSA analysis showed that the major candidate genes of orange-red petals were located in the range of 17.239 ? 21.522 Mb on chromosome A07 of Darmor-bzh reference genome,and the fine mapping region of indel and SSR markers was 164.389 Kb,in which there were 34 genes.The results of transcriptome analysis showed that only BnaA07G0287000 ZS gene was significantly up-regulated expressed in the buds and petals of OrP,but hardly expressed in ZS11,so it was identified as a candidate gene named BnaA07.PAP2.The qRT-PCR results showed that BnaA07.PAP2 was highly expressed in the hypocotyl,cotyledon,young leaves and petals of OrP.1.4 BnaA07.PAP2 induces the expression of structural genes related to anthocyanin synthesis to regulate the formation of orange-red flowersThe candidate gene BnaA07.PAP2 was targeted knocked out in OrP via the CRISPR/Cas9 system,the petals of T1 generation positive individuals were yellow,of which the red colorimetric values ?a were decreased significantly.In B5 stage,the expression of Bna.DFR and Bna.ANS genes involved in anthocyanins synthesis were significantly decreased,which indicated that BnaA07.PAP2 regulates the synthesis of anthocyanins by inducing the expression of Bna.DFR and Bna.ANS genes,anthocyanins and carotenoids make the petals appear orange-red.2 Study on the molecular mechanism of major gene BnaC03.NCED4 regulating white flower formation2.1 The degradation of carotenoids in white petals leads to white petalsThe petals of white-flowered line WP were light green in B3,B4(bud length 4.0?4.9 mm)and B5 stages,turned into yellow gradually in B6(bud length 6.0?6.9 mm)to B7(bud length 7.0?7.9 mm)stages,and faded to white from S1 to S4 stages;ZS11 remained yellow from S1 to S4 stages.The yellow colorimetric value ?b of ZS11 petals in S4 stage was significantly higher than that of WP,indicating that ZS11 was yellowish compared with WP.Metabonomic analysis showed that the total carotenoid content of ZS11 petals was 1.777 and 1.969 times higher than that of WP in S2 and S4 stages,respectively.The total carotenoid content of ZS11 petals increased by 915.253 ?g/g(about 55.463%)from S2 to S4,while WP only increased by 374.597 ?g/g(about 40.346%).In the same period,the total content of the lutein and zeaxanthin in ZS11 decreased by 22.433% ?g/g(about 5.565%),but WP decreased by 83.455% ?g/g(about 34.705%).Therefore,the content of lutein and zeaxanthin in WP petals decreased sharply,which led to the petals fading from pale yellow to white.2.2 Comparative transcriptomics analysis of WP and ZS11For the transcriptomics analysis,the materials included the petals in B5,B7(bud length 7 mm),S1 and S3 stages,as well as the mixed pool petals in S1,S3 stages,which from the extreme phenotype individuals with white and yellow flowers in the F2 population derived from DHM42×WP.Taking ZS11 as control,a total of 9,557 differentially expressed genes(DEGs)were screened,of which 4,593 were upregulated and 5,228 were down-regulated expressed.KEGG enrichment analysis showed that phenylpropane,flavonoids,carotenoids,isoflavones,flavonoids and flavonols,porphyrins and chlorophyll were significantly enriched.2.3 Genetic analysis of white flower characters and screening of candidate genesAccording to the genetic analysis of the F2 population derived from DHM42×WP,the petals of F1 individuals were light yellow or pale white,and the segregation ratio of individuals with white/light yellow and yellow petals in F2 population was 3:1.The yellow colorimetric value ?b of petals in S3 and S4 stages in the F2 population showed multi peak distribution.It is suggested that WP white flower character may be controlled by a pair of dominant major nuclear genes,and may also be affected by minor genes.Two parents and the mixed DNA pools from the F2 population were re-sequenced by 30× whole genome.The results showed that the whole genome coverage of each sample ranged from 92.17% to 95.39%;and the BSA analysis showed that the major candidate genes of white petals were located in the range of 52 ? 54 MB on chromosome C03 of Darmor-bzh reference genome,which was homologous with 68.00 ? 70.14 Mb and 61.35 ? 63.33 Mb of C03 chromosome of ZS11 and No.2127 reference genomes,respectively.Transcriptome analysis showed that only BnaC03G0710000 ZS gene was up-regulated in WP among 215 genes in the candidate region of ZS11 reference genome,so it was identified as BnaC03.NCED4.The qRTPCR results showed that BnaC03.NCED4 gene was highly expressed in cotyledons and petals of WP,but low in roots,young leaves and petals of ZS11.2.4 The candidate gene BnaC03.NCED4 in WP regulates white petalsOverexpression of BnaC03.NCED4 gene in ZS11 resulted in white petals in T1 transgenic positive individuals,and the yellow colorimetric value ?b in S4 stage was decreased significantly,and the expression of BnaC03.NCED4 gene in petals of S3 stage was significantly increased.These results indicated that BnaC03.NCED4 gene could change petals from yellow to white by degrading carotenoids. |
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