Molecular Mechanism Of The Major Ornamental Trait Variations In Kale

Kale(Brassica oleracea L.var.acephala),a variety of Brassica oleracea,is used for food and ornamental purposes,the latter because of its different leaf colors and shapes.Ornamental kale is an extremely popular ornamental plant due to its cold tolerance,wide adaptability and easy cultivation,and has been widely used in the area north of the Yangtze River Basin in China.However,there are some problems in the use of kale.Firstly,Ornamental kale is originated in the Mediterranean region.There are few domestic varieties,and most of the seeds are purchased from Japan and other countries.Secondly,the diversity of kale resources is still insufficient,as the mechanism of some main ornamental traits is not clear,and we therefore cannot create new varieties or alter related genes in plants.We introduced ornamental kale varieties from other countries and created more than 500 DH lines using microspore culture methods since 1998.These DH lines contain wide variation in leaf color and leaf shape,which creates the theoretical foundation for breeding the desired traits and provides materials for the study of the molecular mechanism behind the primary ornamental traits.Red leaf kale and white leaf kale were used as the experimental material to address the mechanism underlying red leaf color;anthocyanin content and distribution were measured,the inheritance pattern was analyzed,and the candidate gene was subjected to fine mapping and sequence analysis.We created a variegated leaf DH line;measured chlorophyll,copper,iron,and JA content,and chlorophyll biosynthesis precursors;and examined the ultrastructure of chloroplasts in kale leaves at three different developmental stages.We analyzed gene transcription in leaves at the different developmental stages to gain an insight into the changes in pigment distribution underlying leaf variegation.We created a bicolor leaf DH line,measured anthocyanin,chlorophyll,IAA,ABA,and GA3 content,and analyzed gene transcription in leaves at the different developmental stages to gain insights into the changes in pigment distribution underlying bicolored leaves.Lobed leaf kale and entire leaf kale were used as the experimental material to explore lobe leaf formation;the inheritance pattern was analyzed,the candidate gene was fine mapped,and three possible mechanisms of lobed leaf shape formation were proposed.1.Fine-mapping of a gene for the red leaf,Re,in ornamental kale(Brassica oleracea L.var.acephala)The red inner leaf phenotype of ornamental kale is caused by anthocyanin accumulation and is an important agronomic trait.Anthocyanin is distributed in seed coats,cotyledons,hypocotyls,young leaves,ornamental inner leaves,buds,and seed pods.The red leaf DH lines,?Y005-15‘ and ?Y005-8,‘ and the white leaf DH lines,?Y011-13-38‘ and ?JC007-2,‘ were used to construct two populations for mapping.The white phenotype is controlled by a single recessive nuclear gene,and the white leaf kales in F2 populations were used for mapping.Re was mapped between C9In-9 and C9In-51 on chromosome 9(C09)at a distance of 0.19 and 1.8 c M,respectively using the first mapping population,and was located between BYIn3 and C09-In51 on chromosome 9(C09)at a distance of 0.003 and 1.8 c M,respectively using the second population.Twenty-six genes were identified in this region,but none were related to the anthocyanin biosynthesis pathway.On the basis of information from Ensemble Plants,transcriptome analysis of the mapping parents,and identification of anthocyanin biosynthetic genes in B.oleracea,we predicted that Bol035269,which encodes dihydroflavonol-4-reductase,is a candidate gene for Re and is located on Scaffold000035_P2.Sequencing of the mapping parents revealed a 2400 bp insertion and a 124 bp deletion in the white leaf line.Quantitative real-time PCR showed that Bol035269 in the red leaf line was expressed at an approximately 1045-fold higher level than that in the white leaf line.The gene was expressed in the organs that contain anthocyanin.DFR activity in the red lines was 6.2-fold higher than that in the white lines.Sequence analysis of DFR showed that the gene in red leaf was similar to that in other plants,and an MYB binding element was located in the promoter region.Based on the results,we inferred that DFR played an important role in red leaf formation.2.Molecular Mechanism of variegated leaf formation in ornamental kaleVariegated leaf is a significantly important trait of ornamental plants and a variegated leaf kale is created,with green margins and white centers.Leaf variegation during development proceeds as follows: S1,leaves are green;S2,white appears at the leaf center,although the margins remain green;and S3,the leaf center is white and the margins are green.We found that the white leaves contained lower chlorophyll content,abnormal chlorophyll biosynthesis precursors content and chloroplasts.Differentially expressed genes(DEGs)involved in chlorophyll metabolism and related to chloroplast organization were identified.In addition,genes involved in photosynthesis were determined through KEGG pathway analysis.Two co-regulated genes(b HLH160 and AOS)may be related with the bicolor leaf formation.The accumulation of Pchlide in white leaves may lead to the white leaves formation,and abnormal photosynthesis and chloroplasts development may also influence the leaf phenotype formation.3.Molecular Mechanism of bicolor leaf formation in ornamental kaleWe identified a peculiar bicolor leaf double haploid line,with green margins and red centers.The development of bicolor leaves can be divided into three stages: S1,S2,and S3.Stage 1(S1),leaves entirely red;Stage 2(S2),red pigment appearing in the centers of leaves and leaf margins slightly green;Stage 3(S3),red leaf centers and green leaf margins.Results showed that the bicolor leaf phenotype is gradually formed with anthocyanin degrading and chlorophyll accumulation.IAA content was positively correlated with anthocyanin content during the bicolor leaf development.During anthocyanin degrading from S1 to S2,cinnamate-4-hydroxylase(C4H)and transport inhibitor response 1(TIR1)were downregulated,while lateral organ boundaries domain 39(LBD39)was upregulated.Two peroxidases,two ?-glucosidases(BGLU),LBD39,LBD37,detoxifying efflux carrier 35(DTX35),three no apical meristem(NAC)transcription factors(TFs),and 15 WRKY DNA-binding protein(WRKY)TFs were downregulated in S3_S vs.S3_C.The bicolor phenotype was mainly linked to anthocyanin degrading and chlorophyll accumulation,and that anthocyanin degrading resulted from reduced anthocyanin biosynthesis and increased anthocyanin degradation.4.Fine-mapping of a gene for the lobed-leaf,Bo Ll,in ornamental kale(Brassica oleracea L.var.acephala)Leaf shape is an important agronomic trait of ornamental kale(Brassica oleracea L.var.acephala).The lobed leaf DH line ?Y005-8‘ and the entire leaf DH line ?JC007-2‘ were used to construct the population for mapping.Genetic analysis indicated that the lobed leaf trait was quantitatively inherited.By QTL-seq analysis,the candidate region for Bo Ll(lobed-leaf)was mapped on chromosome 9(BRAD,Brassica oleracea,chromosome v1.0).The mapping region was confirmed and narrowed by simple sequence repeat(SSR)and insertion-deletion(indel)markers in the F2 population,and Bo Ll was located between markers LYIn39(0.17 c M)and LYIn40(0.11 c M).According to the B.oleracea genome database(chromosome v1.0),the mapped interval(75.3 kb)contained eight genes,and 7 of which were annotated in BRAD and one was not.According to the other B.oleracea genome information on Ensembl Plant,the mapped interval(79.6 kb)contained eleven genes.Two orthologous genes of At LMI1/ATHB5 related to lobed leaves in Arabidopsis,Bol010029/Bo9g181710(BRAD/ Ensembl Plant)and Bol010030/Bo9g1181720,were identified as possible genes for Bo Ll,but sequence analysis revealed that no variations were found in their promoter and coding regions The expression levels of Bol010029/Bo9g181710 and Bol010030/Bo9g1181720 were significantly higher in young lobed leaves than that in young entire leaves.Among the other genes in the interval,only Bol010025 and Bol010031/Bo9g181730 showed co-dominance in the recombinant individuals,and their expression levels were similar in lobed leaves and in entire leaves.Three hypotheses were proposed for the formation of lobed leaf.