Genes Cloning And Molecular Mechanisms Of Orange Flower And White Flower Formation In Chinese Cabbage (Brassica Rapa L.)

In Brassica crops,the flower color is an important trait and has wide variation source and rich variety.The flower color plays important roles in improving hybrid seed purity by removing mixed non-target material plants and testing the reliability of technology for interspecific trait transfer because of the characteristics of genetic stability and easy observation and it also affects the behaviour of pollinator.The flower color of Brassica crops has high ornamental value that can be developed into tourism resources,which will play great potential for promoting the development of regional economy.The studies of genes cloning and molecular mechanism of orange flower and white flower formation in Chinese cabbage were performed,which should lay the foundation for the breeding of flower color and revealing the mechanism of flower color variation in Chinese cabbage.Yellow-flowered inbred line 92S105,orange-flowered inbred line 94C9,white-flowered inbred line 15S1040,F₁ plants obtained from the crossing of the three inbred lines,BC₁F₁,BC₁F₂,and F₂population constructed from backcross or/and self-cross of F₁ plants were selected as study materials.These materials were used for fine mapping of orange flower and white flower genes,identification of the candidate genes for flower color,analysis of the sequence difference and expression pattern of the candidate genes,and analysis of carotenoid content and composition of yellow,orange,and white petals and expression levels of genes associated with carotenoid.The major results are as follows:1.Genetic analysis showed that a single recessive gene,Brof,controlled the orange flower trait.Using BC₁F₁ and BC₁F₂ mapping populations constructed from a cross between94C9（orange flower）and 92S105（yellow flower）,Brof was mapped to a 41.5-kb region on chromosome A09 delimited by InDel409 and dCAPS425 with the genetic distance of 0.4 cM.The mapping interval contained six annotated genes.2.Designed special primers were used to clone the cDNA sequences of six annotated genes in the mapping interval.The results indicated that only Bra037124 and Bra037125,which encode an AP2 domain-containing transcription factor and an SEC-C motif-and OTU domain-containing cysteine protease family protein,respectively,were successfully cloned.The sequence analysis revealed two SNPs resulting in amino acid residue changes in the coding region of Bra037124,as well as seven SNPs and one insertion leading to amino acid residue mutations in the coding region of Bra037125,between orange flower lines（94C9,15S1014,94B6）and yellow flower lines（92S105,09Q5）.The expression levels of six annotated genes located in mapping region were tested.The results showed that Bra037124and Bra037125 had high expression levels in petals and exhibited significant differences in expression level between 92S105 and 94C9,however,other genes had extremely low expression levels and were barely tested in petals,which were consistent with the results of cDNA sequence cloning.The expression pattern analysis of Bra037124 and Bra037125revealed that Bra037124 expressed in all tested tissues,however,expression difference multiple of Bra037124 between the two parents was much higher in roots than in other tissues（stems,cauline leaves,and petals）,and Bra037125 exhibited higher expression level in petals than in other tissues（roots,stems,and cauline leaves）.These results together with the previous study of homologous gene function of Bra037124 and Bra037125 encodes protein with unknown function,indicating that Bra037125 was most likely the candidate gene for orange flower.Based on the DNA sequence difference of Bra037125 between92S105 and 94C9,a co-segregating marker InDel601 was developed.3.Genetic analysis indicated that the white flower trait was controlled by two recessive genes,named Brwf1 and Brwf2.Using F₂ mapping population developed from the cross between 15S1040（white flower）and 92S105（yellow flower）,Brwf1 was mapped to a49.6-kb region on chromosome A01 flanked by S361 and S363 containing 9 annotated genes,and the genetic distance of S361 and S363 was 0.11 cM;Brwf2 was located in a 59.3-kb interval containing 12 annotated genes,and the genetic distance of the two closest markers（S82 and S83）was 0.08 cM.Based on annotated information of genes located in mapping regions of Brwf1 and Brwf2,Bra013602 and Bra031539,which encode a plastid-lipid associated protein（BrPAP）and a carotenoid isomerase（BrCRTISO）,were considered as the candidate genes for white flower.4.The alignment of predicted amino acid sequences of Bra013602 and Bra031539between white flower lines（15S1040,15S1001,17S690）and yellow flower lines（92S105,09Q5）were performed.The results indicated that one SNP led to amino acid residue mutation in the coding region of Bra013602,which was located in the conserved domain of BrPAP;one SNP resulted in the change of amino acid residue and it was located in the conserved domain of BrCRTISO,while one insertion（943 bp）at the 3’end resulted in 15amino acid residue changes,one amino acid residue insertion,and three amino acid residue deletions in the coding region of Bra031539,between white flower lines and yellow flower lines.The expression levels of Bra013602 and Bra031539 were detected in different tissues（roots,stems,cauline leaves,and petals）of 92S105 and 15S1040 using quantitative real-time PCR（qRT-PCR）.The results showed that compared to other tissues,Bra013602 and Bra031539 had high expression levels in petals,and the expression level of Bra013602 in petals of 92S105 was higher than that in 15S1040,while Bra031539 had no significant difference in expression level between the petals of the two parents.The ultrastructural analysis of chromoplasts in the yellow-flowered and white-flowered petals were carried out using transmission electron microscopy（TEM）.The results indicated that yellow-flowered petals had normal chromoplasts with numerous fully developed plastoglobules（PGs）,however,white-flowered petals showed abnormal chromoplasts with few PGs.These results confirmed that Bra013602 and Bra031539 were the candidate genes for white flower.Two markers（W543 and W61）co-segregated with Bra013602 and Bra031539,respectively,were obtained.5.The high-performance liquid chromatography（HPLC）was used to analyze carotenoid content and composition of yellow,orange,and white petals.The results revealed that the total carotenoid contents of yellow,orange,and white petals were 211.69±21.70μg/g,457.33±25.60μg/g,and 10.49±1.21μg/g,respectively,and the main carotenoid in yellow and white petals were violaxanthin and lutein,however,the total carotenoid contents had significant differences between yellow and white petals,which may result in the difference of flower color between yellow and white flowers;whereas orange petals showed considerable levels of lutein andβ-carotene,and the difference of flower color between yellow and orange flowers may be mainly due toβ-carotene accumulation.6.The expression levels of genes associated with carotenoid metabolism in yellow,orange,and white petals were examined.The RNA-seq results suggested that the expression levels ofβ-carotene hydroxylase（CHYB）and zeaxanthin epoxidase（ZEP）decreased in orange petals compared to yellow petals,which may be the reason forβ-carotene accumulation.The results of qRT-PCR showed that the majority of carotenoid biosynthesis pathway genes were down-regulated expression in white petals compared to yellow petals,which may lead to the decrease of carotenoid content in white petals.