Mapping And Candidatade Gene Analysis Of The White-flowered Gene In Brassica Juncea

Rape is one of the important oil crops with a high economic value.It is an important source of edible vegetable oil.Flower color is one of the important traits of rapeseed with the characteristics of phenotype intuitive,genetic stability,and less affected by the environment.Flower color not only has some influence on the growth and variety breeding of rapeseed,but also,a large planting area and a long flowering period for high ornamental value,has great potential in the development of tourism resources,increase the income of farmers,the growth of modern agriculture.In this study,genetic analysis and mapping of the white flower trait JG800-1 in Brassica juncea had been done.A series of molecular markers linked to white flowers were developed.And the white flower genes were isolated by the map-based cloning approach with the candidate gene approach.Our study provides a theoretical basis and practical basis for understanding the mechanism of flower color variation and carring out the flower color breeding.The main results of this study are as follows:1.Genetic analysis and gene isolation Genetic analysis showed two independent recessive genes?Bjpc1 and Bjpc2?control the white flower trait in B.juncea.Subsequently,we successfully generated nine BC₃ lines?designated XG1-9?with a 1:1 segregation at the white locus.Line XG1 was selected for mapping BjPC1.The allelic relationship between XG3 and XG1 was then identified through an assay of flower-color segregation ratio.Our resualt clearly indicated that the segregating loci of XG3 and XG1 were not allelic.So,line XG3 was selected for mapping BjPC2.2.Caritenoid analysis in yellow and white petals using HPLC We analyzed carotenoid profiles in yellow and white petals to investigate whether low pigmentation was due to decreased carotenoid accumulation.Esterified carotenoids were abundant in yellow petals.Furthermore,HPLC analysis without saponification revealed more carotenoid peaks in yellow flowers than white flower.HPLC with saponification showed that violaxanthin,9-cis-violaxanthin and cis-neoxanthin accounted for 91.6%of the total carotenoids in yellow petals,approximately eight times higher than their percentage in white petals.These results indicate that white petals do not accumulate carotenoid esters,thus leading to a decrease in carotenoid accumulation.Moreover,yellow flower petals in B.juncea are the result of heavy carotenoid?particularly violaxanthin?accumulation.3.Ultrastructure analysis of chromoplasts in yellow and white petals We used TEM to determine whether changes in chromoplast morphology contributed to reduced petal pigmentation in white B.juncea flowers.At the bud stage in both yellow-and white-flowered plants,petal plastids exhibited a chloroplastic structure with granal stacking.In yellow flowers only,a few plastoglobules?PGs?appeared as electron-dense granules in plastids.Two days befor anthesis,plastids of yellow petals began differentiating into chromoplasts,and PGs were less electron-dense than the bud stage PGs.However,plastids in white petals were barely visible,and only a few,electron-dense PGs were present.During anthesis,complete chromoplasts were present in yellow petals,filled with numerous,fully developed PGs?as suggested by a low electron density inside and high density on the surface?.In contrast,anthesis white petals did not exhibit further chromoplast development;the plastids appeared flattened,with only a few small PGs surrounded by an incomplete envelope membrane.4.Fine mapping of the BjPC1 gene and candidate gene identification To fine-map the BjPC1 locus,a BC₄ population comprising 2,295 plants was used to construct a map.Finally,the location of the Bj PC1 gene was eventually mapped to a0.13 cM region between the markers SSR4 and SSR11,which corresponded to 40 kb in the syntenic region of the B.juncea A02.An annotation analysis of the candidate interval genes from the BRAD database indicated that BjuA008406,homologous to Bra032956 in B.rapa and AtPES2 in A.thaliana which encodes a protein with phytyl ester synthesis and diacylglycerol acyltransferase activities,was a promising candidate for increasing esterified xanthophyll accumulation.5.Fine mapping of the BjPC2 gene and candidate gene analysis Combined BSA with whole-genome resequencing to fine-map the target gene BjPC2,we successfully detected two significant genomic regions for BjPC2,covering 2.45 Mb on chromosome B04.New SSR markers designed from sequences in these two regions allowed us to fine-map BjPC2,then localize it on to a 31 kb region on chromosome B04,where six genes were annotated.Our qRT-PCR analyses found that most of the six genes were lowly expressed or not detected in both yellow and white petals.Only BjuB027334?homologous to AtPES2 in A.thaliana which encodes a protein with phytyl ester synthesis and diacylglycerol acyltransferase activities?was significantly and differentially expressed,being far higher in yellow B.juncea petals.Additionally,whole-genome resequencing revealed that non-synonymous mutations between the yellow-and the white-flowered traits are present in BjuB027334.The loss of BjuB027334 expression is likely responsible for pigment absence in white B.juncea petals.Further expression profiling and RNA-seq analysis have partially confirmed this hypothesis.