Fine Mapping Of BoVl Conferring A Variegated Leaf Phenotype In Ornamental Kale

Variegated leaves phenotypes are commonly found in plant leaves.So far,researchers have identified hundreds of variegated leaves mutants in Arabidopsis,cucumber,rice,Acer truncatum,and many other species,being the most recurrent ones a type classified as stripe mutants and variegated leaves mutants.The presence of variegated leaves mutant in a species is usually a result of the chloroplast development defect in the non-green region of the leaf.Therefore,this mutant is a suitable resource for studying chloroplast development,photosynthesis,and chlorophyll synthesis.The characters of variegated leaves have high utilization value in garden plants.These materials enrich the variety resources of ornamental plants and promote the breeding and application of excellent and new varieties.The regulation mechanism of ornamental plants with variegated leaves phenotype arose as a new research hotspot in the field of gardens and horticulture.Kale（Brassica oleracea var.acephala）gathered attention as an essential ornamental plant for beautifying and greening in the cold season due to its strong resistance to low temperatures,changeable leaf shape,and colorful leaf tone.Kale is a plant originally found on the Mediterranean coast,and it hasn’t been a long time since this commodity was first introduced to the Chinese market.Currently,there are few varieties of kale sold in China,and they usually vary in color,e.g.,purple,red,pink,and white.By looking into the features of this type of market in China,we have identified that breeding new varieties of kale with independent intellectual property rights and innovation the variation types in their ornamental characters are urgent problems to be solved in the kale sector.In the early stage of this project,our team obtained a variegate mutant with great ornamental value during the creation of a double haploid line of kale.The mutant leaves are green on the edge and white in the center,and the plant leaves are inlaid with white and green.During the ornamental period,the aesthetics of the whole plant resembles a lotus,which has high ornamental value.Based on the mutant phenotype observation,photosynthetic characteristics identification,and genetic characteristics analysis,the mutant gene Bo Vl was located with BSR-seq technology combined with a traditional map-based cloning method.The molecular mechanism of variegated leaves formation was predicted after obtaining the results of these experiments and led to the following conclusions:1.We measured the contents of chlorophyll and carotenoids in leaves at different growth and development stages and concluded at the S2 stage,chl a and chl b contents and total chlorophyll and carotenoids reached the lowest value.The chlorophyll and carotenoid concentration in white and green areas during the period of variegated leaves were also analyzed,and significantly lower rates were found in white areas.The results showed that the amount of photosynthetic pigment in white leaves was low,and the net photosynthetic rate also decreased significantly.Subsequently,the chloroplast ultrastructure of white and green leaves at the spotted leaves stage was examined,showing that the chloroplast stacking degree of white leaves at the variegated leaves stage was low and tended to collapse.2.The F₁,BC₁,and F₂populations were constructed by crossing the variegated leaves DH line’JC007-2B’of kale and the high generation inbred line’BS’.Then,the genetic characteristics of the variegated leaves trait were analyzed,and the results proved that the variegated leaves trait was controlled by a pair of dominant nuclear genes.In the F₂population,50 all-green plants and 50 white-green spotted plants were selected to construct two extreme mixed pools.By combining the results of BSR-seq analysis and the localization analysis results of traditional methods,the variegated leaves gene Bo Vl could be located between the two In Del Markers Q3I-8and Q3I-15 on the C03 chromosome.The genetic distances of the two markers from Bo Vl were 0.08 cm and 0.04 cm,respectively.The final physical distance of the localization interval is 6.737 KB,including three genes:Bo3g002070,Bo3g002080,and Bo3g002090.3.Furthermore,we combined the gene cloning results and deducted that Bo3g002080 is a candidate gene for Bo Vl.Also,Bo3g002080 was deemed as a homologous gene of the Arabidopsis gene At Med4 because it encodes a glycoprotein rich in hydroxyproline.This glycoprotein can be combined with the conserved subunits of transcription factors and RNA polymerase II to form a compound and promote the transcription process of these factors.The total length of the gene sequence of Bo3g002080 is 972bp and contains two exons and one intron.It was also identified that there are two base substitutions G→A and C→T,and one base insertion on the second exon of Bo3g002080 in the parent’JC007-2B’.The mutation of 395^thbase G→A in the second exon causes the 132^thamino acid in the conserved domain to mutate from arginine（Arg,AGA）to lysine（Lys,AAA）.The insertion of base C in the second exon caused frameshift mutation,resulting in the mutation of the179^thamino acid of’JC007-2B’from lysine（Lys,AAG）to glutamine（Gln,CAA）,which further led to the early termination of translation when the 195^thcodon became the termination codon.Bioinformatics analysis showed that JC007-2B’is considered a hydrophilic protein compared with’BS’,and its secondary and tertiary structures had irregular curls and shorter spirals.q RT-PCR analysis showed that Bo3g002080 was significantly overexpressed in the color transition phase（S2）,and its expression was2158.85 times higher than that in the green leaf phase.The expression levels of Bo3g002080 in different parts were also analyzed,with the root as the control,and the results showed that Bo3g002080 was highly expressed in leaves.The overall expression was 209 times higher than that in roots.In addition,the expression of Bo3g002080 was significantly overexpressed in the white region at the variegated leaves stage,which was 26.91 times higher than that in the green region.