Gene Mapping And Candidate Gene Analysis Of Purple-leaf Trait In Brassica Carinata

Anthocyanins are the main pigments of leaves,flowers and fruits in piants,which performs a variety of biological functions in plants and are beneficial to human health.The purple leaves of Brassica carinata（BBCC,2n=34）are brightly colored for their large amounts of anthocyanins.They can be used to breed new Brassica species with higher ornamental and nutritional values and to provide pure natural pigments for food.In this study,the purple and green leaves of Brassica carinata lines were used as the experimental materials for locating the purple pigments of leaves,genetic analysis and gene mapping of the purple-leaf trait,combing analysis of transcriptome sequencing（RNA-seq）and proteome sequencing for screening the genes related to the anthocyanin biosynthetic pathway.The results obtained are:1.Through phenotypic and microscopic observations:it is found that the front and back sides of the purple-leaf parental lines（BC-P01,P₁）are purple.The front side is darker than the back side,the leaves,stems and veins are all purple.Cross-sectional observation of BC-P01 revealed that the upper epidermis had a uniform distribution of purple pigment,and the light purple-red pigments in lower epidermis were also evenly distributed.The front and back sides of the green-leaf parent lines（BC-G02,P₂）are green,all leaves,stems and veins are green,and there is no magenta pigment on the upper and lower epidermis when observed in cross-section.The center of the front and back of the F₁leaves are light purple,the leaves,stems and veins are also light purple.The cross-sectional observation shows that the upper epidermis is scattered with light purple,and the lower epidermis is also very light purple.2.The F₁leaves from the crossing between purple-leaf parental line of BC-P01 and green-leaf parental lines of BC-G02 are all purple.The F₂population had 208 plants,among which the leaves of 149 plants were purple and 59 ones were green,fitting one segregation ratio of 3:1（x²=1.256,p=0.262）.The the BC₁population from F₁and the green-leaf parental line had 167plants,among which the leaves of 92 plants were purple and 75 ones were green,fitting one segregation ratio of 1:1（x²=1.731,p=0.188）.All those results indicated that purple-leaf trait of Brassica carinata is controlled by a single dominant gene.3.The ILP（Intron length polymorphism）markers were used to screen the purple-leaf DNA pool（P-Bulk）and green-leaf DNA pool（G-Bulk）each of which was the DNA mixture of 10 F₂plants.The Purple leaf-trait gene of Bc Purple in Brassica carinata was successfully mapped on the C03 linkage group.There were 3 markers were located on one side of Bc Purple and 12markers were on the other side.The closest markers of Bc Purple were Bnap PIP1267 and Bnap PIP1399,with the genetic distances of 2.7 c M and 0.9 c M,respectively.4.Through next-generation high-throughput RNA-sequencing methods,6682 DEGs were obtained between in the purple-leaf parental line and the green-leaf parental line,including 3239up-regulated genes and 3443 down-regulated genes.Through GO and KEGG enrichment and functional annotation analysis,41 DEGs involved in the anthocyanin biosynthesis were obtained,including 18 structural genes,7 transcription factors,10 transfer genes,2 genes encodingβ-glucosidase,and 4 genes belonging to the H⁺-APTase gene family.Among them,11 genes were enriched in GO and KEGG analysis,including DN11967＿c0＿g2（FLS1,1.78）,DN4934＿c0＿g1（FLS6,2.63）,DN9277＿c0＿g1（F3’H,4.14）,DN18387＿c0＿g2（F3’H,4.82）,DN17695＿c2＿g5（DFR,2.43）,DN13833＿c0＿g3（DFR,3.53）,DN11359＿c0＿g1（DFR,7.12）,DN14763＿c1＿g1（ANS,4.12）,DN8427＿c0＿g1（UF3GT,7.69）,DN5961＿c0＿g2（UGT75C1,6.65）and DN5961＿c0＿g1（UGT75C1,6.55）。5.Proteomic sequencing was performed on the purple-leaf parental line and the green-leaf parental line.As a result,285 DEPs were obtained.Compared with the green-leaf parental line,there were 175 up-regulated DEPs and 110 down-regulated DEPs in the purple-leaf parental line.GO and KEGG analysis enriched 8 differential proteins related to anthocyanin biosynthesis,including DN8578＿c0＿g1（CHI,0.50）,DN8345＿c3＿g1（CHS,0.57）,DN18761＿c0＿g1（CHS,0.27）,DN8938＿c0＿g2（AT5MAT,0.87）,DN8427＿c0＿g1（UF3GT,0.85）,DN5961＿c0＿g2（UGT75C1,1.09）,DN5961＿c0＿g1（UGT75C1,1.05）and DN9891＿c0＿g2（UGT78D2,0.31）.6.The consistency analysis of 6682 DEGs obtained by transcriptome sequencing and 285DEPs obtained by proteome sequencing revealed that 41 of them were differentially expressed at both transcription and protein levels.Among the 41 genes,38 ones had the same expression trend,and the other 3 Gene expression trends are inconsistent.According to the results of gene function annotation,11 genes are obtained,which are corresponding to 9 Arabidopsis genes:CHS,CHI,UF3GT,UGT75C1,AT5MAT,GBSS1,GSTF3,GSTF2 and GSTU20.Combined with the previous enrichment analysis,6 candidate genes were finally screened out.Only the genes of DN18761＿c0＿g1 and DN8345＿c3＿g1 were located in the interval of the Bc Purple gene,which were corresponding to the gene of arabidopsis anthocyanin synthesis:CHS,and the candidate gene that regulates the purple-leaf trait of Brassica carinata was named Bc CHS.The q RT-PCR analysis showed that the expression level of the Bc CHS in purple-leaf parental lines was 3.26 times higher than that in the green-leaf parental lines.We firstly conducted the genetic analysis,gene mapping,combined analysis of transcriptome sequencing and proteome sequencing,candidate gene screening for regulating purple-leaf trait in Brassica carinata.The research could provide reference information for breeding puple-leaf lines in Brassica carinata for sightseeing,the cloning of purple-leaf gene and the molecular marker-assisted breeding of purple-leaf trait in Brassica carinata.